New treatments for infectious diseases

Abstract

The present invention relates to the treatment of infectious diseases. In this study, we identify potential defense mechanisms involved in maintaining brain vascular integrity during bacterial infection and perform RNA analysis of primary endothelial cells isolated from human skin (HDMEC) or brain (HBMEC) microvessels. The cells were either uninfected or infected with Neisseria meningitidis in vitro. We found 40 genes, especially ANGPTL4, that are specifically regulated in brain endothelial cells during infection. In the context of sepsis, ANGPTL4 was found to be the main barrier stabilizing protein with a defensive effect against bacterial infection. ANGPTL4 (or derived peptides) also showed potential as an adjunctive therapy to reduce bacteria-induced vascular dysfunction and mortality. Thus, the present invention relates to peptides derived from the protein ANGPTL4 and their use in the treatment of infectious diseases.

Description

[Technical field]

[0001] The present invention relates to peptides derived from the protein ANGPTL4 and their use in the treatment of infectious diseases. [Background technology]

[0002] Sepsis is one of the most common fatal diseases in the world and is currently recognized by the World Health Organization (WHO) as a global health priority [Reference 1]. Globally, approximately 1,000 people die from sepsis every hour, and the number of sepsis cases continues to rise dramatically. Sepsis is one of the few diseases that ravages resource-poor and developed countries alike. Globally, more than 49 million people are affected by sepsis each year, resulting in 11 million deaths [References 1, 2]. Patients with these conditions are often treated in hospital intensive care units, which are estimated to cost $24 billion annually. The total cost of treating patients hospitalized with this condition is increasing by an average of 19% each year, adjusted for inflation. Despite the high cost of treatment, sepsis is often a life-threatening condition. Patients who recover from severe sepsis are more likely to suffer from permanent organ damage, cognitive impairment, and physical disability.

[0003] Vascular endothelial cells are among the first cells in the body to come into contact with circulating bacteria and / or bacterial molecules, making them the primary target of sepsis-induced events [1-3]. The human vasculature is significantly altered by a mixture of microbial virulence factors and proinflammatory mediators released by activated blood cells. Most of the physiological functions of the endothelium are inhibited, leading to increased vascular permeability, activation of coagulation, and participation in the inflammatory response. Severe endothelial dysfunction contributes to hypoxic injury in various organs, leading to multiple organ failure [3]. Disseminated intravascular coagulation is also a major complication of sepsis.

[0004] Treatment of patients with sepsis consists of a combination of antibiotic treatment, elimination of the source of infection, and hemodynamic, respiratory, and metabolic support. Aggressive fluid resuscitation and hemodynamic support are used to restore tissue perfusion and normalize cellular metabolism [2]. However, mortality remains high in patients with severe sepsis and septic shock. To date, multiple clinical trials aimed at suppressing host inflammatory responses and coagulation abnormalities in severe infections have been performed with limited clinical success [1]. Thus, new strategies aimed at maintaining vascular integrity / barrier function, implementing endothelial cell protective mechanisms, and suppressing deleterious hemostatic and inflammatory processes are urgently needed.

[0005] The blood-brain barrier (BBB) ​​is a critical barrier in tightly regulating the homeostasis of the central nervous system, and also functions in regulating inflammation and hemostasis [4,5]. The BBB is formed by continuous, non-fenestrated specialized endothelial cells expressing intercellular tight junction proteins and specific transporters that limit paracellular diffusion and regulate the selective transport and metabolism of substances from the blood to the brain [6,7]. An extensive coverage of pericytes and smooth muscle cells constitutes an additional barrier [8]. Finally, a thick basement membrane in close contact with the foot processes of astrocytes also forms a limiting membrane [9]. These specific functions help to protect the brain from invading pathogens. However, little is still known about the cytoprotective mechanisms involved in maintaining the integrity of the BBB during infection.

[0006] A representative pathogen of invasive bacterial infections that causes severe sepsis and meningitis is Neisseria meningitidis (Nm). Nm is an extracellular Gram-negative diplococcus restricted to humans, and is usually present as an asymptomatic carrier in the nasopharynx of about 10% of the population. Invasive infection with this bacterium can cause severe septic shock [Reference 10]. Upon entering the blood circulation, Nm attaches to the apical cell surface of endothelial cells, facilitating bacterial attachment, especially in the microcirculation, where blood flow velocity is slow compared to large blood vessels [Reference 11]. The interaction of Nm with human microvasculature can cause endothelial dysfunction syndrome, which in the worst case can lead to fulminant purpura, a life-threatening syndrome with vascular leakage, widespread thrombosis, and septic shock that kills 30% of patients [Reference 4]. In addition, Nm also interacts with cerebral microvascular endothelial cells. By colonizing the subarachnoid space, parenchyma, and cerebral capillaries of the choroid plexus

[12] , bacteria can breach this strong barrier and reach the meninges and cerebrospinal fluid, where they proliferate and cause meningitis. In contrast to what is observed in the periphery, this step occurs without signs of intravascular coagulation or vascular leakage and with the BBB integrity largely intact. Summary of the Invention

[0007] To identify potential defense mechanisms involved in maintaining brain vascular integrity during bacterial infection, the inventors performed RNA analysis of primary endothelial cells isolated from human skin (HDMEC) or brain (HBMEC) microvessels, either left uninfected or infected with N. meningitidis in vitro. They found 40 genes that were specifically regulated in brain endothelial cells during infection. Consistent with their hypothesis, these included negative regulators of the innate immune response that may protect against excessive inflammation, as well as factors with vasostabilizing properties that may contribute to brain vascular protection.

[0008] They searched for paracrine and juxtacrine molecules / signals that may underlie the maintenance of the BBB and focused on ANGPTL4 (angiopoietin-like 4), a secreted glycoprotein with diverse roles in vascular permeability, angiogenesis, glucose homeostasis, lipid metabolism, and inflammation

[13] . ANGPTL4 is a member of the angiogenic factor-like protein family (ANGPTL1-8) and shares a similar structure with an N-terminal helical coiled-coil domain and a C-terminal fibrinogen-like domain separated by a cleavable linker. Notably, ANGPTL4 has high sequence homology and related functions with ANGPTL3 and ANGPTL8. ANGPTL4 has long been considered an orphan receptor because it does not bind to the angiogenic factor tyrosine kinase receptors Tie1 and Tie2, but recent studies have shown that ANGPTL4 has many binding partners, including lipoprotein lipase, integrins, cadherins, and syndecans

[13] . To date, its role in vascular integrity is unclear. Known to be induced by hypoxia and produced by vascular cells in ischemic disease, ANGPTL4 has been shown to exert both angiogenic and antiangiogenic effects [14,15] and to increase or decrease vascular permeability depending on pathophysiological conditions, tissue context, and / or its binding partners, release, and proteolysis [16-20].

[0009] In this study, we show that in the setting of sepsis, ANGPTL4 is a key barrier-stabilizing protein that provides protection against bacterial infection, and we also demonstrate the potential of ANGPTL4 (or derived peptides) as an adjunctive therapy to reduce bacterial-induced vascular dysfunction and mortality.

[0010] The present invention therefore relates to peptides derived from the protein ANGPTL4 and their use in the treatment of infectious diseases. In particular, the present invention is defined by its claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] [Peptide or protein of the present invention] The first aspect of the present invention relates to a method for the preparation of a polypeptide having the amino acid sequence X1SALERRLSACGSX 14 X 15 (SEQ ID NO: 65) or a function-conservative variant thereof, wherein X1 is no amino acid or leucine (L), and X 14 is no amino acid or alanine (A), and X 15 is no amino acid or cysteine ​​(C).

[0012] In one embodiment, X is leucine (L) and X 14 is no amino acid and X 15 is without amino acids (peptide P12). In one embodiment, X is leucine (L) and X 14 is alanine (A) and X 15 is cysteine ​​(C) (peptide P9).

[0013] In one embodiment, X is no amino acid; 14 is alanine (A) and X 15 is cysteine ​​(C) (peptide P13).

[0014] In one embodiment, X is leucine (L) and X 14 is alanine (A) and X 15 is free of amino acids.

[0015] In one embodiment, the peptide derived from protein ANGPTL4 contains or consists of amino acid residues 66 to 78 in SEQ ID NO:1, where the amino acid at position 76 is any naturally occurring amino acid (peptide P12 or peptide P12') or a function-conservative variant thereof.

[0016] In one embodiment, the peptide contains or consists of the amino acid sequence LSALERRLSACGS (SEQ ID NO:55) (Peptide 12).

[0017] In one embodiment, the peptide derived from the protein ANGPTL4 contains or consists of amino acid residues 67-80 of SEQ ID NO:1, with the amino acids at positions 76 and 80 being any naturally occurring amino acid (peptide P13 or peptide P13') or a function-conservative variant thereof.

[0018] In one embodiment, the peptide contains or consists of the amino acid sequence SALERRLSACGSAC (SEQ ID NO:58) (Peptide 13).

[0019] In one embodiment, the peptide derived from the protein ANGPTL4 contains or consists of the amino acid residues 66 to 80 in SEQ ID NO:1, with the amino acids at positions 76 and 80 being any naturally occurring amino acid (peptide P9 or peptide P9') or a function-conservative variant thereof.

[0020] Thus, a second aspect of the present invention relates to a peptide derived from the ANGPTL4 protein comprising or consisting of amino acid residues 66 to 80 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are any naturally occurring amino acid (peptide P9 or peptide P9') or a function-conservative variant thereof.

[0021] In other words, the first aspect of the present invention relates to a method for the preparation of a polypeptide having the amino acid sequence LSALERRLSAX 11 GSAX 15 (SEQ ID NO: 2, peptide P9 or peptide P9') 11 and X 15 is any naturally occurring amino acid or a function-conservative variant thereof.

[0022] According to a particular embodiment and for all peptides or proteins of the invention, the amino acids at positions 76 and 80 may be amino acids selected from the group consisting of cysteine ​​(Cys or C) or alanine (Ala or A) or serine (Ser or S).

[0023] Thus, in a particular embodiment, the invention relates to a peptide derived from the ANGPTL4 protein containing or consisting of amino acid residues 66 to 80 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or function-conservative variants thereof.

[0024] In other words, the present invention relates to the amino acid sequence LSALERRLSAX 11 GSAX 15 (SEQ ID NO: 2), 11 and X 15 is an amino acid selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or a function-conservative variant thereof.

[0025] As used herein, the term "ANGPTL4" means "angiopoietin-like protein 4" and refers to a serum hormone that directly regulates lipid and glucose metabolism. Native full-length ANGPTL4 can form higher-order structures via intermolecular disulfide bonds. ANGPTL4 assembles through its N-terminal region (nANGPTL4). Full-length ANGPTL4 undergoes proteolysis at the linker region, releasing nANGPTL4 and the monomeric C-terminal portion of ANGPTL4 (cANGPTL4). The Entrez Gene reference for the gene sequence is 51129, and the UniProt reference for the protein is Q9BY76.

[0026] Amino acid sequence of human ANGPTL4 (SEQ ID NO:1): MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRVDPEV LHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFE IQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQL TAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS In another specific embodiment, the invention relates to a peptide derived from the ANGPTL4 protein comprising or consisting of the amino acid residues 65 to 80 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are any naturally occurring amino acid (peptide P6 or peptide P6') or a function-conservative variant thereof.

[0027] In other words, and in a particular embodiment, the present invention relates to the amino acid sequence QLSALERRLSAX 12 GSAX 16 (SEQ ID NO: 3, peptide P6 or peptide P6') 12 and X 16 is any naturally occurring amino acid or a function-conservative variant thereof.

[0028] In other specific embodiments, the invention relates to a peptide derived from the ANGPTL4 protein containing or consisting of amino acid residues 65 to 80 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or function-conservative variants thereof.

[0029] In other words, and in a particular embodiment, the present invention relates to the amino acid sequence QLSALERRLSAX 12 GSAX 16 (SEQ ID NO: 3), 12 and X 16 is an amino acid selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or a function-conservative variant thereof.

[0030] In another specific embodiment, the invention relates to a peptide derived from the ANGPTL4 protein comprising or consisting of the amino acid residues at positions 56 to 83 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are any naturally occurring amino acid (peptide P4 or peptide P4') or a function-conservative variant thereof.

[0031] In other words, and in other specific embodiments, the amino acid sequence REHAERTRSQLSALERRLSAX 21 GSAX 25 QGT (SEQ ID NO: 4, peptide P4 or peptide P4') 12 and X 16 is any naturally occurring amino acid or a function-conservative variant thereof.

[0032] In another specific embodiment, the invention relates to a peptide derived from the ANGPTL4 protein containing or consisting of amino acid residues 56 to 83 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or function-conservative variants thereof.

[0033] In other words, and in other specific embodiments, the present invention relates to the amino acid sequence REHAERTRSQLSALERRLSAX 21 GSAX 25 Related to a peptide derived from the ANGPTL4 protein containing or consisting of QGT (SEQ ID NO: 4), 21 and X 25 is an amino acid selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or a function-conservative variant thereof.

[0034] In another specific embodiment, the invention relates to a peptide derived from the ANGPTL4 protein comprising or consisting of the amino acid residues at positions 38 to 83 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are any naturally occurring amino acid (peptide P1 or peptide P1') or a function-conservative variant thereof.

[0035] In other words, and in other specific embodiments, the present invention provides the amino acid sequence WDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAX 39 GSAX 43 QGT (SEQ ID NO: 5, peptide P1 or peptide P1') 39 and X 43 is any naturally occurring amino acid or a function-conservative variant thereof.

[0036] In another specific embodiment, the invention relates to a peptide derived from the ANGPTL4 protein containing or consisting of amino acid residues 38 to 83 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or function-conservative variants thereof.

[0037] In other words, and in other specific embodiments, the present invention provides the amino acid sequence WDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAX 39 GSAX 43 Related to a peptide derived from ANGPTL4 that contains or consists of QGT (SEQ ID NO: 5), 39 and X 43 is an amino acid selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or a function-conservative variant thereof.

[0038] In another specific embodiment, the invention relates to a peptide derived from the ANGPTL4 protein containing or consisting of amino acid residues 26 to 164 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are any naturally occurring amino acid (nANGPTL4 or nANGPTL4') or a function-conservative variant thereof.

[0039] In other words, and in other specific embodiments, the present invention provides the amino acid sequence GPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAX 51 GSAX 55 QGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRK (SEQ ID NO: 6, nANGPTL4 or nANGPTL4'), 51 and X 55is any naturally occurring amino acid or a function-conservative variant thereof.

[0040] In another specific embodiment, the invention relates to a peptide derived from the ANGPTL4 protein containing or consisting of amino acid residues 26 to 164 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or function-conservative variants thereof.

[0041] In other words, and in other specific embodiments, the present invention provides the amino acid sequence GPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAX 51 GSAX 55 QGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRK (SEQ ID NO: 6), 51 and X 55 is an amino acid selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or a function-conservative variant thereof.

[0042] In another specific embodiment, the invention relates to a peptide derived from the protein ANGPTL4, comprising or consisting of the amino acid residues at positions 1 to 406 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are any naturally occurring amino acid (ANGPTL4 or ANGPTL4') or a function-conservative variant thereof.

[0043] In other words, and in other specific embodiments, the present invention provides the amino acid sequence MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAX 76 GSAX 80QGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS (SEQ ID NO: 7, ANGPTL4 or ANGPTL4'), 76 and X 80 is any naturally occurring amino acid or a function-conservative variant thereof.

[0044] In another specific embodiment, the invention relates to a protein derived from the protein ANGPTL4, which contains or consists of amino acid residues 1 to 406 in SEQ ID NO:1, wherein the amino acids at positions 76 and 80 are selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A) or serine (Ser or S), or function-conservative variants thereof.

[0045] In other words, and in other specific embodiments, the present invention provides the amino acid sequence MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAX 76 GSAX 80QGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS (SEQ ID NO: 7), 76 and X 80 is an amino acid selected from the group consisting of cysteine ​​(Cys or C), or alanine (Ala or A), or serine (S), or function-conservative variants thereof.

[0046] In certain embodiments, the peptide or protein of the present invention comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 1, 8-30 and 37-64.

[0047] Peptide P9: LSALERRLSACGSAC (SEQ ID NO: 8) Peptide P9 C76A,C80A: LSALERRLSAAGSAA (SEQ ID NO: 9) Peptide P9 C76A: LSALERRLSAAGSAC (SEQ ID NO: 10) Peptide P9 C80A: LSALERRLSACGSAA (SEQ ID NO: 11) Peptide P9 C76S,C80S: LSALERRLSASGSAS (SEQ ID NO:37) Peptide P9 C76S: LSALERRLSASGSAC (SEQ ID NO: 38) Peptide P9 C80S: LSALERRLSACGSAS (SEQ ID NO: 39) Peptide P6: QLSALERRLSACGSAC (SEQ ID NO: 12) Peptide P6 C76A,C80A: QLSALERRLSAAGSAA (SEQ ID NO: 13) Peptide P6: C76A: QLSALERRLSAAGSAC (SEQ ID NO: 14) Peptide P6 C80A: QLSALERRLSACGSAA (SEQ ID NO: 15) Peptide P6 C76S,C80S: QLSALERRLSASGSAS (SEQ ID NO: 40) Peptide P6 C76S: QLSALERRLSASGSAC (SEQ ID NO: 41) Peptide P6 C80S:QLSALERRLSACGSAS (SEQ ID NO:42) Peptide P4: REHAERTRSQLSALERRLSACGSACQGT (SEQ ID NO: 16) Peptide P4 C76A,C80A: REHAERTRSQLSALERRLSAAGSAAQGT (SEQ ID NO: 17) Peptide P4 C76A: REHAERTRSQLSALERRLSAAGSACQGT (SEQ ID NO: 18) Peptide P4 C80A: REHAERTRSQLSALERRLSACGSAAQGT (SEQ ID NO: 19) Peptide P4 C76S,C80S: REHAERTRSQLSALERRLSASGSASQGT (SEQ ID NO: 43) Peptide P4C76S:REHAERTRSQLSALERRLSASGSACQGT (SEQ ID NO:44) Peptide P4C80S:REHAERTRSQLSALERRLSACGSASQGT (SEQ ID NO: 45) Peptide P1: WDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSACQGT (SEQ ID NO: 20) Peptide P1 C76A,C80A: WDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAAGSAAQGT (SEQ ID NO: 21) Peptide P1 C76A: WDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAAGSACQGT (SEQ ID NO: 22) Peptide P1 C80A: WDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSAAQGT (SEQ ID NO: 23) Peptide P1 C76S,C80S: WDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSASGSASQGT (SEQ ID NO: 46) Peptide P1 C76S: WDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSASGSACQGT (SEQ ID NO: 47) Peptide P1 C80S: WDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSASQGT (SEQ ID NO: 48) Peptide nANGPTL4: GPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRK (SEQ ID NO: 24) Peptide nANGPTL4 C76A,C80A: GPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAAGSAAQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRK (SEQ ID NO: 25) Peptide nANGPTL4 C76A: GPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAAGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRK (SEQ ID NO: 26) Peptide nANGPTL4 C80A: GPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSAAQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRK (SEQ ID NO: 27) Peptide nANGPTL4 C76S,C80S: GPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSASGSASQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRK (SEQ ID NO: 49) Peptide nANGPTL4 C76S: GPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSASGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRK (SEQ ID NO: 50) Peptide nANGPTL4 C80S: GPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSASQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRK (SEQ ID NO: 51) Protein ANGPTL4 C76A,C80A: MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAAGSAAQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS (SEQ ID NO: 28) Protein ANGPTL4 C76A: MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSAAGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS (SEQ ID NO: 29) Protein ANGPTL4 C80A: MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSAAQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS (SEQ ID NO: 30) Protein ANGPTL4 C76S,C80S: MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSASGSASQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS (SEQ ID NO: 52) Protein ANGPTL4 C76S: MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSASGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS (SEQ ID NO: 53) Protein ANGPTL4 C80S: MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSASQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS (SEQ ID NO: 54) Peptide 12: LSALERRLSACGS (SEQ ID NO:55) Peptide 12 C76A: LSALERRLSAAGS (SEQ ID NO:56) Peptide 12 C76S: LSALERRLSASGS (SEQ ID NO:57) Peptide 13: SALERRLSACGSAC (SEQ ID NO:58) Peptide 13 C76A,C80A: SALERRLSAAGSAA (SEQ ID NO:59) Peptide 13 C76A: SALERRLSAAGSAC (SEQ ID NO: 60) Peptide 13 C80A: SALERRLSACGSAA (SEQ ID NO: 61) Peptide 13 C76S,C80S: SALERRLSASGSAS (SEQ ID NO:62) Peptide 13 C76S: SALERRLSASGSAC (SEQ ID NO: 63) Peptide 13 C80S: SALERRLSACGSAS (SEQ ID NO: 64) According to the invention, the peptide or protein of the invention may also have the mutations S67R and / or R72L.

[0048] As used herein, the term "function-conservative variant" refers to a change (insertion, deletion, substitution) in a specific amino acid residue in a protein or enzyme without changing the overall conformation and function of the polypeptide. Such variants include proteins with amino acid changes such as deletion, insertion, and / or substitution. "Deletion" refers to the absence of one or more amino acids in a protein. "Insertion" refers to the addition of one or more amino acids in a protein. "Substitution" refers to the replacement of one or more amino acids in a protein with another amino acid residue. Usually, a specific amino acid is replaced with an amino acid with similar properties (e.g., polarity, hydrogen bonding ability, acidic, basic, hydrophobic, aromatic, etc.). Amino acids not shown as conserved amino acids may differ in proteins, and the similarity of protein or amino acid sequences between any two proteins with similar functions may vary and may range from 70% to 99%, for example, as determined according to an alignment scheme such as the cluster method with similarity based on the MEGALIGN algorithm. "Function-conserving variants" also include polypeptides that have an amino acid identity of at least 60%, particularly at least 75%, more particularly at least 85%, even more particularly at least 90%, and even more particularly at least 95% as determined by the BLAST or FASTA algorithm, and have the same or substantially similar properties or functions as the native or parent protein to which they are compared. Two amino acid sequences are "substantially homologous" or "substantially similar" if the amino acids are more than 80%, particularly more than 85%, particularly more than 90% identical, or more than about 90%, particularly more than 95% similar (functionally identical) over the entire length of the shorter sequence. Two amino acid sequences, particularly similar or homologous sequences, are identified by aligning them using, for example, the pileup program of GCG (Genetics Computer Group, Program Manual GCG Package, Version 7, Madison, Wis.) or any sequence comparison algorithm such as BLAST, FASTA, etc.

[0049] In certain embodiments, the peptides of the present invention are 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 or 13 amino acids.

[0050] In certain embodiments, the peptides of the present invention are at least 70% identical, more particularly at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the peptides or proteins described in the present invention (SEQ ID NOs: 1-30 and 37-64, in particular 1, 8-30 and 37-64), and can still efficiently reduce the incidence of intravascular coagulation, vascular degeneration, and bacterial infections in sepsis, and can reduce the damage caused by the major bacterial infections.

[0051] In general, the present invention encompasses peptides or proteins containing or consisting of the amino acid sequences set forth in SEQ ID NOs: 1-30 and 37-64, in particular 1, 8-30 and 37-64, in which one or more residues have been conservatively replaced with functionally similar residues, while exhibiting the functional profile of the peptides or proteins of the invention (i.e., retaining the ability to reduce the incidence of vascular degeneration and intravascular coagulation in bacterial infections and mitigating the high toll of sepsis).

[0052] Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue, such as isoleucine, valine, leucine, or methionine, for another amino acid residue; the substitution of a polar (aqueous) residue, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine, for another amino acid residue; the substitution of a basic residue, such as lysine, arginine, or histidine, for another amino acid residue; or the substitution of an acidic residue, such as aspartic acid or glutamic acid, for another amino acid residue.

[0053] The term "conservative substitution" also includes the use of a chemically derivatized residue in place of a residue that is not chemically derivatized. "Chemical derivative" refers to a peptide having one or more residues that are chemically derivatized by reaction of a functional side chain. Examples of such derivatized molecules include molecules in which free amino groups are derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups, or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters, or other esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-imi-benzylhistidine. Chemical derivatives also include peptides that contain one or more naturally occurring amino acid derivatives of the 20 common amino acids. For example, 4-hydroxyproline may be replaced by proline, 5-hydroxylysine by lysine, 3-methylhistidine by histidine, homoserine by serine, and ornithine by lysine.

[0054] In one embodiment, the peptide or protein of the present invention may comprise a tag. The tag is an epitope-containing sequence and is useful for purifying the peptide. The tag can be attached by various techniques, such as affinity chromatography, and used to locate the peptide or polypeptide in a cell or tissue sample using immunolabeling techniques, or to detect the peptide or polypeptide, such as by immunoblotting. Examples of tags commonly used in the art include GST (glutathione-S-transferase) tag, FLAG™ tag, Strep-tag™, V5 tag, myc tag, His tag, etc.

[0055] In one embodiment, the peptide or protein of the present invention can be labeled with a fluorescent dye. Dye-labeled fluorescent peptides are important tools in cell research. Peptides can be labeled at the N-terminus or C-terminus.

[0056] N-Terminal Peptide Labeling with Amine-Reactive Fluorescent Dyes: Amine-reactive fluorescent probes have been widely used to modify the N-terminus or lysine residues of peptides or proteins. Many amino-group-reactive fluorescent dyes have been developed to label various peptides, and the resulting conjugates are widely used in biological applications. The three major classes of amine-reactive fluorescent reagents currently used to label peptides are succinimidyl esters (SEs), isothiocyanates, and sulfonyl chlorides.

[0057] C-Terminal Labeling with Amine-Containing Fluorescent Dyes: Amine-containing dyes are used to modify peptides using water-soluble carbodiimides (such as EDC) to convert the peptide's carboxyl groups into amide groups. Either NHS or NHSS can be used to improve the coupling efficiency of EDC-mediated protein-carboxylic acid conjugation.

[0058] In certain embodiments, the peptide or protein used in the therapeutic method of the present invention may be modified to enhance its therapeutic effect. Such modifications of therapeutic compounds may be used to reduce toxicity, extend circulation time, or change biodistribution. For example, by combining with various drug carrier vehicles that change biodistribution, the toxicity of potentially important therapeutic compounds can be significantly reduced.

[0059] Strategies for improving drug efficacy include the use of water-soluble polymers.Various water-soluble polymers have been shown to change biodistribution, improve cellular uptake patterns, change the permeability of physiological barriers, and change clearance rates from the body.To achieve targeting or sustained release effects, water-soluble polymers have been synthesized to contain drug moieties as terminal groups, as part of the backbone, or as pendant groups on the polymer chain.

[0060] Polyethylene glycol (PEG) is widely used as a drug carrier due to its high biocompatibility and easy modifiability. Attachment to a variety of drugs, proteins, and liposomes has been shown to improve residence time and reduce toxicity. PEG can be conjugated to active agents through hydroxyl groups at the ends of the chains and other chemical methods, but PEG itself is limited to a maximum of two active agents per molecule. Alternatively, copolymers of PEG and amino acids have been investigated as new biomaterials that maintain the biocompatibility of PEG while offering the advantage of a large number of conjugation sites per molecule (capable of loading more drugs), and can also be synthetically engineered for a variety of applications.

[0061] Those skilled in the art are aware of PEGylation techniques that effectively modify drugs. For example, drug delivery polymers consisting of alternating copolymers of PEG and trifunctional monomers such as lysine are used by VectraMed (Plainboro, NJ). PEG chains (usually 2000 daltons or less) are linked to the α- and ε-amino groups of lysine through stable urethane bonds. Such copolymers retain the desirable properties of PEG while providing reactive pendant groups (the carboxylic acid groups of lysine) at tightly controlled and predefined intervals along the polymer chain. The reactive pendant groups can be used for derivatization, crosslinking, or linking to other molecules. These polymers are useful for producing stable, long-circulating prodrugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkages between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug / linking group conjugates and the amount of drug per molecular weight of the conjugate (the smaller the PEG segment, the higher the drug loading). In general, increasing the overall molecular weight of the block copolymer conjugate increases the circulating half-life of the conjugate. However, the conjugate must be readily degraded or have a molecular weight below the threshold limiting glomerular filtration rate (eg, less than 45 kDa).

[0062] Furthermore, in addition to the importance of the polymer backbone in maintaining circulatory half-life and biodistribution, linkers can also be used to maintain the therapeutic agent in a prodrug form until it is released from the backbone polymer by a specific trigger, usually enzymatic activity, in the target tissue. For example, this type of tissue-activated drug delivery is particularly useful when delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of a lesion. Linker libraries for use in activated drug delivery are well known to those skilled in the art and may be based on enzyme kinetics, the abundance of active enzymes, and the cleavage specificity of selected disease-specific enzymes (see, for example, technology established by VectraMed, Inc., Plainboro, NJ). Such linkers can be used to modify peptides derived from those described herein for therapeutic delivery.

[0063] In certain embodiments, the peptides of the invention may be peptidomimetics. As used herein, the term "peptidomimetic" refers to a polypeptide designed to mimic a peptide, such as the peptides of the invention. The term "peptidomimetic" may also refer to a non-peptide chemical moiety. A peptide is a short chain of amino acid monomers linked by peptide (amide) bonds, which are covalent bonds formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid. The shortest peptides are dipeptides, two amino acids joined by one peptide bond, followed by tripeptides, tetrapeptides, etc. A peptidomimetic chemical moiety may contain chemical moieties other than amino acids. A peptidomimetic chemical moiety may also contain one or more amino acids separated by one or more non-amino acid chemical units. A peptidomimetic chemical moiety does not contain two or more adjacent amino acids linked by peptide bonds in any part of its chemical structure. The term "amino acid" as used herein means glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, and citrulline.

[0064] According to the invention, peptides or proteins can be produced by conventional automated peptide / protein synthesis methods or by recombinant expression. The general principles of peptide / protein design and production are well known to those skilled in the art.

[0065] The peptide or protein of the present invention can be synthesized according to conventional techniques in solution or on solid support. A variety of automated synthesizers are commercially available and can be used according to known protocols described in Stewart and Young; Tam et al., 1983; Merrifield, 1986, and Barany and Merrifield, Gross and Meienhofer, 1979. The peptide of the present invention can also be synthesized by solid-phase techniques using an exemplary peptide synthesizer such as the Model 433A from Applied Biosystems Inc. The purity of any protein produced by automated peptide synthesis or recombinant methods can be determined using reverse-phase HPLC analysis. The chemical authenticity of each peptide can be established by any method known to those skilled in the art.

[0066] As an alternative to automated peptide / protein synthesis, recombinant DNA techniques may be used in which a nucleotide sequence encoding the protein of interest is inserted into an expression vector and transformed or transfected into a suitable host cell and cultured under conditions suitable for expression, as described below. Recombinant methods are particularly preferred for producing longer polypeptides.

[0067] A variety of expression vector / host systems can be utilized to contain and express sequences encoding peptides or proteins, including, but not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., 1999); insect cells infected with viral expression vectors (e.g., baculovirus, Ghosh et al., 2002); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids; see, e.g., Babe et al., 2000); or animal cell systems. Those skilled in the art are aware of a variety of techniques for optimizing mammalian expression of proteins, see, e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells useful for recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese Hamster Ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, 293 cells, and the like. Representative protocols for recombinant expression of peptide substrates or fusion polypeptides in bacteria, yeast, and other invertebrates are well known to those of skill in the art and are briefly described below. U.S. Patent No. 6,569,645, U.S. Patent No. 6,043,344, U.S. Patent No. 6,074,849, and U.S. Patent No. 6,579,520 provide specific examples of recombinant production of peptides, and these patents are expressly incorporated herein by reference for their teachings. Mammalian host systems for the expression of recombinant proteins are also well known to those of skill in the art. Host cell lines can be selected based on their particular capabilities for processing the expressed protein or for generating certain post-translational modifications useful for conferring activity to the protein. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation, and the like.Post-translational processing, which cleaves the "prepro" form of a protein, may also be important for correct insertion, folding, and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be selected to ensure correct modification and processing of the introduced foreign protein.

[0068] In the recombinant production of the peptide or protein of the present invention, it will be necessary to use a vector that encodes a polynucleotide molecule derived from the peptide. Methods for preparing such vectors and producing host cells transformed with such vectors are well known to those skilled in the art. The polynucleotide molecules used in such endeavors can generally be linked to vectors that contain a selection marker and an origin of replication, allowing propagation in a host. The elements of such expression constructs are well known to those skilled in the art. In general, an expression vector contains DNA encoding a given protein operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from mammalian, microbial, viral, or insect genes. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and suitable sequences that control transcription and translation.

[0069] The terms "expression vector," "expression construct," or "expression cassette" are used interchangeably throughout this specification and are meant to include any type of genetic construct capable of transcribing part or all of a nucleic acid sequence encoding a gene product.

[0070] Of course, the selection of an appropriate expression vector for expressing the peptide or polypeptide of the present invention depends on the particular host cell used and is within the skill of the ordinary technician. Methods for constructing mammalian expression vectors are disclosed, for example, in Okayama and Berg, 1983; Cosman et al., 1986; Cosman et al., 1984; EP-A-0367566; and WO91 / 18982. Other considerations for constructing expression vectors are described in detail, for example, in Makrides et al., 1999; Kost et al., 1999. Wurm et al., 1999 is incorporated herein as a teaching for considering large-scale transient expression in mammalian cells for recombinant protein production.

[0071] Expression requires that the vector is provided with appropriate signals, such as viral and mammalian enhancers / promoters, which can be used to promote expression of the nucleic acid of interest in the host cell. Usually, the nucleic acid to be expressed is under the transcriptional control of the promoter. "Promoter" refers to a DNA sequence that is recognized by the synthetic machinery of the cell or introduced synthetic machinery necessary to initiate the specific transcription of a gene. A nucleotide sequence is operably linked when the regulatory sequence is functionally related to the DNA encoding the peptide of interest (i.e., 4N1K, variants, etc.). Thus, a promoter nucleotide sequence is operably linked to a given DNA sequence if the promoter nucleotide sequence directs the transcription of the sequence.

[0072] Similarly, the phrase "under transcriptional control" means that the promoter is in the appropriate position and orientation relative to the nucleic acid to control the initiation of RNA polymerase and expression of the gene. Any promoter that promotes expression of the nucleic acid can be used. The particular promoter employed to control expression of the nucleic acid sequence of interest is not believed to be critical, provided it is capable of directing expression of the nucleic acid in the target cell. Thus, when targeting human cells, it is preferred to place the coding region of the nucleic acid under the control of a promoter that is expressible in human cells. Generally, such promoters may include either human or viral promoters. Common promoters include, for example, the immediate early gene promoter of human cytomegalovirus (CMV), the SV40 early promoter, the Rous sarcoma virus long terminal repeat, [β]-actin, the rat insulin promoter, the phosphoglycerol kinase promoter, and the glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are well known and readily available to those skilled in the art, can be used to obtain high levels of expression of the coding sequence of interest. It is also conceivable that other viral, mammalian or bacteriophage promoters known in the art can be used to achieve expression of the coding sequence of interest, provided that the expression level is sufficient to produce a recoverable yield of the protein of interest.By using a promoter with well-known properties, the expression level and pattern of the protein of interest after transfection or transformation can be optimized.Inducible promoters can also be used.

[0073] Another control element used in protein expression is an enhancer. This is a genetic element that increases transcription from a promoter located at a distant location on the same DNA molecule. When an expression construct uses a cDNA insert, it is usually desirable to include a polyadenylation signal sequence to facilitate proper polyadenylation of the gene transcript. Any polyadenylation signal sequence recognized by the cells of the selected transgenic animal species, such as human or bovine growth hormone and SV40 polyadenylation signals, is suitable for carrying out the present invention.

[0074] In one embodiment, the peptide or protein of the present invention is linked to at least one cell membrane penetrating peptide.

[0075] The term "cell membrane permeable peptide" or "CPP" is used interchangeably and refers to a cationic cell membrane permeable peptide, also referred to as a transport peptide, carrier peptide, or peptide transduction domain. As shown herein, CPPs are capable of inducing cell membrane permeability of a peptide fused to the CPP in 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (including all integers therebetween) of the cells of any cell culture population, allowing the translocation of macromolecules in multiple tissues in vivo upon systemic administration. A cell membrane permeable peptide may also refer to a peptide that, when contacted with cells under appropriate conditions, penetrates from the extracellular environment to the intracellular environment (including the cytoplasm, organelles such as mitochondria, or the nucleus of the cell) under conditions much higher than passive diffusion. Such penetrating peptides include those described in Fonseca SB et al., Advanced Drug Delivery Reviews, 2009, 61:953-964; Johansson et al., Methods in Molecular Biology, 2011, Vol. 683, Chapter 17; WO2004 / 011595 and WO2003 / 011898.

[0076] In one embodiment, the CPP is selected from the group consisting of, but not limited to, Tat peptide, polyarginine peptide, HA2-R9 peptide, penetratin peptide, transportan peptide, Vectocel® peptide, maurocalcine peptide, decalidine peptide, HIV-Tat derived PTD4 peptide, Hepatitis B virus transport motif (PTM) peptide, mPrP1-28 peptide, POD, pVEC, EB1, Rath, CADY, histatin 5, Antp peptide, and Cyt86-101 peptide.

[0077] Tests can be carried out with each peptide to verify whether the peptide or protein of the present invention can still reduce the incidence of vascular degeneration, intravascular coagulation in bacterial disease, and reduce the severe damage caused by sepsis.For example, those skilled in the art can test the ability of peptide or protein to protect the integrity of primary human dermal microvascular endothelial cells (HDMEC) from meningococcal infection in vitro, and / or prevent the formation of vascular lesions, thrombosis, and the progression to organ failure in vivo using the in vivo model of lipopolysaccharide (LPS)-induced endotoxemia and / or meningococcal infection.

[0078] [Nucleic acids, vectors, and recombinant host cells] A third aspect of the invention relates to a nucleic acid sequence encoding a peptide or protein according to the invention (such as ANGPTL4).

[0079] A fourth aspect of the present invention relates to an expression vector comprising a nucleic acid sequence encoding a peptide or protein according to the present invention (ANGPTL4).

[0080] According to the present invention, an expression vector suitable for use in the present invention may include at least one expression control element operably linked to the nucleic acid sequence. The expression control element is inserted into the vector to control and regulate the expression of the nucleic acid sequence. Examples of expression control elements include, but are not limited to, the lac system, the operator and promoter regions of phage lambda, yeast promoters, and promoters from polyoma, adenovirus, retrovirus, lentivirus, or SV40. Further preferred or essential operational elements include, but are not limited to, leader sequences, stop codons, polyadenylation signals, and other sequences necessary or preferred for proper transcription and subsequent translation of the nucleic acid sequence in the host system. Those skilled in the art will understand that the correct combination of essential or preferred expression control elements will depend on the host system selected. In addition, it will be understood that the expression vector should also include additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selection markers. In addition, those skilled in the art will understand that such vectors can be readily constructed using conventional methods or commercially available products.

[0081] A fifth aspect of the present invention is a host cell comprising an expression vector as described above.

[0082] According to the present invention, examples of host cells that can be used include eukaryotic cells such as animals, plants, insects and yeast, as well as prokaryotic cells such as E. coli. Methods for introducing a vector carrying a gene into a cell include, but are not limited to, microinjection, electroporation, transduction, or transfection using DEAE-dextran, lipofection, calcium phosphate, or other procedures well known to those skilled in the art.

[0083] In certain embodiments, eukaryotic expression vectors that function in eukaryotic cells are used.Examples of such vectors include, but are not limited to, viral vectors such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, etc.; lentivirus, bacterial expression vector, plasmid such as pcDNA3, or baculovirus transfer vector, etc.Preferred eukaryotic cell lines include, but are not limited to, COS cells, CHO cells, HeLa cells, NIH / 3T3 cells, 293 cells (ATCC number CRL1573), T2 cells, dendritic cells, or monocytes, etc.

[0084] [Use of the peptide or protein of the present invention] A sixth aspect of the invention relates to a peptide, protein, nucleic acid, vector or host cell according to the invention for use in the treatment of an infectious disease in a subject in need thereof.

[0085] According to the present invention, the infectious disease may be caused by pathogens such as bacteria, viruses, protozoa, prions, viroids, or fungi. In particular, infectious diseases promoted by any pathogen that induces vascular degeneration may be treatable by the peptides or proteins of the present invention.

[0086] In particular, the protozoan may be Plasmodium falciparum.

[0087] In particular, the virus may be an influenza virus, such as influenza A virus (IAV) or influenza B virus (IAB), an adenovirus, a metapneumovirus, a cytomegalovirus, a parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), a human rhinovirus (HRV), a human respiratory syncytial virus (HRSV), or a coronavirus.

[0088] The term "coronavirus" as used herein has its general meaning in the art and refers to any virus that constitutes the Coronaviridae family. Coronaviruses are viruses with genomes that are positive-stranded RNA, ranging in length from about 27 kb to about 33 kb, depending on the particular virus. The virus particle RNA is capped at the 5' end and has a polyA tail at the 3' end. This length of RNA makes coronaviruses the largest of the RNA virus genomes. In particular, coronavirus RNA encodes (1) an RNA-dependent RNA polymerase, (2) an N protein, (3) three envelope glycoproteins, and (4) three nonstructural proteins. In particular, coronavirus particles are composed of at least four canonical structural proteins: E (envelope protein), M (membrane protein), N (nucleocapsid protein), and S (spike protein). The S protein is cleaved into three strands: spike protein S1, spike protein S2, and spike protein S2'. Through translation of ORF1a and ORF1ab via the -1 ribosomal frameshift mechanism, the production of replicase proteins is initiated. This mechanism results in the generation of two large viral polyproteins, pp1a and pp1ab, which are further processed by two virus-encoded cysteine ​​proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), sometimes referred to as main protease (Mpro). Coronaviruses infect a variety of mammals and birds. They can cause respiratory infections (common), enteric infections (mainly in infants over 12 months), and neurological syndromes. Coronaviruses are transmitted by aerosol through respiratory secretions. Coronaviruses include, but are not limited to, human enteric coV (ATCC accession number VR-1475), human coV229E (ATCC accession number VR-740), human coV OC43 (ATCC accession number VR-920), Middle East Respiratory Syndrome-related coronavirus (MERS-Cov), and severe acute respiratory syndrome (SARS) coronavirus (Centers for Disease Control), specifically SARS-Cov1 and SARS-Cov2.

[0089] According to the present invention, the coronavirus may be MERS-CoV, SARS-CoV, SARS-CoV-2, or any new family member that may emerge in the future.

[0090] In particular, the pathogen may be a bacterium, and in this case the invention therefore relates to a peptide, a protein, a nucleic acid, a vector or a host cell according to the invention for use in the treatment of a bacterial infection (or bacterial disease) in a subject in need thereof.

[0091] The inventors have shown that bacterial infection, particularly infection with Neisseria meningitidis, causes vascular dysfunction (or vascular damage or vascular degeneration) (see the Examples section).The peptide or protein of the present invention (ANGPTL4 or a peptide or protein derived from ANGPTL4) exerts a protective effect on the integrity of endothelial cell monolayers infected with bacteria (particularly N. meningitidis), maintains the integrity of endothelial cell junctions, and prevents vascular degeneration, thrombosis and inflammation, and progression to organ failure and death during bacterial infection (such as meningococcal sepsis), and can therefore be used as a key vascular stabilizing factor that provides protection against bacterial infection and sepsis due to infection such as bacterial infection.

[0092] The present invention therefore also relates to a peptide, a protein, a nucleic acid, a vector or a host cell according to the invention for use in maintaining the integrity of endothelial cell junctions and in preventing the occurrence of vascular degeneration, thrombosis and inflammation during bacterial infection.

[0093] In a particular embodiment, the present invention also relates to a peptide, a protein, a nucleic acid, a vector or a host cell according to the invention for use in the protection of primary human dermal microvascular endothelial cells (HDMEC) or primary human brain microvascular endothelial cells (HBMEC).

[0094] In particular, the invention relates to a peptide, a protein, a nucleic acid, a vector or a host cell according to the invention for use in the treatment of sepsis, in particular due to (or induced by) a bacterial infection.

[0095] As used herein, the term "sepsis" refers to a pathological condition caused by a mixture of microbial virulence factors (such as toxins) and proinflammatory mediators caused by infection or trauma, and includes early stage sepsis, severe sepsis, and the acute phase of septic shock.

[0096] "Early stage sepsis" refers to the stage of the disease when typical clinical signs of severe infection appear, such as chills, profuse sweating, irregular intermittent fever, and weakness.

[0097] "Severe sepsis" refers to the stage of the disease characterized by persistent fever, lymphopenia, disseminated intravascular coagulation, respiratory distress syndrome, multiple organ failure, hypotension leading to shock, as well as early clinical signs of sepsis.

[0098] "Acute septic shock" refers to the collapse of peripheral circulation resulting in hemodynamic, metabolic, and visceral dysfunction that is almost always fatal.

[0099] In particular, peptides used in the treatment of sepsis may be peptides as defined in SEQ ID NOs: 1-30 and 37-64, in particular 1, 8-30 and 37-64.

[0100] According to the invention, the bacterial infection is a gram-negative or gram-positive infection.

[0101] "Gram-negative" bacterial infections refer to local or systemic infections with gram-negative bacteria. Proteobacteria are the major group of gram-negative bacteria and include Escherichia coli (E. coli), Salmonella, Shigella, other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Acetobacteria, and Legionella. Other notable groups of gram-negative bacteria include cyanobacteria, spirochetes, green sulfur bacteria, and green nonsulfur bacteria. There are four types of medically relevant gram-negative cocci that cause sexually transmitted diseases (Neisseria gonorrhoeae), meningitis (Neisseria meningitidis), and respiratory symptoms (Moraxella catarrhalis and Haemophilus influenzae). Medically relevant gram-negative bacilli include numerous species. Some of them cause mainly respiratory problems (Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), mainly urinary problems (Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescen), and mainly digestive problems (Helicobacter pylori, Salmonella Enteritidis, Salmonella Typhimurium). Gram-negative bacteria associated with hospital-acquired infections include Acinetobacter baumannii, which can cause bacteremia, secondary meningitis, and ventilator-associated pneumonia in hospital intensive care units.

[0102] In another particular embodiment of the invention, the Gram-negative bacterium according to the invention is selected from the group consisting of Escherichia coli, Pseudomonas spp, Salmonella spp, Klebsiella spp, Acinetobacter spp, E. corrodens, and Haemophilus influenza.

[0103] In a more specific embodiment of the invention, the Gram-negative bacteria according to the invention are Pseudomonas spp, Acinetobacter spp, and Klebsiella spp.

[0104] The term "Pseudomonas bacteria" has its general meaning in the art and refers to bacteria that are normally or pathogenic in the lungs of humans and other animals. The term "Pseudomonas" refers to, but is not limited to, the Gram-negative bacteria Pseudomonas, for example, bacteria of the Pseudomonas aeruginosa group, e.g., the P. aeruginosa group.

[0105] In particular, the Pseudomonas bacterium according to the present invention is Pseudomonas aeruginosa.

[0106] Pseudomonas aeruginosa is a common Gram-negative bacterium that can cause disease in animals, including humans. P. aeruginosa is citrate, catalase, and oxidase positive. P. aeruginosa is present in soil, water, skin flora, and most artificial environments worldwide. P. aeruginosa thrives in normal as well as low-oxygen atmospheres, allowing it to colonize many natural and artificial environments. P. aeruginosa utilizes a wide range of organic matter as sustenance, and in animals, its adaptive capabilities allow it to infect damaged or immunocompromised tissues. Symptoms of such infections are systemic inflammation and sepsis. If such colonization occurs in vital organs such as the lungs, urinary tract, or kidneys, it can be fatal (Balcht et al., Informa Health Care, 1994). As the bacterium thrives on moist surfaces, it can also be present on and in medical devices such as catheters, causing cross-infection in hospitals and clinics.

[0107] The term "Klebsiella bacteria" has its general meaning in the art and refers to bacteria that normally or pathogenically reside in the lungs of humans and other animals. The term "Klebsiella" refers to, but is not limited to, the gram-negative bacterium Klebsiella, including, for example, bacteria of the Klebsiella pneumoniae group, such as K. pneumoniae group, Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella michiganensis, Klebsiella pneumoniae (type species), Klebsiella pneumoniae subsp. Ozaenae, Klebsiella pneumoniae subsp. Pneumoniae, Klebsiella pneumoniae subsp. Rhinoscleromatis, Klebsiella quasipneumoniae, Klebsiella quasipneumoniae subsp. Quasipneumoniae, Klebsiella quasipneumoniae subsp. Similipneumoniae, and Klebsiella variicola.

[0108] In particular, the Klebsiella according to the present invention is Klebsiella pneumoniae.

[0109] The term "Acinetobacter bacteria" has its general meaning in the art and refers to bacteria that are normally or pathogenic in the lungs of humans and other animals. The term "Acinetobacter bacteria" refers to, but is not limited to, the gram-negative bacteria Acinetobacter, including, for example, bacteria of the Acinetobacter baumannii group, such as Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter genomospecies 3, and Acinetobacter genomospecies 13 (Ingela Tjernberg and Jan Ursing), which are grouped together as "Acinetobacter calcoaceticus-baumannii comple."

[0110] In particular, the Acinetobacter according to the present invention is Acinetobacter baumannii.

[0111] The term "gram-positive" bacterial infection refers to a localized or systemic infection with gram-positive bacteria. Gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their cell walls. Gram-positive bacteria absorb the crystal violet stain used in the test and appear purple when viewed under a microscope. This is because the thick peptidoglycan layer of the bacterial cell wall retains the stain even after it is washed off from the rest of the sample during the decolorization phase of the test.

[0112] In the classical sense, six genera of Gram-positive bacteria are usually pathogenic to humans. Of these, two genera, Streptococcus and Staphylococcus, are cocci (spherical). The remaining bacteria are bacilli (rod-shaped) and can be further subdivided based on their ability to form spores. Non-spore-forming bacteria include Corynebacterium and Listeria (coccobacilli), whereas Bacillus and Clostridium form spores (Gladwin et al., 2007). Miami, Florida: MedMaster. pp. 4-5. ISBN 978-0-940780-81-1).

[0113] In a particular embodiment of the invention, the Gram-positive bacterium according to the invention is selected from the group consisting of Staphylococcus, Streptococcus, Clostridium, Listeria, Bacillus and Corynebacterium.

[0114] In another particular embodiment of the invention, the Gram-positive bacteria according to the invention are S. aureus group (S. argenteus, S. aureus, S. schweitzeri, S. simiae), S. auricularis group (S. auricularis), S. epidermidis group (S. capitis, S. caprae, S. epidermidis, S. saccharolyticus), S. haemolyticus group(S. borealis, S. devriesei, S. haemolyticus, S. hominis); S. hyicus‐intermedius group(S.agnetis, S.chromogenes, S.cornubiensis, S.felis, S.delphini, S.hyicus, S.intermedius, S.lutrae, S.microti, S.muscae, S.pseudintermedius, S.rostri, S.schleiferi), S.lugdunensis group(S.lugdunensis); S.sciuri S. fleurettii, S. lentus, S. sciuri, S. stepanovicii, S. vitulinus), S. simulans group (S. simulans), and S. warneri group (S. pasteuri, S. warneri).

[0115] In a more specific embodiment, the Gram-positive bacterium according to the invention is of the S. aureus group.

[0116] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment, including treatment of subjects at risk of contracting or suspected of contracting a disease, and subjects suffering from or diagnosed with a disease or medical condition, and therapeutic or disease-modifying treatment, including suppression of clinical recurrence. Treatment may be administered to subjects with a medical disease or subjects who may eventually suffer from the disease, to prevent, treat, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disease or recurrent disease, or to extend the subject's survival beyond that expected in the absence of such treatment. "Treatment regimen" refers to a pattern of treatment of a disease, e.g., a dosing pattern used during treatment. A treatment regimen may include induction therapy and maintenance therapy. The term "induction therapy" or "induction period" refers to a treatment regimen (or a portion of a treatment regimen) used for the initial treatment of a disease. The general goal of induction therapy is to administer a high dose of a drug to a subject in the early stages of the treatment regimen. Induction therapy may employ a "loading regimen" in part or in whole, which may include administering a higher dose of drug than the dose employed by the physician in maintenance therapy, administering a drug more frequently than the physician in maintenance therapy, or both. The term "maintenance therapy" or "maintenance period" refers to a treatment plan (or a portion of a treatment plan) used to maintain a subject's condition during disease treatment, for example, to maintain the subject in remission for an extended period of time (months or years). Maintenance therapy may employ continuous therapy (e.g., a regimen in which a drug is administered periodically, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interruption therapy, intermittent therapy, treatment upon relapse, or treatment upon the achievement of certain predefined criteria (e.g., symptoms of disease, etc.).

[0117] The present invention also relates to a method for treating an infectious disease comprising administering to a subject in need thereof a therapeutically effective amount of the peptide, protein, nucleic acid, vector or host cell.

[0118] [Kit set] A seventh aspect of the present invention relates to i) a peptide, protein, nucleic acid, vector or host cell and ii) at least one anti-pathogen infection agent, used as a combined preparation for simultaneous, separate or sequential use in the treatment of an infection in a subject in need thereof.

[0119] In particular, the anti-pathogen infection agent may be an anti-viral infection agent, an anti-bacterial infection agent, an anti-protozoan infection agent, an anti-prion infection agent, an anti-viroid infection agent, an anti-fungal infection agent.

[0120] Thus, the present invention also relates to i) a peptide, protein, nucleic acid, vector or host cell according to the present invention and ii) at least one anti-bacterial infection agent as a combined preparation for simultaneous, separate or sequential use for the treatment of a bacterial infection in a subject in need thereof.

[0121] According to antibacterial infection agents, it may be an antibiotic.

[0122] As used herein, the terms "antibiotic" and "antimicrobial compound" are used interchangeably and refer to compounds that reduce the viability or inhibit the growth or reproduction of microorganisms. The term "antibiotic agent" has its general meaning in the art and refers to antimicrobial agents as described in U.S. Patent 2013 / 0029981.

[0123] Suitable antibiotic agents include, but are not limited to:

[0124] 1. β-lactam antibiotics (β-lactam antibiotics) are antibiotics that contain a β-lactam ring in their molecular structure and contain a β-lactam functional group. These β-lactam antibiotics include penicillins and derivatives (penams), cephalosporins (cephems), monobactams, carbapenems, and carbacephems. Most β-lactam antibiotics act by inhibiting the biosynthesis of bacterial cell walls and are the most widely used group of antibiotics (in 2003, more than half of all antibiotics on the market were β-lactam compounds).

[0125] "Cephalosporin (cephalomycin)" refers to a subgroup of beta-lactam antibiotics originally derived from the fungus Acremonium. Cephalosporins, along with cephamycins, make up a subgroup of beta-lactam antibiotics called cephalomycins. Cephalosporins include ceftazide.

[0126] As used herein, "monobactam" refers to a type of beta-lactam antibiotic that is monocyclic and in which the beta-lactam ring is not fused to another ring. Monobactams include aztreonam.

[0127] "Carbapenem" refers to a type of beta-lactam antibiotic that exerts a bactericidal effect by binding to penicillin-binding protein (CBP) and inhibiting the synthesis of bacterial cell walls. This antibiotic is usually used to treat multidrug-resistant (MDR) bacterial infections. Carbapenems include imipenem.

[0128] As used herein, "penicillin" and "penicillin derivatives (penams)" refer to a class of beta-lactam antibiotics derived from common molds known as Penicillium; these include penicillin G (administered intravenously), penicillin V (administered orally), procaine penicillin, and benzathine penicillin (administered intramuscularly). Penicillin antibiotics were among the first to be effective against many bacterial infections caused by staphylococci and streptococci. They are still widely used today, but their widespread use has led to the development of resistance in many types of bacteria. There are several enhanced penicillins that are effective against even more bacteria, including antistaphylococcal penicillins, aminopenicillins, and antipseudomonal penicillins. They are produced by the Penicillium fungi.

[0129] Examples of natural penicillins: Penicillin G, Penicillin K, Penicillin N, Penicillin O, Penicillin V.

[0130] Examples of β-lactamase-resistant penicillin derivatives: methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin.

[0131] Examples of aminopenicillin antibiotics: ampicillin, amoxicillin, pivampicillin, hetacillin, bacampicillin, methampicillin, talampicillin, epicillin.

[0132] Examples of carboxypenicillins: carbenicillin, ticarcillin, temocillin.

[0133] Examples of ureidopenicillins: mezlocillin, piperacillin, azlocillin.

[0134] Examples of β-lactamase inhibitor penicillin derivatives: clavulanic acid, sulbactam, tazobactam.

[0135] 2. Aminoglycosides are protein synthesis inhibitors (targeting the 30S ribosomal subunit) that target gram-negative bacteria and are antibiotics that contain an amino-modified glycoside as part of the molecule (Mingeot-Leclercq MP et al. 1999. Antimicrob. Agents Chemother. 43(4):727-37). The term "aminoglycoside" may also refer more generally to organic molecules that contain an amino sugar substructure. Aminoglycoside antibiotics are bactericidal against gram-negative aerobic bacteria and some anaerobic bacilli to which resistance has not yet developed, but are generally inactive against gram-positive and anaerobic gram-negative bacteria.

[0136] Streptomycin was the first of the aminoglycoside antibiotics. It was extracted from Streptomyces griseus and was the earliest modern drug used to treat tuberculosis. Streptomycin does not contain the common 2-deoxystreptamine moiety found in most other drugs in this class. Other examples of aminoglycoside antibiotics include the deoxystrepamine-containing agents, kanamycin, tobramycin, gentamicin, and neomycin.

[0137] 3. Antibiotics that inhibit oxidative nuclear synthesis -Antibiotics that inhibit DNA gyrase (a topoisomerase specific to bacteria): aminocoumarins, quinolones.

[0138] -Antibiotics that inhibit bacterial RNA polymerase: rifampicin.

[0139] 4. Antibiotics that inhibit protein synthesis (other than aminoglycosides) -Antibiotics that inhibit the formation of peptide bonds: amphenicols (e.g. chloramphenicol, thiamphenicol, azidamphenicol, florfenicol) -Antibiotics that inhibit the elongation of the polypeptide chain: tetracyclines (e.g. tetracycline, doxycycline, aureomycin, eravacycline, sarecycline, omadacycline), macrolides (e.g. erythromycin, azithromycin) and ketolides (e.g. telithromycin, cethromycin, solithromycin).

[0140] 5. Antibiotics that inhibit folic acid metabolism Sulfonamides are also called sulphonamides, sulfa drugs or sulpha drugs (e.g., sulfamethoxazole), and sulfanilamides.

[0141] 6. Glycopeptide antibiotics such as vancomycin.

[0142] 7. A new class of antibiotic compounds In the late 2000s and early 2010s, four new antibiotics entered clinical use: cyclic lipopeptides (e.g., daptomycin), glycylcyclines (e.g., tigecycline), oxazolidinones (e.g., linezolid), and lipiarmycins (e.g., fidaxomicin).

[0143] In a particular embodiment, the antibiotics of the present invention are cefotaxime and aminoglycosides (amikacin).

[0144] According to the invention, in case of sepsis, an anti-sepsis agent can be used.

[0145] Thus, in this case, the invention relates to i) a peptide, a protein, a nucleic acid, a vector or a host cell according to the invention and ii) at least one anti-sepsis agent, used as a combined preparation for simultaneous, separate or sequential use in the treatment of sepsis in a subject in need thereof.

[0146] As used herein, the term "anti-sepsis agent" refers to a compound capable of combating sepsis, such as antibiotics, vasoconstrictors (such as norepinephrine or dopamine), hydrocortisone, fludrocortisone, and the like.

[0147] As used herein, the term "concurrent use" means that the use of a peptide, protein, nucleic acid, vector or host cell according to the invention occurs simultaneously with the use of at least one antimicrobial infectious agent.

[0148] As used herein, the term "separate use" means the use of a peptide, protein, nucleic acid, vector or host cell according to the invention and at least one antimicrobial infectious agent in the absence of each other.

[0149] As used herein, the term "sequential use" refers to the sequential use of a peptide, protein, nucleic acid, vector or host cell according to the invention and at least one antimicrobial infectious agent.

[0150] [Therapeutic composition] Another object of the invention relates to a therapeutic composition comprising an antibody or a peptide, a protein, a nucleic acid, a vector or a host cell according to the invention for use in the treatment of an infectious disease in a subject in need thereof.

[0151] In a particular embodiment, the invention relates to a therapeutic composition according to the invention comprising a peptide, protein, nucleic acid, vector or host cell for use in treating sepsis in a subject in need thereof.

[0152] The therapeutic agents of the present invention can be combined with pharma- ceutically acceptable excipients, and optionally with sustained release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0153] "Pharmaceutical" or "Pharmaceutically acceptable" refers to molecular substances and compositions that do not produce adverse, allergic, or other untoward reactions when administered to a mammal, especially a human. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0154] The form of the pharmaceutical composition, the route of administration, the dose and the administration regimen will, of course, depend on the condition to be treated, the severity of the disease, the age, weight, sex, etc. of the patient.

[0155] The pharmaceutical compositions of the present invention can be adapted for a variety of routes of administration, including topical, oral, nasal, parenteral, ocular, intravenous, intrathecal, intramuscular, subcutaneous, and pulmonary nebulizer administration.

[0156] Desirably, the pharmaceutical composition comprises a vehicle pharma- ceutical acceptable for injectable preparations, which may in particular be an isotonic sterile saline solution (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixed salts thereof) or a dried, in particular lyophilized, composition that can be made into an injectable solution, if necessary by adding sterile water or saline.

[0157] The doses used for administration can be adapted depending on various parameters, in particular the method of administration used, the disease involved or the desired duration of treatment.

[0158] Additionally, other pharma- ceutical acceptable forms, such as tablets or other solids for oral administration, time-release capsules, and other forms currently in use may also be used.

[0159] The pharmaceutical compositions of the invention may comprise further therapeutically active agents. The invention also relates to kits comprising an agonist, antagonist or inhibitor of expression according to the invention and a further therapeutically active agent.

[0160] For example, anti-infective agents such as antibiotics and anti-septic agents (discussed above) may be added to the pharmaceutical compositions as described below. [Brief description of the drawings]

[0161] The present invention is further illustrated by the figures and examples given below, which should not be construed as limiting the scope of the invention in any way.

[0162] [Chart] Figure 1: Exogenous ANGPTL4 exerts a protective effect on dermal microvascular endothelial cells infected with N. meningitidis. Figure 1A: HDMECs were infected with Nm 2C4.3 for 30 min and treated with 1 μg / mL recombinant human ANGPTL4 for 2 h 30 min. Cells were fixed, stained for intracellular actin, VE-cadherin, and DAPI, and analyzed by fluorescence microscopy. Quantification of VE-cadherin enrichment at cell-cell junctions and the area of ​​intercellular spaces was performed in 30 fields using Image J software. Kruskal-Wallis test **P<0.001; ***P<0.0001. Figure 1B: HDMECs were either uninfected or infected with N. meningitidis 2C4.3 for 30 min and treated with 1 μg / mL recombinant human ANGPTL4, its N-terminal part (nANGPTL4), or its C-terminal part (cANGPTL4) for 2 h 30 min. Caspase-3 / 7 reagent was added and apoptosis was detected in real time over a period of 9 hours using the IncuCyte® Live Cell Analysis System.

[0163] Figure 2: Vascular protection is mediated by the N-terminal portion of ANGPTL4. Figure 2A: Schematic diagram of the structure of ANGPTL4. ANGPTL4 contains an N-terminal portion (nANGPTL4, aa 26-164) linked to a C-terminal portion (cANGPTL4, aa 164-406) by a cleavable linker. nANGPTL4 contains a highly conserved lipoprotein lipase (LPL)-binding domain (aa 44-55), two cysteines at positions 76 and 80 involved in protein oligomerization, and a coiled-coil domain (aa 100-143). The C-terminal portion encodes a fibrinogen-like domain. Figure 2B: HDMECs were infected with Nm 2C4.3 for 30 min and treated with 1 μg / mL recombinant human ANGPTL4, nANGPTL4, or cANGPTL4 for 2 h 30 min. Cells were fixed, stained for intracellular actin, VE-cadherin, and DAPI, and analyzed by fluorescence microscopy. Figure 2B: Quantification of VE-cadherin enrichment at cell-cell junctions and the area of ​​intercellular space was performed in 30 fields using Image J software. Four independent experiments were performed in triplicate, Kruskal-Wallis test, **P<0.001, ***P<0.0001.

[0164] Figure 3: Vascular protection is mediated by a novel binding motif (aa 66-80) present in the N-terminal part of ANGPTL4. Figure 3A: Peptides derived from nANGPTL4 are shown. Figure 3B: HDMECs were infected with Nm 2C4.3 for 30 min and treated with each peptide at 1 μg / mL. Quantification of VE-cadherin enhancement at cell-cell junctions was performed in 30 fields of view using Image J software.

[0165] Figure 4: Interaction of nANGPTL4 with syndecan-4 confers vascular protection. Figure 4A: HDMECs were infected with Nm 2C4.3 for 30 min and treated with 1 μg / mL recombinant human ANGPTL4, nANGPTL4, or cANGPTL4 for 2 h 30 min in the absence or presence of anti-SDC4 antibody. Cells were fixed, stained for intracellular actin, VE-cadherin, and DAPI, and analyzed by fluorescence microscopy. Quantification of VE-cadherin enhancement at cell-cell junctions and the area of ​​intercellular space was performed in 30 fields using Image J software. Four independent experiments were performed in triplicate, with Kruskal-Wallis test showing **P<0.001, ****P<0.00001, ns1 P=0.2172; ns2P>0.999. Figure 4B: HDMECs were transfected with small interfering RNA (siRNA) against SDC4 or non-targeting siRNA control for 48 h, infected with Nm 2C4.3 for 30 min, and treated with 1 μg / mL recombinant human ANGPTL4 for 2 h 30 min. Quantification of VE-cadherin enrichment at cell-cell junctions and the area of ​​intercellular space was performed in 30 fields using Image J software. Two independent experiments were performed in triplicate, **P<0.001, ***P<0.0001 by Kruskal-Wallis test.

[0166] Figure 5: Neutralizing effect of antibodies against SDC4 in brain endothelial cells induces loss of endothelial cell integrity in response to meningococcal infection. HCMEC / D3 were infected with Nm 2C4.3 for 30 min and treated with 10 μg / mL of anti-ANGPTL4 antibody for 2 h 30 min in the presence or absence of anti-SDC4 antibody or anti-ANGPTL4 antibody as a positive control. Figure 5A: Quantification of VE-cadherin reinforcement at cell-cell junctions and the area of ​​intercellular space was performed in 30 fields using Image J software. Kruskal-Wallis test **P<0.001, ***P<0.0001. Figure 5B: Human primary human brain microvascular endothelial cells (HBMEC) were cultured on cell filters. Cells were infected with Nm 2C4.3 for 30 min and treated with 10 μg / mL of anti-ANGPTL4 antibody for 2 h 30 min in the presence or absence of anti-SDC4 or anti-ANGPTL4 antibodies. Transendothelial electrical resistance (TEER) was measured. Results are shown from one representative experiment out of two independent experiments performed in triplicate. **P<0.001 by Kruskal-Wallis test.

[0167] Figure 6: ANGPTL4 protects dermal microvascular endothelial cells from infection by S. pneumoniae. HDMECs were infected with S. pneumoniae TIGR4 for 30 min and treated with 1 μg / mL recombinant human ANGPTL4 for 2 h. Cells were fixed, stained for intracellular actin, VE-cadherin, and DAPI, and analyzed by fluorescence microscopy. Quantification of VE-cadherin enhancement at cell-cell junctions was performed in 30 fields using Image J software. ***P < 0.0001 by Kruskal-Wallis test.

[0168] Figure 7: ANGPTL4 reduces signs of thrombosis, vascular injury, and inflammation. Figure 7A-D: SCID mice grafted with human skin were treated with the Nm 2C4.3 wild-type strain (5 × 10 6Mice were either intravenously infected with ANGPTL4, nANGPTL4 or cANGPTL4, or not infected with ANGPTL4 or cANGPTL4. Thirty minutes or two hours after bacterial infection, mice were intravenously administered 1 μg of recombinant human ANGPTL4, nANGPTL4 or cANGPTL4, or vehicle as a control. Mice were sacrificed 4 hours after infection. The experiment was performed twice with skin from two donors, with n=3 or 4 mice per group. Figure 7A: Schematic of the protocol used in vivo. Figure 7B: Control sepsis 4 hours after infection (mean ± SEM). Figure 7C: Bacterial colonization within the skin grafts, assessed by immunofluorescence analysis, was quantified using ImageJ software as the ratio of bacterial surface colonization to vessel lumen surface. A total of 30–50 vessels were analyzed per mouse. Error bars indicate mean ± SEM. Figure 7D: Thrombus formation within the skin grafts was assessed by immunofluorescence analysis and quantified using ImageJ as the ratio of thrombus surface to vessel lumen surface. A total of 30-50 vessels were analyzed per mouse. Error bars indicate mean ± SEM; ***P < 0.0001; ns P = 0.0541 by two-tailed Student's t test.

[0169] Figure 8: ANGPTL4 improves the outcome of meningococcal infection, alone or in combination with antibiotics. Figure 8A-D: SCID mice grafted with human skin were infected with Nm 2C4.3 wild-type strain (5 × 10 6Mice were infected intravenously with ANGPTL4 (1 μg, intravenous), cefotaxime (200 mg / kg, intraperitoneal) alone or in combination with ANGPTL4 or vehicle as a control 2 h after bacterial infection. ANGPTL4 (1 μg) and cefotaxime (200 mg / kg) were re-administered 18 h after bacterial infection. The experiment was performed twice with skin harvested from two different donors, with n=3 or 4 mice in each group. Figure 8A shows a schematic of the protocol used in vivo. Figure 8B shows bacteremia (mean ± SEM) at 4, 18, 48, and 72 h post-infection. Figure 8C shows survival curves: *P=0.0275; two-sample log-rank-Mantel-Cox survival analysis. In Fig. 8D , thrombosis in skin grafts 4 h postinfection was assessed by immunofluorescence analysis and quantified as shown in Fig. 8 (error bars indicate the mean ± standard deviation; 40 vessels were analyzed per mouse; n = 4 mice per group; *** P < 0.001 by two-tailed Student's t test).

[0170] Figure 9: ANGPTL4 prevents LPS-induced endotoxia by preventing vascular dysfunction and organ failure. Figure 9A-B: Balb / c mice were administered LPS (5 mg / kg, i.p.); 30 min after LPS administration, mice were administered recombinant human ANGPTL4 (2 μg, i.v.) or vehicle as a control. Figure 9A: Schematic of the protocol used. Figure 9B: Survival curves (n=16 mice per group): ****P<0.0001; two-sample log-rank-Mantel-Cox survival analysis. Figure 8C,D: Balb / c mice were administered LPS (5 mg / kg, i.p.); 30 min after LPS administration, mice were administered nANGPTL4 (2 μg, i.v.) or vehicle as a control. Figure 8C: Schematic of the protocol used. D: Survival curves (n=6 mice per group): **P=0.0033; two-sample log-rank-Mantel-Cox survival analysis. E,F: Balb / c mice were administered LPS (5 mg / kg, i.p.); 30 min after LPS administration, mice were administered peptide 1 (2 μg, i.v.) or vehicle as control. Figure 9E: Schematic of the protocol used. Figure 9F, Survival curves (n=6 mice per group): *P=0.0163; two-sample log-rank-Mantel-Cox survival analysis. Figure 9G: Balb / c mice were administered LPS (5 mg / kg, i.p.) and 30 min after LPS administration, mice were administered recombinant human peptide P9 (2 μg, i.v.) or vehicle as control. Survival curves (n=6 mice per group). ***P<0.001; two-sample log-rank-Mantel-Cox survival analysis. Figure 9H: Balb / c mice were administered LPS (4 mg / kg, intraperitoneally) together with anti-ANGPTL4 blocking antibody (10 μg / kg, intravenously) or vehicle as a control. 18 h after LPS administration, mice were perfused with Evans blue for 1 h, sacrificed, and their brains were removed. Quantification analysis of Evans blue exudation (n = 4 mice per group). Bar graphs and error bars show the mean ± standard deviation of two independent experiments, respectively, and one-way ANOVA with Tukey's multiple comparison test showed ***P < 0.001; NS (not significant: P > 0.05).

[0171] [Table 1] EXAMPLES

[0172] [material and method] <Antibodies, recombinant proteins and reagents> Anti-collagen IV (ab6311) mouse monoclonal antibody and anti-CD41 (ab33611) rat monoclonal antibody were purchased from Abcam. Anti-VE-cadherin F-8 Alexa Fluor® 647 mouse monoclonal antibody (sc-9989) was purchased from Santa Cruz. Anti-syndecan 4 rabbit polyclonal antibody was purchased from Invitrogen (36-3100). Anti-Angptl4 rabbit polyclonal antibody was purchased from Thermo scientific (40-9800). Polyclonal antiserum raised against Neisseria meningitidis strain 2C4.3 was previously reported

[22] . Secondary antibodies used for immunofluorescence labeling and Western blotting were obtained from Jackson ImmunoResearch Laboratories and Thermoscientific Lab.

[0173] Recombinant human ANGPTL4 (rhANGPTL4, 44487-AN), C-terminal fragment (cANGPTL4, 3485-AN), N-terminal fragment (nANGPTL4, 8249-AN), rhANGPTL3 (3829-AN) or rhANGPTL8 (9983-AN) were purchased from R&D Systems. Lipopolysaccharide (LPS) from Escherichia coli (0111:B4) was purchased from Sigma-Aldrich. All peptides were synthesized at Covalab (Lyon, France) and were ≥98% pure.

[0174] [Bacterial strains and infections] Neisseria meningitidis (Nm) strain 2C4.3 (previously called clone 12) is a encapsulated Opa-Opc variant of group C meningococcal clinical isolate 8013 that has been previously described [Ref. 23]. Bacterial strains were stored frozen at −80°C and routinely cultured on GC agar (Difco) with Kellogg's supplements at 37°C in 5% CO2.

[0175] On the day of infection, bacterial suspensions from overnight cultures on GCB agar were adjusted to OD600 = 0.05 and incubated for 2 h at 37 °C in pre-warmed cell culture medium. Cells were infected with bacteria at a multiplicity of infection (MOI) of 100 bacteria per cell (OD = 0.1) for 30 min, washed twice to remove non-adherent bacteria, and allowed to continue infection for various times. Cells were then washed and fixed with 4% paraformaldehyde for immunofluorescence analysis.

[0176] [Cell line] Human brain microvascular endothelial cells (HCMEC / D3) are a fully differentiated brain endothelial cell line derived from human brain capillaries that were generated in our laboratory and recapitulate the major phenotypes of the blood-brain barrier [13,24]. HCMEC / D3 were cultured on Cultrex dishes (R&D) coated with rat collagen type I in Endothelial Cell Basal Medium-2 (Lonza) supplemented with 5% FCS, 1.4 μM hydrocortisone (Lonza), 5 μg / mL ascorbic acid (Lonza), and 1 ng / mL b-FGF (Lonza) at 37°C under 5% CO2.

[0177] Primary human brain microvascular endothelial cells (HBMECs) were isolated from brain biopsies in healthy areas of pediatric brain tumors and cultured in their specific endothelial cell growth medium at 37°C and 5% CO2.

[0178] Primary human dermal microvascular endothelial cells (HDMECs) were isolated from the dermis of young foreskin and adult skin (different sites) and cultured in specific endothelial cell growth medium purchased from PromoCell at 5% CO2 and 37 °C. When required, HDMECs were seeded on channel slides (μ-slide 0.4 I Luer, ibidi) and cultured for 2 days to reach confluence, after which they were cultured under laminar shear stress (10 dyn / cm 2 ) was administered for four days (ibidi Pump System, ibidi).

[0179] [Confocal immunofluorescence microscopy] Cells were grown on Thermonox coverslips (Thermo Fischer Scientific) until confluent. After infection, cells were fixed with 4% paraformaldehyde for 10 min, washed three times with PBS, and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were blocked with 3% BSA in PBS for 30 min and incubated with primary antibodies for 2 h. After washing three times with PBS, cells were incubated with CY3-conjugated anti-mouse IgG and CY2-conjugated anti-rabbit IgG (Jackson Immunochemicals) for 1 h. Images were acquired using a confocal microscope (spinning disk Leica DMI6000, ×20). Images were analyzed using ImageJ software (NIH). Each experiment was repeated at least three times.

[0180] [Infection of SCID mice with human skin transplants] Six-week-old CB17 / Icr‐Prkdcscid female mice were obtained from Janvier Labs. Human skin tissue was obtained from adult patients undergoing plastic surgery at the Service de Chirurgie Plastique et Reconstructive of the Groupe Hospitalier Saint‐Joseph. In accordance with French law, patients were informed and did not refuse to participate in the study. The experimental procedures were carried out according to the guidelines of the French National Agency for Health and Medical Research as previously described

[25] and in accordance with the European ethical regulations (Directive 2010 / 63 / EU). The experimental protocol was approved by the Animal Experiments Ethics Committee of the University Paris Descartes (consent forms CEEA34.OJL039.12 and 2018012515596498). Briefly, after intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg), the back and abdomen were shaved and the mice were prepared for transplantation. Skin flap was prepared and a full-thickness human skin graft was placed on the wound bed. The graft was secured with 6-0 nonabsorbable monofilament sutures, and then the skin flap was sutured over the graft. The transplanted mice were infected 4-6 weeks after human skin grafting. N. meningitidis was cultured overnight at 37°C on iron-free GCB agar medium, and bacterial colonies were harvested with the addition of 15 μM deferoxamine (Desferal, Novartis) and statically cultured in RPMI medium containing 1% BSA medium and 0.06 μM deferoxamine until they reached the logarithmic phase of growth. The bacteria were then resuspended in saline. Mice were administered 200 μl (5 × 10 6Mice were infected intravenously with 10 mg of human phorotransferrin (R&D Systems) immediately prior to infection. Four hours after infection, mice were sacrificed. Human skin grafts were carefully harvested using a sterile cutter. Tissues were washed with PBS and fixed overnight at 4°C with 4% paraformaldehyde in phosphate buffer. After washing with phosphate buffer, specimens were embedded in OCT medium and frozen at -80°C. Sections of dermis (7 μm thick) were fixed on Superfrost Plus microscope slides and subjected to immunofluorescence analysis. Sections were incubated with primary antibodies in PBS / 3% BSA for 1 h, followed by an additional hour of incubation with Alexa-conjugated secondary antibodies with DAPI. After additional washing, coverslips were mounted in glycergel (Dako) and further analyzed on a slide scanner (Lamina, PerkinElmer). Images were analyzed using ImageJ software (NIH).

[0181] [LPS-induced sepsis in mice] Female BALB / c mice (9–10 weeks old) obtained from Janvier Labs were intraperitoneally administered 5 mg / kg LPS and monitored for survival for 72 h (n=12 per group). BALB / c mice were randomly divided into two groups: LPS and LPS+Angptl4. Angptl4 (2 μg / mouse) or vehicle (saline) was administered intravenously 30 min after LPS administration. In some experiments, blood, kidneys, and lungs were harvested 18 h after LPS administration. Kidneys and lungs were fixed in 4% PFA and embedded in paraffin, and 5 μm sections were cut from each block and stained with H&E. Stained sections were observed and acquired with a slide scanner (Lamina, PerkinElmer). Images were analyzed using ImageJ software (NIH).

[0182] [Statistical analysis] The significance of the data was verified using Prism Software (GraphPad Software). Statistical significance was assessed by Student's t-test and one-way analysis of variance. Quantitative analysis was performed on three independent experiments using ImageJ software.

[0183] [result] Endogenous ANGPTL4 protects brain microvascular endothelial cells against infection with Neisseria meningitidis. Transcriptional analysis of primary human skin (HDMEC) or primary human brain (HBMEC) microvessels, either uninfected or infected in vitro with N. meningitidis, revealed that ANGPTL4 mRNA levels were higher in HBMEC compared with HDMEC and were further increased by N. meningitidis infection, whereas no change was observed in HDMEC (data not shown). These differences were also confirmed at the protein level (data not shown).

[0184] We therefore analyzed the effect of endogenous production of ANGPTL4 on the integrity of brain endothelial cells using hCMEC / D3 cells, a human endothelial cell line derived from brain microvessels that retains most of the structural and functional characteristics of primary brain endothelial cells [12,13]. Similar to primary brain endothelial cells, HCMEC / D3 cells constitutively express ANGPTL4, and this production was further induced by meningococcal infection (data not shown). These cells resisted meningococcal infection for 3 h while maintaining the connective organization of VE-cadherin, a major component of adherens junctions that control vascular permeability, but the addition of anti-ANGPTL4 antibodies led to a loss of distribution of VE-cadherin binding sites and monolayer integrity (data not shown). These results suggest that ANGPTL4 secreted from brain microvascular cells maintains vascular endothelial integrity against N. meningitidis infection.

[0185] Exogenous ANGPTL4 protects human dermal microvascular endothelial cells against meningococcal infection. In contrast to brain microvascular endothelial cells, which are resistant to meningococcal infection, the monolayer of human dermal microvascular endothelial cells (HDMEC) significantly lost its integrity upon infection, with loss of adherens junctions and cell detachment (data not shown). As HDMEC produce less ANGPTL4 compared to HBMEC (data not shown), we analyzed whether the addition of human ANGPTL4 would have a protective effect on these cells. Treatment with ANGPTL4 did not significantly affect the organization of adherens junctions formed by HDMEC, but the addition of ANGPTL4 30 min after the start of infection fully maintained the integrity of this monolayer, significantly reduced phosphorylation of Src kinase and VE-cadherin (Figure 1A), and inhibited cell apoptosis induced by meningococcal infection (Figure 1B). Furthermore, infection was accompanied by a massive release of proinflammatory cytokines (TNF-α, IL-6, IL-8), markers of vascular inflammation (E-selectin, ICAM-1, VCAM-1), and markers of endothelial cell degeneration (endoglin, thrombomodulin), all of which were reduced by 35–80% upon administration of ANGPTL4 (data not shown). Although ANGPTL4 shares sequence homology and biochemical and functional properties with ANGPTL3 and ANGPTL8 [6], addition of rhANGPTL3 or rhANGPTL8 did not provide a protective effect on infected HDMECs (data not shown), indicating that this effect was specific to ANGPTL4. Taken together, these results demonstrate that ANGPTL4 has a potent cytoprotective effect against meningococcal-induced vascular endothelial injury.

[0186] Vascular protection is mediated by a novel binding motif in the N-terminal portion of ANGPTL4. ANGPTL4 produced in endothelial cells undergoes proteolysis by proprotein convertase at the linker region (lysine 164) [Reference 14], releasing the N-terminal portion (nANGPTL4, aa 26-164) and the C-terminal portion (cANGPTL4, aa 164-406) (Figure 2A) (amino acid numbering is based on the complete sequence of ANGPTL4 (SEQ ID NO: 1)). nANGPTL4 contains a highly conserved lipoprotein lipase (LPL)-binding domain (aa 44-55) that allows inhibition of LPL activity, which regulates lipid metabolism

[15] , two cysteines at positions 76 and 80 involved in protein oligomerization through intramolecular disulfide bonds required for functional LPL inhibition, and a coiled-coil domain (aa 100-143) of unknown function, while the monomeric C-terminal portion encodes a fibrinogen-like domain involved in protein interactions with various receptors (extracellular matrix proteins, integrins) and has been shown to increase angiogenesis, vascular permeability, and ROS (reactive oxygen species) production

[16] . We next analyzed the role of each of these domains in the protective effect of ANGPTL4 against the degeneration of endothelial cells caused by infection. Upon infection, the addition of recombinant human nANGPTL4 was sufficient to maintain the integrity of the cell monolayer structure, as observed with the full-length protein (Figure 2B). Conversely, addition of recombinant human cANGPTL4 had no significant effect on protecting monolayer integrity, maintaining the infection-altered state (Fig. 2B).

[0187] To identify the minimal interacting motif of nANGPTL4 required to promote vascular protective effects, we analyzed the effects of derived peptides (Figure 3A). First, we tested two peptides: P1 (aa 38-83), which contains the conserved LPL-binding domain and two cysteine ​​residues (positions 76 and 80), and P2 (aa 99-149), which contains a coiled-coil domain. P1 was sufficient to prevent infection-induced loss of adherens junctions and cell detachment as efficiently as the full-length protein, whereas P2 had no significant effect (Figure 3B). Interestingly, the P1 peptide in which the two cysteine ​​residues were replaced by alanines (P1C76A, C80A) also efficiently exhibited cytoprotective effects against the damage caused by infection, indicating that peptide multimerization is not required to exert this function (Figure 3B). Next, one of the two peptides derived from P1, P3 (aa 38-55), showed no protective effect, indicating that the conserved LPL-binding motif is not involved in this process, while the other, P4 (aa 56-83), showed a protective effect equivalent to that of P1 (Fig. 3B). Sequence plot analysis of P4 using PSIPRED predicted a potential membrane-interacting domain containing aa 65-80. P6 (aa 65-80) and P9 (aa 66-80), which contain this potential domain, completely inhibited the damage caused by infection, whereas P5 (aa 56-73), P7 (60-75), or two shorter peptides P8 (aa 66-75) and P10 (aa 67-78) had no effect (Fig. 3B), and the synthesis of peptide P11 (aa 69-80) failed, probably because the hydrophobicity in the sequence causes inter- or intramolecular aggregation or the formation of secondary structures. Thus, peptides P12 (aa 66-78) and P13 (aa 67-80) were also shown to suppress damage caused by infection.

[0188] These results (summarized in Table 1 ) provide evidence that exogenous ANGPTL4 exerts a major protective effect on the integrity of endothelial cell monolayers infected with N. meningitidis and that this effect is mediated by a novel binding motif (aa 66-80) that we identified in the N-terminal part of the protein.

[0189] <The vascular protective effect is mediated by the interaction between nANGPTL4 and syndecan-4.> The N-terminal portion of ANGPTL4 has previously been shown to bind heparan sulfate proteoglycans

[14] , particularly syndecan-4 (SDC4)

[17] , so we investigated the potential role of SDC4 in the protective effects of ANGPTL4. Interestingly, SDC4 mRNA and protein are expressed in both brain (HBMEC, hCMEC / D3) and skin (HDMEC) microvascular endothelial cells and are increased by N. meningitis infection and are more strongly induced in HDMEC (data not shown), which is associated with an increased interaction of SDC4 with ANGPTL4, as revealed by enrichment coimmunoprecipitation (data not shown).

[0190] Co-treatment of HDMECs with ANGPTL4, nANGPTL4, in combination with anti-SDC4 antibody abolished the protective effects of both ANGPTL4 and nANGPTL4 against vascular degeneration caused by infection, whereas anti-SDC4 antibody alone had no deleterious effect on uninfected cells (Fig. 4A). Anti-SDC4 antibody also abolished the protective effects of peptides P1 and P6 (data not shown), whereas anti-SDC1 antibody showed no antagonistic effect on ANGPTL4, nANGPTL4, or HDMECs treated with these peptides (data not shown), indicating that this effect was specific to SDC4. Furthermore, reduction of SDC4 using siRNA in HDMECs prior to infection also abolished the protective effect of ANGPTL4 (Fig. 4B). These results revealed that nANGPTL4 exerts a protective effect on the integrity of endothelial cell monolayers infected with N. meningitidis by interacting with SDC4 via its binding motif (aa 66-80).

[0191] Similarly, when anti-SDC4 antibody was added to HCMEC / D3 cells, the loss of VE-cadherin junction distribution and the loss of monolayer integrity were induced as a response to meningococcal infection, but no harmful effects were observed on uninfected monolayer structures (Figure 5A). These effects were further confirmed using primary brain endothelial cells. Infection with meningococci for 3 hours only partially affected the strength and integrity of the barrier formed by HBMECs, as evaluated by only a 50% decrease in transendothelial electrical resistance (TEER) and maintenance of VE-cadherin junctional tissue, and addition of anti-ANGPTL4 or anti-SDC4 blocking antibodies 30 minutes after the start of infection similarly decreased the electrical resistance by 80% and induced disruption of the junctions (Figure 5B). This further indicates that the functional interaction between SDC4 and ANGPTL4 in brain endothelial cells provides a defense function against bacterial infection.

[0192] These data indicate the protective effect of ANGPTL4 against endothelial cell degeneration promoted by meningococcal infection. To investigate whether ANGPTL-4 can also protect blood vessels from Gram-positive bacteria, we examined the effect of ANGPTL4 on HDMECs exposed to infection with Streptococcus pneumoniae, a Gram-positive bacterium that is one of the main causative agents of pneumonia, sepsis, meningitis, and other diseases. While endothelial cell junctional tissue was lost upon S. pneumoniae infection, addition of human ANGPTL4 maintained endothelial integrity (Figure 6). These results suggest that ANGPTL4 is a potent vascular stabilizing factor that has the potential to protect against infection with both Gram-positive and Gram-negative bacteria.

[0193] <ANGPTL4 prevents the formation of vascular lesions in vivo.> We therefore investigated the potential in vivo protective effect of ANGPTL4 against vascular degeneration caused by N. meningitidis using a validated and robust humanized mouse model of meningococcal infection consisting of severe combined immunodeficient (SCID) mice grafted with human skin that recapitulates vascular lesions similar to the purpuric lesions observed in patients [18,19] (Figure 7A). 6 Intravenous injection of bacteria from the mouse AngPTL4-treated mice (Fig. 7B) resulted in extensive colonization of human dermal blood vessels in the skin grafts 4 h after infection (Fig. 7C). As previously reported [20,21], this intravascular colonization was associated with signs of compromised endothelial integrity, as assessed by massive platelet and erythrocyte aggregation, vascular occlusion (data not shown), and loss of the binding marker VE-cadherin (data not shown). Intravenous administration of human ANGPTL4 protein (1 μg / mouse) 30 min or 2 h after the onset of infection (Fig. 7A) resulted in similar bacteremia and intravascular colonization 4 h after infection in ANGPTL4-treated and control mice (Fig. 7B). However, ANGPTL4 treatment significantly reduced vascular thrombosis (Fig. 7D) and maintained the integrity of endothelial cell junctions, as assessed by sequential staining for VE-cadherin (data not shown). Furthermore, infection released proinflammatory factors (TNF-α, IL-6, IL-8) and elevated biomarkers of vascular inflammation (soluble E-selectin, ICAM-1, VCAM-1) and vascular degeneration (angiopoietin-2, thrombomodulin) in mouse serum, whereas administration of ANGPTL4 reduced serum levels of all of these markers by 60-80% (data not shown). Administration of ANGPTL4 30 min or 2 h postinfection similarly reduced vascular lesions and thrombosis, but early administration was necessary to prevent the release of inflammatory factors, and no effect on these inflammatory markers was observed with administration at 2 h (data not shown).

[0194] Interestingly, administration of the N-terminal and C-terminal domains of ANGPTL4 (1 μg / mouse) did not affect vascular colonization (Figure 7C), but was found to reduce infection-induced vascular thrombosis (Figure 7D). On the other hand, administration of nANGPTL4 maintained the integrity of inter-epithelial cell junctions (data not shown), and administration of cANGPTL4 most efficiently suppressed the production of inflammatory factors, which are biomarkers of vascular inflammation (data not shown), indicating two functions that depend on two different functional domains of ANGPTL4. As expected, administration of ANGPTL3 or ANGPTL8 did not show a protective effect against vascular degeneration, thrombosis, or release of inflammatory markers (data not shown), indicating that these effects are specific to ANGPTL4. These data demonstrate a clear protective effect of ANGPTL4 against vascular degeneration, thrombosis, and inflammation in meningococcal sepsis.

[0195] <ANGPTL4 improves the outcome of meningococcal infection.> Next, we investigated whether ANGPTL4 improves the outcome of infected mice (Figure 8A). In control mice, intravenous infection with 1 × 10 6 bacteria resulted in persistent bacteremia with an average of 10 6 ~5 × 10 6 colony-forming units / ml at 18 hours post-infection (Figure 8B), and all mice died within 2 days (20 - 40 hours post-infection) (Figure 8C). Administration of recombinant human ANGPTL4 (1 μg / mouse, intravenous) 2 hours after infection resulted in bacteremia at 4 and 18 hours post-infection that was similar to that of vehicle-administered control mice (Figure 8B), but mice administered ANGPTL4 died at later time points (30 - 50 hours later), and only 15% survived the infection (Figure 8C). Infected control grafts showed extensive colonization of human skin blood vessels, formation of lumen-occluding thrombi, and signs of impaired endothelial integrity at death (20 - 40 hours post-infection), whereas human skin blood vessels in ANGPTL4-treated grafts showed reduced signs of thrombosis and vascular leakage (data not shown).

[0196] Next, we analyzed whether ANGPTL4 confers beneficial effects when administered in combination with antibiotics. Similar to the previously reported observations [Reference 20], treatment with an antibiotic (200 mg / kg cefotaxime) exerted a strong bactericidal effect (Figure 8B), improved mouse survival (Figure 8C), but could not prevent thrombus induction at 4 hours post-infection (Figure 8D) or vascular degeneration associated with extravascular leakage of erythrocytes and massive infiltration of polymorphonuclear leukocytes at 72 hours post-infection (data not shown). Antibiotic treatment in combination with ANGPTL4 resulted in reduced thrombus size, macrophage infiltration, and signs of fibrinolysis associated with preserved vascular integrity in infected blood vessels, which are typical features of thrombolysis (data not shown). These data indicate the beneficial effects of ANGPTL4 in restoring vascular permeability and reducing the morbidity and mortality associated with meningococcal infection. It also shows that ANGPTL4 confers an adjuvant effect to antibiotic treatment by reducing vascular degeneration.

[0197] <ANGPTL4 prevents lethal sepsis by preventing vascular dysfunction and organ failure.> Since severe vascular endothelial injury leads to coagulation dysregulation and vascular dysfunction and plays a central role in the progression of organ failure during sepsis, we used lipopolysaccharide (LPS)-induced endotoxemia to analyze whether ANGPTL4 may play a protective role during sepsis (Figure 9A). After LPS administration (5 mg / kg, intraperitoneal), all mice died within 2 days (Figure 9B). Administration of a recombinant human ANGPTL4 (2 μg / mouse, intravenous) resulted in 80% survival after infection, showing a significant reduction in sepsis-related mortality compared to vehicle-administered control mice (Figure 9B). Although there was no significant decrease in overall weight loss in the ANGPTL4-administered group compared to the vehicle-administered control group, body temperature rapidly stabilized in the ANGPTL4-administered group (data not shown).

[0198] We next evaluated the effect of ANGPTL4 on acute lung and kidney injury 18 h after LPS administration. LPS induced significant histopathological changes, such as vascular wall thickening, collapse of alveolar sacs, and intraalveolar hemorrhage, in the lungs of vehicle-treated control mice, whereas no pathological signs were observed in the lungs of ANGPTL4-treated mice (data not shown). Similarly, LPS induced severe renal hemorrhage in vehicle-treated control mice, whereas no signs of hemorrhage were detected in ANGPTL4-treated mice (data not shown). These data indicate that ANGPTL4 provides an efficient protective effect against LPS-induced acute lung and kidney injury by preventing progression to severe endothelial dysfunction and organ failure as well as death. As expected, administration of nANGPTL4 (2 μg / mouse, i.v.) and derived peptides P1 or P9 efficiently reduced sepsis-associated mortality similar to the full-length protein (Figures ​(Figures​(C-F,​G),​cANGPTL4​or​peptide​P10​(2 μg / mouse, i.v.)​ did not significantly increase survival (data not shown), further supporting the dependency of this effect on the ability of the N-terminal portion of ANGPTL4 to maintain vascular integrity. In contrast, administration of LPS (4 mg / kg, i.p.)​ did not result in any leakage of Evans blue in the brains of LPS-treated mice, indicating that BBB integrity was intact (Figure​(Figure​(C-F,​G),​). When anti-ANGPTL4 inhibitory antibody (10 μg / kg, i.v.) was administered together with LPS, significant exudation of Evans blue was observed ( Fig. 9H ), indicating that endogenous production of ANGPTL4 at the central nervous system level prevents LPS-induced BBB disruption.

[0199] Taken together, these results indicate that ANGPTL4 is a key vascular stabilizer that provides protection against bacterial infection. The use of human ANGPTL4 (or derived peptides) may reduce the incidence of vascular degeneration and intravascular coagulation in bacterial infections, potentially reducing the costly effects of sepsis.

[0200] [References] Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into this disclosure.

[0201] 1. Cavaillon, JM, M. Singer, and T. Skirecki, Sepsis therapies: learning from 30 years of failure of translational research to propose new leads. EMBO Mol Med, 2020. 12(4): p. e10128. 2. Jarczak, D., S. Kluge, and A. Nierhaus, Sepsis-Pathophysiology and Therapeutic Concepts. Front Med (Lausanne), 2021. 8: p. 628302. 3. Angus, DC and T. van der Poll, Severe sepsis and septic shock. N Engl J Med, 2013. 369(21): p. 2063. 4. Coureuil, M., et al., Invasive meningococcal disease: a disease of the endothelial cells. Trends Mol Med, 2014. 20(10): p. 571-8. 5. Coureuil, M., et al., A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers. Nat Rev Microbiol, 2017. 15(3): p. 149-159. 6. Wolburg, H. and A. Lippoldt, Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol, 2002. 38(6): p. 323-37. 7. Abbott, N.J., et al., Structure and function of the blood-brain barrier. Neurobiol Dis, 2010. 37(1): p. 13-25. 8. Daneman, R., et al., Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature, 2010. 468(7323): p. 562-6. 9. Abbott, N.J., L. Ronnback, and E. Hansson, Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci, 2006. 7(1): p. 41-53. 10. Caugant, D.A. and M.C. Maiden, Meningococcal carriage and disease--population biology and evolution. Vaccine, 2009. 27 Suppl 2: p. B64-70. 11. Mairey, E., et al., Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier. J Exp Med, 2006. 203(8): p. 1939-50. 12. Pron, B., et al., Interaction of Neisseria maningitidis with the components of the blood- brain barrier correlates with an increased expression of PilC. J Infect Dis, 1997. 176(5): p. 1285-92. 13. Yang, X., Y. Cheng, and G. Su, A review of the multifunctionality of angiopoietin-like 4 in eye disease. Biosci Rep, 2018. 38(5). 14. Chong, H.C., et al., Angiopoietin-like 4 stimulates STAT3-mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice. Mol Ther, 2014. 22(9): p. 1593-604. 15. Le Jan, S., et al., Angiopoietin-like 4 is a proangiogenic factor produced during ischemia and in conventional renal cell carcinoma. Am J Pathol, 2003. 162(5): p. 1521-8. 16. Huang, R.L., et al., ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters. Blood, 2011. 118(14): p. 3990-4002. 17. Li, L., et al., Angiopoietin-like 4 Increases Pulmonary Tissue Leakiness and Damage during Influenza Pneumonia. Cell Rep, 2015. 10(5): p. 654-663. 18. Zhang, B., et al., Protective effects of angiopoietin-like 4 on the blood-brain barrier in acute ischemic stroke treated with thrombolysis in mice. Neurosci Lett, 2017. 645: p. 113-120. 19. Galaup, A., et al., Angiopoietin-like 4 prevents metastasis through inhibition of vascular permeability and tumor cell motility and invasiveness. Proc Natl Acad Sci U S A, 2006. 103(49): p. 18721-6. 20. Bouleti, C., et al., Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke. Eur Heart J, 2013. 34(47): p. 3657-68.

Claims

1. Amino acid sequence X １ SALERRLSACGSX １４ X １５ or includes or consists of functionally preserving variants thereof, X １ It is either amino acid-free or leucine (L), X １４ is either amino acid-free or alanine (A), and X １５ It is either amino acid-free or contains cysteine ​​(C). A peptide derived from the ANGPTL4 protein.

2. The molecule contains or consists of amino acid residues from position 66 to 80 in SEQ ID NO: 1, where the amino acids at positions 76 and 80 are any natural amino acids (peptide P9 or peptide P9') or their function-conserving variants. The peptide derived from the ANGPTL4 protein according to claim 1.

3. The amino acid residues in SEQ ID NO: 1 include or consist of amino acid residues from position 65 to 80, with the amino acids at positions 76 and 80 being any natural amino acid (peptide P6 or peptide P6') or a function-conserving variant thereof. The peptide derived from the ANGPTL4 protein according to claim 2.

4. The molecule comprises or consists of amino acid residues from position 56 to 83 in SEQ ID NO: 1, wherein the amino acids at positions 76 and 80 are any natural amino acids (peptide P4 or peptide P4') or functionally conserved variants thereof. The peptide derived from the ANGPTL4 protein according to claim 2.

5. The amino acid residues in SEQ ID NO: 1 include or consist of amino acid residues from position 38 to 83, with the amino acids at positions 76 and 80 being any natural amino acid (peptide P1 or peptide P1') or a function-conserving variant thereof. The peptide derived from the ANGPTL4 protein according to claim 2.

6. The amino acid residues in SEQ ID NO: 1 include or consist of amino acid residues from position 26 to 164, with the amino acids at positions 76 and 80 being any natural amino acid (nANGPTL4 or nANGPTL4') or a function-conserving variant thereof. The peptide derived from the ANGPTL4 protein according to claim 2.

7. The amino acid residue in SEQ ID NO: 1 comprises or consists of amino acid residues from position 1 to position 406, wherein the amino acids at positions 76 and 80 are any natural amino acid (ANGPTL4 or ANGPTL4') or a function-conserving variant thereof. The peptide derived from the ANGPTL4 protein according to claim 2.

8. The amino acids at positions 76 and 80 may be amino acids selected from the group consisting of cysteine ​​(Cys or C), alanine (Ala or A), or serine (Ser or S). The peptide derived from the ANGPTL4 protein according to claim 1.

9. Containing an amino acid sequence defined in SEQ ID NOs: 1 or 8-30 or 37-64 The peptide derived from the ANGPTL4 protein according to claim 1.

10. A nucleic acid sequence encoding the peptide according to any one of claims 1 to 9.

11. A peptide according to any one of claims 1 to 9, or a nucleic acid encoding said peptide, for use in treating an infectious disease requiring treatment.

12. For use in the treatment of bacterial infections, The peptide or nucleic acid according to claim 11.

13. For use in the treatment of sepsis caused by bacterial infection, The peptide or nucleic acid according to claim 12.

14. i) a peptide according to any one of claims 1 to 9 or a nucleic acid encoding such peptide, and ii) a formulation for simultaneous, separate or sequential use for the treatment of an infection in a subject requiring treatment of an infection, comprising:

15. A therapeutic agent for use in treating an infectious disease of a target requiring treatment for the infectious disease, comprising a peptide according to any one of claims 1 to 9, an antibody of the peptide, a protein containing the peptide, a nucleic acid encoding the peptide, a vector containing the nucleic acid, or a host cell containing the vector.

Images