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1. (WO2018224548) ANALYSES DE BIOLOGIE DÉLOCALISÉE DE PRO-THYMOSINE 100-109 POUR DÉTECTER UNE SEPSIE
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POINT-OF-CA E TESTING OF PRO-THYMOSIN 100-109 FOR DETECTING SEPSIS

FIELD OF THE INVENTION

[001] The present invention relates to an in vitro method for determination of a life-threatening condition of patients due to bacterial infections.

BACKGROUND OF THE INVENTION

[002] Sepsis (from Greek σήψις, decay) is a life-threatening response to infection, leading to excessive tissue damage, organ failure, and death. Sepsis represents a life threatening condition and common cause of death in intensive care units worldwide. Infection severity and patient's response to infection are crucial factors in sepsis development and mortality rates. Sepsis is associated with factors related to invading pathogens, most commonly Gram-negative bacteria, as well as the status of the host's immune system. Excessive immune cell activation leads to release of inflammatory mediators, which are usually determined in blood samples and used as biomarkers of infection.

[003] Different classes of molecules and processes are involved in alerting an organism of the presence of dangers that threaten survival. Damage-associated molecular patterns in inflammation and tissue repair as well as immune responses, play an essential role in the recognition of sepsis by the own organism. Current sepsis biomarkers include cytokines (e.g. interleukin 6 and tumor necrosis factor alpha TNF-a), lactate, acute-phase C-reactive protein, procalcitonin, damage-associated molecular patterns (DAMPs), cell surface receptors, e.g. triggering receptor expressed on myeloid cells-1 (Biron BM et al, Biomarkers for sepsis: what is and what might be? Biomark Insights. 2015; 10: 7-17).

[004] Among DAMPs, the prototype alarmin high-mobility group box 1 protein (HMGB1 ), an ubiquitous nuclear protein, has been extensively studied both as a biomarker and a therapeutic target (Fontaine M et al, Innate danger signals in acute injury: From bench to bedside. Anaesth Crit Care Pain Med. 2016; 35: 283-92). HMGB1 is secreted as a cytokine mediator of inflammation by activated macrophages and monocytes. HMGB1 plays a critical role at the intersection of the host inflammatory response to sterile and infectious threat. HMGB1 is actively released by stimulation of the innate immune system with exogenous pathogen-derived molecules and is passively released by ischemia or cell injury in the absence of invasion. Molecular mechanisms of HMGB1 binding and signaling through TLR4 reveal signaling pathways that mediate cytokine release and tissue damage. Accordingly, antibodies that neutralize HMGB1 may confer protection against damage and tissue injury during sepsis.

(Andersson U et al, HMGB1 is a therapeutic target for sterile inflammation and infection. Annu Rev Immunol. 2011; 29: 139-62).

[005] Prothymosin alpha (proTa) is an immunoactive polypeptide with a dual role, intracellular^, as a survival and proliferation mediator and, extracellularly, as a biological response modifier (loannou K et al. ProTa: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy. Cancer Immunol Immunother. 2012; 61: 599-614). US 4,716,148 describes a role for proTa in reconstituting immune function. EP 3 153 523 A2 discloses proTa as a therapeutically effective molecule for treating viral, bacterial and fungal infections as well as proliferative blood disorders. Further, diagnosis of proliferative disorders by determination of proTa levels has been described in US 5,248,591.

[006] Nevertheless, sepsis diagnosis and assessment of its severity is rather complex because of its heterogeneity. An incomplete understanding of the underlying pathobiology, having a large number of clinical signs and symptoms, makes it difficult to define a uniform clinical condition. The complexity in number of mediators and pathways involved in sepsis has contributed negatively to the defnition of a reliable single biomarker of this condition. In addition, sensitive and specific assays discriminating infectious from non-infectious cases are not available. Since sepsis cannot be detected at an early stadium, by the time symptoms are readily detectable, therapeutic approaches are no longer effective and tissue damage is irreversible. Consequently, novel surrogate biomarkers of sepsis are needed. Therefore, the state of the art represents a problem.

SUMMARY OF THE INVENTION

[007] The present disclosure relates to an in vitro method for determining the severity of a life-threatening condition of a patient due to bacterial infection, comprising the steps of: a) performing an assay method for detecting the presence of a decapeptide comprising C-terminal amino acids 100 to 109 of human prothymosin alpha (proTa) in a body sample obtained from said patient; b) measuring the concentration of said decapeptide comprising amino acids 100 to 109 of human proTa in the body sample; c) comparing said measured concentration of said decapeptide comprising amino acids 100 to 109 of human proTa to a predetermined reference upper value predictive of patient survival; and d) assessing of said patient of suffering a life-threatening condition by assigning an increased likelihood of an adverse outcome when said measured concentration of said decapeptide comprising amino acids 100 to 109 of human proTa is higher than said predetermined value early post-infection, or by assigning a decreased likelihood of an adverse outcome when said measured concentration of said decapeptide comprising amino acids 100 to 109 of human proTa is lower than said predetermined value early post- infection. The decapeptide according to the invention displays amino acid sequence SEQ ID NO: TKKQKTDEDD.

[008] The described method may provide quick determination of the severity of a life-threatening condition of a patient due to bacterial infection from diverse body samples at clinical laboratories with automated assay devices. The advantageous method gives clinicians a reliable parameter for effective assessment of patient survival in a septic condition at the early onset of an infection.

[009] According to the present disclosure, an adverse outcome may be sepsis or sepsis-related mortality. In one preferred embodiment, a predetermined value, which can be used as a reference upper value to evaluate the likelihood of survival of a patient suffering a bacterial infection, may be in the range from 4 to 6 ng/mL, preferably from 4.5 to 5.5 ng/mL.

[0010] According to the disclosure, higher levels of measured decapeptide comprising

C-terminal amino acids 100 to 109 of human proTa in said body sample early post-infection, in relation to a predetermined reference upper value predictive of patient survival, may be indicative of augmented degree of cell necrosis and increased likelihood of sepsis-related mortality.

[0011] According to the inventive method, lower levels of measured decapeptide comprising C-terminal amino acids 100 to 109 of human proTa in said body sample early postinfection, in relation to a predetermined reference upper value predictive of patient survival, may be indicative of augmented degree of cell apoptosis and reduced likelihood of sepsis-related mortality.

[0012] In a preferred embodiment, the determination of a decapeptide comprising C-terminal amino acids 100 to 109 of human proTa sample may be performed in a test sample selected from blood, serum or plasma. In another embodiment, a plurality of samples may obtained in a time-dependent manner, wherein test samples may be collected in intervals of time in the range of minutes, hours and days from the onset of bacterial infection.

[0013] According to the present method, the level of caspase-3 activation in said body sample may be further determined, preferably by immunocytochemistry.

[0014] In a preferred embodiment, the assay method for detecting the presence of said decapeptide comprising C-terminal amino acids 100 to 109 of human proTa in a body sample may be an immunoassay.

[0015] In one embodiment, said immunoassay may comprise the steps of contacting said body sample with a solid phase having at least one antibody that specifically binds a decapeptide comprising C-terminal amino acids 100 to 109 of human proTa; and detecting the binding of said antibody.

[0016] A kit for determining a life-threatening condition of a patient due to bacterial infection according to the invention may comprise a solid phase having antibodies capable of binding a decapeptide comprising C-terminal amino acids 100 to 109 of human proTa; and

reagents for the detection of said antibody-decapeptide complex, which may be used in accordance with the inventive method.

[0017] The kit of the disclosure may further comprise blocking buffer containing 2 % w/v BSA; washing buffer containing 150 mM NaCI, 10 mM phosphate buffer, 0.05 % Tween 20; and coloring buffer containing 1 % 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 30% hydrogen peroxide.

[0018] The necessity of introducing novel biomarkers for prognosis and early prediction of the outcome of septic patients is highly critical. According to the disclosure, the decapeptide proTa(100-109) can be used as a biomarker to monitor the progression of sepsis, as suggested from the in vivo experiments performed in mice infected with K. pneumoniae. ProTa(100-109) is the C-terminal fragment of proTa, a polypeptide with reported immunostimulatory activity and considered a danger signal or alarmin.

[0019] We infected mice with two K. pneumoniae strains: the clinical isolate L-78 and the prototype ATCC 43816. Infection of mice with either strain, increased serum levels of proTa(100-109), obtaining a differential pattern of increase over time. In the L-78 model, the concentration of proTa(100-109) rose in a time-dependent manner after the onset of sepsis.

[0020] Mice developed visible signs of bacteremia (lethargy, hunched posture, increased breathing rate and shiver), but as L-78 was phagocytosed and presumably successfully cleared, infection was resolved within 48 h. In contrast, in the 106 CFU ATCC 43816 model, the levels of proTa(100-109) significantly increased early post-infection (at 3 hours) and remained high until death of all animals at 15 hours post- infection. In this case, the bacteria rapidly reached the spleen and rose exponentially, as confirmed in spleen cell cultures.

[0021] To verify that the levels of proTa(100-109) correlate with apoptosis, we assessed the cell death pattern of murine splenocytes following K. pneumoniae infection. L-78 infection induced mostly apoptosis, which was more apparent in splenic monocytes/macrophages, while infection with ATCC 43816 induced necrosis, predominantly at later time points post- infection.

[0022] Without being bound by theory, we suggest cellular apoptosis and necrosis as causative elements for the presence of proTa(100-109) and, probably, intact proTa in blood serum of K. pneumoniae- infected mice, respectively. Activation of apoptotic pathways directly relates to alterations in procaspase-3/caspase-3 levels, and results in degradation of cellular components (Nicholson DW et al, Cell Death Differ 1999), including cleavage of proTa (Evstafieva AG et al, Exp Cell Res. 2003). Indeed, in L-78 infection, the levels of procaspase-3 were significantly reduced at the early stages post-infection indicating activation of caspase-3, whereas in the ATCC 43816 model, procaspase-3 levels decrease in a lesser extent. Our results suggest that L-78 infection massively induces caspase-3-mediated apoptosis at early stages post- infection in vivo.

[0023] To extend the aforementioned host-pathogen interactions in man, we exposed human monocytes/macrophages to the two K. pneumoniae strains. Monocytes infected with L-78 died mainly by apoptosis, whereas those infected with ATCC 43816 did mostly by necrosis. Quantification of proTa(100-109) levels in infected monocyte and macrophage culture supernatants showed significantly elevated proTa(100-109) concentration in supernatants of L-78-infected monocytes. In agreement with in vivo results, high proTa(100-109) levels corresponded to increased percentages of apoptotic cells. Confocal microscopy confirmed that L-78 was mostly internalized early post- infection, whereas at the same time point, ATCC 43816 was present, almost exclusively, extracellularly.

[0024] Finally, we tested the predictive ability of our assay in a "moderate" model of sepsis induced by K. pneumoniae ATCC 43816 strain. We infected mice with an inoculum corresponding to the LD50 of ATCC 43816 and quantified serum levels of proTa(100-109) over 48 hours. High levels of the decapeptide in the serum of animals which finally died due to sepsis were determined as early as 3 hours post-infection, indicating that proTa(100-109) serves as an early surrogate biomarker for sepsis outcome.

[0025] To our knowledge, this is the first report showing that the levels of proTa(100-109) can serve as a biomarker for monitoring the outcome of bacteria-induced sepsis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In the figures and drawings appended hereto:

Fig. 1 shows dot plots comparing post-infection concentrations of proTa(100-109) in serum of mice infected with Klebsiella pneumoniae strains L-78 (A) and ATCC 43816 (B,C) .

Fig. 2 shows flow cytometry dot plots of total splenocytes and spleen monocytes/macrophages isolated from mice infected with Klebsiella pneumoniae strains L-78 (A) and ATCC 43816 (B).

Fig. 3 shows bar diagrams comparing percentages of apoptotic/necrotic human monocytes (A, B) and in wfro-differentiated macrophages (D, E) infected with Klebsiella pneumoniae strains L-78 (A, D) and ATCC 43816 (B, E); and diagrams comparing post- infection concentrations of proTa(100-109) from infected human monocytes (C) and macrophages (F).

Fig. 4 shows diagrams comparing percentages of phagocytosis post-infection in human monocytes (A) and in vitro-ά iff e re nti ated macrophages (B) infected with Klebsiella pneumoniae strains L-78 and ATCC 43816.

Fig. 5 shows a mechanistic model for an in vitro method for determining a life-threatening condition due to bacterial infection with the use of proTa(100-109) as an early surrogate biomarker of sepsis according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Sepsis is a life-threatening condition that requires urgent care. Thus, the identification of specific and sensitive biomarkers for its early diagnosis and management are of clinical importance. The alarmin prothymosin alpha (proTa) and its decapeptide proTa(100-109) are immunostimulatory peptides related to cell death. We generated bacterial models of sepsis in mice using two Klebsiella pneumoniae strains (L-78 and ATCC 43816) and monitored sepsis progression using proTa(100-109) as a biomarker. Serum concentration of proTa(100-109) gradually increased with sepsis progression in mice infected with L-78, a strain which, unlike ATCC 43816, was phagocytosed by monocytes/macrophages. Analysis of splenocytes from L-78-infected animals revealed that post-infection spleen monocytes/macrophages were gradually driven to caspase-3-mediated apoptosis. These results were verified in vitro in L-78-infected human monocytes/macrophages. Efficient phagocytosis of L-78 by monocytes stimulated their apoptosis and the concentration of proTa(100-109) in culture supernatants increased. Human macrophages strongly phagocytosed L-78, but resisted cell death.

[0028] The present invention discloses a reference upper value of proTa(100-109) indicative of patient survival, allowing reliable assessment of sepsis progression and likelihood of patient mortality. No prior art disclosure suggests that higher levels of proTa(100-109) early post- infection, compared to said reference value, correlate, both in vitro and in vivo, with increased percentages of cell apoptosis. Moreover, we show that lower levels of proTa(100-109) early post-infection, in relation with said predetermined value predictive of survival, correlate with sepsis resolution and patient survival. Thus, the decapeptide of the disclosure provides an early surrogate biomarker for predicting bacteria-induced sepsis outcome.

[0029] In vitro methods are performed using mammalian cells, microorganisms or biological molecules in assays outside their normal biological context. In contrast, in vivo methods are conducted in living animals, including humans. According to the present application, results obtained from in vivo and in vitro experiments may be extrapolated into complex systems occurring in an entire organism and can be used to predict life-threatening clinical conditions in human patients.

[0030] A bacterial infection takes place when pathogenic bacteria cause a change in the normal functioning of an organism, leading to a pathological condition including systemic inflammatory response, resulting in shock and death. Examples of pathogenic bacteria belong to genera Bacillus, Campylobacter, Chlamydia, Clostridium, Enterococcus, Escherichia, Helicobacter, Klebsiella, Legionella, Listeria, Mycoplasma, Salmonella, Staphylococcus, Streptococcus, Vibrio, Yersinia etc.

[0031] According to the present invention, a body sample is a specimen for diagnostic examination, evaluation, and identification of a disease or clinical condition, including blood, plama, serum, saliva, excreta, body tissue and tissue fluids. In particular, test samples can be arterial blood, venous blood, cerebrospinal fluid, pleural fluid, amniotic fluid.

[0032] The decapeptide derived from human proTa according to the present disclosure has been previously described in Samara P et al, Development of an ELISA for the quantification of the C-terminal decapeptide prothymosin o(100-109) in sera of mice infected with bacteria. J Immunol Methods. 2013, wherein C-terminal amino acids 100-109 display amino acid sequence TKKQKTDEDD.

[0033] According to the present invention, a predetermined value refers to a standard or reference state of a function, as a basis for comparison. A predetermined upper value predictive of patient survival refers to the maximum value of a relevant parameter or substance concentration, which is not exceeded by an average human subject that survives a clinically pathogen-related condition; and it is usually obtained by statistical analysis of a relevant population of comparable subjects. According to the invention, a reference upper value of proTa(100-109) is used as a limit concentration, above which, values obtained from clinical samples are interpreted as an indication of sepsis related mortality. In contrast, lower values indicate patient survival.

[0034] An adverse outcome relates to any clinical condition originated by bacterial infections, in which the life of a patient is in danger. According to the present disclosure, an adverse outcome may be sepsis or sepsis-related mortality.

[0035] In accordance with the inventive method, early post-infection is considered any time point from the onset of infection, in which the symptoms of such bacterial infection are essentially not detectable or not indicative of sepsis progression, preferably from 0 hours to 24 hours, more preferred from 0 hours to 12 hours, most preferred from 0 hours to 3 hours.

[0036] Cell necrosis is a form of cell injury which results in premature autolysis-mediated cell death in living tissue. Necrosis may be caused by external factors such as infection, toxins, or trauma. Necrosis is, in general, detrimental to the organism and may lead to fatal outcomes. In contrast, apoptosis relates to the naturally occurring programmed cellular death, which is often associated with beneficial effects to the organism.

[0037] Cell death due to necrosis is independent from the apoptotic signal transduction pathway, resulting in loss of cell membrane integrity and an uncontrolled release of cellular components into the extracellular space. An inflammatory response is initiated in the surrounding tissue, which attracts leukocytes and nearby phagocytes that remove cellular material by phagocytosis. However, leukocytes release antimicrobial molecules, which also may lead to tissue damage, inhibiting the healing process. Thus, untreated necrosis results in accumulation of decomposing tissue and cell debris around the site of cellular death. It is therefore necessary to remove surgically necrotic tissue. A classic example is gangrene.

[0038] According to the present disclosure, collection of test samples in a time-dependent manner relates to the intervals of time in which samples are obtained from a patient, starting from time point 0 hours or onset of infection.

[0039] The term splenocyte can refer to any white blood cell type located in the spleen or purified from splenic tissue. Splenocytes consist of a variety of cell populations such as T and B lymphocytes, monocytes, macrophages and dendritic cells, having different immune functions.

[0040] Monocytes are the largest type of leukocyte, representing 2% to 10% of all leukocytes in humans, and can differentiate into macrophages and myeloid lineage dendritic cells. Monocytes are produced in bone marrow from precursors called monoblasts, bipotent cells that differentiated from hematopoietic stem cells. In adults, half of the total monocyte population is stored in the spleen. Three subclasses of monocytes have been described in human blood based on their phenotypic receptors. They are amoeboid in appearance and have granulated cytoplasm with an ellipsoidal nucleus. Monocytes circulate in the bloodstream for one to three days and, then, typically move into tissues throughout the body, where they differentiate into macrophages and dendritic cells. In response to insult or infection, inflammation signals promote monocyte migration within 8-12 hours.

[0041] Monocytes and their macrophage and dendritic-cell progeny are responsible for phagocytosis, antigen presentation, and cytokine production in immune responses. Phagocytosis is the process of uptake, digestion and disruption of microorganisms and foreign particles. Monocytes can perform phagocytosis using intermediary proteins, such as antibodies or complement, thus, coating the pathogen. They can also bind directly via pattern-recognition receptors present on invading microorganisms. Digested microbial fragments can serve as antigens and be incorporated into MHC molecules on the cell surface of monocytes (and macrophages and dendritic cells). Antigen presentation leads to activation of T lymphocytes, which then initiate a specific immune response against the antigen-bearing microorganism.

[0042] Macrophages (from Greek μακρος (makros) = large, φαγειν (phagein) = to eat) are white blood cells able to capture, engulf and digest foreign substances and microorganisms, cellular debris, cancer cells, etc. by phagocytosis. Macrophages are ubiquitous throughout the body, searching for potential pathogen threats. Besides phagocytosis, they play a critical role in non-specific, innate immunity and collaborate to initiate specific, adaptive immunity by recruiting other immune cells, such as lymphocytes, as well as presenting antigens to T cells. Macrophages also play an important anti-inflammatory role, decreasing immune reactions through the release of cytokines. Macrophages participating in inflammation are called M 1 macrophages, whereas those involved in decrease of inflammation and promotion of tissue repair are M2 macrophages.

[0043] Klebsiella pneumonia is a Gram-negative, non-motile, encapsulated, rod-shaped bacterium and the most significant member of the Klebsiella genus of the Enterobacteriaceae. It naturally occurs in soil and has been shown to increase crop yields through its ability to fix nitrogen in anaerobic conditions. It can also be found in the normal flora of the mouth, skin, and intestines. If inhaled, however, it may become an aggressive pathogen, causing lung tissue damage in humans. In recent years, Klebsiella species have become relevant pathogens in nosocomial infections, in which a patient is treated in a hospital for a specific reason, however, getting in contact with an unrelated pathogen leading to an additional clinical indication. In general, Klebsiella infections mostly occur in patients with a weakened immune system or suffering from debilitating diseases, including alcoholism, diabetes, liver disease, chronic obstructive pulmonary diseases, renal failure, glucocorticoid therapy, etc. The most common condition caused by Klebsiella outside hospitals is pneumonia, having a death rate of 50%, even with antibiotic treatment. The mortality rate can be close to 100% in patients suffering from bacteremia (presence of bacteria in the blood). Sepsis and septic shock usually follow upon entry of bacteria into the blood stream. Klebsiella can also cause infections in the urinary tract, lower biliary tract, and surgical wound sites.

[0044] Summarizing, Klebsiella may be responsible for a wide range of clinical diseases including pneumonia, upper respiratory tract infection, septicemia, bacteremia, thrombophlebitis, urinary tract infection, cholecystitis, diarrhea, osteomyelitis, meningitis and wound infection. In particular, patients in intensive care units with invasive devices in their bodies, such respiratory support equipment and urinary catheters, are exposed to bacterial contamination and increased health risk. The use of antibiotics may be responsible for an increased nosocomial infection risk by Klebsiella.

[0045] The thymus is a central lymphoid organ with crucial role in generating T cells and maintaining homeostasis of the immune system. More than 30 peptides, initially referred to as "thymic hormones," are produced by this gland. Although the majority of them have not been proven to be thymus-specific, thymic peptides comprise an effective group of regulators, mediating important immune functions. Thymosin fraction five (TFV) was the first thymic extract shown to stimulate lymphocyte proliferation and differentiation. Subsequent fractionation of TFV led to the isolation and characterization of a series of immunoactive peptides/polypeptides, members of the thymosin family. Extensive research on prothymosin alpha (proTa) and thymosin alpha 1 (Ta1 ) has shown that they may be of clinical significance and potential medical use. They may serve as molecular markers for cancer prognosis and/or as therapeutic agents for treating immunodeficiencies, autoimmune diseases and malignancies. Although the molecular mechanisms underlying their effect are yet not fully elucidated, proTa and Ta1 could be considered as candidates for cancer immunotherapy.

[0046] It has been suggested that the variable activity of proTa might be exerted through different parts of the molecule. In particular, the main immunoactive region of proTa is the carboxy- terminal decapeptide proTa(100-109). However, no potential use of decapeptide proTa(100-109) as a biomarker of sepsis progression and prediction of patient survival has so far been suggested.

[0047] Human prothymosin alpha (proTa) is a proliferation-related nuclear protein undergoing caspase-mediated fragmentation in apoptotic cells. Caspase-3 is the principal executor of proTa fragmentation in vivo. In apoptotic HeLa cells and in vitro, caspase-3 cleaves proTa at one major carboxy terminal (DDVD(99)) and several suboptimal sites. ProTa cleavage at two amino-terminal sites (AAVD(6) and NGRD(31 )) contributes significantly to the final pattern of proTa fragmentation. The major caspase cleavage at D(99) disrupts the nuclear localization signal of proTa, which leads to a profound alteration in subcellular localization of the truncated protein. Nuclear escape and cell surface exposure of endogenous proTa occurs in apoptotic, but not in normal cells. Ectopic production of human proTa confers increased resistance of HeLa cells toward the tumor necrosis factor-induced apoptosis (Evstafieva AG et al, Apoptosis-related fragmentation, translocation, and properties of human proTo. Exp Cell Res. 2003).

[0048] Increased levels of proTa(100-109) in serum can be directly correlated with the induction of massive cell apoptosis resulting from a severe bacterial infection. High-affinity-purified polyclonal antibodies (Abs) raised in rabbits are available in conjunction with a competitive ELISA assay for proTa(100-109) with a sensitivity of 0.1 ng/mL to 10 μg/mL (Samara P et al. Development of an ELISA for the quantification of the C-terminal decapeptide prothymosin o(100-109) in sera of mice infected with bacteria. J Immunol Methods. 2013), which according to the inventive method is acceptable for the quantification of the decapeptide in serum samples. In silico analysis suggests that such polyclonal antibodies are unlikely to cross-react with any other unrelated mouse or bacterial protein.

[0049] The peptide proTa(100-109) is generated upon truncation of polypeptide proTa (proTa) by activated caspase-3 in cells undergoing apoptosis (Samara P et al, ProTo and immune responses: are we close to potential clinical applications? Vit Horm. 2016; Evstafieva AG et al, Apoptosis-rela ted fragmentation, translocation, and properties of human proTo. Exp Ceil Res. 2003). Both molecules extracellularly act as alarmins, ligate TLR-4, induce the maturation of dendritic cells, and mount TH1 -biased immune responses in vitro (Omotuyi O et al, Evidence for Pro To- TLR4/MD-2 binding: molecular dynamics and gravimetnc assay studies. Expert Opin Biol Ther. 2015).

[0050] Without being bound by theory, we hypothesized that, if proTa and proTa(100-109) are released from damaged cells, they could serve as immune stimuli, acting similarly to HMGB1. To detect and quantify the extracellular release of these peptides, an ELISA test for proTa(100-109) has been developed (Samara P et al, Development of an ELISA for the quantification of the C-terminal decapeptide prothymosin o(100-109) in sera of mice infected with bacteria. J Immunol Methods. 2013). A significant increase in the concentration of

proTa(100-109) in serum has been described in Streptococcus pyogenes (S. pyogenes)-infected mice during the progress of infection.

[0051] To further evaluate the role of proTa(100-109) as a sepsis biomarker, we used Klebsiella pneumoniae {K. pneumoniae) as challenge-microorganism, a Gram-negative bacterium associated with aggressive infections (e.g. septicemia and pneumonia) and numerous nosocomial outbreaks (Pitout JD et al, Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance. Antimicrob Agents Chemother. 2015). We selected two K. pneumoniae strains of diverse virulence and properties (Tzouvelekis LS et al, KPC-producing, multidrug-resistant Klebsiella pneumoniae sequence type 258 as a typical opportunistic pathogen. Antimicrob Agents Chemother. 2013) and analyzed K. pneumoniae infection in mice in vivo and in human cells in vitro, focusing on the bacterial mechanisms of innate immune-related cell death, which leads to the generation of proTa(100-109). We further validated our results in a "moderate" model of sepsis in mice, and showed that early post-infection (pi) proTa(100-109) serum levels can predict mortality of individual mice infected with K. pneumoniae.

[0052] Experimental animal studies are essential in identifying and characterizing the pathophysiological stages of sepsis and for developing new therapeutic strategies and diagnostic tools. Bacterial infusion in mice is the most widely used model, as introduction of a single pathogen under controlled conditions provides reproducibility of the infection (Fink MP. Animal models of sepsis. Virulence 2014).

[0053] The disclosure provides a functional biomarker of sepsis, which can be successfully translated from the laboratory to a clinical setting. ProTa(100-109) is, according to the present invention, a specific and sensitive biomarker, which is easy to interpret and cost effective, so that it can be used routinely in diagnosis of sepsis and sepsis-related diagnosis.

[0054] Animals were infected with the clinically isolated strain L-78, which is of low virulence (50% lethal dose [LD50] > 108 colony-forming units [CFU]), highly resistant to antibiotics and endocytosed by monocytes/macrophages, and with the ATCC 43816 strain, which is highly infective (LD50 = 5 x 103 CFU), sensitive to antibiotics and not endocytosed. Based on the Kaplan-Meier survival curves of Tzouvelekis LS et al, Antimicrob Agents Chemother. 2013, for immunocompetent mice, animals were intraperitoneally (ip) infected with 108 CFU of L-78, and 5x103 or 106 CFU of ATCC 43816. As shown in Fig. 1 , low serum levels of proTa(100-109) were detected early post-infection with Klebsiella strain L-78.

[0055] In detail, CD-1 mice were intraperitoneally infected with L-78 (Fig. 1A) and ATCC 43816 (Fig. 1 B, C), respectively. Serum samples were collected shortly prior infection (0 hours), at 3, 6, 12, 24, and 48 hours post-infection (pi) for the L-78 and the 5x103 CFU ATCC 43816 groups (Fig. 1A, C), and at 3, 6, 12, and 15 hours pi for the 106 CFU ATCC 43816 group (Fig. 1 B). Each circle represents the concentration of proTa(100-109) from a single mouse at the

relative time point. In Fig. 1 C, gray circles depict mice that died between 30-45 h. Horizontal bars indicate mean values. *, p<0.05; ***, p<0.001 after one-way ANOVA followed by Dunnett's test (vs 0 hours).

[0056] Mice sera were analyzed by ELISA for proTa(100-109) as shown in Samara P et al, J Immunol Methods 2013. To rule out obvious non-specific interactions between proTa(100-109)-specific antibodies and any cross-reacting substance present in murine serum, we assessed if the primary structure of the decapeptide can be depicted within the amino acid sequence of currently known mouse proteins or proteins known to be encoded in K. pneumoniae (data not shown). A scan with the regular expression pattern against the proteins encoded in the most current version of the mouse genome showed that cross-reactivity was unlikely to occur.

[0057] In animals infected with 108 CFU of L-78, serum concentration of proTa(100-109) gradually increased during the course of infection with maximum quantity (5.72 ng/mL) detected at 48 hours post-infection (pi) (Fig. 1 A), which further reduced to background levels at 96 hours (data not shown). Mice infected with 106 CFU of ATCC 43816 exhibited a sharp increase in serum concentration of proTa(100-109) already in the first three hours post-infection (4.53 ng/mL) which remained relatively constant up to 15 hours post-infection (6.29 ng/mL) (Fig. 1 B). The distinct patterns of rise in the levels of proTa(100-109) among the two murine groups could be attributed to the diverse virulence of the strains. Specifically, animals injected with L-78 showed transient signs of infection and all resisted infection and recovered within 48 hours, while animals administered 106 CFU of ATCC 43816 died at 15 hours post-infection, due to generalized sepsis. Mice infected with 5x103 CFU of ATCC 43816 ("moderate" model of sepsis), showed a different pattern of increase in serum proTa(100-109) levels, with some animals showing high and others low concentrations of the decapeptide early post-infection (Fig. 1 C). This group of mice was used to validate our results and data are further analyzed and discussed.

[0058] To evaluate the impact of traumatic injections on proTa(100-109) levels, we additionally collected serum from mice punctured ip and injected with saline. No differences in proTa(100-109) levels prior and 3 h after traumatic injection were observed.

[0059] At the same time points to blood collection, spleens from two mice of the L-78 and 106 CFU ATCC 43816 groups were removed and bacterial load, expressed in CFU, was determined. Spleens were not contaminated with intestinal or other external bacteria during isolation, and the individual colonies on the plates were typical for Klebsiella. We sacrificed only two mice per time point per model based on the 3R principle (Replacement, Reduction, Refinement) for laboratory animal use, to verify similar results available in our laboratory from previous experiments.

[0060] It is well documented that intraperitoneal (ip) administration of bacteria leads to rapid and effective delivery to the bloodstream resulting in systemic infection (Franklin GA et al, Am J Surg. 2003). In animals infected with 108 CFU of L-78, the number of bacteria that entered the spleen between 3-12 hours was approximately 106 CFU, increased to ~107 CFU at 24 hours and decreased thereafter. Two mice from each group were sacrificed at the same time points as in Fig. 1A and 1 B and for an additional time point (96 hours) for L-78 infected animals to confirm bacterial clearance. Spleen homogenates were plated on agar and CFU counts were determined. In contrast, in animals infected with 106 CFU of ATCC 43816, the bacterial load in spleen significantly increased between 6-12 hours (up to -7x108 CFU at 12 hours) and sharply decreased afterward, as animals died due to generalized sepsis at 15 hours (p=0.0338). Apparently, ATCC 43816, although injected at a lower inoculum, multiplied rapidly in vivo, and led to multi-organ failure within 15 hours post-infection. At this time point, generalized sepsis was accompanied by extensive necrosis of vital internal organs (including spleen), and lower bacterial spleen load was detected (~104 at 15 hours).

[0061] The type and extent of cell death in spleen cells were evaluated by flow cytometry, using Annexin V (V) and propidium iodide (PI) staining. We analyzed total splenocytes, as well as spleen monocytes/macrophages (CD1 1 b+). L-78 infection induced apoptosis and ATCC 43816 necrosis in mouse spleen cells, as shown in Fig. 2. Total splenocytes and spleen monocytes/macrophages isolated from K. pneumoniae infected-mice at the indicated time points post-infection [0-48 hours for L-78 (Fig. 2A) and 0-15 h for ATCC 43816 (Fig. 2B)] were stained with Annexin V/PI and analyzed by flow cytometry. Percentages in lower right quadrants are apoptotic cells (Annexin V+PI-), and in upper quadrants necrotic cells (PI+). Prior to infection (0 h), few mouse splenocytes were apoptotic (-2% Annexin V+PI-). During the course of L-78 infection (3-48 hours), percentages of apoptotic splenocytes gradually increased (up to 12.9%) (Fig. 2A, left panel). In spleen monocytes/macrophages, percentages of apoptotic cells significantly increased up to 24 hours (34.9%) and then decreased (Fig. 2A, right panel). In animals infected with 106 CFU of ATCC 43816, the percentages of apoptotic total splenocytes were low and remained unaltered (Fig. 3B, left panel), but necrotic splenocytes significantly increased from 3 to 12 hours post-infection (Fig. 2B, right panel). High percentages of necrotic monocytes/macrophages particularly at 12 hours post-infection (69.6%) were also detected.

[0062] Combining the ELISA values (Fig. 1A) with the results of flow cytometry (Fig. 2A) in L-78-infected mice, a positive correlation between serum proTa(100-109) concentration and the percentage of apoptotic splenic monocytes/macrophages was observed. Before infection both were low, whereas post- infection the levels of the decapeptide gradually increased following the increase in the percentage of apoptotic cells.

[0063] To verify activation of the apoptotic pathway during K. pneumoniae infection, we determined procaspase-3 levels in lysates of mouse splenocytes by Western blotting (data not shown). Total protein extracts of pooled splenocytes from L-78- and ATCC 43816-infected mice were immunoblotted against procaspase-3. Apoptotic and necrotic HeLa cells were used as controls. β-Actin was used as a loading control. All samples were run on and cut from the same gel. We treated HeLa cells with TNF-a and emetine which led the majority of cells to apoptosis, or high concentration of doxorubicin which induced massive necrosis. Western blot analysis showed that the levels of procaspase-3 in HeLa lysates were significantly reduced only under apoptotic conditions (p=0.001 ), and remained unaltered when spleen cells were driven to necrosis.

[0064] In our in vivo models, early after infection (3 and 6 hours) with L-78, the relative level of procaspase-3 was reduced by half compared to pre-infection levels (p=0.0046 and p=0.0056, respectively) and was restored to background levels at 12-24 hours post-infection in parallel with bacterial clearance (p=0.0407 at 12 h; p=0.0061 at 24 h). In mice infected with 106 CFU of ATCC 43816, procaspase-3 protein levels decreased early post-infection (p=0.0066 at 3 h) and progressively increased from 6 to 15 hours (p=0.0037 at 6 h; p=0.0055 at 12 h).

[0065] In another set of experiments shown in Fig. 3, human monocytes (Fig. 3A, B) and in w'fra-differentiated macrophages (Fig. 3D, E) were infected with L-78 (Fig. 3A, D) and ATCC 43816 (Fig. 3B, E), and harvested at 0-180 min, stained with Annexin V/PI and analyzed by flow cytometry. Dark columns show percentages of apoptotic cells; white columns, necrotic cells. Mean values ± SD from 5 different healthy blood donors are shown. ***, p<0.0001 for L-78-infected apoptotic and necrotic monocytes (Fig. 3A), for ATCC 43816-infected apoptotic and necrotic monocytes (Fig. 3B) and for L-78-infected necrotic macrophages (D). *, p=0.0106 for L-78-infected apoptotic macrophages (Fig. 3D) and p=0.0229 for ATCC 43816-infected apoptotic macrophages (Fig. 3E) (one-way ANOVA). Supernatants from the same cultures were analyzed by ELISA and proTa(100-109) concentration is expressed as mean fold-increase ± SD from three independent experiments (Fig. 3C, F). *, p=0.0015 for L-78-infected monocytes (C) and p=0.0324 for L-78-infected macrophage (Fig. 3F) (one-way ANOVA).

[0066] Monocytes infected with L-78 showed a gradual increase in the percentage of apoptotic cells (from 6.85% at 0 min to 36.05% at 180 min) (Fig. 3A). On the contrary, monocytes infected with ATCC 43816 showed lower percentages of apoptotic cells (18.15% at 180 min), but the percentage of necrotic cells increased early post-infection (18.30% at 15 min) and remained high (30.80% at 120 min post-infection) (Fig. 3B). Unlike monocytes, human macrophages resisted infection with both K. pneumoniae strains, and much lower percentages of dead cells were recorded (<10%). Specifically, few macrophages incubated with L-78 were apoptotic (1-2%) (Fig. 3D). Macrophages incubated with ATCC 43816 showed a similar pattern of cell death, characterized by few apoptotic (<2%) and necrotic (<5%) cells (Fig. 3E). In parallel, supernatants of the aforementioned co-cultures were collected and analyzed by our ELISA. Gradually increasing concentrations of proTa(100-109), expressed as fold-increase, paralleled the increase of apoptotic cells from 30 to 180 min post- infection in the supernatants of L-78-infected monocytes (p=0.0015). The concentration of proTa(100-109) in the supernatants of ATCC 43816-infected monocytes remained low and relatively constant up to 180 min postinfection, as less monocytes were driven to apoptosis (Fig. 3C). On the contrary, as

macrophages infected with either L-78 or ATCC 43816 remained alive, the quantity of proTa(100-109) detected in their culture supernatant was low (fold-increase <3 for both bacterial strains, at all time points) (Fig. 3F). Nevertheless, the differences observed in L-78-infected macrophages over time were statistically significant (p=0.0324).

[0067] Only L-78 was efficiently phagocytosed by human monocytes and macrophages, as shown in Fig. 4. Human monocytes (Fig. 4A) and macrophages (Fig. 4) were incubated with the two K. pneumoniae strains labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) to visualize and monitor phagocytosis by flow cytometry. Monocytes incubated with L-78 showed a moderate gradual increase in bacterial phagocytosis up to 60 min post- infection, which was significantly reduced between 120-180 min. Conversely, monocytes co-incubated with ATCC 43816 for the same time did not phagocytose the bacteria, and minimum internalization was recorded (0.9-5.6%). In contrast to monocytes, macrophages rapidly and strongly phagocytosed L-78 up to 120 min post-infection and only at 180 min postinfection, L-78 phagocytosis was reduced by half. A very low percentage of ATCC 43816 was phagocytosed by macrophages (<10% over 180 min).

[0068] Microscopy analysis was conducted by examining coverslips on which monocytes and macrophages were co-incubated with CFSE-labeled bacteria for 30 min, a time point where high percentages of phagocytosis were recorded by flow cytometry. Confocal microscopy confirmed the intracellular localization of L-78 in monocytes/macrophages, excluding the possibility of bacterial adherence on the cell surface (data not shown). ATCC 43816 and L-78 were stained green with CFSE. Nuclei were stained blue with DAPI. In monocytes and especially macrophages infected with L-78, their typical ellipsoidal and round nuclei were surrounded by green fluorescence, suggesting that L-78 was internalized and digested by both cell subpopulations. On the contrary, monocytes and macrophages infected with ATCC 43816 emitted blue fluorescence accumulated in their characteristic nuclei, and less green fluorescence, whereas non-phagocytosed bacteria appeared in the extracellular space.

[0069] To validate the significance of our results, we generated a "moderate" model of infection, by infecting mice with 5x103 CFU ATCC 43816 (LD50). In this case 50% of the animals were expected to die due to sepsis between 30-45 hours post-infection. We were not able to generate a similar "moderate" mouse model of sepsis using higher inocula of L-78, as bacterial preparations corresponding to >1010 CFU/mL resulted in difficult to handle highly viscous preparations.

[0070] Mice that developed lethal septicemia and died had increased serum concentrations of proTa(100-109) as early as 3 hours post-infection (Fig. 1 C, light grey circles). The differences were more pronounced at 6 and 12 hours post-infection, where the levels of proTa(100-109) in serum of infected animals that died at 30-45 hours were similar (>5 ng/mL) to those of mice infected with the high (lethal) inoculum of ATCC 43816 (106 CFU) (Fig. 1 B). On the contrary, mice that recovered infection and survived had lower proTa(100-109) levels in their serum at 6 and 12 hours post-infection, and these resembled more the levels of the decapeptide in the serum of L-78-infected animals (<5 ng/mL; Fig 1A). Overall, 4 infected mice with increased proTa(100-109) concentration in serum between 3-12 hours post-infection died at 30-45 h, suggesting that very early determination of the levels of the decapeptide in their peripheral blood may predict sepsis-induced death.

[0071] Without willing to be bound by theory, we propose a scenario on the significance of extracellular proTa(100-109) quantification relevant to diverse mechanisms of sepsis induced by the two Klebsiella pneumoniae strains, and it is summarized in Fig. 5. K. pneumoniae strains L-78 (left) and ATCC 43816 (right) injected intraperitoneal^ in mice enter the bloodstream through the microcirculation of the peritoneum, infect internal organs, e.g. spleen, and multiply. In spleen, monocytes (m0)/macrophages (mcp) phagocytose L-78 (rectangles) early postinfection (3 hours) and caspase-3 levels are increased, driving cells to apoptosis. In apoptotic cells, proTa is cleaved by activated caspase-3 and the decapeptide proTa(100-109) is gradually exocytosed. Finally, L-78 is cleared; mice control the infection (at -24 hours) and recover. On the contrary, ATCC 43816 is minimally phagocytosed by spleen monocytes (m0)/macrophages (mcp), thereby remaining in the extracellular space where they rapidly multiply. In this case, monocytes/macrophages are led to necrosis (12 hours) and caspase-3 is activated in a lesser extent. High levels of DAMPs are extracellularly released; bacteria are not cleared; sepsis and multiple organ failure occur; and by 15 hours all mice die.

[0072] L-78 is phagocytosed by innate immune cells, caspase-3 is activated and cells are driven to apoptosis. Apoptotic cells gradually excrete proTa(100-109), resulting in progressive stimulation of immune responses. Therefore, no toxicity occurs, for instance, due to excess cytokine secretion. L-78 clearance by monocytes/macrophages leads to recovery from sepsis. On the contrary, infection with the non-phagocytosed ATCC 43816 leads to massive cell necrosis, abrupt release of high concentrations of alarmins, including proTa, excess immune system activation, irreversible septic shock, and death. In any case, determination of proTa(100-109) at the early phase of sepsis can be used as an early sepsis biomarker in humans. Clinical studies in humans are currently in progress.

[0073] Quantification of proTa(100-109) in serum is advantageous compared to HMGB1 determination, which has been thoroughly studied in sepsis. Firstly, generation of proTa(100-109) is a very early apoptotic event, occurring within the first 2 hours (Evstafieva AG et al, Exp Cell Res. 2003). ProTa(100-109) can thus serve as a very early sepsis biomarker, in contrast to HMGB1 , which is a late sepsis mediator. Secondly, although the exact mechanism of proTa(100-109) release from cells is yet unknown, the presence of proTa(100-109) extracellularly is strongly associated with massive cell apoptosis induced by an infectious agent. Preliminary results in humans show that serum proTa(100-109) levels increase in septic patients, but not in cases of "sterile inflammation", occurring in patients with autoinflammatory diseases, for instance. In contrast, increased levels of HMGB1 have been reported to occur also in several non-infectious conditions (Andersson U et al, Annu Rev Immunol. 2011).

[0074] Experiments involving animals were performed in strict accordance with the guidelines of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. All animal experiments were approved by the Animal Care and Use Committee of the Hellenic Pasteur Institute and by National Authorities (Veterinary Section of the Greek Republic). The study involving human samples was approved by the Ethical Committee of the 2nd Peripheral Blood Transfusion Unit and Hemophilia Center, "Laikon" General Hospital, Athens, Greece and informed consent was obtained from all subjects. The study was carried out in accordance with the Declaration of Helsinki, as revised in 2013.

EXAMPLES

Example 1 - Bacterial strains and culture conditions

[0075] Two strains of the Gram-negative bacillus K. pneumoniae of different virulence were used. K. pneumoniae KPC-Kp ST258 (L-78) is a clinical isolate derived from patients with bacteremia in Greek hospitals during 2009-201 1 and its identity has been previously confirmed by standard techniques (Tzouvelekis LS et al, KPC-producing, multidrug-resistant Klebsiella pneumoniae sequence type 258 as a typical opportunistic pathogen. Antimicrob Agents Chemother. 2013; 57: 5144-6). K. pneumoniae ATCC 43816 is a K2 strain used in animal models of infection. Both bacterial strains were stored at -80°C in medium containing 20% glycerol and were grown in freshly prepared Luria-Bertani (LB) agar (Sigma-Aldrich, St Louis, MO). Bacteria were incubated at 37°C in ambient air in order to reach the log phase of their growth. Bacterial growth was spectrophotometrically monitored and adjusted to an initial optical density (OD 580nm) of 0.3. Prior animal infection, bacterial preparations were adjusted to 109 CFU/mL for the L-78 strain and 5x104 or 107 CFU/mL for the ATCC 43816 strain.

Example 2 - In silico analysis of K. pneumoniae and mouse genomes

[0076] We retrieved K. pneumoniae protein sequence data available in publicly accessible sequence databases. In particular, data regarding K. pneumoniae complete genomes were retrieved from the NCBI website (http://www. ncbi. nlm. nih.gov/ genome/genomes/815; accessed 9 June 2016). In total, the predicted protein complements for 58 genomes were retrieved. In addition, we retrieved protein sequence sets from the same source for 7 yet incomplete genomes for K. pneumoniae strains which are related to the strains studied in this work for completeness. Proteins encoded in the mouse genome were downloaded from ENSEMBL, accessed 8 June 2016; build GRCm38.p4 (Flicek P et al. Nucleic Acids Res.

2012). For representing the proTa(100-109)-like peptides, we used the same sequence set and alignments described in Samara P et al, J Immunol Methods. 2013.

Example 3 - In vivo models of sepsis

[0077] Female ICR (CD-1 ) mice aged 6 to 8 weeks with a weight of 25-30 g were maintained in the Department of Animal Models for Biomedical Research of the Hellenic Pasteur Institute under pathogen-free conditions. Mice were intraperitoneally (ip) injected with 108 CFU per mouse of the L-78 strain (n=18), 5x103 or 106 CFU/mouse of the ATCC 43816 strain (n=10 and n=16, respectively) and monitored every 2 hours for clinical signs of bacteremia (lethargy, hunched posture, increased breathing rate or shiver). Blood was collected by retro-orbital sinus puncture using a glass Pasteur pipette before infection (control, 0 h), at 3, 6, 12, 24, and 48 h pi for L-78 and 5x103 ATCC 43816 groups, and at 3, 6, 12, and 15 h post infection (pi) for 106 ATCC 43816 group. At each time point, two animals from the L-78 and 106 ATCC 43816 groups were euthanized by cervical dislocation. Spleens were aseptically removed, weighed and homogenized in sterile Dulbecco's phosphate-buffered saline (DPBS; Lonza, Cologne, Germany). An aliquot of the homogenate was analyzed by flow cytometry and Western blotting, while the remaining homogenate was diluted and plated onto LB agar to determine the number of viable microorganism CFUs.

Example 4 - Isolation of peripheral blood mononuclear cells (PBMCs)

[0078] Human buffy coat preparations were used as a source of peripheral blood mononuclear cells (PBMCs) isolated by centrifugation over Ficoll-Histopaque (Sigma-Aldrich) density gradient. PBMCs were resuspended (10-15x106/mL) in DMEM, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 imM Hepes, 5 μg/mL gentamycin and 102 U/mL penicillin/streptomycin (all from Lonza). Highly enriched monocytes (>85% CD14+ as verified by flow cytometry following staining with FITC anti-human CD14 antibody [BioLegend, London, UK]) were obtained from PBMCs by plastic adherence for 2 h at 37°C in a humidified 5% CO2 incubator. Macrophages were differentiated from monocytes by incubation with 100 ng/mL recombinant human granulocyte macrophage colony-stimulating factor (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) for 5 days. Differentiation was confirmed by flow cytometry, by staining macrophages with APC-Cy7 anti-human CD206 antibody (BioLegend). Both monocytes and macrophages were in vitro infected with L-78 and ATCC 43816. The levels of proTa(100-109) were quantified in supernatants by ELISA, whereas the type of induced cell death and their phagocytic ability were analyzed by flow cytometry and confocal microscopy.

Example 5 - Antibody production

[0079] ProTa(100-109) TKKQKTDEDD peptide was synthesized using standard Fmoc/tBu chemistry with TBTU as coupling reagent on a synthesizer Syro II (MultiSynTech,

Witten, Germany) as described in Zaidi et al, FEBS 2007. Peptide Cys-TKKQKTDEDD was synthesized by manual amino(N)-terminal extension. Peptides were purififed using RP-HPLC and their identity confirmed by ESI-MS and MALDI-TOF-MS. For antibody production, rabbits were injected intradermally with 0.5 mg Cys-protTa (100-109) coupled to keyhole limpet hemocyanin and emulsified in Complete Freunds adjuvant. Antisera were collected and IgG fraction purified using a PROTEUS protein G midi purification spin column pack (AbD Serotec, Morphosys) following the manufacturer's instructions. Anti-proTa (100-109) specific antibodies were isolated using the cysteine-modified peptide with Sulfo-Link Resin (Pierce, Rockford, IL, USA) as described in Samara et al, J Immunol Methods. 2013.

Example 5 - Enzyme-linked immunosorbent assay (ELISA) for proTo(100-109)

[0080] Blood samples from each mouse were individually treated as previously described in Samara et al, J Immunol Methods. 2013. Precipitated mouse sera or supernatants from K. pneumoniae- infected human monocytes or macrophages were analyzed in duplicates by our in-house developed ELISA, and the quantity of proTa(100-109) was expressed in ng/mL using the linear part of the standard curve (Samara P et al, J Immunol Methods. 2013).

Example 6 - Evaluation of cell apoptosis

[0081] To evaluate morphological changes in splenocytes of Klebsiella-infected mice, as well as in Klebsiella-infected human monocytes/macrophages, cells were stained with Annexin V and PI fas shown in Bauer M et al, Proc Natl Acad Sci, 2011). Annexin V binds to phosphatidylserine exposed on the outer leaflet of the cell membrane of apoptotic cells, while PI is a DNA-binding dye, which enters cells when their membranes are ruptured. For cell labeling, the FITC Annexin V apoptosis kit with PI (BioLegend) was used and staining was performed as recommended by the manufacturer. For adherent cells, i.e., human monocytes and macrophages, detachment via 1X trypsin/EDTA solution (Lonza) was used and viability was >90% as confirmed by Trypan Blue exclusion (as shown in Jin X et al, J Vis Exp. 2016). Cells were washed twice with PBS and resuspended in Annexin V binding buffer at a concentration of 106 cells/mL. 100 μΙ_ cell suspension were transferred in FACS tubes, where 5 μΙ_ FITC Annexin V and 10 μΙ_ PI were added. Cells were gently vortexed and incubated for 15 min at room temperature in the dark. Four hundred μΙ_ of Annexin V binding buffer were added to each tube and cells were analyzed by a FACSCanto II flow cytometer (Becton-Dickinson [BD] Biosciences, Erembodegem, Belgium) equipped with FACSDiva software (BD Biosciences). Monocytes/macrophages from mouse spleens were gated using phycoerythrin anti-mouse CD1 1 b antibody (BioLegend) and analyzed separately.

Example 7 - Western blotting

[0082] Splenocytes were lysed in lysis buffer (50 mM Tris-HCI, 150 rtiM NaCI, 2 rtiM EDTA, pH 7.6, 1 % Triton X-100 [v/v]) supplemented with Complete® protease inhibitor cocktail (Roche Life Science, Manheim, Germany) for 30 min on ice. Cell lysates were centrifuged at 10,000g for 10 min at 4°C and protein concentration in the supernatants was determined using the Bradford protein assay (SERVA Electrophoresis GmbH, Heidelberg, Germany). In parallel, HeLa cells (ATCC CCL-2), led in vitro to apoptosis (4 h incubation with 5 ng/mL TNF-a and 1 μg/mL emetine [both from Sigma-Aldrich]) or necrosis (4 h incubation with 100 μg/mL doxorubicin [Sigma-Aldrich]), were used as controls (Evstafieva AG et al, Exp Cell Res. 2003). Equal amounts of protein (20 μg/lane) were separated by SDS-PAGE on 15% (w/v) gels and transferred onto nitrocellulose membranes. The blots were blocked in a solution of 5% (w/v) nonfat dry milk in Tris-buffered saline containing Tween-20 (TBS-T; 20 mM Tris-HCI, pH 7.5, 137 mM NaCI, 0.05% [v/v] Tween-20) for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies against procaspase-3 (1 :500 in TBS-T; ab44976, Abeam, Cambridge, UK) or β-actin (1 :2500; Sigma-Aldrich). After washing with TBS-T (4x5 min), blots were incubated with the appropriate HRP-conjugated secondary antibody (1 :5000 in TBS-T containing 1 % [w/v] nonfat dry milk) for 1 h at room temperature, washed again with TBS-T (4x5 min), developed using the enhanced chemiluminescence reagent (ECL reagent; Amersham Biosciences, Buckinghamshire, UK) and quantified by densitometry (Gel Analyzer v.1.0, Biosure, Athens, Greece). All densitometric values were normalized against β-actin levels. Procaspase-3 levels in control samples were set at one, and the respective values of treated samples are expressed as fold-induction over control samples.

Example 8 - Analysis of phagocytosis by flow cytometry

[0083] The two strains of K. pneumoniae were labeled with the fluorescent dye CFSE (Sigma-Aldrich) (according to Sokolovska et al, Curr Protoc Immunol, 2012). Human monocytes or macrophages were incubated with the bacteria following the protocol described in Sokolovska et al, Curr Protoc Immunol, 2012 with slight modifications. In brief, 4.5-5x105 monocytes or macrophages were plated per well of a 6-well culture plate (Greiner Bio-One GmbH, Frickenhausen, Germany) in 2 ml. DMEM-1 % FBS-without antibiotics (w/a). The medium was replaced with 1.5 imL ice-cold DMEM-1 % FBS-w/a containing the CFSE-labeled bacterial particles at a multiplicity of infection (MOI) of 25. Plates were centrifuged (100g, 4°C, 5 min) to allow bacteria to settle onto the cells and incubated at 37°C for 15, 30, 60, 120, and 180 min. Wells placed on ice (4°C) were used as controls. Phagocytosis was stopped by adding 1.5 imL of ice-cold DPBS containing 5 mM EDTA (Lonza) to each well. Cells were washed twice with DPBS/EDTA, once with DPBS and were detached using 1X trypsin/EDTA solution. Cells were then centrifuged, resuspended in 400 μΙ_ DPBS, transferred to FACS tubes placed on ice, and

immediately analyzed by flow cytometry. The results are presented as percentage of monocytes or macrophages bearing internalized bacteria (% phagocytosis) versus time.

Example 9 - Analysis of phagocytosis by confocal microscopy

[0084] Human monocytes or macrophages were grown on sterilized 12x12 mm coverslips (Sigma-Aldrich) precoated with poly-L-lysine (50 μg/mL for 1 h at 37°C; Sigma-Aldrich) and placed in 6-well culture plates at 37°C in a humidified 5% CO2 incubator. Cells were infected with the two Klebsiella strains for 30 min, as described in the previous paragraph, fixed with 4% paraformaldehyde diluted in DPBS for 15 min at room temperature and washed three times with DPBS. Each coverslip was placed on a single slide containing mounting medium with DAPI (Sigma-Aldrich) and protected from light. Specimens were examined on a multiphoton confocal microscope Leica TCS SP8 MP (Wetzlar, Germany) equipped with an Argon laser (excitation lines at 458, 476, 488, 496, and 514nm) and an IR MaiTai DeepSee Ti:Sapphire laser (Spectra-Physics, Santa Clara, CA, USA) for multiphoton applications. Images were acquired with the spectral detector of the microscope using appropriate emission wavelength ranges; CFSE (green) at 500-550nm and DAPI (blue) at 420-500nm. Acquisition was performed with the LAS X software (Leica Microsystems CMS GmbH, Wetzlar, Germany) using the same parameters (laser power, gain, pinhole, speed and analysis) for all specimens. Images were acquired as stacks of 6 to 20 optical sections with a Z-step of 0.50 μιη. Average or Maximum Projections of the stacks were created with the LAS X software and minor adjustments to contrast, brightness and levels with Adobe Photoshop CC (Adobe Systems Inc., San Jose, CA, USA) for better representation.

Statistical Analysis

[0085] Each in vitro and in vivo experiment was conducted at least three times. All data were analyzed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). Results are expressed as means ± standard deviation (SD). For statistics, we used Student's t-test and one-way analysis of variance (ANOVA) followed, where indicated, by Dunnett's test. P-values <0.05 were considered statistically significant.