WO2010060636A1 - Antimicrobial therapy - Google Patents

Abstract

The invention relates to polypeptides for the prevention and treatment of conditions associated with bacterial infection. In particular the invention is concerned with a peptide derived from the protein high molecular weight kininogen (HK) which can be used to block the activation of the contact system and to prevent or treat the onset of serious conditions associated with bacterial infection.

Description

ANTIMICROBIAL THERAPY Field of the invention

The invention relates to polypeptides for the prevention and treatment of conditions associated with bacterial infection.

Background of the invention

Although many bacterial infections are treated without complications, they can develop into serious and life-threatening conditions such as sepsis, toxic shock syndrome (TSS) or toxic shock like syndrome (TSLS). Conditions of this type, which are associated with bacterial infection, are a serious threat to health and have high mortality rates. For example, sepsis has been reported to be the most common cause of death in the non-coronary intensive care unit. An effective means to prevent and treat such conditions is therefore needed.

Summary of the invention

The pathogenesis of conditions associated with bacterial infection, such as sepsis and TSS, has typically been poorly understood. However, the present inventors hypothesised that they are the result of an uncontrolled host inflammation response induced by the pathogen, in particular a systemic activation of proteolytic cascades such as the contact system. The present inventors have demonstrated that a peptide with the amino acid sequence HKHGHGHGKHKNKGKKNGKH (HKH20; SEQ ID NO:1) derived from the protein high molecular weight kininogen (HK) can be used to inhibit the activation of the contact system and to prevent or treat the onset of serious conditions associated with bacterial infection. According to the invention there is thus provided:

A polypeptide for use in the prevention or treatment of a condition associated with bacterial infection in an individual, wherein the polypeptide is 10 to 40 amino acids in length and comprises:

(a) at least 10 contiguous amino acids from the sequence of HKHGHGHGKHKNKGKKNGKH (SEQ ID NO: 1); or

(b) a variant of (a) having the ability to inhibit activation of the contact system, which comprises upto four substitutions within the sequence from SEQ ID NO:1.

Additionally, there is provided a polypeptide according to the invention and at least one of: i) an antibiotic; ii) activated protein C (APC) for simultaneous, separate or sequential use in the treatment of a condition associated with bacterial infection in an individual. Additionally, there is provided a pharmaceutical composition comprising a polypeptide according to the invention and a pharmaceutically acceptable carrier or diluent.

Description of the Figures Figure 1 shows HK breakdown in plasma samples from patients with S. pyogenes infection.

A) Plasma samples from a patient with septic shock caused by S. pyogenes were taken 6h (lane 3), 18h (lane 4), 30h (lane 5), 44h (lane 6), 52h (lane 6), and 93h

(lane 7) after hospitalization and subjected to Western blot analysis followed by immunodetection with antibodies against HK and LK. Plasma from a healthy volunteer was used a positive control (lane 1) and complete HK breakdown in this sample was induced upon ex vivo treatment of this sample with dextran sulfate (lane 2).

B) HK breakdown was studied in plasma samples from patients with necrotizing fasciitis (lane 1) and erysipelas (lane 2), and suspected toxic shock syndrome (lane 3) all caused by S. pyogenes.

Figure 2 shows that HKH20 interferes with the intrinsic pathway of coagulation.

A) aPTT test results for human or BALB/c plasma incubated with 50 μM HKH20, GCP28 or buffer alone (control) for 60 s.

B) aPTT test results for human plasma incubated with increasing amounts of HKH20.

C) PT and the TCT test results for human plasma incubated with 50 μM HKH20, GCP28 or buffer alone (control) for 60 s. Values are mean ± SD (n=3) for Figs. A-C.

D) shows plasma kallikrein activity in human plasma measured by substrate assay after incubation with kaolin in the presence of 50 μM HKH20, GCP28 or buffer alone (control) for 15 min. Data are presented as percent activity compared to the control, values are means ± standard deviations (n=3). E) shows a representative SDS-PAGE Western blot stained with antibodies to HK and LK. Samples were human plasma incubated with buffer (lane 1), kaolin (lane 2), or kaolin and 50 μM HKH20 (lane 3) for 15 min.

Figure 3 shows the effect of HKH20 on S. pyogenes-induced contact activation A) shows plasma kallikrein activity in human plasma measured by substrate assay after incubation with S. pyogenes bacteria in the presence of 50 μM HKH20,GCP28 or buffer alone (control) for 15 min. Data are presented as percent activity compared to the control, values are mean ± SD (n=3, *p < 0.05 by t test). B) shows a representative SDS-PAGE Western blot stained with antibodies to HK and LK. Samples were 1) normal plasma, 2) kaolin-treated plasma, 3) plasma proteins absorbed and released by S. pyogenes, 4) plasma proteins absorbed and released by S. pyogenes in the presence of 50 μM HKH20, 5) or 100 μM HKH20, 6) plasma proteins absorbed and released by S.pyogenes in the presence of 100 μM GCP28. Figure 4: shows analysis of lung tissue from BALB/c mice infected with S. pyogenes. Light microscopy (left) and scanning electron microscopy (right) of representative mouse lung tissue sections are shown. Mice were injected i.p. A) with 200 μl PBS buffer, B) 5x10⁶ CFU S. pyogenes, C) 5x10⁶ CFU S. pyogenes and 200 μg HKH20, and D) 5x10⁶ CFU S. pyogenes and 275 μg GCP28. Bars represent 250μm (light microscopy) and 50μm (scanning electron microscopy)

Figure 5: shows that Leukocyte recruitment is not impaired by HKH20 FACS analysis of peritoneal lavage from non-infected mice injected with PBS (a) or HKH20 (b), mice infected i.p. with 5xlO⁶ CFU S. pyogenes in the absence (c) or presence (d) of 200 μg HKH20. Peritoneal lavage was analyzed 18 h after infection. Neutrophil populations are red and are the population shown between 10² and 10 for CD45 and above 200 for Side Scatter; monocyte populations are green and are the population shown between 10² and 10³ for CD45 and below 200 for Side Scatter, e- g) Scanning electron microscopy of representative mouse lung tissue sections are shown. Neutropenic mice were injected i.p. with e) 200 μl PBS, f) 5x106 CFU S. pyogenes, g) 5x106 CFU S. pyogenes and 200 μg HKH20.

Figure 6: shows A) Activated partial thromboplastin time (aPTT) and B) Prothrombin time (PT) of BALB/c plasma samples following subcutaneous infection with S. Pyogenes. Animals were injected subcutaneously in the neck with 2xlO⁷ CFU S. pyogenes bacteria, plasma was taken at 0, 4, 6, 10, 12, 18, 24, and 42 hours after infection (n=2-6/group) and clotting times were measured immediately (*P<0.05; ***P<0.0001). Figure 7: shows analysis of lung tissue from BALB/c mice infected s.c. with

S. pyogenes. Scanning electron microscopy of representative mouse lung tissue sections are shown. Mice were injected with A) 2x10⁷ CFU S. pyogenes s.c. and 100 μl PBS i.p. 8 hours after infection B) 2x10⁷ CFU S. pyogenes and 100 μl HKH20 (2 mg/ml) i.p. 8 hours after infection. The bar represents 50 μm. Figure 8: shows the effect of HKH20 on the survival of BALB/c mice after subcutaneous infection with S. pyogenes. Mice were injected s.c. in the neck with 2x10⁷ CFU S. pyogenes bacteria and treated with 100 μl HKH20 (2mg/ml) or 100 μl PBS (control) i.p. 8 hours after infection (n=4-5/group). Mortality was recorded for a period of 5 days. The experiment was repeated four times and the results from a total of 17 animals per group are shown.

Figure 9: shows the effect of HKH20 in combination with clindamycin treatment on the survival of BALB/c mice after subcutaneous infection with S. pyogenes. Mice were injected s.c. in the neck with 2xlO⁷ CFU S. pyogenes bacteria and treated with 200 μg HKH20 and 10mg/kg clindamycin or 10 mg/kg clindamycin in a volume of 200μl PBS. Treatment occurred i.p. 18, 42, 48 and 72 hours after infection (n=5/group). Mortality was recorded for a period of 7 days. The experiment was repeated two times and the results from a total of 10 animals per group are shown.

Description of the Sequences

SEQ ID NO:1 is the amino acid sequence of the HKH20 peptide, HKHGHGHGKHKNKGKKNGKH, which corresponds to residues 497 to 516 of full- length HK (high molecular weigh kininogen) as set out in SEQ ID NO: 3.

SEQ ID NO: 2 is the nucleic acid sequence of the gene encoding HK. This sequence has been made available to the public as NCBI accession no. NMJ)01102416

SEQ ID NO: 3 is the amino acid sequence of full-length HK which is encoded by residues 213 to 2147 of SEQ ID NO: 2. This sequence has also been made available to the public as NCBI accession no. NP 001095886. SEQ ID NO: 4 is the amino acid sequence of the GCP28 peptide, GCPRDIPTNSPELEETLTHTITKLNAEN, which corresponds to residues 266 to 293 of full-length HK as set out in SEQ ID NO: 3.

Detailed description of the invention

The contact system

The contact system, also known as the intrinsic pathway of coagulation or the kallikrein-kinin system, is involved in normal hemostasis and inflammation. It comprises four components: Factor XI (FXI), FXII, plasma kallikrein (PK) and high molecular weight kininogen (HK). Under physiological conditions, these factors circulate in their inactive forms in the bloodstream or are bound to the surface of different cell types, such as endothelial cells, platelets and polymorph nuclear neutrophils (PMNs). Contact activation can occur upon the conversion of the endothelium from an anti-coagulant to a pro-coagulant state, which is seen for instance during arterial vessel injury. Apart from cellular surfaces, the contact system is also assembled and activated on many non-physiological, negatively charged surfaces for instance glass, dextran and kaolin.

The initial step is an auto-catalytically driven activation of FXII to FXIIa, which then converts PK and FXI into their active proteolytic forms. FXIa triggers the endogenous clotting cascade (or common coagulation pathway) via the activation of FX and ultimately results in the conversion of fibrinogen to fibrin. Activated PK cleaves HK and releases bradykinin (BK) and antibacterial peptides. BK, a peptide consisting of nine amino acids, is a potent proinflammatory mediator. BK has been shown to evoke the generation of nitric oxide (NO) and other inflammatory substances (for instance prostaglandins and leukotrienes) and induce fever. Notably, and probably more important in respect to infectious diseases, BK also induces increased vascular permeability and capillary leakage, causing pain, edema and hypotension. While the local activation of the contact system is considered to have a beneficial effect to the human host, i.e. via generation of HK-derived antibacterial peptides, a systemic contact activation may lead to severe complications such as kinin induced vascular leakage and bleeding disorders.

Such a systemic contact activation can occur due to assembly and activation of the contact system on the surface of bacterial cells. Bacterial cells typically bind HK, leading to its cleavage and the downstream events in the contact pathway. HK binding to the bacterial cell surface is typically mediated by one or more bacterial proteins with affinity for HK. Examples of such bacterial proteins include a bacterial M protein, a bacterial curli protein, or a homologue of either thereof. It has been shown in vitro and in vivo that certain types of bacteria, such as Streptococcus spp. , Staphylococcus spp., E. coli and Salmonella spp., in particular S. pyogenes or S. aureus, are able to assemble and activate the contact system on the bacterial cell surface. Furthermore, the products of HK cleavage, e.g. BK, are often significantly increased in patients with sepsis and septic shock.

Disorders arising from inappropriate activation of the contact system must not be confused with disorders arising from activation of the extrinsic pathway of coagulation. In particular, certain (usually hereditary) bleeding disorders may result from problems with activation of the extrinsic pathway of coagulation rather than the contact system. It is therefore necessary to distinguish between the two coagulation pathways. Although the contact system and the extrinsic pathway of coagulation do share common elements in the later stages of coagulation, the two pathways are clearly distinguishable in that they are activated in different ways.

The extrinsic pathway is activated in response to tissue injury, which leads to the formation of an active complex between tissue factor (TF) and FVII. This triggers the endogenous clotting cascade (common coagulation pathway) via the activation of FX and ultimately results in the conversion of fibrinogen to fibrin. Thus, the extrinsic pathway is so-called because the key initiating component, TF, is extrinsic to the plasma. By contrast, the contact system is also known as the "intrinsic coagulation pathway", because (as set out above) its activation is auto-catalytic and once an activation surface is provided all other necessary components are intrinsic to the plasma.

The present invention is concerned only with the contact system (the intrinsic pathway of coagulation).

Peptides The present invention provides a peptide for the prevention and treatment of conditions associated with bacterial infection. The peptide of the invention inhibits activation of the contact system, preferably activation of the contact system on a bacterial cell surface. The peptide of the invention typically does not inhibit activation of the extrinsic coagulation pathway. The peptide of the invention preferably has no significant direct effect on the downstream components involved in coagulation which are common to the intrinsic and extrinsic pathways. That is, the peptide of the invention does not act directly upon the components of the common coagulation pathway. The peptide of the invention is typically derived from HK (high molecular weigh kininogen). The protein sequence of human HK is publically available (see NCBI accession number NP_001095886; SEQ ID NO: 3). The peptide of the invention is preferably from a region of HK which is capable of interaction with a bacterial cell surface. This interaction may be mediated by a protein, for example a bacterial cell protein or a homologue thereof. The bacterial cell protein is typically a bacterial M protein or a bacterial curli protein, or a homologue of either thereof.. Alternatively, the interaction may be mediated by non-proteinous substances such as peptidoglycan, lipoteichoic acid, hyaluronic acid, or lipopolysaccharide.

The peptide of the invention typically has a minimum length. Thus the peptide is typically at least about 10 amino acids in length, more preferably at least about 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.

The peptide also typically has a maximum length. Thus the peptide is typically no greater than about 40 amino acids in length, more preferably no greater than about 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20 amino acids in length.

It will be appreciated that the peptide may be of a length defined by any of the minimum lengths set out above in combination with any of the maximum lengths above. For example, the peptide is typically 10 to 40 amino acids in length, but may be 11 to 40, 12 to 40, 13 to 40, 14 to 40, 15 to 40, 16 to 40, 17 to 40, 18 to 40, 19 to 40 or 20 to 40 amino acids in length. Alternatively, the peptide may be, for example, 10 to 39, 10 to 38, 10 to 37, 10 to 36, 10 to 35, 10 to 34, 10 to 33, 10 to 32, 10 to 31, 10 to 30, 10 to 29, 10 to 28, 10 to 27, 10 to 26, 10 to 25, 10 to 24, 10 to 23, 10 to 22, 10 to 21 or 10 to 20 amino acids in length.

The peptide typically comprises at least 10 amino acids from the sequence of HKHGHGHGKHKNKGKKNGKH (HKH20; SEQ ID NO: 1 ), which corresponds to residues 497 to 516 of human HK (SEQ ID NO: 3; See NCBI Accession No: NP_001095886). More preferably the peptide comprises at least 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids from the sequence of HKHGHGHGKHKNKGKKNGKH (HKH20; SEQ ID NO:1). The peptide may comprise a variant of HKHGHGHGKHKNKGKKNGKH (HKH20; SEQ ID NO:1) having the ability to inhibit activation of the contact system, said variant comprising upto four substitutions within the sequence from SEQ ID NO:1. The variant preferably inhibits activation of the contact system on the surface of a bacterial cell. The variant typically does not inhibit activation of the extrinsic coagulation pathway. The variant preferably has no significant direct effect on the downstream components involved in coagulation which are common to the intrinsic and extrinsic pathways.

Methods to assess inhibition of activation of the contact system, the extrinsic coagulation pathway and the common coagulation pathway

The ability of a peptide or variant to inhibit activation of the contact system may be assessed by any suitable method.

One such method involves direct measurement of activation of the contact system on the surface of a bacterial cell. For example, cultures of S. pyogenes bacteria (typically strain API) may be prepared by any suitable method, and then incubated with a test peptide prior to the addition of human plasma. This mixture is then incubated further, before centrifugation and collection of pellets. The resuspended pellets may then be assayed for PK activity by incubation with a chromogenic substrate such as S-2302, prior to centrifugation and measurement of absorbance. A peptide or variant which is able to inhibit activation of the contact system at the surface of the bacterial cell will produce a significant decrease in PK activity, relative to the PK activity measured in the absence of the peptide or variant. In other methods, the ability of a peptide or variant to inhibit activation of the contact system may be assessed by reference to its effects on coagulation. Coagulation is typically monitored using a coagulometer. These devices are well known in the art, and typically monitor clotting in a sample of blood or plasma by detecting changes in the properties of a sample over time in order to determine when clotting is complete. For example, many coagulometers measure the optical properties of a sample, such as its absorbance of a particular wavelength of light.

As described above, the contact system is also known as the intrinsic coagulation pathway. A suitable peptide or variant of the invention typically inhibits activation of coagulation via the intrinsic coagulation pathway and does not inhibit activation of coagulation via the extrinsic coagulation pathway. A suitable method of determining whether or not a peptide or variant inhibits activation of the intrinsic coagulation pathway involves an assay which measures clotting time in human plasma via this pathway. One such assay measures the activated Partial Thromboplastin time (aPTT). This typically involves initiating clotting in a plasma or blood sample in an aPTT specific manner, and then measuring the time taken for the sample to clot in the presence and absence of a test peptide/variant using a coagulometer. Thus, a standard method for measuring aPTT is as follows:

30 μl of test peptide/variant diluted in sterile water is incubated with 100 μl human citrate-treated plasma for 1 min followed by the addition of 100 μl aPTT reagent (aPTT Automate: Diagnostica Stago, Asnieres, France) for 60 s at 37⁰C. Clotting is initiated by the addition of 100 μl of a 25 mM CaCl₂ solution. The time taken to clot is monitored using a coagulometer. The aPTT time in the absence of the test peptide/variant is established by carrying out the same method with 30 μl of sterile water/buffer in place of the test peptide/variant, preferably using a further 100 μl from the same human citrate-treated plasma sample.

A peptide or variant which inhibits activation of the intrinsic coagulation pathway will result in an increased aPTT time relative to the aPTT time in the absence of the peptide. A suitable method of determining whether or not a peptide or variant inhibits activation of the extrinsic coagulation pathway involves an assay which measures clotting time in human plasma via this pathway. One such assay measures the Prothrombin time (PT). This typically involves initiating clotting in a plasma or blood sample in a PT-specific manner, and then measuring the time taken for the sample to clot in the presence and absence of a test agent using a coagulometer. Thus, a standard method for measuring PT is as follows:

30 μl of test peptide/variant diluted in sterile water is incubated with 100 μl human citrate-treated plasma for 1 min and clotting is initiated by the addition of 100 μl Thrombomax with calcium (PT reagent: Sigma- Aldrich). The time taken to clot is monitored using a coagulometer. The PT time in the absence of the test peptide/variant is established by carrying out the same method with 30 μl of sterile water/buffer in place of the test peptide/variant, preferably using a further 100 μl from the same human citrate-treated plasma sample. A peptide or variant which inhibits activation of the extrinsic coagulation pathway will result in an increased PT time relative to the PT time in the absence of the peptide or variant. A peptide or variant which does not inhibit activation of the extrinsic coagulation pathway will produce no significant change in PT time relative to the PT time in the absence of the peptide or variant.

Therefore, when tested according to the above methods, a suitable peptide or variant of the invention will typically:

- produce an increased aPTT time relative to the aPTT time when measured in the absence of the peptide or variant; and - produce no significant change in PT time relative to the PT time when measured in the absence of the peptide or variant.

Preferred peptides or variants of the invention also have no significant direct effect on the downstream components involved in coagulation which are common to the intrinsic and extrinsic pathways. A suitable method for assessing this is with an assay which measures thrombin-induced fibrin-network formation. One such assay measures the Thrombin Clotting Time (TCT). This typically involves initiating clotting in a plasma or blood sample using a TCT-specific manner, and then measuring the time taken for the sample to clot in the presence and absence of a test agent using a coagulometer. Thus, a standard method for measuring TCT is as follows:

30 μl of test peptide/variant diluted in sterile water is incubated with 100 μl human citrate-treated plasma for 1 min and clotting is initiated by the addition of 100 μl Accuclot thrombin time reagent (TCT reagent: Sigma-Aldrich). The time taken to clot is monitored using a coagulometer. The TCT time in the absence of the test peptide/variant is established by carrying out the same method with 30 μl of sterile water / buffer in place of the test peptide/variant, preferably using a further 100 μl from the same human citrate-treated plasma sample.

A peptide or variant which inhibits the downstream components of coagulation will result in an increased TCT time relative to the TCT time in the absence of the peptide or variant. A peptide or variant which has no significant effect on these components will produce no significant change in TCT time relative to the TCT time in the absence of the peptide or variant. Accordingly, preferred peptides or variants of the invention will produce no significant change in TCT time relative to the TCT time in the absence of the peptide or variant. Variants

A suitable variant of a peptide of the invention may typically comprise one, two, three or four substitutions within the sequence from SEQ ID NO:1.

The substitutions are typically conservative. By "conservative" it will be understood that the amino acid inserted by a substitution will have similar physio- chemical properties and characteristics to the amino acid that it replaces, i.e. the inserted amino acid will be similar in terms of its chemical, structural, charge and hydrophobic / hydrophilic properties. In particular, where the amino acid to be replaced has a polarity of charge, the amino acid inserted will preferably have the same polarity of charge, i.e. a positively (+) charged amino acid will be replaced by another (+) charged amino acid. For example Histidine (His, H) may preferably be replaced by Lysine (Lys, K) or Arginine (Arg, R) (in order of preference), and Lys may preferably be replaced by His or Arg (in order of preference). Where the amino acid to be replaced has neutral charge, the amino acid inserted will preferably be a neutral or a (+) charged amino acid. Thus, Glycine (GIy, G) may preferably be replaced by any neutral amino acid, or by His or Lys or less preferably by Arg. Similarly, Asparagine (Asn, N may preferably be replaced by any neutral amino acid, or by His or Lys or less preferably by Arg. In summary, the conservative substitutions will typically maintain or increase the positive charge of the peptide of the invention. A summary of the amino acids and their properties is provided in the table below.

Peptide synthesis

The peptides of the invention comprise at least 10 amino acids from the sequence of HKHGHGHGKHKNKGKKNGKH (HKH20; SEQ ID NO:1). Peptides according to the invention may be produced synthetically, or may be expressed using recombinant techniques. Peptides may be synthesised using methods well known in the art.

Preferred methods include solid-phase peptide synthesis techniques and most preferably an automated or semiautomated peptide synthesizer. Typically, using such techniques, an α-N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidinone or methylene chloride in the presence of coupling agents such as dicyclohexylcarbodiimide and 1- hydroxybenzotriazole in the presence of a base such as diisopropyl-ethylamine. The α-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, and include t- butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc). The term "peptide" includes not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example, by making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.

Similarly, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond. It will also be appreciated that the peptide may conveniently be blocked at its N-or C-terminus so as to help reduce susceptibility to exoproteolytic digestion. For example, the N-terminal amino group of the peptides may be protected by reacting with a carboxylic acid and the C-terminal carboxyl group of the peptide may be protected by reacting with an amine. Other examples of modifications include glycosylation and phosphorylation. Another potential modification is that hydrogens on the side chain amines of R or K may be replaced with methylene groups (-NH₂ — > - NH(Me) or -N(Me)₂). Analogues of peptides according to the invention may also include peptide variants that increase or decrease the peptide's half-life in vivo. Examples of analogues capable of increasing the half-life of peptides used according to the invention include peptoid analogues of the peptides, D-amino acid derivatives of the peptides, and peptide-peptoid hybrids. A further embodiment of the variant polypeptides used according to the invention comprises D-amino acid forms of the polypeptide. The preparation of polypeptides using D-amino acids rather than L- amino acids greatly decreases any unwanted breakdown of such an agent by normal metabolic processes, decreasing the amounts of agent which needs to be administered, along with the frequency of its administration.

The peptides of the invention may also be produced by recombinant techniques using the polynucleotides, vectors or host cells as described below.

Polynucleotides, vectors and cells The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide of the invention may be provided in isolated or purified form. A nucleic acid sequence which "encodes" a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For the purposes of the invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence. Such nucleic acid sequences encode a peptide of the invention.

Polynucleotides of the invention can be synthesised according to methods well known in the art. The polynucleotide molecules of the present invention may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the peptide of the invention in vivo in a targeted subject or in vitro for the production of the peptides of the invention for subsequent administration. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors) which are suitable for use as reagents for nucleic acid immunization or for expression of the peptides in suitable host cells in vitro. Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide of the invention may be administered to a host subject. Preferably the polynucleotide is prepared and/or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a peptide of the invention.

The present invention thus includes expression vectors that comprise such polynucleotide sequences. Thus, the present invention provides a vector for use in preventing or treating a condition associated with a bacterial infection in an individual.

Furthermore, it will be appreciated that the compositions and products of the invention may comprise a mixture of polypeptides and polynucleotides. Accordingly, the invention provides a composition or product as defined herein, wherein in place of any one of the polypeptide is a polynucleotide capable of expressing said polypeptide. Expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for expression of a peptide of the invention. Other suitable vectors would be apparent to persons skilled in the art.

Thus, a polypeptide of the invention may be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.

"Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given regulatory sequence, such as a promoter, operably linked to a nucleic acid sequence is capable of effecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the nucleic acid sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

A number of expression systems have been described in the art, each of which typically consists of a vector containing a gene or nucleotide sequence of interest operably linked to expression control sequences. These control sequences include transcriptional promoter sequences and transcriptional start and termination sequences. The vectors of the invention may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. A "plasmid" is a vector in the form of an extrachromosomal genetic element. The vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example to allow in vivo expression of the polypeptide. A "promoter" is a nucleotide sequence which initiates and regulates transcription of a polypeptide-encoding polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. It is intended that the term "promoter" or "control element" includes full-length promoter regions and functional (e.g., controls transcription or translation) segments of these regions.

A polynucleotide, expression cassette or vector according to the present invention may additionally comprise a signal peptide sequence. The signal peptide sequence is generally inserted in operable linkage with the promoter such that the signal peptide is expressed and facilitates secretion of a polypeptide encoded by coding sequence also in operable linkage with the promoter. Typically a signal peptide sequence encodes a peptide of 10 to 30 amino acids for example 15 to 20 amino acids. Often the amino acids are predominantly hydrophobic. In a typical situation, a signal peptide targets a growing polypeptide chain bearing the signal peptide to the endoplasmic reticulum of the expressing cell. The signal peptide is cleaved off in the endoplasmic reticulum, allowing for secretion of the polypeptide via the Golgi apparatus. Thus, a peptide of the invention may be provided to an individual by expression from cells within the individual, and secretion from those cells.

Polynucleotides of interest may be used in vitro, ex vivo or in vivo in the production of a peptide of the invention. Such polynucleotides may be administered or used in the prevention or treatment of a condition associated with a bacterial infection.

Methods for gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859 and 5,589,466. The nucleic acid molecule can be introduced directly into the recipient subject, such as by standard intramuscular or intradermal injection; transdermal particle delivery; inhalation; topically, or by oral, intranasal or mucosal modes of administration. The molecule alternatively can be introduced ex vivo into cells that have been removed from a subject. Cells containing the nucleic acid molecule of interest are re-introduced into the subject such that an immune response can be mounted against the peptide encoded by the nucleic acid molecule. The nucleic acid molecules used in such immunization are generally referred to herein as "nucleic acid vaccines."

The polypeptides, polynucleotides, vectors or cells of the invention may be present in a substantially isolated form. They may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of the proteins, polynucleotides, cells or dry mass of the preparation.

Formulations and compositions

The peptides, polynucleotides, vectors and cells of the invention may be provided to an individual either singly or in combination. Each molecule or cell of the invention may be provided to an individual in an isolated, substantially isolated, purified or substantially purified form. For example, a peptide of the invention may be provided to an individual substantially free from other peptides.

Whilst it may be possible for the peptides, polynucleotides or compositions according to the invention to be presented in raw form, it is preferable to present them as a pharmaceutical formulation. Thus, according to a further aspect of the invention, the present invention provides a pharmaceutical formulation for preventing or treating a condition associated with a bacterial infection, comprising a composition, vector or product according to the invention together with one or more pharmaceutically acceptable carriers or diluents and optionally one or more other therapeutic ingredients.

The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active substance, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride. Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

Typically, carriers for injection, and the final formulation, are sterile and pyrogen free. Formulation of a composition comprising the peptide, polynucleotide or cell of the invention can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan.

For example, compositions containing one or more molecules or cells of the invention can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or vehicle. These excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen- free water) prior to parenteral administration of the reconstituted composition. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3 -butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.

Other parentally-administrable compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Alternatively, the peptides or polynucleotides of the present invention may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules.

The formulation of any of the peptides, polynucleotides or cells mentioned herein will depend upon factors such as the nature of the substance and the method of delivery. Any such substance may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, by inhalation, intravenously, intramuscularly, intrasternally, transdermally, intradermally, sublingually, intranasally, buccally or by infusion techniques. The substance may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular individual. The compositions or formulations of the invention will comprise a suitable concentration of each peptide/polynucleotide/cell to be effective without causing adverse reaction. A therapeutically effective amount of a substance used in the prevention or treatment of a condition associated with bacterial infection may be determined according to various parameters, especially according to the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific antibiotic, the age, weight and conditions of the subject to be treated and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g. That dose may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily.

The composition or formulations should have a purity of greater than 95% or 98% or a purity of at least 99%.

A composition may be formulated which comprises a molecule and/or cell of the invention and also one or more other therapeutic agent or composition. A composition of the invention may alternatively be used simultaneously, sequentially or separately with one or more other therapeutic agents as part of a combined treatment. For example, the invention provides a polypeptide according to the invention and at least one of: i) an antibiotic; ii) activated protein C (APC) for simultaneous, separate or sequential use in the treatment of a condition associated with bacterial infection in an individual.

The antibiotic is typically a broad spectrum antibiotic selected from one or more aminoglycosides, cephalosporins, fluoroquinolones, lincosamides, macrolides, penicillins, sulfonamides, or tetracyclins. For example, suitable antibiotics include, but are not limited to, Gentamicin, Kanamycin, Neomycin, Streptomycin, Tobramycin, Cefazolin, Cephalexin, Cephapirin, Cephradine, Cefuroxime, Cefixime, Cefotaxime, Ceftazidime, Ceftizoxime, Ceftriaxone, Ciprofloxacin, Levofloxacin, Ofloxacin, Clindamycin, Azithromycin, Clarithromycin, Erythromycin, Amoxicillin, Ampicillin, Ampicillin-Sulbactam, Cloxacillin, Dicloxacillin, Mezlocillin, Nafcillin, Oxacillin, Penicillin G Benzathine, Penicillin G Potassium, Penicillin G Procaine, Penicillin V Potassium, Piperacillin, Ticarcillin, Ticarcillin-Clavulanate potassium, Pyrimethamine-Sulfadoxine, Sulfadizine, Sulfisoxazole, Sulfmethoxazole, Trimethoprim-sulfamethoxazole, Chlortetracycline, Doxycycline, and Tetracycline.

Protein C is present in the circulation in inactive form, and is activated (usually at the site of a clot) by a complex of thrombin and an endothelial protein called thrombomodulin. Activated protein C (APC) is thought to have several effects which may be beneficial in the treatment of conditions associated with bacterial infection. In particular: (i) Inhibition of clotting. APC has potent inhibitory effects on factors Va and Villa (and a lesser effect on the unactivated forms); (ii) Promotion of fibrinolysis. APC inhibits PAI-I (plasminogen activator inhibitor 1), and by so doing, promotes plasmin activity, and thus clot lysis; (iii) Anti-inflammatory effects; and (iv) Inhibition of endothelial cell apoptosis.

Conditions to be treated Serious conditions associated with bacterial infection include sepsis (in particular severe sepsis or septic shock) and toxic shock syndrome (TSS, also known as toxic shock like syndrome, TSLS).

Sepsis is a systemic inflammatory response to infection, which causes organ failure and death in severe cases. It is an increasingly common cause of morbidity and mortality, particularly in elderly, immuno-compromised, and critically ill individuals. Sepsis has been reported to be the most common cause of death in the non-coronary intensive care unit. It occurs in 1-2% of all hospitalizations and mortality rates range from 20% for sepsis to 40% for severe sepsis to >60% for septic shock (a sub-category of severe sepsis). The clinical definition of sepsis is the presence of two or more of the following conditions:

(1) fever (temperature >38°C) or hypothermia (temperature <36°C);

(2) heart rate >90 beats per minute;

(3) respiratory rate >20 breaths per minute or PaCO₂ <32 mm Hg; and (4) white blood cell count > 12 (x 10⁹ cells/L) or <4 (x 10⁹ cells/1), at the same time as a confirmed or suspected infection.

Symptoms (1) to (4) are known as the SIRS (Severe Inflammatory Response Syndrome) criteria and are a recognised international standard for diagnosis of severe inflammation. An individual exhibiting two or more of the SIRS criteria without a confirmed or suspected infection is classified as having non-infection associated SIRS. For the purposes of the present invention, it will be understood that the peptide of the invention is intended for the prevention or treatment of a condition characterised in that the individual has a confirmed or suspected infection caused by a bacterial cell, and/or displays one or more of the SIRS criteria, symptoms (1) to (4) defined above.

The clinical definition of severe sepsis is sepsis as defined above, associated with sepsis-induced hypotension, organ dysfunction or perfusion abnormalities. Sepsis-induced hypotension is defined as a systolic blood pressure of <90 mm Hg or a reduction of <40 mm Hg from baseline in the absence of other causes of hypotension. Perfusion abnormalities may include, but are not limited to hypoperfusion, lactic acidosis, oliguria, or an acute alteration in mental status. Severe sepsis includes as a sub-category the condition of septic shock. This condition is specifically defined by the presence of sepsis-induced hypotension despite adequate fluid resuscitation along with the presence of perfusion abnormalities. Individuals who are receiving inotropic or vasopressor agents may not be hypotensive at the time that perfusion abnormalities are measured.

Toxic shock syndrome (TSS) is a severe inflammatory response to various bacterial toxins which act as superantigens. It has been reported to have mortality rates approaching 20%, even when antibiotic treatment and intensive care are provided. It is typically caused by Staphylococcus or Streptococcus bacteria. Streptococcal toxic shock syndrome is sometimes referred to as Toxic Shock Like Syndrome (TSLS). Symptoms of TSS vary depending on the underlying cause. However, in all cases, diagnosis is based upon internationally recognised criteria:

1) Body temperature > 38.9 °C

2) Systolic blood pressure < 90 mmHg

3) Diffuse rash, intense erythroderma, blanching with subsequent desquamation, especially of the palms and soles; and 4) Involvement of three or more organ systems selected from: gastrointestinal, muscular, mucous membrane, renal, hepatic, hematologic, and neurologic. The presence of three or more of the above symptoms in the presence of a confirmed or suspected bacterial infection is sufficient to diagnose TSS. For the purposes of the present invention, it will be understood that the peptide of the invention is intended for the prevention or treatment of a condition characterised in that the individual has a confirmed or suspected infection caused by a bacterial cell, and/or displays one or more of the TSS criteria 1) to 4) defined above. Individual to be treated

The individual is typically a mammal. The mammal is typically a human or a domestic mammal such as a horse, a cow, a sheep, a dog or a cat. The individual is preferably a human. The individual may have or be at risk of a condition associated with bacterial infection because they have a confirmed or suspected infection, and/or display the symptoms of SIRS or TSS as outlined above.

The confirmed or suspected infection is typically a bacterial infection. A bacterial infection may be caused by one or more Gram negative or Gram positive bacteria. The one or more Gram negative bacteria may be selected from Escherichia coli, Klebsiella spp. (typically K.pneumoniae or K. oxytoca), Enterobacter spp (typically E. cloacae or E. aerogenes), Bordetella spp. (typically B. bronchiseptica, B. pertussis or B. parapertussis), Chlamydia spp. (typically C. trachomatis), Legionella spp. (typically L. pneumophilia), Pseudemonas spp. (typically P. aeruginosa), Mycoplasma spp. (typically M. pneumoniae), Haemophilus influenza, Serratia marcescens, Proteus mirabilis, , Acinetobacter baumannii, Stenotrophomonas maltophilia and Neisseria meningitidis (typically of serogroup A, B, C, H, I, K, L, X, Y, Z, 29E or Wl 35). The one or more Gram positive bacteria may be selected from Staphylococcus spp. (typically S. aureus or Coagulase negative Staphylococci), Streptococcus spp. (typically S. pneumoniae or S.pyogenes) and Enterococcus spp. (typically E.faecium or E.faecalis). The bacterial is preferably selected from Streptococcus spp. , Staphylococcus spp., E. coli or Salmonella spp, and most preferably S. pyogenes or S. aureus.

The confirmed or suspected infection may affect any part of the body. Typical examples include an infection which affects the lungs; the respiratory tract; the liver; the kidneys; the urinary tract; the skin (cutaneous and subcutaneous); the heart; the stomach; the intestines; the blood; the bones; the joints or any combination thereof. Most preferably, the infection affects the lungs, typically causing lung haemorrhage. The infection may be confirmed by diagnostic practices known in the art, for example microbial culture of samples taken from the individual, antigen testing of urine or other fluid samples taken from the individual (especially for S. pneumoniae and Legionella sp. infections) or PCR analysis (especially for atypical pneumonia caused by bacterial infection e.g. Mycoplasma, Legionella, Chlamydia, and B. pertussis). More recently multiplex PCR techniques have been developed which enable simultaneous testing for multiple bacterial and fungal infections. The infection may be suspected because of the presence of one or more of the following general symptoms: fever higher than 38⁰C; chills; pain; an ache or tenderness; general feeling of tiredness; night sweats; and a wound or incision with associated redness, heat, swelling or pain, or that exudes a fluid that is white, yellowish or greenish.

The individual may have one or more additional risk factors and/or one or more predispositions toward the development of a condition associated with bacterial infection. Risk factors that predispose towards development of such conditions typically include any factor which increases susceptibility to infection. These factors can include a weakened immune system (i.e. the individual is immuno-compromised), or the presence in a hospitalised patient of an intravenous line, surgical wound, surgical drain, or a site of skin breakdown known as decubitus ulcers or bedsores. A diabetic individual is more prone to developing severe sepsis or TSS.

The following Example illustrates the invention: Example 1

The present study was undertaken to investigate whether a peptide (HKH20) derived from a region of HK could be used to block the activation of the contact system and to treat experimental S. pyogenes infections. The results show that HKH20 is a potent inhibitor of the contact system in vitro.

Material and Methods

Bacteria, growth conditions and plasma sources The S. pyogenes strain API (40/58) of serotype Ml was from the World

Health Organization Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic. Bacteria were grown in Todd-Hewitt broth at 37°C in the presence of 5% CO2. Fresh frozen plasma from healthy individuals was obtained from the blood bank at Lund University Hospital, Lund, Sweden, and kept frozen at -80⁰C until use.

Patient material

Plasma samples were obtained from a 64 year old male with a previous history of chronic obstructive lung disease and cardiac failure. The patient was admitted directly from the emergency room to the intensive care unit due to a severe deep tissue infection in the lower limb and septic shock with multiorgan failure. Blood cultures and wound cultures revealed growth of S. pyogenes T4. The Acute Physiology and Chronic Health Evaluation II score (APACHE II score) at the time for the first plasma collection was 39. The patient was treated with Penicillin G,

Clindamycin and intravenous gammaglobulin. The patient survived, although the right lower limb had to be amputated. Plasma samples were collected at 6, 18, 30, 44, 52, 93 hours after hospitalization. Blood samples were also collected from another three previously healthy patients with infections due to S. pyogenes. One 41 year old male with necrotizing fasciitis and S. pyogenes bacteremiae, one 68 year old female with erysipelas and one 43 year old male with a wound infection and a suspected STSS. The two latter had negative blood cultures. The ethics committee of Lund University approved of the study, and all samples were taken with the informed consent of the patients.

Peptides

The synthetic peptides based on sequences in human HK were produced as shown below:

Peptide SEQ aa-sequence Position in

ID SEQ ID NOj NO: 3

HKH20 1 HKHGHGHGKHKNKGKKNGKH 497 - 516

GCP28 4 GCPRDIPTNSPELEETLTHTITKLNAEN 266 - 293

Clotting assays

All clotting times were measured using an Amelung coagulometer. Activated partial thromboplastin time (aPTT) was measured by incubating 30 μl of HKH20 or GCP28 (50 μM final concentration) diluted in sterile water, with 100 μl human citrate-treated plasma for 1 min followed by the addition of 100 μl aPTT reagent

(aPTT Automate: Diagnostica Stago, Asnieres, France) for 60 s at 37oC. Clotting was initiated by the addition of 100 μl of a 25 mM CaC12 solution. In the Prothombrin time assay (PT), 30 μl of HKH20 or GCP28 (50 μM final concentration) was incubated with 100 μl human citrate-treated plasma for 1 min and clotting was initiated by the addition of 100 μl Thrombomax with calcium (PT reagent: Sigma- Aldrich). Thrombin clotting time (TCT) was measured by incubating 30 μl of HKH20 or GCP28 (50 μM final concentration) with 100 μl human citrate-treated plasma for 1 min and clotting was initiated by the addition of 100 μl Accuclot thrombin time reagent (TCT reagent: Sigma-Aldrich).

Chromogenic substrate assay

Overnight cultures of S. pyogenes API bacteria were washed three times with 50 mM Tris-HCl (pH 7.5), resuspended and diluted to a final concentration of 2 x 10¹⁰ CFU/ml in 50 mM Tris-HCl/ 50 μM ZnSO₄ buffer. Two hundred microliters of bacteria were incubated with 60 μl of HKH20 or GCP28 (final concentration 50 or 100 μM) for 30 s prior to the addition of 200 μl human plasma. Samples were incubated for 30 min at 37°C with shaking. After centrifugation, pellets were washed two times in 50 mM Tris (pH 7,5) centrifuged, resuspended in 100 μl 50 mM Tris- HCl/ 50 μM ZnSO₄ buffer containing 1 mM of the chromogenic substrate S-2302 and incubated for 30 min at 37°C. The samples were centrifuged and the absorbance of the supernatants was measured at 405 nm.

Bactericidal assay

Bacteria were grown to mid-log phase (OD approximately 0.4 at 620 nm) in TH broth, washed and diluted in 50 mM Tris-HCl (pH 7.5). Fifty microliters of bacteria (2 x 10⁶ CFU/ml) were incubated together with HKH20 at various concentrations for 1 h at 37°C. To quantify the bactericidal activity, serial dilutions of the incubation mixtures were plated on TH agar, incubated overnight at 37°C, and the number of CFU were determined.

Incubation of bacteria in plasma

Overnight cultures of S. pyogenes API bacteria were washed three times with 50 mM Tris-HCl (pH 7,5), resuspended and diluted to a final concentration of 2xlO¹⁰ CFU/ml in 50 mM Tris-HCl/ 50 μM ZnSO₄ buffer. One hundred microliters of bacteria were incubated with 30 μl of HKH20 or GCP28 (final concentration 50 or 100 μM) for 30 s prior to the addition of 100 μl human plasma. Samples were incubated on a rotator at room temperature for 15 min unless indicated otherwise. After incubation the bacteria were washed two times in 50 mM Tris (pH 7,5) centrifuged and resuspended in 50 μl 50 mM Tris-HCl/ 50 μM ZnSO₄ buffer. The suspensions were allowed to stay at room temperature for 15 min, followed by centrifugation at 10,000 rpm. Supernatants were collected and kept at -20°C until Western blot analysis.

Electrophoresis and Western blot analysis

SDS-PAGE was performed according to standard protocols. Proteins in the supernatants from bacterium-plasma incubations were separated on gels of 10% total acrylamide with 3% bisacrylamide. Plasma samples diluted 1/100, untreated or treated with kaolin for 15 min, served as controls. Before loading, samples were boiled in sample buffer containing 2% (w/v) SDS and 5% (v/v) beta-mercaptoethanol for 10 min. For Western blot analyses, proteins were transferred to nitrocellulose membranes, with a semidry transfer cell. Subsequently, nitrocellulose membranes were blocked in phosphate-buffered saline-Tween (PBST) containing 5% (w/v) nonfat dry milk at room temperature for 30 min, washed three times with PBST for 5 min, and incubated with sheep antibodies against HK and its degradation products (1 :6,000 in the blocking buffer) at room temperature for 30 min. After a washing step, membranes were incubated with peroxidase-coηjugated secondary donkey antibodies against goat IgG (MP Biomedicals) at room temperature for 30 min. Bound secondary antibodies were detected by the chemiluminescence method.

Animal experiments

API bacteria grown to early logarithmic phase were washed and diluted in PBS to a concentration of 2 x 10⁸ CFU/ml. Female BALB/c mice, 10 - 12 weeks old were injected intraperitoneal (i.p.) with 50 μl of the bacterial solution diluted in 50 μl PBS, or with 50 μl of bacteria together with 50 μl HKH20 (4 mg/ml which corresponds to 200 μg/mouse) that were mixed directly before injection, or with 100 μl PBS alone (control group). After 18 h mice were killed and spleens and lungs were removed. The spleens were kept on ice until homogenization in PBS and the number of CFU in the spleen was quantified by plating serial dilutions of the homogenized material on blood agar plates. Plates were incubated over night at 37°C. Lungs were further processed for microscopical analysis. Alternatively, mice were injected with 2 x 10⁷ CFU API bacteria in an air-pouch on the neck (ref). Mice showed systemic signs of being unwell 8 - 12 hours after infection. Bacteremia was diagnosed in all cases, and death occurred between 24 - 64 hours after challenge with bacteria. To measure clotting times, 8 groups of female B ALB/c mice were infected, and after various time points (Ohr, 4hr, 6hr, lOhr 12hr, 18hr, 24hr and 42hr), the animals were anesthetized with isofiuorane and terminal blood samples were taken. Approximately 0.5 ml of blood was drawn by cardiac puncture into polypropylene tubes containing 1/10 volume of 3.8% trisodium citrate. Plasma was separated by centrifugation and clotting times were measured as described earlier. In order to determine bacterial dissemination, spleens were removed and bacterial counts were determined as described above. Statistical analysis was performed. The P-value was determined by using the unpaired t-test (comparison of two groups) or the log-rank test (comparison of survival curves). All animal experiments were approved by the regional ethical committee for animal experimentation (permit M209-06).

Histochemistry and histo-pathological evaluation

Mice were sacrificed, lungs rapidly removed and fixed at 4°C for 24 h in buffered 4% formalin (pH 7.4). Tissues were dehydrated and imbedded in paraffin, cut into 4 μm sections, and mounted. After removal of the paraffin, tissues were stained with Mayers hematoxylin and eosin. Mouse organs were fixed in 4% paraformaldehyde (in PBS, pH 7.4) and processed for routine histo-pathological evaluation.

Scanning Electron Microscopy

Lung samples from the mice were fixed according to standard protocols. After fixation, samples were washed, dehydrated, critical point dried, and sputtered with palladium/gold. Samples were examined in a Jeol JSM-350 scanning electron microscope.

Results

Contact system activation in the plasma of a patient with S. pyogenes sepsis

An important role for the contact system in severe streptococcal infections is indicated by the observation that patients with STSS exhibit prolonged activated partial thromboplastin time (aPTT), whereas the extrinsic tissue factor-driven coagulation pathway is not affected. The results of this study also suggest a massive activation of the contact system leading to the degradation of HK and release of BK in these patients, contributes to the pain, vascular leakage, and severe hypotension, which are characteristic symptoms of STSS. To investigate contact activation during GAS infection, plasma samples from a patient suffering from STSS were subjected to Western blot analysis followed by immuno-detection with antibodies against HK and low molecular weight kininogen (LK; LK is a shorter splice variant of HK). Intact HK has a molecular weight at 120 kD (Fig. Ia, lane 1) and the addition of dextran sulfate to human plasma (Fig. 1, lane 2), results in the cleavage and a conversion of HK from a single-chain molecule into a two-chain molecule consisting of a heavy (55 kDa) and light (46 kDa) chain (Fig. Ia, lane 2). Analysis of plasma samples from the patient 6 and 18 hs after hospitalization revealed complete and partial degradation of HK, respectively (Fig. Ia, lane 3 and 4), while in subsequent plasma samples the HK signal reverts and the degradation products become weaker (Fig. Ia lane 5 to 8). These data support the notion that contact activation occurs early during the course of disease and leads to a systemic and almost complete activation of the system. HK degradation was also observed in patients with necrotizing fasciitis (Fig. Ib, lane 1) and erysipelas (Fig. Ib, lane 2), but not in a patient with suspected toxic shock syndrome in the absence of bacteremia (Fig. Ib, lane 3).

HKH20 inhibits the intrinsic, but not the extrinsic, pathway of coagulation Previous studies have shown that domain 5 of HK is a potent adhesin with high affinity for negatively charged surfaces, including cellular membranes (for instance endothelial cells and neutrophils) and artificial substances, such as kaolin or dextran sulfate. The present inventors hypothesised that HKH20 could block such interactions, and thereby inhibit contact activation in human plasma. Various clotting assays were assessed and it was found that HKH20 impairs the intrinsic pathway of coagulation in normal human plasma and in mouse (BALB/c) plasma. Figure 2a shows that incubation of plasma with HKH20 led to a 4-fold increase of the activated partial thromboplastin time (aPTT) when compared with GCP28, a peptide derived from domain D3 of HK, and with buffer alone (control). The effect of HKH20, as depicted in Figure 2b, was dose dependent. In contrast other components of the coagulation system as represented by the prothrombin time (PT), monitoring the extrinsic pathway of coagulation, and the thrombin clotting time (TCT), measuring thrombin-induced fibrin-network formation were not affected in normal human plasma (Fig. 2c) or mouse (BALB/c) plasma (not shown). The results demonstrate that HKH20 targets exclusively the intrinsic pathway of coagulation.

The inventors next investigated whether HKH20 interferes with the activation of PK at negatively charged surfaces. For these experiments, kaolin was pre-incubated with HKH20, GCP28, or buffer alone, followed by incubation with human plasma. After 15 min., unbound plasma proteins were removed by a centrifugation step and the PK activity at the surface of kaolin was determined using a specific chromogenic 5 substrate for PK (S-2302). HKH20 efficiently blocks PK activity, whereas the control peptide GCP28 has no influence on the enzymatic activity (Fig. 2d). Since HK is a substrate for activated PK, Western blot was used to analyze, whether inhibition of PK activity prevents HK degradation. Kaolin was pre-incubated with HKH20 and then mixed with human plasma for 15 min. Plasma alone or plasma treated with kaolin served as negative and positive controls, respectively. Samples were diluted in PBS, separated by SDS-PAGE, transfered onto nitrocellulose, and finally immunostained with antibodies directed against HK and low molecular weight kininogen (LK). Notably, LK is a shorter splice variant of HK, and a polyclonal antiserum against HK will also react with LK. Figure 2e depicts intact HK (Fig. 2e, lane 1) and processed HK following kaolin treatment (Fig. 2e, lane 2). Contact activation resulted in the cleavage and a conversion of HK from a single-chain molecule into a two-chain molecule consisting of a heavy (55 kDa) and light (46 kDa) chain (Fig. 2e, lane 2). It should be noted that previous studies have shown that this processing results in a complete release of BK from the HK precursor. When plasma was incubated with kaolin in the presence of HKH20, cleavage of HK was blocked and no degradation products were detected (Fig. 2e, lane 3). Taken together, the results demonstrate that HKH20 inhibits kaolin-induced activation of the contact system in human plasma.

HKH20 prevents contact activation at the surface of S. pyogenes bacteria

To determine whether treatment with HKH20 inhibits PK activity not only at the surface of kaolin, but also at bacterial surfaces, S. pyogenes bacteria of strain API were incubated with HKH20, followed by the addition of plasma. After 30 min, bacteria were washed and the PK activity at the bacterial surface was determined by measuring hydrolysis of substrate S-2302. The results show that treatment with HKH20 evoked a significant decrease in PK activity as compared to controls incubated with buffer alone or with the GCP28 peptide (Fig. 3a). Next the inventors analysed whether the co-application of HKH20 prevents HK processing under these experimental conditions. Thus, the bacteria of the API strain were preincubated with HKH20 for 1 min, followed by the addition of plasma for 15 min. Bacteria were then washed and resuspended in buffer, followed by an additional incubation step for 15 min to allow the dissociation of bacteria-bound plasma proteins from the bacterial surface. The bacteria were spun down and proteins in the supernatant were separated by SDS-PAGE, transferred to nitrocellulose, and immunostained with antibodies against HK and LK (Fig. 3b). Non-treated and kaolin-treated plasma served as negative and positive controls, respectively (Figure 3b, lanes 1 and 2). Western blot analysis of plasma proteins bound to and released from the streptococcal surface, revealed that HK was degraded (Fig. 3b, lane 3). However, when bacteria were pre-incubated with 50 or 100 μM HKH20 before adding plasma, the degradation HK bound to the bacteria was significantly decreased (Fig. 3b, lane 4 and 5). As a control, S. pyogenes was treated with peptide GCP28, which did not prevent HK cleavage (Fig. 3b, lane 6). The results show that the contact system is assembled and activated at the surface of S. pyogenes, and that HKH20 interferes with these molecular events. HKH20 blocks contact activation ex vivo and in vivo in mouse plasma To test the effect of HKH20 in a mouse model of invasive S. pyogenes infection, the inventors first tested the ex vivo effect of the peptide in mouse plasma. As, in the experiments with human plasma (Fig. 1), HKH20 significantly prolonged the aPTT when added to BALB/c plasma (Fig. 4), but the peptide had no effect on the TCT and PT (data not shown). In the next series of experiments, HKH20 (200 μg/animal) was given intraperitoneally to BALB/c mice (n=5/group). Plasma samples were collected 30 or 90 min after injection and the aPTT was measured. The analysis of the clotting times revealed that 60% of the mice treated with HKH20 showed significant prolongation of aPTT after 30 min, while normal clotting times were measured 90 min after injection (data not shown), suggesting that the peptide is rapidly cleared from the circulation.

HKH20 prevents lung lesions in mice infected with S. pyogenes bacteria

After testing the effect of HKH20 on un-infected mice, the inventors investigated its mode of action in a mouse model of S. pyogenes infection. Mice were intraperitoneally infected with 5x10⁶ CFU S. pyogenes and different groups (n=5/group) were injected with peptides HKH20, GCP28, or with PBS). Uninfected animals were used as healthy controls. Eighteen hours after challenge with API bacteria, all infected animals showed clear signs of sickness, like ruffled fur and less activity. Mice were sacrificed, lungs removed and examined by light and scanning electron microscopy. Figure 4 depicts micrographs of lungs from non-infected mice, showing no indication of pulmonary damage (Fig. 4a and e), whereas mice infected with S. pyogenes suffered from severe hemorrhage and massive tissue destruction (Fig. 4b and f). Lung lesions were almost completely prevented when bacteria were injected together with HKH20 (Fig. 4c and g), but not with peptide GCP28 (Fig. 4d and h). These in vivo data suggest that activation of the contact system by S. pyogenes induces vascular leakage, bleeding and destruction of the lung tissue, and that these effects can be prevented by administration of the HKH20 peptide.

Analysis of the mode of action ofHKH20 in vivo It has previously been reported that HKH20 may act by impairing polymorphonuclear neutrophil (PMN) recruitment, or by having antibacterial activity. To test whether any of these properties contribute to the protective effect of the peptide, we first investigated the effect of HKH20 on neutrophil influx. Mice were infected i.p. with the bacteria in the presence or absence of HKH20 as described above. Eighteen hours after infection, the animals were sacrificed and leukocyte recruitment into the peritoneum was monitored by FACS analysis. Figure 5 shows that the infection induced a massive invasion of PMNs, which was not significantly changed when mice were treated with HKH20 (14,5 x 10⁵ ± 3,2 xlO⁵ PMNs/ml in the untreated group versus 14,7 xl O⁵ ± 2,7 xlO⁵ PMNs/ml in the HKH20 treated group). Injection of HKH20 in uninfected mice did not cause a significant influx of PMNs. In a next series of experiments we tested whether PMNs contribute to the vascular leakage in the lungs of the infected animals. Mice were made neutropenic by injecting a monoclonal antibody against a neutrophil surface antigen (Ag GR-I) 16, which removed about 97% of all PMNs in the blood of the mice as determined by FACS analysis and white blood cell count. The neutropenic mice were then infected i.p. in the presence or absence of HKH20 as described above (n=3/group). Ten hours after challenge, mice were sacrificed and the lungs were removed and examined by light and scanning electron microscopy. Figure 5e depicts a representative electron micrograph showing that PMN depletion did not affect the lung architecture of uninfected animals, while infection with API bacteria caused severe lung lesions (Fig. 5f). As seen in non-neutropenic mice, lung damage was significantly reduced when infected mice were treated with HKH20 (Fig. 5g). The same pattern was observed when the lungs were analyzed by light microscopy (data not shown). These findings suggest that the effect of HKH20 relies on the inhibition of contact system activation rather then on preventing PMN recruitment. This notion was further supported by the observation that there was no significant difference in the number of CFUs from the spleens of the neutropenic animals, regardless of HKH20 treatment (data not shown). Finally, the fact that the number of CFUs from the spleens of normal and neutropenic mice showed no significant difference in the PBS, HKH20, and GCP groups, demonstrates that the concentration of HKH20 in the mouse experiments performed in this study, is too low to be antibacterial. Indeed, when the inventors directly tested the effect of HKH20 on S. pyogenes API bacteria in a bacterial assay, efficient killing of bacteria was recorded only at higher concentrations of HKH20 (38 % of the bacteria were killed when incubated with HKH20 at a concentration 0,5 mM for 60 min; data not shown).

Thus, the inventors have demonstrated that the protective effect of HKH20 cannot be due to antibacterial or PNM recruitment activities, and must be due to the inhibition of the contact system.

HKH20 prolongs the survival time of mice subcutaneously infected with S. pyogenes

In order to establish a patho-physiologically more relevant and conclusive infection model, mice were injected subcutaneously (s.c.) with a lethal dose of S. pyogenes. The animals were challenged with S. pyogenes bacteria administered s.c. into air pouches in the neck. Bacterial spreading to the bloodstream was followed by plating homogenates of the spleens, and was earliest detected 10 hours after infection (data not shown). Infected animals showed severe signs of sickness after eighteen hours and death occurred between 29 to 64 hours after infection. To determine the earliest time point of contact activation, plasma samples were collected from infected animals at different time points and the clotting times of the samples were determined. Figure 6a shows that the aPTT increased in a time dependent manner starting 10 hours after infection. The prothombin time, on the other hand, was not prolonged during the first 24 hours of infection, and increased for the first time after 42 h (Fig. 6b). Based on these results the mice were treated with the HKH20 peptide eight hours after infection. Electron microscopical analysis of lung biopsies from these animals revealed a significant reduction of lung lesions as compared with infected mice that were treated with the buffer control (Fig. 7 A = control, Fig. 7B = HKH20). A beneficial effect of HKH20 was also reflected in survival studies. Figure 8 shows that 41 % of infected mice died during the first 42 hours when they were not treated with HKH20 (median survival 43 hours). In the HKH20 treated group all animals were alive after 42 hours and statistical analysis revealed that HKH20 treatment caused a reproducible significant prolonged survival (P=0.0171). Comparing the overall mortality rate in the HKH20 and PBS groups the HKH20 treated animals showed a tendency toward prolonged survival but this was not statistical significant (P=0,0539) when the experiment was terminated after 96 hours. The results show that that a single injection of HKH20 8 h after infection delays the infection when mice are challenged with S. pyogenes bacteria (mortality rate > 95%).

HKH20 in combination with clindamycin treatment significantly increases survival in S. pyogenes infected mice

In the absence of an additional antimicrobial treatment, HKH20 administration resulted in a reversion of S. pyogenes-evoked lung lesions and prolonged survival times, but it did not significantly affect overall survival. The inventors therefore concluded that these animals die because they cannot contain the overwhelming bacterial proliferation. Thus, in the next series of animal experiments the inventors tested the effect of HKH20 in combination with an antibiotic (clindamycin). This approach resembles a more clinical situation, since clindamycin is the treatment of choice for patients with an invasive streptococcal infection. Initial experiments revealed that S. pyogenes strain API is clindamycin sensitive (MIC < 0,064 mg/1, E- test, not shown). Moreover, when API bacteria were grown in plasma together with 10 mg/ml clindamycin (156 x MIC cone.) for 24 hours, a 97% bacterial decrease of CFU was recorded, which was not affected when HKH20 was added to the plasma (not shown).

To test the effect of HKH20 in combination with a clindamycin treatment, mice were subcutaneously infected with S. pyogenes bacteria of the API strain. After 18 h, all animals showed clear signs of advanced sepsis (bacteremiae, ruffled fur, average 5% weight loss) and a slight but significant increase in the aPTT. This time point was then chosen for the first treatment with clindamycin (10mg/kg) and HKH20 (200μg/mouse). Both substances were given three times i.p. (42, 48 and 72 hours after infection) when the severity of sepsis reached a maximum. As control, mice were injected with clindamycin only (lOmg/kg): within the first 72 h, 80% of the animals in this control group had died, while all mice, injected with a combination of clindamycin and HKH20 were still alive (Fig 9). The overall mortality in the control group was 100% after 96 h, while 30% of the animals receiving a combination of clindamycin and HKH20 were completely recovered after 168 h. Statistical analysis revealed a median survival of 52,5 h in the clindamycin-treated group versus 103 h in the clindamycin/HKH20-treated group, which is highly significant (P=O₅OOOl). These data show that HKH20 has a strong effect on the survival time and significantly decreases the mortality rate in S. pyogenes infected mice, when given simultaneously with clindamycin.

Discussion

Over the last forty years the role of the contact system in infectious diseases has attracted considerable attention. Several studies have shown that a massive activation of the system can trigger the generation of pathological kinin levels, and lead to a consumption of contact factors followed by impaired hemostasis. For instance, it was demonstrated as early as 1970 by Mason and colleagues that patients with hypotensive septicemia have significantly decreased levels of contact factors whereas Pixley and colleagues published that low levels of FXII and HK in patients with SIRS (systemic inflammatory response syndrome) correlate with a fatal outcome of the disease. These and other findings support the notion that a systemic activation of the contact system contributes to the pathopysiology of conditions associated with severe and invasive infectious diseases. The contact system has been the target in animal models of infection. Thus, inhibition of FXII in a lethal experimental baboon model of bacteremia protects the animals from hypotension and prolongs their survival, and a synthetic plasma kallikrein inhibitor prevents infected rats from developing severe lung lesions in a Salmonella sepsis model. So far, only one clinical trial targeting the contact system has been conducted. In this study, deltibant, a B2R antagonist that blocks the effect of BK, was tested on patients with SIRS and presumed sepsis. The drug had no significant effect on risk-adjusted 28-day survival, even though posthoc analysis revealed a trend toward improvement. As the drug targets BK released as a consequence of contact activation, the drug acts on only one of the two kinin receptors. Hypothetically substances interfering earlier during contact activation could be more effective. An important role for the contact system in severe streptococcal infections is indicated by the observation that patients with streptococcal toxic shock syndrome exhibit prolonged aPTT, whereas the extrinsic tissue factor driven coagulation is not affected. The results suggest that contact activation in these patients contributes to the pain, vascular leakage, and severe hypotension which are characteristic symptoms of this syndrome. Sriskandan and colleagues have reported similar findings when analyzing the activation of the contact system in a mouse model of streptococcal myositis/fasciitis, hi the present study the inventors used a subcutaneously infection model in mouse for S. pyogenes sepsis. As described for the myositis/fasciitis mouse model, the inventors recorded a massive activation of the contact system occurring approximately 18 hours before the extrinsic pathway of coagulation was affected. To inhibit contact activation the inventors introduced a novel peptide inhibitor spanning a region of HK responsible for its binding to bacterial and eukaryotic cell surfaces and by interfering with this binding the assembly and activation of the contact system was blocked. In previous studies the inventors have utilized an irreversible contact system inhibitor. This tri- peptide derivate (H-D-Pro-Phe-Arg-CMK) forms a covalent link with the catalytic pocket of PK and FXII (25, 26). As the CMKgroup (chloromethylketone) is toxic and the substance may inhibit also other serine proteases when given at therapeutic doses, it is an unrealistic drug candidate. HKH20 has a completely different mode of action. The peptide displaces HK from its binding to negatively charged surfaces but does not influence the enzymatic activity of PK and FXII. The interference with a defined protein interaction should also enhance the specificity of HKH20. Moreover, HKH20 is not cytotoxic when tested against eukaryotic cells in vitro, and ongoing toxicity studies show that the peptide is well tolerated when administered to mice at high and repeated doses (200 μg/mouse two times daily for 5 days).

It is estimated that invasive 5. pyogenes infections cause more than 150000 deaths annually worldwide, despite the fact that the bacterium is fully sensitive to penicillin. The prognosis for patients with severe S. pyogenes infection is especially poor in cases with lung hemorrhage. In the mouse model of infection used in this investigation, lung bleedings occur in later stages of the disease. These were almost completely prevented by HKH20 treatment when the peptide was given eight hours after the infection was initiated. In the absence of other treatments, HKH20 did not significantly influence the overall survival of the mice, but significantly prolonged their survival time. However, in combination with clindamycin, HKH20 significantly increased overall survival, even eighteen hours after infection with an otherwise lethal dose of S. pyogenes bacteria. In patients with septic shock, the average survival rate is reduced by 7.6 percent every hour that adequate antibacterial therapy is delayed. Thus, the interference with the assembly and thereby the activation of the contact system at the surface of pathogenic bacteria slows down the progression of conditions associated with infections, and thus HKH20 and derivatives of HKH20 are novel and interesting therapeutic options.

This is particularly significant when it is considered that only one new drug has recently been launched for the treatment of patients with severe sepsis. However, this drug, activated protein C (APC), is only recommended in patients at high risk of death (sepsis-induced multiple organ failure, septic shock, or sepsis-induced acute respiratory distress syndrome). Moreover, due to the anti-coagulative properties of APC, the protein should not be given to patients at a risk of bleeding. Thus, there is an urgent need for novel therapies with a broader clinical indication and an improved safety profile. The contact system is activated at an early stage of the infection and it plays only a secondary role in hemostasis. This and the fact that its inactivation is combined with an inhibition of powerful inflammatory host responses, including vascular leakage, make HKH20 and derivates of HKH20 particularly interesting.

Claims

1. A polypeptide for use in the prevention or treatment of a condition associated with bacterial infection in an individual, wherein the polypeptide is 10 to 40 amino acids in length and comprises:

(a) at least 10 contiguous amino acids from the sequence of HKHGHGHGKHKNKGKKNGKH (SEQ ID NO: 1); or

(b) a variant of (a) having the ability to inhibit activation of the contact system and comprising upto four substitutions within the sequence from SEQ ID NO:1.

2. A polypeptide according to claim 1, wherein the variant comprises one, two, three or four substitutions within the sequence from SEQ ID NO:1.

3. A polypeptide according to claim 2, wherein the one, two, three or four substitutions are conservative.

4. A polypeptide according to any of the preceding claims wherein the bacterial infection is caused by a bacterial cell which is capable of activating the contact system at its cell surface.

5. A polypeptide according to claim 4, wherein high molecular weight kininogen (HK) binds to the bacterial cell surface.

6. A polypeptide according to claim 5 wherein the binding of HK on the bacterial cell surface is mediated by a protein.

7. A polypeptide according to claim 6 wherein the protein is a bacterial M protein, a bacterial curli protein, or a homologue of either thereof.

8. A polypeptide according to claim 5 wherein the binding of HK on the bacterial cell surface is mediated by a non-protein.

9. A polypeptide according to claim 8 wherein the non-protein is peptidoglycan, lipoteichoic acid, hyaluronic acid, or lipopolysaccharide

10. A polypeptide according to any one of claims 4 to 9 wherein the bacterial cell is a cell of a Gram negative or a Gram positive bacteria.

11. A polypeptide according to claim 10 wherein:

- the Gram negative bacteria is selected from Escherichia coli, Klebsiella spp., Enterobacter spp, Bordetella spp, Chlamydia spp. , Legionella spp. , Pseudemonas spp. , Mycoplasma spp., Haemophilus influenza, Serratia marcescens, Proteus mirabilis, , Acinetobacter baumannii, Stenotrophomonas maltophilia and Neisseria meningitides;

- the Gram positive bacteria is selected from Staphylococcus spp, Streptococcus spp. and Enterococcus spp.

12. A polypeptide according to claim 10 or 11 wherein the bacteria is selected from Streptococcus spp. , Staphylococcus spp., E. coli and Salmonella spp.

13. A polypeptide according to any one of claims 10 to 12 wherein the bacterial cell is from Streptococcus pyogenes or Staphylococcus aureus.

14. A polypeptide according to any one of the preceding claims wherein the individual is a mammal.

15. A polypeptide according to claim 14 wherein the individual is a human.

16. A polypeptide according to any one of the preceding claims, wherein the condition is characterised in that the individual has a confirmed or suspected infection caused by a bacterial cell as defined in any one of claims 4 to 13, and/or displays one or more of the following symptoms: i) fever (temperature >38⁰C) or hypothermia (temperature <36⁰C); ii) heart rate >90 beats per minute; iii) respiratory rate >20 breaths per minute or PaCO₂ <32 mm Hg; and iv) white blood cell count >12 (xl0⁹cells/L) or <4 (xl O⁹ cells/1),

17. A polypeptide according to any one of claims 1 to 16, wherein the condition is characterised in that the individual has a confirmed or suspected infection caused by a bacterial cell as defined in any one of claims 4 to 13, and displays one or more of the following symptoms: i) Body temperature > 38.9 ⁰C ii) Systolic blood pressure < 90 mmHg iii) Diffuse rash, intense erythroderma, blanching with subsequent desquamation, especially of the palms and soles; and iv) Involvement of three or more organ systems selected from: gastrointestinal, muscular, mucous membrane, renal, hepatic, hematologic, and neurologic.

18. A polypeptide according to any one of the preceding claims wherein the condition is sepsis, severe sepsis, toxic shock syndrome (TSS) or toxic shock like syndrome (TSLS).

19. A polypeptide according to any one of claims 15 to 18 wherein the individual is: irnmuno-compromised; a diabetic; a hospitalised patient with an intravenous line, a surgical wound, a surgical drain or a bedsore; or any combination thereof.

20. A polypeptide according to any one of claims 1 to 19 and at least one of: i) an antibiotic; ii) activated protein C (APC) for simultaneous, separate or sequential use in the treatment of a condition associated with bacterial infection in an individual.

21. A polypeptide according to claim 20 wherein the antibiotic is a broad- spectrum antibiotic.

22. A polypeptide according to claim 20 wherein the antibiotic is clindamycin.

23. A polynucleotide encoding a polypeptide according to any one of the preceding claims.

24. A pharmaceutical composition comprising a polypeptide or polynucleotide according to any one of the preceding claims and a pharmaceutically acceptable carrier or diluent.

25. A polypeptide according to any one of claims 1 to 20, a polynucleotide according to claim 23 or a composition according to claim 24 formulated for oral administration, nasal administration, subcutaneous administration, sublingual administration, intradermal administration, buccal administration or for administration by inhalation or by injection.

26. A method of treating an individual for a condition associated with bacterial infection comprising administering a therapeutically effective amount of a polypeptide according to any one of claims 1 to 20, or a polynucleotide according to claim 23, or a composition according to claim 24.