WO2018114986A1 - Methods for treating gram negative bacterial infection - Google Patents

Abstract

The present invention relates to plasma phospholipid transfer protein (PLTP) for use in the treatment of gram negative bacterial infection. More specifically, it concerns use of rhPLTP for the treatment of gram negative bacterial infection in order to prevent gram negative sepsis. rhPLTP is successfully used as an exogenous therapeutic protein to treat endotoxemia and sepsis. In mouse models with either purified lipopolysaccharides injection or with polymicrobial infection, it is demonstrated that rhPLTP prevented bacterial growth and detoxified LPS. In further support of the antimicrobial effect of PLTP, PLTP -knocked out mice were found to be less able than wild-type mice to fight against sepsis. The production of rhPLTP to counter infection and to reduce endotoxemia and its harmful consequences is reported here for the first time. It paves the way for a novel strategy to satisfy long-felt, but unmet needs to prevent sepsis.

Description

METHODS FOR TREATING GRAM NEGATIVE BACTERIAL INFECTION

FIELD OF THE INVENTION

The present invention relates to plasma phospholipid transfer protein (PLTP) for use in the treatment of gram negative bacterial infection. More specifically, it concerns use of rhPLTP for the treatment of gram negative bacterial infection in order to prevent gram negative sepsis. BACKGROUND OF THE INVENTION

Sepsis is a syndrome which includes a heterogeneous constellation of symptoms, including physiologic derangements and organ dysfunction, which accounts for the greatest mortality in the intensive care unit. Sepsis causes millions of deaths globally each year and is the most common cause of death in people who have been hospitalized (Deutschman, CS et al. Immunity (2014). 40 (4): 463-75). The worldwide incidence of sepsis is estimated to be 18 million cases per year (Lyle, NH; et al. Annals of the New York Academy of Sciences. 1323 (2014): 101-14). It is the 13th leading cause of death in America, with estimates of > 750,000 cases per year (Angus DC et al. Crit. Care Med. (2001) 29: 1303-1310). Despite its prevalence and high associated mortality, upwards of 40 - 80%, attempts to alter these abysmal statistics have met with relatively little success in the preceding decades. Any improvement in survival may be more likely attributed to improvements in supportive management rather than therapies directed at the underlying etiology.

Sepsis-inducing toxins have been found associated with pathogenic bacteria, viruses, plants and venoms. Among the well described bacterial toxins are the endotoxins or lipopolysaccharides (LPS) of the gram-negative bacteria. Lipopolysaccharides (LPS) are amphipathic molecules which are localized in the outer leaflet of the outer membranes of Gram-negative bacteria. They activate the innate immune system through a complex process involving Toll-like receptors (TLR) and the MD-2, CD 14 and lipopolysaccharide-binding protein (LBP) accessory proteins (Miller, S.I. et al. Nat. Rev. Microbiol. (2005) 3, 36-46). In the first step of endotoxic shock, LPS binds to plasma LBP and then complexes with CD14 antigen which can behave either as a membrane-anchored or a soluble protein. After the TLR4-MD2-CD14 complex has formed, cell signalling results in the production of proinflammatory cytokines (TNFalpha, IL-6, IFNgamma,...) and nitric oxide, which leads to shock-like symptoms (Miller, S.I. et al. (2005)). Over the last decade most strategies to develop pharmacological interventions to treat sepsis have involved downregulation of the inflammatory process and mediators (noticeably through treatments with corticosteroids or with agents targeting LPS, Toll-like receptor 4 (TLR4), tumor necrosis factor (TNF), interleukin (IL-1) as well as platelet-activating factor (PAF) (Fink, M.P. et al. Nat Rev Drug Discov 13, 741-758 (2014), Lehmann, C. et al. Expert Opin Drug Discov 9, 523-531 (2014)). All these strategies for the treatment of sepsis, concern sepsis patient with a inflammatory process well developed.

Accordingly, there is a medical need to identify new therapeutical tools to inhibit gram negative infection in order to prevent sepsis development.

SUMMARY OF THE INVENTION

Inventors discovered that PLTP targets the very early, LPS-mediated initiating step, thus providing a unique strategy for a dual beneficial outcome: 1) the limitation of Gram(-) bacterium growth and 2) the neutralization and detoxification of LPS, the culprit initiator of the inflammatory response.

Accordingly, the present invention relates to a method of treating gram negative bacterial infections using plasma phospholipid transfer protein (PLTP). More specifically, it concerns the discovery of the unexpected bacteriostatic properties of the plasma phospholipid transfer protein (PLTP), useful for the treatment of gram negative bacterial infections and also useful to prevent harmful consequence of such gram negative bacterial infection such as endotoxemia and sepsis.

The present invention relates to a phospholipid transfer protein (PLTP) for use in the treatment of gram negative bacterial infection.

The invention also provides a phospholipid transfer protein (PLTP) for use in the prevention of gram negative sepsis. The invention also provides a phospholipid transfer protein (PLTP) for use in the prevention of gram negative bacterial infection in a patient at risk to develop sepsis.

The present invention also relates to a phospholipid transfer protein (PLTP) for use as a bacteriostatic agent in a method of treatment. DETAILED DESCRIPTION OF THE INVENTION

In the present study, inventors produced fully active recombinant human PLTP (rhPLTP) in the milk of new lines of transgenic rabbits. They successfully used rhPLTP as an exogenous therapeutic protein to treat endotoxemia and sepsis. In mouse models with either purified LPS injection or with polymicrobial infection, inventors demonstrated that rhPLTP prevents bacterial growth and detoxifies LPS. In further support of the antimicrobial effect of PLTP, PLTP -knocked out mice were found to be less able than wild-type mice to fight against bacterial infection and sepsis. To our knowledge, the use of PLTP to counter bacterial infection and to reduce endotoxemia and its harmful consequences is reported here for the first time. It paves the way for a novel strategy to satisfy long-felt, but unmet needs to prevent and treat gram negative bacterial infection together with sepsis. Accordingly by treating with PLTP gram- negative bacterial infection in a patient, it also allows to prevent the onset of sepsis condition

In an additional study (example 2), inventors also demonstrate 1) the diffusion into the peritoneal cavity of intravenously injected rhPLTP, 2) less tissue damage in the liver and kidney with rhPLTP treatment, 3) no effect of rhPLTP on gram (+) bacteria, unlike gram- negative bacteria (-), 4) rhPLTP efficacy demonstrated in a wild-type / C57B16 gene pool naturally expressing PLTP at a high level.

The present invention relates to a phospholipid transfer protein (PLTP) for use in the treatment of gram negative bacterial infection. PLTP (also called "Lipid transfer protein Π") is a member of the lipid transfer/lipopolysaccharide binding protein (LT/LBP) gene family. It includes cholesteryl ester transfer protein (CETP), lipopolysaccharide binding protein (LBP), and bactericidal permeability increasing protein (BPI) in addition to PLTP. Plasma phospholipid transfer protein (PLTP) is ubiquituously expressed in vertebrate species. It has the ability to bind and transfer a number of amphipathic lipid compounds, including phospholipids, unesterified cholesterol, diacylglycerides and tocopherols (Lagrost 1998). Phylogenetic analyses revealed that PLTP belongs to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) gene family as do cholesteryl ester transfer protein (CETP), lipopolysaccharide binding protein (LBP) and the bactericidal permeability increasing protein (BPI) which are involved in innate immunity (Bingle 2004, Cheung 2010). Like most other members of the related palate-lung epithelial clone (PLUNC) superfamily of genes (known to constitute the first barrier of defense against pathogens in the upper airways), PLTP was recently found to bind and transfer lipopolysaccharides (LPS) which are located in the outer wall of gram negative bacteria (Hailman 1996, Levels 2005, Gautier 2008 and 2011, Yu 2016). Consistently, recent studies came in support of a key role of PLTP in the lipoprotein-mediated reverse LPS transport pathway through which purified LPS aggregates can be disrupted, transferred to lipoprotein vehicles and transported to the liver for elimination in the bile (Gautier 2008, 2011). Earlier in vitro and in vivo studies revealed that the PLTP-mediated transfer of LPS to lipoproteins results in the neutralization of the proinflammatory properties of LPS and in its elimination from the body. In further support of the pathophysiological relevance of the PLTP-mediated detoxification of LPS, PLTP -knocked out mice were less able than wild-type mice with naturally elevated PLTP expression level to get rid of purified LPS (Gautier 2008).

The term "PLTP" should be understood broadly, it encompasses the native PLTP, variants and fragments thereof having bacteriostatic activity.

Typically native PLTP variants and fragments thereof have also Phospholipid transfer activity. Typically the PLTP, can be from pig, rabbit, mouse or human origin. Preferably, if the patient is human, the PLTP is a human PLTP (UniProtKB / Swiss-Prot P55058). More preferably, the human PLTP is a human recombinant PLTP.

The protein sequence of said human PLTP, and its isoforms, may be found in NCBI database with the following access numbers:

PLTP isoform a precursor mRNA NM 006227, and protein id: NP 006218 (this variant encodes the predominant isoform (a)),

PLTP isoform b precursor mRNA NM_182676 and protein_id: NP_872617

PLTP isoform c precursor: mRNA NM 001242920, and protein id: NP 001229849,

PLTP isoform d mRNA NM 001242921, and protein id: NP 001229850.

Typically a variant of PLTP has at least 80%, preferably, at least 85%, more preferably at least 90%, more preferably at least 95% and even more preferably at least 99% identity with native PLTP. In a particular embodiment said PLTP is PLTP isoform a.

Typically, identity may be determined by BLAST or FAST A algorithms. Bacteriostic activity of PLTP can be measured for example by bacterial growth assessment using the broth microdilution assay (see experimental section and figure 3). Broth microdilution is a method used to test the susceptibility of bacteria to antibiotics. During testing, multiple microtiter plates are filled with a broth composed of bacteria and supplements of blood (Lorian, Victor (2005). Antibiotics in Laboratory Medicine. Lippincott Williams & Wilkins. p. 149. Retrieved 16 November 2014). Varying concentrations of the antibiotics and the bacteria to be tested are then added to the plate. The plate is then placed into a non-C02 incubator and heated at thirty-five degrees Celsius for sixteen to twenty hours. Following the allotted time, the plate is removed and checked for bacterial growth. If the broth became cloudy or a layer of cells formed at the bottom, then bacterial growth has occurred. The results of the broth microdilution method are reported in Minimum Inhibitory Concentration (MIC), or the lowest concentration of antibiotics that stopped bacterial expansion (Engelkirk, Paul; Duben-Engelkirk, Janet (2008). Laboratory Diagnosis of Infectious Diseases. Lippincott Williams & Wilkins. p. 168. Retrieved 16 November 2014.). Phospholipid transfer activity of PLTP (PLTP activity) can be measured for example as the rate of radiolabeled phosphatidylcholine transferred from liposomes to human HDL3 (see Damen J et al. Biochim Biophys Acta 1982;12:444-52). Commercially available kits (Cardiovascular targets, New York, NY, USA) are available for fluorescent assays of plasma phospholipid transfer activity. Typically, a fluorescent phospholipid (NBD-labelled phospholipid) is present in a self-quenched state when associated with the donor. The PLTP- mediated transfer is determined by the increase in fluorescence intensity as the fluorescent lipid is removed from the donor and transferred to the acceptor (Masson D et al. Mol Human Reprod 2003;9:457).

Methods for producing PLTP are well known. For example, PLTP can be obtained by classical sequential procedures for PLTP purification from total human plasma. Starting from large volumes of human plasma, Tollefson et al {J Lipid Res 1988;29:1593-602) purified human PLTP by using a complex/6-step process including ultracentrifugation, hydrophobic- interaction chromatography, anion exchange chromatography, affinity chromatography, cation exchange chromatography, and adsorption chromatography. By using another sequential procedure (ultracentrifugation, hydrophobic-interaction chromatography, affinity chromatography, and anion exchange chromatography), a highly active, purified preparation of human PLTP was obtained (Lagrost L et al. J Lipid Res 1994;35:825-35).

Alternatively, PLTP can be a recombinant PLTP. Typically, recombinant human, mouse or pig PLTP were produced in trans fected cells:

- transfected baby hamster kidney (BHK) cells: production of human and mouse PLTP

(Albers JJ et al. Biochim Biophys Acta 1995 ; 1258 :27-34)

COS-1 cells (CV-1 (simian) in Origin, and carrying the SV40 genetic material) cells: transient production of pig PLTP (Pussinen PJ et al. J Lipid Res 1997;38: 1473-81).

NB: the protein appeared as a multiple band in the molecular weight region of 75-83 kDa. This size is similar to the apparent molecular weight of PLTP purified from pig plasma, 81 kDa which, however, runs as a single band. Underway large scale production, purification, and detailed functional characterization of recombinant PLTP

(with the COS system) is claimed in the article.

- production of recombinant PLTP using a baculovirus/insect cell (Sf-9) system (Huuskonen et al. Biochim Biophys Acta 1998; 1391 : 181 -92).

production in a stably transfected HeLa (human cervical cancer) cell line (Huuskonen

J et al J Lipid Res 1998;39:2021-30).

More recently inventors describes method of production of fully active recombinant human PLTP (rhPLTP) in the milk of new lines of transgenic rabbits with the human PLTP sequence placed under the control of the WAP promoter (WO2011125034). The resulting purified rhPLTP was found to display the main features of the native protein. In this model, human PLTP is produced in the mammary gland and is secreted in the milk of rabbit does. The presence of human PLTP in milk, was a common feature of transgenic rabbits and was observed in several lineages. Human PLTP transgene expression in rabbit mammary glands resulted in the production of transgenic milk with elevated PLTP activity levels related to the copy number of the PLTP gene

The term "gram-negative" bacterial infection refers to a local or systemic infection with gram- negative bacteria. The proteobacteria are a major group of gram-negative bacteria, including Escherichia coli (E. coli), Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc. Other notable groups of gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria. Medically relevant gram-negative cocci include the four types that cause a sexually transmitted disease (Neisseria gonorrhoeae), a meningitis (Neisseria meningitidis), and respiratory symptoms (Moraxella catarrhalis, Haemophilus influenzae). Medically relevant gram-negative bacilli include a multitude of species. Some of them cause primarily respiratory problems (Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary problems (Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), and primarily gastrointestinal problems (Helicobacter pylori, Salmonella enteritidis, Salmonella typhi). Gram-negative bacteria associated with hospital-acquired infection include Acinetobacter baumannii, which cause bacteremia, secondary meningitis, and ventilator-associated pneumonia in hospital intensive- care units.

In another particular embodiment of the invention, the Gram-negative bacteria according to the invention are selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella specie, E. corrodens, and Haemophilus influenza.

As previously described, the treatment with PLTP of a patient with gram negative infection allow to prevent harmful consequence of such gram negative bacterial infection such as endotoxemia and sepsis. Accordingly, the invention also provides a phospholipid transfer protein (PLTP) for use in the prevention of gram negative sepsis.

In a particular embodiment, PLTP is used preventively on patient in need thereof with a gram negative bacterial infection. As used herein, the term "patient" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human.

In a particular embodiment of the invention, said patient is suffering from gram negative bacterial infections

In a specific embodiment, the patient is at risk to develop a sepsis, such as a patient before a medical or surgical intervention, very young and very old subject (infants and seniors), people with chronic or serious illnesses (including diabetes and cancer), and immunocompromised patients.

In a specific embodiment, very young subject (infant), means subject with less than 5, 4, 3, 2, 1 years old. In a specific embodiment, very old subject (senior) , means subject more than ,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100 years old. The invention also provides a phospholipid transfer protein (PLTP) for use in the prevention of gram negative bacterial infection in a patient at risk to develop a sepsis.

The term "sepsis" means morbid condition frequently induced by a toxin, the introduction or accumulation of which is most commonly caused by infection or trauma. The initial symptoms of sepsis or septic shock typically include chills, profuse sweat, irregularly remittent fever, prostration and the like, followed by persistent fever, hypotension leading to shock, neutropenia, leukopenia, disseminated intravascular coagulation, adult respiratory distress syndrome and multiple organ failure. These final symptoms, generally referred to as acute phase septic shock, almost invariably lead to death.

Sepsis-inducing toxins have been found associated with pathogenic bacteria, viruses, plants and venoms. Among the well described bacterial toxins are the endotoxins or lipopolysaccharides (LPS) of the gram-negative bacteria. These molecules are glycolipids that are ubiquitous in the outer membrane of all gram-negative bacteria. While the chemical structure of most of the LPS molecule is complex and diverse, a common feature is the lipid A region of LPS (Rietschel et al., 1984, In Handbook of Endotoxins, eds. R. A. Proctor and E. Th. Rietschel, Elsevier, Amsterdam 1 : 187-214). Recognition of lipid A in biologic systems initiates many, if not all, of the pathophysiologic changes of sepsis. Because lipid A structure is highly conserved among all types of gram-negative organisms, common pathophysiologic changes characterize gram-negative sepsis.

The present invention also relates to a phospholipid transfer protein (PLTP) for use as a bacteriostatic agent in a method of treatment.

A bacteriostatic agent or bacteriostat, is a biological or chemical agent that stops bacteria from reproducing, while not necessarily killing them otherwise. When bacteriostatic antimicrobials are used, the duration of therapy must be sufficient to allow host defense mechanisms to eradicate the bacteria.

Typically, the bacteria is a Gram(-) bacteria. The present invention also relates to a method for treating gram negative bacterial infection in a patient, such method involving the step of administering to a patient in need thereof a therapeutically effective amount of PLTP. The present invention also relates to a method for preventing gram negative gram negative sepsis in a patient, such method involving the step of administering to a patient in need thereof a therapeutically effective amount of PLTP.

The present invention also relates to a method for preventing gram negative bacterial infection in a patient at risk to develop sepsis, such method involving the step of administering to a patient in need thereof a therapeutically effective amount of PLTP.

By a "therapeutically effective amount" is meant a sufficient amount to be effective, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient in need thereof will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, the duration of the treatment; drugs used in combination or coincidental with the and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The PLTP can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, rectal, nasal, buccal, ocular, sublingual, transdermal, rectal, topical, vaginal, parenteral (including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intraperitoneal. It will be appreciated that the preferred route may vary with for example the condition of the recipient. In a preferred embodiment PLTP is administred by parenteral way (including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural).

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies. Typically medicaments according to the invention comprise a pharmaceutically-acceptable carrier. A person skilled in the art will be aware of suitable carriers. Suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to well-known text such as Remington; The Science and Practice of Pharmacy.

FIGURE LEGENDS

Figure 1. Endogenous PLTP expression impacts on bacterial burden and LPS concentration in the bloodstream of mice after CLP surgery (a) PLTP expression decreases bacterial burden. Colony-forming units (CFU) were determined in blood samples harvested from WT and Pltp-/- mice before CLP, 8h and 24h following CLP. Data are means ± sem (n = 11-13).

(b,c) PLTP expression protects mice from polymicrobial sepsis by limiting the concentration of LPS in the bloodstream, and increasing its biliary excretion. LPS concentrations were determined by direct quantitation of 3-hydroxymyristate (3HM), over a 24h period following CLP, in plasma and blood cells (b) and in bile (c) from WT and Pltp-/- mice. Data are means ± sem (plasma n = 5-6; bile n = 12).

Figure 2 - PLTP expression protects mice against inflammation and mortality associated with polymicrobial sepsis.

(a) PLTP deficiency increases mortality. Aged-matched WT and Pltp^~'^~ male mice underwent CLP using 21G needle. The mice were observed twice daily for 10 days. Data were analyzed using Kaplan-Meier method, with statistical significance determined using the log rank test. Data are means ± sem (n=18-20 mice).

(b) PLTP deficiency induces hyperglycemia post CLP. Blood glucose concentration was determined with a glucose meter before and at 2h, 4h, 6h, 8h and 24h following CLP. Data are means ± sem (n=5). * P < 0.05

(c) PLTP deficiency increases levels of IL-6, MCP-1, and IL-Ιβ and decreases level of IFN-γ. Plasma samples, harvested from WT and Pltp ^1' mice before CLP, 8h and 24h after CLP, were assayed using a Milliplex mouse cytokine panel. Data are means ± sem (n=5).

Figure 3. Purified recombinant human PLTP has bacteriostatic properties in vitro (a) rhPLTP inhibits bacterial growth in vitro to a greater extent than rhBPI. The effect of rhPLTP and rhBPI on E. coli growth was examined using broth microdilution assay in nutrient MHB. rhPLTP and rhBPI (40 μg/ml) were added to a 96-well sterile microplate in order to get two-fold serial dilutions in MHB. Inoculum containing 10⁶ CFU/ml of ATCC25922 E. coli was added to each well. Microplates were incubated at 37°C. MHB with and without bacteria were used as growth and sterility controls, respectively. Bacterial growth was determined after 6h of incubation by measuring absorbance at 600 nm and expressed as a percentage of control growth (without rhPLTP and rhBPI). Data are means ± sem (n = 5).^a' P < 0.05, ^b P < 0.01 Significantly different from control. (b) Transmission electronic microscopy images of E. coli after 6h incubation in MHB in the absence (Control) or in the presence of rhPLTP. Normal ATCC25922 E. coli (fig 3b-Control) display typical Gram-negative structures with intact membrane and high-density cytoplasm. E. coli exposed to rhPLTP for 6h (fig 3b-rhPLTP) displayed condensation of the cytoplasmic contents. Significant changes in the morphology of cell membrane occurred. The most apparent phenomenon was plasmo lysis involving the outflow of intracellular constituents suggesting alteration in the fluidity and integrity of cell membranes but with no visible pores.

Figure 4 - rhPLTP injection reduces the bacterial burdens and increases the clearance of LPS in the bloodstream. (a,b) Intravenous rhPLTP administration reduces bacterial burdens in blood samples and bacterial load in peritoneal lavages of Pltp^~'^~ mice with polymicrobial sepsis. Pltp-/- mice received several i.v. injections of vehicle (control) or rhPLTP (25 μg in a volume of 200 μΐ) 2h, 4h, 7h, and 23h after CLP. Colony-forming units (CFU) were determined in blood samples before CLP, 8h and 24h following CLP (a) and in peritoneal lavage fluids 24h after CLP (b). Data are means ± sem (n=15).

(c,d) Intravenous rhPLTP administration reduces 3HM levels in plasma and blood cells by increasing its biliary excretion in Pltp ^' mice after CLP. Pltp-/- mice received several i.v. injections of vehicle (control) or rhPLTP (25 μg in a volume of 200 μΐ) 2h, 4h, 7h, and 23h after CLP. LPS concentrations were determined by direct quantitation of 3-hydroxymyristate (3HM) over a 24h period following CLP in plasma and blood cells (c) and in bile (d) from Pltp-/- mice. Data are means ± sem (n=14-18). Figure 5 - rhPLTP protects against inflammation and death in mice with polymicrobial sepsis.

a) Repeated i.v. injections of rhPLTP increase survival after CLP. Pltp-/- mice received several intravenous injections of rhPLTP (25 μg in a volume of 200 μΐ) or vehicle at time 2h,

4h, 7h, 23h, 32h, 48h, 56h, 72h, 80h, 96h, 104h after CLP. Survival rates were analyzed by the Kaplan-Meier method and compared using log rank test. Data are means ± sem (n = 16- 20).

b) Repeated i.v. injections of rhPLTP prevent the production of inflammatory cytokines (IL-6, MCP-1, TNF-a, IL-Ιβ) after CLP. Pltp-/- mice received several i.v. injections of vehicle

(control) or rhPLTP (25 μg in a volume of 200 μΐ) 2h, 4h, 7h, and 23h after CLP. Plasma samples, harvested from Pltp-/- mice before CLP, 8h and 24h after CLP, were assayed using a Milliplex mouse cytokine panel. Data are means ± sem (n = 15-19). Figure 6 - Activity and distribution of rhPLTP following intravenous injection (a)

Repeated iv injections of rhPLTP after CLP produced a cumulative effect on plasma PLTP activity. Pltp-/- mice received iv injections of vehicle (control) or rhPLTP (25 μg in a volume of 200 μΐ) 2h, 4h, 7h and 23h after CLP. PLTP activity was measured in plasma before and 8h and 24h after CLP and expressed as a percentage of WT mouse plasma activity used as a reference (n = 8 mice). Data are means ± sem. *** P < 0.001.

(b) Repeated iv injections of rhPLTP after CLP had an impact on the PLTP activity of peritoneal fluid. Pltp-/- mice received iv injections of vehicle (control) or 2h, 4h, 7h and 23h after CLP. PLTP activity was measured in plasma and peritoneal 24h after CLP and expressed in fluorescence units (n=4 mice). Data are means ± sem. * < 0.05.

Figure 7 - rhPLTP has no effect on gram (+) bacteria growth

rhPLTP had no effect on S. aureus (ATCC25923) growth. The effect of rhPLTP on bacterial growth was examined using broth microdilution assay in nutrient MHB. Bacterial growth was determined after 6h of incubation and expressed as a percentage of control growth (dashed line) i.e. without rhPLTP (S. aureus: n = 4 independent experiments).

Figure 8 - rhPLTP retained its protective effects in WT mice with polymicrobial sepsis.

WT mice received iv injections of vehicle (control) or rhPLTP (25 μg in a volume of 200 μΐ) 2h, 4h, 7h and 23h after CLP. (a) Repeated iv injections of rhPLTP after CLP induced a significant increase in plasma PLTP activity 24h after CLP. PLTP activity was measured in plasma before, 8h and 24h after CLP and expressed as a percentage of TO mouse plasma activity (n=12-14 mice per group, Kruskal-Wallis test with Dunn's multiple comparisons test).

(b) Repeated iv injections of rhPLTP increased mouse survival after CLP. Numbers of alive and dead animals were compared using the χ² test (n = 14-15 mice per group).

(c) Repeated iv injections of rhPLTP prevented the production of cytokines (IL-6, MCP-1, TNF-a, IL-Ιβ, IL-10) after CLP. Plasma samples, harvested from WT mice before CLP, 8h and 24h after CLP, were assayed using a Milliplex mouse cytokine panel (n = 12-14 mice per group, Mann- Whitney test).

(d and e) Intravenous rhPLTP administration reduced bacterial burdens in blood and peritoneal lavages of WT mice with polymicrobial sepsis. Colony-forming units (CFU) were determined in blood samples (d) before CLP, 8h and 24h following CLP and in peritoneal lavage fluids (e) 24h after CLP (n = 12-14 mice per group, Mann- Whitney test),

(f) Intravenous rhPLTP administration significantly reduced LPS levels in WT plasma after CLP. LPS concentrations were determined by direct quantitation of 3-hydroxymyristate (3HM) over a 24h period following CLP in plasma from WT mice (n = 12-14 mice per group, Mann- Whitney test). Data are means ± sem. *P < 0.05, **P < 0.01. EXAMPLE 1

MATERIALS AND METHODS

Polymicrobial sepsis after cecal ligation and puncture

Cecal ligation and puncture was performed as previously described (Rittirsch 2009). Mice were anesthetized with isoflurane then placed on a heating pad during the surgical procedure. A midline laparotomy was made after the abdomen had been shaved and prepared with alcohol. The cecum was then exteriorized and ligated with a 4-0 suture below the ileocecal valve without causing bowel obstruction. The ligated cecum was subsequently perforated by a single through and through puncture with a 21 -gauge needle and gently squeezed to extrude a small amount of fecal content. The cecum was placed back into the peritoneal cavity, and the incision was closed in two layers with 6-0 sutures and wound clips. The mice were then resuscitated with 0.4 ml of saline injected subcutaneously to compensate fluid loss that occurred during the procedure. At the end of the chirurgical procedure the mice were returned to their cages and survival was monitored twice a day for 10 days. Separate groups of animals underwent the same procedure. Blood samples were collected before (TO) and at different time during the 24h after CLP.

Blood sampling

All materials were of pyrogen- free grade or made apyrogenic by overnight heating at 150°C, and all of the reagents used were of "endotoxin-free" grade.

Blood was collected at the indicated times via retroorbital or cardiac puncture in anesthetized mice. Plasma was obtained by blood centrifugation (10 min, 6000 g at 4 °C)

Measurements of glycemia

Blood was drawn from the tail vein at 0, 2, 4, 6, 8 and 24h following the CLP, and glucose concentration was determined with a glucose meter (One Touch Ultra®).

Measurements of plasma concentrations of cytokines

Plasma concentrations of IL-6, TNFa, MCP-1, IL-Ιβ, IFN-γ, IL-10 were quantified using a Milliplex MAP Mouse Cytokine/Chemokine Panel (# MCYTOMAG-70K, Millipore, Billerica, MA). The assays were performed according to the manufacturer's instructions. Standards and samples were analyzed on a LuminexR® apparatus (Bio-Plex 200, BioRad, Munchen, Germany) using the BioPlex Manager Software (Version 5, BioRad, Hercules, CA).

LPS mass quantitation

LPS mass concentration was determined by direct quantitation of 3-hydroxytetradecanoic acid (or 3HM) by LC/MS/MS as previously described (Pais de Barros 2015). Briefly, samples were spiked with 5 ng of internal standard (3 -hydroxytridecanoic acid 10 mg/ml in ethanol), and then hydrolyzed with 300 μΐ of HC1 8 M for 4h at 90°C or with 75 μΐ of NaCl 150 mM, respectively. Free fatty acids were then extracted with 600 μΐ of distilled water and 5 ml of hexane. After vacuum evaporation of the hexane phase, fatty acids were dissolved in 100 μΐ of a 40% A/60% B eluent mixture (eluent A, ammonium acetate 5 mM pH 5.0; eluent B, acetonitrile/acetate ammonium 5 mM pH 7.3 96.7%/3.3%). Fatty acid separation was performed in an Infinity 1200 HPLC binary system (Agilent) equipped with a Poroshell 120 EC C18 100 x 4.6 mm 2.7 μιη column (Agilent) set at 30°C. The sample volume injected was 10 μΐ. A 7 min eluent gradient was established as follows: from 0 to 0.5 min, the flow was maintained constant at 1 ml/min of 80% B; then the proportion of B was increased linearly up to 100% in 1 min; concomitantly the flow rate was decreased to 0.5 ml/min; these conditions were maintained constant for 1 min; then the flow rate was increased to 1 ml/min for an additional 2.5 min; finally, the column was reequilibrated with 80% B at 1 ml/min for 2.5 min.

MS/MS detection was performed using a QQQ 6460 triple quadruple mass spectrometer (Agilent) equipped with a JetStream ESI source in the negative mode (gas temperature 300°C, gas flow 10 1/min, nebulizer 20 psi, sheath gas temperature 200°C, sheath gas flow 11 1/min, capillary 3,500 V). Nitrogen was used as the collision gas. The mass spectrometer was set up in the selected reaction monitoring (SRM) mode for the quantification of selected ions as follows: for 3-hydroxytetradecanoic acid, precursor ion 243.2 Da, product ion 59 Da, fragmentor 93 V, collision cell 9 eV; for 3-hydroxytridecanoic acid, precursor ion 229.2Da, product ion 59 Da, fragmentor 110 V, collision cell 10 eV.

Evaluation of bacterial burden

Blood samples were obtained from anesthetized mice via retro-orbital puncture before or 8h after CLP and by cardiac puncture using a sterile technique 24h after the CLP procedure. Peritoneal lavage fluids were obtained from mice 24h after CLP by injection of sterile PBS (3 ml) through the fascia into the peritonea! cavity a d gentle aspiration. For bacterial ciilturing, peritoneal lavage fluid or blood was diluted with PBS 1 : 10- 1 : 10⁶ and 30-100μ1 o each dilution was cultured on a Columbia agar medium supplemented with 5% sheep blood. Plates were incubated at 35 ± 2°C in anaerobic conditions for 24-48h and then the number o colony-forming units (CFU/ml) was counted.

In vitro evaluation of antimicrobial activity of purified recombinant proteins

E. Coli growth was examined in vitro using a broth microdilution assay in nutrient Mueller Hinton Broth (MHB, BD 275730). E. Coli (ATCC 25922) were plated on Tryptic soy agar (BD236950) overnight at 37 °C. An isolated bacterial colony was used to inoculate Mueller Hinton Broth (MHB, BD 275730), and the bacterial cultures were allowed to grow overnight at 37°C. 100 iL of culture was used to freshly inoculate 3 ml of MHB. The suspension was then allowed to grow at 37 °C with shaking at 225 rpm for ~2 h, where a final bacterial concentration of ~ 10⁸ colony forming units/mL (CFU/mL) was reached (OD600 ~0.1). rhPLTP was obtained as described above and rhBPI was purchased from R&D Systems (Catalog number 7468-BP). Recombinant proteins were diluted in MHB and were added to a 96-well sterile microplate in order to get two-fold serial dilutions in MHB. Next, 50 μΐ of inoculum containing 10⁶ CFU/ml of E Coli ATCC 25922 was added to each well, so that each well contained 50 μΐ of rhPLTP or rhBPI solution and 50 μΐ of cell suspension. Then, the microplates were incubated at 37°C for 24h. MHB with and without bacteria were used as growth and sterility controls, respectively. Bacterial growth was determined 6h after incubation by measuring absorbance at 600 nm (OD600).

Transmission electron microscopy (TEM)

Cells were pelleted by centrifugation 5 min, at 3000 g and 4°C. After washing with Sorensen's phosphate buffer (0.1 M, pH 7.4), the pellets were re-suspended with a solution of

4% [v/v] paraformaldehyde and 1.5% [v/v] glutaraldehyde in Sorensen's phosphate buffer.

After fixation for 45 min at room temperature, samples were washed four times with

Sorensen's phosphate buffer at 4°C and embedded in low-melting-point agarose (1.5-2%).

The agarose pellet was solidified at 4°C and cut into small cubes of about 1 mm edge length. Samples were post- fixed with 1% osmium tetroxide in Sorensen's phosphate buffer in the dark for lh at room temperature. Samples were dehydrated by graded ethanol and propylene oxide and embedded in EMBed-812 resin. Ultrathin sections were cut with an ultramicrotome

(Reichert Ultracut E, Leica, Rueil-Malmaison, France), transferred to copper/palladium grids.

The grids were contrasted with uranyl acetate and lead citrate and observed on a Hitachi H7500 TEM (Hitachi Scientific Instruments Co., Tokyo, Japan) operating at 80 kV and equipped with an AMT camera driven by AMT software (AMT, Danvers, USA).

Statistical Analysis

Data are presented as the mean ± sem. All data were analyzed using GraphPad Prism 7 software (GraphPad Software Inc.). The differences in survival rates were analyzed by Kaplan-Meier plot and the statistical significance was determined using log-rank test. For pairwise comparison of experimental groups, unpaired t test or Mann-Whitney test was performed. For all statistical analyses, a P value of 0.05 was considered significant.

RESULTS

Endogenous PLTP expression impacts on bacterial burden in the bloodstream of mice after CLP surgery

Cecal ligation and puncture (CLP) is recognized as a clinically relevant animal model of sepsis (Dejager 2011). Since CLP surgery causes the release of intestinal content including viable bacteria, we investigated whether PLTP expression impacts on bacterial burden in the bloodstream. Bacterial colony forming units (CFU) detected in blood of PLTP-deficient (Pltp^{~ A}) mice were significantly higher than in WT mice at 8h and 24h after CLP (Fig. la). Since blood cultures obtained after CLP results mainly from translocation of gram negative bacteria (Merx 2004), level of 3hydroxymyristate (3HM) (ie the most abundant hydroxylated fatty acid of the lipid A moiety of most LPS molecules) was quantitated by LC-MS/MS (Pais de Barros 2015). The amount of LPS detected in plasma and blood cells of Pltp^~'^~ mice 24 h after CLP was clearly higher than in WT counterparts (Fig. lb). Bile levels of 3HM measured 24h after CLP were significantly lower in Pltp-/- than in WT mice, indicating a weaker LPS excretion capacity in mice lacking PLTP (Fig. lc).

Endogenous PLTP expression protects against polymicrobial sepsis

Whereas 61% of WT mice survived after CLP, the overwhelming majority (70%) of Pltp ^' mice died (Fig. 2a). Consistent with the increased mortality of the PLTP-deficient mice, their glycaemia was significantly higher at 2h and 4h after CLP (Fig. 2b) as well as their inflammatory cytokines/chemokines plasma levels (Fig. 2c) relative to WT mice. Secretions of IL-6, MCP-1 and IL-Ι β in plasma peaked at 8h after CLP and were substantially higher in PLTP-deficient mice than in WT controls.

Purified recombinant human PLTP has bacteriostatic properties in vitro

BPI has longer been designated as the bacteriostatic member of the LT/LBP family through its ability to bind LPS in the outer wall of gram(-) bacteria, thus increasing permeability. Surprisingly, and as shown in Figure 3 a, recombinant human PLTP (rhPLTP) prepared from milk of PLTP transgenic rabbit females was able to inhibit bacterial growth more efficiently than recombinant human BPI (rhBPI) in vitro. Six hours after addition of recombinant proteins to incubation mixtures, growth of E. coli ATCC25922 was decreased by 65 ± 1% in the presence of rhPLTP as compared to 18 ± 8 % only in the presence of rhBPI. Based on this bacteriostatic activity of rhPLTP on E. coli, transmission electronic microscopy was used to detect damage to bacterial cells. The normal shape of E. coli ATCC25922 is shown in fig 3b (control). It displays typical Gram-negative structure with intact membrane and high density cytoplasm. E. coli exposed to rhPLTP for 6h displayed condensation of the cytoplasmic contents. Significant changes of the morphology of cell membrane occurred. The most apparent phenomenon was plasmolysis involving the outflow of intracellular constituents, suggesting alteration in the fluidity and integrity of cell membranes but with no visible pores. Injection of exogenous rhPLTP reduces bacterial burden after CLP surgery

Bacterial CFU from blood and peritoneal lavage fluid were determined in mice subjected to CLP and treated or not with rhPLTP. Repeated administration of rhPLTP in mice was able to markedly reduce bacterial burden which was almost undetectable in blood at 8h and 24h time points following CLP surgery (Fig. 4a). Consistently, bacterial burden was also markedly reduced in peritoneal lavage fluid of rhPLTP -treated mice as compared with vehicle-treated mice (Fig. 4b). In further support of bacteriostatic properties of rhPLTP, lower levels of 3HM were measured in the bloodstream of Pltp^~'^~ mice treated with rhPLTP as compared to vehicle controls. A significant decrease in 3HM was first observed in the blood cells at 8h and in the plasma at 24h after CLP (Fig. 4c). Biliary excretion of 3HM was significantly improved by PLTP as illustrated by the nearly two-fold increase in the amounts of 3HM detected in the bile of mice treated with rhPLTP as compared to mice receiving the vehicle only (Fig.4d).

Injection of exogenous rhPLTP to prevent sepsis

Repeated iv injections of rhPLTP after CLP in PLTP-deficient mice produced a significant reduction in mortality (Fig. 5a). Ten days after CLP, only 20% of mice receiving the vehicle survived against 50% of survival in the rhPLTP-treated group. Importantly, injection of exogenous rhPLTP provided the same level of protection against lethal sepsis as observed in WT animals with constitutive ly high PLTP expression (Fig.2a).

Significant reduction in the circulating concentrations of IL-6, MCP-1, and IL-Ιβ as well as a downward trend for TNF-a was observed at 24h after CLP in the mice receiving recombinant PLTP compared with the mice receiving the vehicle (Fig.5b). Thus, repeated injections of rhPLTP during the first days following CLP notably reduced the production of inflammatory cytokines, extended survival, and overall significantly contributed to fight against septic shock and its harmful consequences.

EXAMPLE 2

MATERIALS AND METHODS PLTP Activity measurements

PLTP activity was measured using a commercially available fluorescence activity assay (Roar Biomedical Inc., New York, NY) according to the manufacturer's instructions. Briefly, samples (5 μΐ), fluorescent-labelled donors (3 μΐ) and unlabeled acceptors (50 μΐ), were incubated at 37°C in a final volume of 100 μΐ of assay buffer in 96-well microplates. Changes in fluorescence were monitored using a Victor³ multilabel counter (PerkinElmer Life Sciences) with a 465 nm excitation and a 535 nm emission wavelength. This fluorimetric assay measures the transfer (unquenching) of fluorescent phospholipids from donor to acceptor synthetic liposomes. Phospholipid transfer activity was calculated from the delta of fluorescence between 1 and 20 minutes and expressed as the increase in fluorescence (arbitrary units of fluorescence) or as a percentage of WT mouse plasma activity used as a reference.

Histological Examination Liver, and kidney segments were fixed in 10% (v/v) phosphate -buffered formalin (pH 7.4) for 48 h and then embedded in paraffin. Five^m-thick sections were stained with hematoxylin and eosin (HE) and viewed with a digital slide scanner (NanoZoomer, Hamamatsu, Japan) at x400 magnification. All tissue sections were evaluated blindly by a pathologist.

In vitro evaluation of antimicrobial activity of purified recombinant proteins on gram (+) bacteria growth

S. aureus (ATCC 25923) growth was examined in vitro using a broth microdilution assay in nutrient Mueller Hinton Broth. S. aureus were plated on Tryptic soy agar overnight at 37 °C. An isolated bacterial colony was used to inoculate Mueller Hinton Broth, and the bacterial cultures were allowed to grow overnight at 37°C. 100 iL of culture was used to freshly inoculate 3 ml of MHB. The suspension was then allowed to grow at 37 °C with shaking at 225 rpm for ~2 h, where a final bacterial concentration of ~ 10⁸ colony forming units/mL (CFU/mL) was reached (OD600 -0.1). rhPLTP was obtained as described above. Recombinant protein was dialyzed in MHB and was added to a 96-well sterile microplate in order to get two-fold serial dilutions in MHB. Next, 50 μΐ of inoculum containing 10⁶ CFU/ml of S. aureus (ATCC 25923) was added to each well, so that each well contained 50 μΐ of rhPLTP solution and 50 μΐ of cell suspension. Then, the microplates were incubated at 37°C. MHB with and without bacteria were used as growth and sterility controls, respectively. Bacterial growth was determined 6h after incubation by measuring absorbance at 600 nm (OD600).

Statistical analysis

Data are presented as the mean ± sem. All data were analyzed using GraphPad Prism 7 software (GraphPad Software Inc.). The survival rates were compared using the χ² test as indicated. For pairwise comparison of experimental groups Mann-Whitney test was performed. Differences between multiple groups were analyzed by one way ANOVA with Tukey's multiple comparisons test for normally distributed data or by the Kruskal-Wallis non- parametric test followed by Dunn's multiple comparison test. For all statistical analyses, a P value of 0.05 was considered significant.

RESULTS

Intravenously injected rhPLTP diffuses to the (intra)peritoneal cavity and prevents tissue damages, in Pltp-I- mice with polymicrobial infection In Pltp-/- mice, we observed that repeated intravenous (iv) injections of rhPLTP after CLP resulted in incremental increases in plasma PLTP activity, even exceeding by 40% the activity measured in the plasma of WT mice 24 hours after CLP (Fig. 6a). Interestingly, the repeated iv injections of rhPLTP in Pltp-/- mice also had a significant impact (P = 0.016) on the PLTP activity of peritoneal fluid which reached 32 ± 5% of that detected in homologous mice plasma (Fig. 6b). Liver histology performed 24 hours after CLP on Pltp-/- mice showed more damage in livers from vehicle-injected animals, especially with substantial acidification of hepatocytes undergoing cell death, than in the livers of mice injected with rhPLTP. Kidney histology revealed a higher amount of injured glomeruli with congestion, hyper-cellularity, compression of capillary loops and reduction of Bowman's space in vehicle-injected animals than in rhPLTP injected mice.

These results showed that intravenously injected rhPLTP gains access to the site of polymicrobial infection from the bloodstream and prevents organ damage.

rhPLTP has no effect on gram (+) bacteria growth

The effect of rhPLTP on gram (+) bacteria (S. aureus) growth was examined using the broth microdilution assay. Six hours after the addition of recombinant PLTP to incubation mixtures, growth of S. aureus (ATCC25923) was not inhibited (Fig. 7).

Exogenous rhPLTP maintains its therapeutic properties when injected into WT mice with polymicrobial infection

In WT mice, and in agreement with observations in Pltp-/- mice (see above), repeated injections of rhPLTP after CLP resulted in increased plasma PLTP activity (visible after 24h, Fig. 8a), reduced mortality 24 hours after CLP (Fig. 8b) and a marked reduction in the circulating concentrations of IL-6, MCP-1, and IL-Ιβ, and in addition here with reductions in TNF-a and IL-10 (Fig. 8c). Again, recurrent administration of rhPLTP in mice diminished bacterial concentrations in the blood and peritoneal lavage 24h post CLP to levels 250 times lower than those in mice injected with vehicle (Fig. 8d-8e). Accordingly, the plasma 3HM level detected in the bloodstream of WT mice treated with rhPLTP was significantly lower (P=0.006) at 24h after CLP (Fig. 8f).

REFERENCES Throughout this application, various references describe the state of the art to which the invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. Bingle, CD. & Gorr, S.U. Host defense in oral and airway epithelia: chromosome 20 contributes a new protein family. IntJ Biochem Cell Biol 36, 2144-2152 (2004).

Cheung, M.C., Vaisar, T., Han, X., Heinecke, J.W. & Albers, J.J. Phospholipid transfer protein in human plasma associates with proteins linked to immunity and inflammation. Biochemistry 49, 7314-7322 (2010).

Dejager, L., Pinheiro, I., Dejonckheere, E. & Libert, C. Cecal ligation and puncture: the gold standard model for polymicrobial sepsis? Trends Microbiol 19, 198-208 (2011). Fink, M.P. & Warren, H.S. Strategies to improve drug development for sepsis. Nat Rev Drug Discov 13, 741-758 (2014)

Gautier, T. et al. Effect of plasma phospholipid transfer protein deficiency on lethal endotoxemia in mice. J Biol Chem 283, 18702-18710 (2008).

Gautier, T. & Lagrost, L. Plasma PLTP (phospholipid-transfer protein): an emerging role in 'reverse lipopolysaccharide transport' and innate immunity. Biochem Soc Trans 39, 984-988 (2011). Hailman, E., Albers, J.J., Wolfbauer, G., Tu, A.Y. & Wright, S.D. Neutralization and transfer of lipopolysaccharide by phospholipid transfer protein. J Biol Chem 271, 12172-12178 (1996).

Lagrost, L., Desrumaux, C, Masson, D., Deckert, V. & Gambert, P. Structure and function of the plasma phospholipid transfer protein. Curr Opin Lipidol 9, 203-209 (1998).

Lehmann, C. et al. Novel approaches to the development of anti-sepsis drugs. Expert Opin Drug Discov 9, 523-531 (2014) Levels, J.H. et al. Lipopolysaccharide is transferred from high-density to low-density lipoproteins by lipopolysaccharide -binding protein and phospholipid transfer protein. Infect Immun 73, 2321-2326 (2005)

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Claims

1. A plasma phospholipid transfer protein (PLTP) for use in the treatment of gram negative bacterial infection.

2. The PLTP for use according to claim 1, wherein said PLTP is a human recombinant PLTP.

3. The PLTP for use according to any one of claims 1 to 2, wherein PLTP is PLTP isoform a.

4. The PLTP for use according to any one of claims 1 to 3 wherein gram negative bacteria is selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella specie, E. corrodens, and Haemophilus influenza.

5. A plasma phospholipid transfer protein (PLTP) for use in the prevention of gram negative sepsis.

6. The PLTP for use according to claim 5, wherein said PLTP is a human recombinant PLTP.

7. The PLTP for use according to any one of claims 5 to 6, wherein PLTP is PLTP isoform a.

8. The PLTP for use according to any one of claims 5 to 7 wherein Gram- bacteria is selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella specie, E. corrodens, and Haemophilus influenza.

9. A plasma phospholipid transfer protein (PLTP) for use in the treatment of gram negative bacterial infection in a patient at risk of developing sepsis.

10. The PLTP for use according to claim 9, wherein said PLTP is a human recombinant PLTP.

11. The PLTP for use according to any one of claims 9 to 10, wherein PLTP is PLTP isoform a.

12. The PLTP for use according to any one of claims 9 to 11 wherein gram negative bacteria is selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella specie, E. corrodens, and Haemophilus influenza.

13. The PLTP for use according to any one of claims 9 to 12, wherein the patient at risk to develop a sepsis is selected from the group consisting of a patient before a medical or surgical intervention, very young and very old subject (infants and seniors), people with chronic or serious illnesses (including diabetes and cancer), and immunocompromised patients.

14. A phospholipid transfer protein (PLTP) for use as a bacteriostatic agent in a method of treatment, wherein the bacteria is a Gram negative bacteria.

15. The PLTP for use according to claim 14, wherein said PLTP is a human recombinant PLTP.