Combination for treating bacterial infection due to pseudomonas aeruginosa antibiotic-resistant strain

Abstract

The present invention relates to a combination of (i) a flagellin polypeptide and (ii) an antibiotic, for the simultaneous or sequential use in the treatment of bacterial infections due to Pseudomonas aeruginosa antibiotic-resistant strain. The present invention also provides a flagellin polypeptide, for use in a method for enhancing sensitivity to an antibiotic of a patient suffering from bacterial infection, due to Pseudomonas aeruginosa antibiotic-resistant strain. Indeed, using an in vivo mouse model, inventors show that while standalone prophylactic flagellin attenuates infection caused by a MDR strain of PA (PAMDR), its combination with an antibiotic such as gentamicin (GNT) leads to a strong decrease in bacterial load in the lung and a significant reduction in cell infiltration and inflammatory cytokines. Moreover, mice receiving flagellin in combination with GNT showed a 100% survival rate, a result that GNT alone was unable to provide against PAMDR.

Description

[0001] COMBINATION FOR TREATING BACTERIAL INFECTION DUE TO

[0002] PSEUDOMONAS AERUGINOSA ANTIBIOTIC-RESISTANT STRAIN

[0003] FIELD OF THE INVENTION:

[0004] The present invention relates to a combination of (i) a flagellin polypeptide and (ii) an antibiotic (e.g gentamicin), for the simultaneous or sequential use in the treatment of bacterial infections especially due to Pseudomonas aeruginosa antibiotic-resistant strain. The present invention also provides a flagellin polypeptide, for use in a method for enhancing sensitivity to an antibiotic of a patient suffering from bacterial infection, especially due to Pseudomonas aeruginosa antibiotic-resistant strain.

[0005] BACKGROUND OF THE INVENTION:

[0006] Since their discovery, antibiotics have revolutionized the medical treatment of patients with bacterial infections by saving numerous lives. However, antimicrobial resistance (AMR) has been observed at dangerously high levels worldwide (Spellberg et al. (2013) Engl. J. Med. 368:299-302) and alternative therapeutic strategies are urgently needed. For instance, resistance broad-spectrum cephalosporins, one of the major class of antibiotic used worldwide, has become a major public health issue (Rossolini et al. (2008) Clin. Microbiol. Infect. 14(suppl 1):33-41). Among this P-lactam family, the main resistance mechanism in enterobacteria involves the production of Extended-Spectrum P-lactamases (ESBLs) with CTX-M-15 being the most prevalent type in European countries (Bevan et al. (2017) J. Antimicrob. 72:2145- 2155). However, AMR mechanisms are diverse, ranging from antibiotic inactivation to preventing their action at the target site, thereby impacting chronic bacterial infections such as pulmonary infections.

[0007] Hence, there is an important need in identifying new solutions for efficiently targeting and fighting AMR.

[0008] The present invention meets this need.

[0009] Pseudomonas aeruginosa (PA) is one of the leading causes of hospital-acquired infections globally and the most common pathogen associated with respiratory chronic illnesses, such as cystic fibrosis (CF) (Reynolds and Kollef, 2021; Saleem et al., 2019; Biswas and Gotz, 2021). Overuse of antimicrobial agents during the past decades has resulted in the emergence of PA resistant strains, particularly to P-lactams, carbapenems, and aminoglycosides antibiotics (Pang et al., 2019). Multidrug-resistant (MDR) strains of PA are even harder to eradicate and cause higher mortality rates (Matos et al., 2018; Ikuta et al., 2022). As a consequence, the World Health Organization (WHO) has included MDR strains of PA in a list of pathogens requiring urgent attention for the development of new therapeutic strategies (Tacconelli etal., 2018).

[0010] Approaches that can enhance and rescue antibiotic efficacy are of significant interest and are increasingly being studied (Horcajada et al., 2019). In this context, host-directed molecules in combination with antibiotics are a promising strategy for preventing infections and restoring antibiotic effectiveness. Flagellin, a structural component of the flagellum of Gram-negative bacteria, has emerged as a promising host-directed immunomodulatory drug (Hajam et al., 2017). In particular, the recombinant form of flagellin (flagellinAi74-4oo), which lacks its antigenic part, has been described for its immuno-stimulatory capacity and antimicrobial properties against intestinal infections caused by Enterococcus faecium, Clostridium difficile, and Escherichia coli (Andersen-Nissen et al., 2007; Kinnebrew et al., 2010; Jarchum et al., 2011), and against respiratory infections caused by Streptococcus pneumoniae (Matarazzo et al., 2019).

[0011] In the airways, the mechanism of action of flagellinAi74-4oo (hereafter only designated as flagellin) is well-described. Flagellin binds to Toll-like receptor 5 (TLR5), a pattern recognition receptor (PRR) present in epithelial cells (ECs), alveolar macrophages (AM), conventional dendritic cells (eDCs), and lymphocytes (Yoon et al., 2012). This interaction strongly activates the transcription factor nuclear factor-kappa B (NFKB), Mitogen-activated protein kinases (MAPK), and interferon (IFN)-regulatory factor (IRF) pathways, resulting in the transcription of genes encoding several immune mediators (Vijayan et al., 2018). This includes chemokines such as CXCL1, CXCL2, CXCL5, CCL2 and CCL20, cytokines such as Interleukin-6 (IL-6), Granulocyte-macrophage colony-stimulating factor (GM-CSF), and Interleukin-1 beta (IL-1 ) as well as mucins and several antimicrobial peptides such as 0-defensins (Janot etal., 2009; Yu et al., 2010; Anas et al., 2016).

[0012] It has been demonstrated that flagellin triggers a short-lived response (peaking 2 hours after administration and decaying 2-3 hours after the peak) due to strong feedback regulatory mechanisms (Lopez-Galvez et al., 2021). This controlled response is crucial because it allows the inflammation to resolve towards homeostasis. Interestingly, the same study also shows that a second stimuli with flagellin (either with soluble protein or PA infection) leads to a less intense proinflammatory state, suggesting that a single prophylactic flagellin instillation can reduce the inflammatory response to PA (Lopez-Galvez et al., 2021). Previous studies have also shown that nasal administration of flagellin has a synergistic effect with the antibiotic amoxicillin in controlling antibiotic-resistant pneumococcal infections (Matarazzo et al., 2019). Other data have suggested that prophylactic nasal administration of flagellin can decrease the bacterial load of pigs infected with the P. aeruginosa (PAK) strain (Lopez-Galvez et al., 2021). However, flagellin has never been tested in combination with an antibiotic to fight PA and was never evaluated against a MDR strain of PA.

[0013] Here, using an in vivo mouse model, inventors show that while standalone prophylactic flagellin attenuates infection caused by a MDR strain of PA (PAMDR), its combination with the antibiotic gentamicin (GNT) leads to a strong decrease in bacterial load in the lung and a significant reduction in cell infiltration and inflammatory cytokines. Moreover, mice receiving flagellin in combination with GNT showed a 100% survival rate, a result that GNT alone was unable to provide against PAMDR.

[0014] SUMMARY OF THE INVENTION:

[0015] Therefore, the present invention relates to a combination of (i) a flagellin polypeptide and (ii) an antibiotic, for the simultaneous or sequential use in the treatment of bacterial infections due to Pseudomonas aeruginosa drug resistance strain:

[0016] DETAILED DESCRIPTION OF THE INVENTION:

[0017] In the present invention, inventors evaluated the prophylactic intranasal administration of flagellin against a multi drug-resistant strain of PA (PAMDR) in mice and assessed the possible synergy with the antibiotic gentamicin (GNT). The results indicated that flagellin treatment before infection decreased bacterial load in the lungs, likely due to an increase in neutrophil recruitment, and reduced signs of inflammation, such as tissue redness and proinflammatory cytokines. The combination of flagellin and antibiotic such as gentamicin showed a synergistic effect, decreasing bacterial load and increasing mice survival by 100%. These findings suggest that preventive nasal administration of flagellin could restore the effect of GNT against MDR strains of PA, paving the way for the use of flagellin in vulnerable patients with chronic respiratory diseases.

[0018] Altogether, these results provide new insights for treating Pseudomonas aeruginosa- related bacterial infection, especially resistant and multi -resistant Pseudomonas aeruginosa responsible of respiratory infection, using flagellin polypeptide of the invention as main active principle ingredient in combination with antibiotic agent. Combination and uses of the invention

[0019] A first aspect of the invention relates to a combination of (i) a flagellin polypeptide according to the invention, and (ii) an antibiotic, for the simultaneous or sequential use in the treatment of bacterial infections due to Pseudomonas aeruginosa antibiotic-resistant strain.

[0020] In particular, embodiment, the flagellin polypeptide is administered by intranasal route.

[0021] The term "Pseudomonas bacteria" is commonly understood in the field to encompass bacteria that typically inhabit or cause infections primarily in the lungs of humans and other animals. Additionally, they may affect other areas such as the eyes, ears, urinary tract, among others. The term "Pseudomonas bacteria" encompasses a variety of Gram-negative bacterial species, including but not limited to Pseudomonas aeruginosa and others such as P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, and P. straminea within the Pseudomonas aeruginosa group.

[0022] In particular, the Pseudomonas according to the invention is Pseudomonas aeruginosa.

[0023] Pseudomonas aeruginosa is a common Gram-negative bacteria that can cause disease in animals, including humans. It is citrate, catalase, and oxidase positive. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized many natural and artificial environments. It uses a wide range of organic material for food; in animals, its versatility enables the organism to infect damaged tissues or those with reduced immunity. The symptoms of such infections are generalized inflammation and sepsis. If such colonization occurs in critical body organs, such as the lungs, the urinary tract, and kidneys, the results can be fatal (Balcht, et al., Informa Health Care, 1994). Because it thrives on moist surfaces, this bacterium is also found on and in medical equipment, including catheters, causing crossinfections in hospitals and clinics.

[0024] The present invention aims in particular at fighting antimicrobial resistance, in particular antibiotic resistance.

[0025] Accordingly, in a particular embodiment bacterial infection is due to antibiotic-multi resistant bacteria.

[0026] In another particular embodiment bacterial infection is a lung bacterial infection due to antibiotic-multi resistant bacteria.

[0027] By “antimicrobial resistance” or “AMR” is meant herein the phenomenon that a microorganism does not exhibit decreased viability or inhibited growth or reproduction when exposed to concentrations of the antimicrobial agent that can be attained with normal therapeutic dosage regimes in patients. It implies that an infection caused by this microorganism cannot be successfully treated with this antimicrobial agent.

[0028] As used herein, the terms "antibiotic" and "antimicrobial compound" are used interchangeably and refer to a compound which decreases the viability of a microorganism, or which inhibits the growth or reproduction of a microorganism. The term “antibiotic agent” has its general meaning in the art and refers to antibacterial agent, such as described in US2013 / 0029981.

[0029] Suitable main class of antibiotic agents include, without limitation:

[0030] 1. P-lactam antibiotics (beta-lactam antibiotic) are the antibiotic agents that contain a beta-lactam ring in their molecular structure and containing a beta-lactam functionality. Those P-lactam antibiotics include penicillin and derivatives (penams), cephalosporins (cephems), monobactams, carbapenems and carbacephems. Most P-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism and are the most widely used group of antibiotics (in 2003 more than half of all commercially available antibiotics in use were P- lactam compounds)

[0031] By “cephalosporins” (cephems) is meant herein a subgroup of P-lactam antibiotics originally derived from the fungus Acremonium. Together with cephamycins, they constitute a subgroup of P-lactam antibiotics called cephems. Cephalosporins include ceftazidime.

[0032] By “monobactam” is meant herein a subgroup of P-lactam antibiotics, which are monocyclic and wherein the P-lactam ring is not fused to another ring. Monobactam include aztreonam.

[0033] By “carbapenems” is meant herein a subgroup of P-lactam antibiotics, which have a bactericide effect by binding to penicillin-binding proteins (CBPs) thus inhibiting bacterial cell wall synthesis This class of antibiotics is usually reserved for known or suspected multidrugresistant (MDR) bacterial infections. Carbapenem include imipenem.

[0034] By “penicillin” and “penicylin derivatives” (penams) is meant herein a subgroup of P- lactam antibiotics, derived originally from common moulds known as Penicillium moulds; which includes penicillin G (intravenous use), penicillin V (use by mouth), procaine penicillin, and benzathine penicillin (intramuscular use). Penicillin antibiotics were among the first medications to be effective against many bacterial infections caused by staphylococci and streptococci. They are still widely used today, though many types of bacteria have developed resistance following extensive use. There are several enhanced penicillin families which are effective against additional bacteria; these include the anti staphylococcal penicillins, aminopenicillins and the antipseudomonal penicillins. They are derived from Penicillium fungi Example of Natural penicillin :Penicillin G, Penicillin K, Penicillin N, Penicillin O, Penicillin V.

[0035] Example of P-lactamase-resistant penicylin derivatives: Methicillin, Nafcillin, Oxacillin, Cioxacillin, Dicloxacillin, Flucloxacillin.

[0036] Example of Aminopenicillins: Ampicillin, Amoxicillin, Pivampicillin, Hetacillin, Bacampicillin, Metampicillin, Talampicillin, Epicillin.

[0037] Example of Carboxypenicillins: Carbenicillin, Ticarcillin, Temocillin.

[0038] Example of Ureidopenicillins: Mezlocillin, Piperacillin, Azlocillin.

[0039] Example of P-lactamase inhibitors penicylin derivatives: Clavulanic acid, Sulbactam, Tazobactam.

[0040] 2. Aminoglycoside are the antibiotic agents directed to Gram negative bacteria that inhibit protein synthesis (targeting the small ribosome sub-unit of (30 Svedberg)) and contain as a portion of the molecule an amino-modified glycoside (Mingeot-Leclercq MP, et al (1999). Antimicrob. Agents Chemother. 43 (4): 727-37). The term “Aminoglycoside” can also refer more generally to any organic molecule that contains amino sugar substructures. Aminoglycoside antibiotics display bactericidal activity against Gram-negative aerobes and some anaerobic bacilli where resistance has not yet arisen but generally not against Grampositive and anaerobic Gram-negative bacteria.

[0041] Streptomycin is the first-in-class aminoglycoside antibiotic. It is derived from Streptomyces griseus and is the earliest modem agent used against tuberculosis. Streptomycin lacks the common 2-deoxystreptamine moiety present in most other members of this class. Other examples of aminoglycosides include the deoxystreptamine-containing agents, kanamycin, tobramycin, gentamicin, and neomycin.

[0042] 3. Antibiotic agents which inhibit acid nucleic synthesis

[0043] Antibiotic agents which block DNA gyrase (topoisomerase specific to bacteria) : aminocoumarines, and quinolones.

[0044] Antibiotic agents which block the bacterial RNA polymerase: rifampicine.

[0045] 4. Antibiotics which inhibit protein synthesis (other than Aminoglycoside)

[0046] Antibiotic agents which block the formation of the peptide bond: amphenicols (examples: chloramphenicol, thiamphenicol azidamfenicol and florfenicol)

[0047] Antibiotic agents which block elongation of the polypeptide chain: Tetracyclins (examples: tetracycline, doxycycline, aureomycine, eravacycline, sarecycline ,omadacy cline) macrolides (examples : erythromycin, azithromycin) and ketolides (examples : telithromycin, cethromycin and solithromycin). 4. Antibiotics which inhibit folate metabolism

[0048] Sulfonamides also called sulphonamides, sulfa drugs or sulpha drugs (examples: Sulfamethoxazole) and sulfanilamides.

[0049] 5. New classes of antibiotics compounds

[0050] Four new classes of antibiotics have been brought into clinical use in the late 2000s and early 2010s: cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), and lipiarmycins (such as fidaxomicin)

[0051] In a particular embodiment, the combination according to the invention, and pharmaceutical compositions of the invention aims at fighting bacterial resistance against Aminoglycoside (i.e- gentamicin).

[0052] In a particular embodiment, the combination according to the invention, and pharmaceutical compositions of the invention aims at fighting multi-resistance bacterial infection especially bacterial infections due to Pseudomonas aeruginosa antibiotic-resistant strain.

[0053] Bacteria are said to be multidrug-resistant (MDR) to antibiotics when, due to the accumulation of acquired resistance to several families of antibiotics, they are only sensitive to a small number of antibiotics usable in therapy (resistance to more than 3 different families).

[0054] As used herein, the term "flagellin" has its general meaning in the art and refers to the flagellin contained in a variety of Gram-positive or Gram-negative bacterial species. Nonlimiting sources of flagellins include but are not limited to Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella enterica serovar Typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces . These examples are illustrative rather than limiting. The amino acid sequences and nucleotide sequences of flagellins are publically available in the NCBI Genbank, see for example Accession Nos. AAL20871, NP_310689, BAB58984, AAO85383, AAA27090, NP_461698, AAK58560, YP_001217666, YP_002151351, YP_001250079, AAA99807, CAL35450, AAN74969, and BAC44986. The flagellin sequences from these and other species are intended to be encompassed by the term flagellin as used herein. Therefore, the sequence differences between species are included within the meaning of the term.

[0055] The term "flagellin polypeptide" is intended to a flagellin or a fragment thereof that retains the ability to bind and activate TLR5. As used herein the term "toll-like receptor 5" or "TLR5" has its general meaning in the art and is intended to mean a toll-like receptor 5 of any species, but preferably a human toll-like receptor 5. Upon activation, a TLR5 induces a cellular response by transducing an intracellular signal that is propagated through a series of signaling molecules from the cell surface to the nucleus. Typically, the intracellular domain of TLR5 recruits the adaptor protein, MyD88, which recruits the serine / threonine kinases IRAK (IRAK- 1 and IRAK-4). IRAKs form a complex with TRAF6, which then interacts with various molecules that participate in transducing the TLR signal. These molecules and other TLR5 signal transduction pathway components stimulate the activity of transcription factors, such as fos, jun and NF-kB, and the corresponding induction of gene products of fos-, jun- and NF-kB- regulated genes, such as, for example, IL-6, TNF-alpha, CXCL1, CXCL2 and CCL20. Typically, the flagellin polypeptide of the present invention comprises the domains of flagellin involved in TLR5 signaling. The term “domain of flagellin” includes naturally occurring domain of flagellin and function conservative variants thereof. "Function conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence identity between any two proteins of similar function may vary and may be, for example, from 70 % to 99 %. Thus, a "function-conservative variant" also includes a polypeptide that has at least 70 % amino acid identity with the native sequence of flagellin or fragment thereof. According to the invention, a first amino acid sequence having at least 70% of identity with a second amino acid sequence, means that the first sequence has 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; or 99, or 100% of identity with the second amino acid sequence. In the same manner, a first amino acid sequence having at least 90% of identity with a second amino acid sequence, means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99, or 100% of identity with the second amino acid sequence. Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990). The domains of flagellin that are involved in TLR5 signaling are well known in the art, see for example Smith et al. (2003) Nat. Immunol. 4: 1247-1253 (e.g., amino acids 78-129, 135-173 and 394-444 of 5. typhimurium flagellin or homologs or modified forms thereof).

[0056] Examples of flagellin polypeptides include but are not limited to those described in U.S. Pat. Nos. 6,585,980; 6,130,082; 5,888,810; 5,618,533; and 4,886,748; U.S. Patent Publication No. US 2003 / 0044429 Al; and in the International Patent Application Publications n°W0 2008097016 and WO 2009156405 which are incorporated by reference. An exemplary E. coli O157:H7 flagellin is SEQD ID NO:1. An exemplary S. typhimurium flagellin is SEQ ID NO:2 or SEQ ID NO:3

[0057] Polypeptide numbering starts at the first amino acid after the eventual N-terminal methionine (not shown in SEQ ID N°3), which is typically excised by methionine aminopeptidase in bacteria host cells as under-mentioned.

[0058] In some embodiments, amino acid sequences having at least 70% of identity with SEQ ID NO: 1 SEQ ID NO:2 or SEQ ID NO:3 can be used as flagellin polypeptides according to the invention. In some embodiments, amino acid sequences having at least 90% of identity with SEQ ID NO: 1 SEQ ID NO:2 or SEQ ID NO:3 can be used as flagellin polypeptides according to the invention. In some embodiments, amino acid sequences having at least 70% of identity with SEQ ID NO:3 can be used as flagellin polypeptides according to the invention provided that the residues 89-96 (i.e. the residues that are involved in TLR5 detection) are not mutated (i.e. not substituted or not deleted). In some embodiments, amino acid sequences having at least 90% of identity with SEQ ID NO: 1 SEQ ID NO:2 or SEQ ID NO:3 can be used as flagellin polypeptides according to the invention provided that the residues 89-96 (i.e. the residues that are involved in TLR5 detection) are not mutated (i.e. not substituted or not deleted).

[0059] In some embodiments, the present encompasses use of the flagellin recombinant polypeptides described in the International Patent Applications n° WO 2009156405, and n° WO 2016 / 102536 which are incorporated by reference in its entirely.

[0060] In some embodiments, the flagellin polypeptide of the present invention comprises: a) a N-terminal peptide having at least 90% amino acid identity with the amino acid sequence starting from the amino acid residue located at position 1 of SEQ ID NO: 3 and ending at an amino acid residue selected from the group consisting of any one of the amino acid residues located at positions 99 to 173 of SEQ ID NO:3 ; and b) a C-terminal peptide having at least 90% amino acid identity with the amino acid sequence starting at an amino acid residue selected from the group consisting of any one of the amino acid residues located at positions 401 to 406 of SEQ ID NO:3 and ending at the amino acid residue located at position 494 of SEQ ID NO:3 , wherein : the said N-terminal peptide is directly linked to the said C-terminal peptide, or the said N-terminal peptide and the said C-terminal peptide are indirectly linked, one to the other, through a spacer chain.

[0061] In some embodiments, said N-terminal peptide is selected from the group consisting of the amino acid sequences 1-99, 1-137, 1-160 and 1-173 of SEQ ID NO:3. In some embodiments, said C-terminal peptide is selected from the group consisting of the amino acid sequences 401-494 and 406-494 of SEQ ID NO:3.

[0062] In some embodiments, said N- terminal and C-terminal peptides consist of the amino acid sequences 1-173 and 401-494 of SEQ ID NO:3, respectively.

[0063] In some embodiments, said N- terminal and C-terminal peptides consist of the amino acid sequences 1-160 and 406-494 of SEQ ID NO:3 , respectively.

[0064] In some embodiments, said N- terminal and C-terminal peptides consist of the amino acid sequences 1-137 and 406-494 of SEQ ID NO:3 , respectively.

[0065] In some embodiments, said N-terminal peptide and the said C-terminal peptide are indirectly linked, one to the other, through an intermediate spacer chain consisting of a NH2- Gly-AIa-AIa-GIy-COOH (SEQ ID NO:4) peptide sequence.

[0066] In some embodiments, the asparagine amino acid residue located at position 488 of SEQ ID NO: 3 is replaced by a serine.

[0067] In some embodiments, the flagellin polypeptide as above described comprises an additional methionine residue at the N-terminal end (regarding flagellin polypeptide of SEQ ID N°3).

[0068] In some embodiments, the flagellin polypeptide as above described comprises one additional methionine residue (M) and one additional lysin residue (L) at the N-terminal end (amino acid residues ML) (regarding flagellin polypeptide of SEQ ID N°3).

[0069] Accordingly, an example of the flagellin polypeptide corresponding to a modified recombinant flagellin (FLAMOD : see SEQ ID N°5), wherein N- terminal and C-terminal peptides consist of the amino acid sequences 1-173 and 401-494 of SEQ ID NO:3, said N- terminal peptide and the said C-terminal peptide are indirectly linked, one to the other, through an intermediate spacer chain consisting of a NH2-GIy-AIa-AIa-GIy-COOH (SEQ ID NO:4) peptide sequence and wherein said polypeptide comprises one additional methionine residue (M) and one additional lysine residue (L) at the N-terminal end.

[0070] In one embodiment, the flagellin polypeptide is the recombinant polypeptide having the amino-acid sequence of SEQ ID N°5.

[0071] The flagellin polypeptide of the present invention is produced by any method well known in the art. In some embodiments, the flagellin polypeptide of the present invention is typically recombinantly produced by recombinant cells that have been transfected with a nucleic acid that encodes its amino acid sequence and allows its effective production within the transfected cells. The nucleic acid sequence encoding the flagellin polypeptide of the invention, may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence if the sequence is to be secreted, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques that are known to the skilled artisan. Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the nucleic acid encoding the flagellin polypeptide of the invention such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity. Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding the flagellin polypeptide to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the betalactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S. D.) sequence operably linked to the DNA encoding the flagellin polypeptide of the invention. Host cells are transfected or transformed with expression or cloning vectors described herein for flagellin polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH, and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991). Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Grampositive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31 ,446); E. coli XI 776 (ATCC 31 ,537); E. coli strain W3110 (ATCC 27,325); and K5772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella enterica serovar Typhimurium, Serratia, e.g., Serratia marcescens, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces . These examples are illustrative rather than limiting. Strain SIN41 of Salmonella Typhimurium (fliC fljB), is particularly interesting for the production of flagellin polypeptides of the invention, since these prokaryotic host cells do not secrete any flagellins (Proc Natl Acad Sci U S A. 2001 ;98: 13722- 7). However, flagellins are secreted through specialized secretion system: the so-called "Type III secretion system". Interestingly, strain SIN41 produces all components of the type III secretion system required for optimal flagellin secretion. Cloning sequence coding new flagellin peptides under fliC promoter enables secretion in large amounts of the flagellin polypeptides of interest in strain SIN41. Strain W3110 is also interesting because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1 A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan.sup.r; E. coli W31 10 strain 37D6, which has the complete genotype tona ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r; E. coli W31 10 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990. The E. coli strains MG1655, MG1655 AfimA-H or MKS12, a fliD- and -f / m>A- / - / -deleted MG1655 strain are also interesting candidates for production of recombinant flagellins as secreted proteins (Nat Biotechnol. 2005; (4):475-81). Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable. Flagellin polypeptide of the invention may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., TRITON- XTM. 100) or by enzymatic cleavage. In some embodiments, the flagellin polypeptide is purified from the supernatant of recombinant S. Typhimurium SIN41 (fliC fljB), as disclosed in Nempont et al. (Nempont, C. C , D ; Rumbo, M ; Bompard, C ; Villeret, V ; Sirard, J.C. 2008 Deletion of flagellin's hypervariable region abrogates antibody-mediated neutralization and systemic activation of TLR5 -dependent immunity. J Immunol 181 :2036-2043.). In particular, Salmonella were grown in Luria-Bertani (LB) broth for 6-18 hours at 37°C with agitation. The supernatant was filtered and saturated with 60% ammonium sulfate (Sigma Aldrich, USA). The precipitated materials were recovered by centrifugation, solubilization in 20 mM Tris / HCI pH7.5 and then dialysis. The proteins were further purified by successive rounds of hydroxyapatite, anion exchange, and size exlusion chromatography (Bio-Rad Laboratories, USA; GE Healthcare, Sweden). Lastly, the proteins were depleted of lipopolysaccharide (LPS) using a polymyxin B column (Pierce, USA). Using the Limulus assay (Associates of Cape Cod Inc., USA), the residual LPS concentration was determined to be less than 30 pg LPS per pg recombinant flagellin. Constructs encoding the flagellins may be generated by PCR and cloned into the expression vector pET22b+. The plasmids can be introduced in Escherichia coli BL21(DE3) and protein production can be induced by adding IPTG ImM. After disruption on French press, the soluble fraction was depleted of lipopolysaccharide (LPS) using Triton X-l 14 extraction. If flagellins are found in the insoluble fraction after the French-press, inclusion bodies are denatured in presence of Urea 8M followed by dialysis and Triton X-l 14 extraction. The proteins can then be purified on anion exchange chromatography and gel filtration. Finally, proteins can be again depleted of LPS using a polymyxin B column (Pierce, USA).

[0072] As used herein, the terms "combination" refers to a "kit-of-parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e. simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners to be administered in the combined preparation can vary. The combination partners can be administered by the same route or by different routes. When the administration is sequential, the first partner may be for instance administered 1, 2, 3, 4, 5, 6, 7, days before the second partner.

[0073] In particular embodiment, the flagellin polypeptide is administered between 24 and 48 hours before antibiotic agent administration.

[0074] In particular embodiment, the flagellin polypeptide is administered by intranasal route. The present invention also provides the flagellin polypeptide according to the invention, for use in a method for enhancing sensitivity to an antibiotic (such as gentamicin) of a patient suffering from antibiotic-resistant bacterial infections especially infections due to a Pseudomonas aeruginosa antibiotic-resistant strain.

[0075] Pharmaceutical compositions according to the invention

[0076] The present invention also provides a pharmaceutical composition comprising: i. a flagellin polypeptide (as defined here above), ii. an antiobiotic agent (as defined here above); and iii. a pharmaceutically acceptable carrier. for use in the prevention or the treatment of antibiotic-resistant bacterial infection due to Pseudomonas aeruginosa antibiotic-resistant strain in a patient in need thereof.

[0077] Pharmaceutical compositions formulated in a manner suitable for administration to humans are known to the skilled in the art. The pharmaceutical composition of the invention may further comprise stabilizers, buffers, etc.

[0078] The compositions of the present invention may, for example, be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for administration by injection (i.e. antibiotic such as gentamycin) and / or by intranasal route (flagellin polypeptide).

[0079] The choice of the formulation ultimately depends on the intended way of administration, such as e.g. an intravenous, intraperitoneal, subcutaneous or oral way of administration, or a local administration (intranasal).

[0080] The pharmaceutical composition according to the invention may be a solution or suspension, e.g. an intravenous / intraperitoneal / intramuscular solution or suspension (ie for antibiotic administration) and an intranasal solution or suspension (i.e. for flagellin polypeptide administration). It may for example be packaged in dosage unit form.

[0081] In a preferred embodiment, the flagellin polypetide of the invention is preferably administered by intranasal route and the antibiotic agent intravenous / intraperitoneal / intramuscular route.

[0082] 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.

[0083] In another aspect, the present invention relates to the pharmaceutical composition of the invention as defined above optionally in combination with at least one antibiotic for use in a method of treating or preventing a lung disease in a subject, wherein the composition is administered to the subject by inhalation or by intranasal route.

[0084] In one embodiment, the lung disease is a lung infectious disease (i.e. lung bacterial infection).

[0085] The term “lung infectious disease” (also referred to as “pulmonary infectious disease” herein) refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent (e.g. common cold) that affect the lungs of a subject. Examples of bacterial infectious diseases, such as Legionnaire's disease (Legionella), tuberculosis, infections by E. coll, Staphylococci, , Pseudomonas, Streptococci Hemophilus influenzae, Klebsiella pneumoniae, ....

[0086] In the present invention the lung infectious disease is a lung infection due to Pseudomonas aeruginosa antibiotic-resistant strain

[0087] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0088] FIGURES:

[0089] Figure 1: C57B1 / 6 mice were divided in groups according to treatment regimen (A). Tweenty-four hours before infection mice in group “Untreated” and “GNT” received NaPi buffer and mice in groups “Flagellin” and “Flagellin+GNT” received FliC (10 pg), resuspended in NaPi buffer, by the nasal route. At TO all animals (with the exception of controls in “Healthy” group) were intranasally infected with 5x105 CFU of a MDR strain of P. aeruginosa. One hour after infection, mice in group GNT and Flagellin+GNT received GNT 15mg / Kg by intraperitoneal injection. Healthy mice were used as control (group “Healthy”). All mice were sacrificed 16h p.i.. Colony forming units (CFU) / lung lobe were counted in all infected mice (B). Each symbol represents an individual animal in 2 independent experiments. Differences between groups were analysed with Mann- Whitney test. Data are reported as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ,****p < 0,0001..

[0090] Figure 2:. Tweenty-four hours before infection, C57B1 / 6 mice received FliC (lOpg) (red and orange groups) resuspended in NaPi buffer, or NaPi buffer (grey, purple and green groups) by the nasal route. At TO all animals (with the exception of controls in healthy group in grey), were intranasally infected with 5xlO5CFU of a MDR strain of P. aeruginosa. One hour after infection mice received GNT 15mg / Kg by intraperitoneal injection (green and orange groups) or PBS (grey, purple and red groups). All mice were sacrificed 16h p.i.. BAL fluids were collected to determine the number and activation of immune and inflammatory cells by flow cytometry (A) and the levels of GM-CSF, TNF-a, IL-1 and IL-6 (B). All data are represented as the mean ± SEM and are cumulative of 2 independent experiments. Statistical analysis was performed using the Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0091] Figure 3: Tweenty-four hours before infection, C57B1 / 6 mice received flagellin (lOpg) (red and orange groups) resuspended in NaPi buffer, or only NaPi buffer (purple and green groups) by the nasal route. At TO all animals were intranasally infected with 8x105 CFU of a MDR strain of P. aeruginosa. One hour after infection mice received GNT 15mg / kg by intraperitoneal injection (green and orange groups) or PBS (purple and red groups). Animal survival were monitored daily. All data are represented are cumulative of 2 independent experiments. Statistical analysis was performed using the Log-rank (Mantel-Cox) test. *p < 0.05, **p < 0.01, ***p < 0.001, ,****p < 0.0001.

[0092] EXAMPLE:

[0093] Material & Methods

[0094] 1.1 Microbial strain and culture conditions

[0095] Pseudomonas aeruginosa strain was isolated from a clinical patient and kindly provided by Dr. Katy Jeannot (CHRU Besanpon, France). This strain is multidrug resistant (denominated in this study as PAMDR) and its characterization (by microbiology service of CHRU Bretonneau (Tours, France)) is in Supplementary Table 1 and Supplementary Figure 1.

[0096] 1.2 Flagellin

[0097] The custom-designed flagellin primarily used in the present study (recombinant flagellin FliCA174-400) derives from Salmonella enterica serovar Typhimurium FliC (GenBank accession no. AAL20871) as described previously (Didierlaurent et al., 2008; Nempont et al., 2008) . The recombinant flagellin was resuspended in NaPi buffer (10 mM phosphate buffer, pH 6.5, 145 mM NaCl and Tween 80 0.02 % w / v), for intranasal administration.

[0098] 1.3 Susceptibility testing

[0099] Broth microdilution method was used for the determination of minimal inhibitory (MIC) of gentamicin against PAMDR. Minimal inhibitory concentrations were determined in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CSLI). Briefly, PAMDR strain was cultured on TSA plates overnight. Three isolated colonies were suspended in 3 mL of LB medium and grown overnight at 37°C, under agitation (200 rpm). Then several dilutions of this fresh suspension were prepared and incubated at 37°C for 4 hours, under agitation (200 rpm). The suspension with OD600 between 0.3 and 0.6, representing the exponential phase of growth, was centrifuged 10 minutes at 3000 g. Bacteria were suspended in MH medium to obtain approximately 2x105 CFU / mL (CFU for colony forming unit). The inoculum size was verified by plating 5-fold dilutions on TSA plates and incubating overnight at 37°C for CFU counts. Hundred microliters / well of the bacterial suspension were inoculated into 96-well microtiter plate and 100 pL / well of MH (control) or gentamicin were added in duplicate for each condition. The microtiter plate was incubated in a plate reader (TECAN Infinite 200, Lyon, France) for 24 h at 37°C. The absorbance at OD600 was read at 30-min intervals.

[0100] 1.4 Animal care and handling

[0101] C57B1 / 6 female mice were purchased from the Centre d'Elevage R. Janvier (Le Genest Saint-Isle, France) and were used at about 8 weeks of age. All procedures were in accordance with the European animal welfare regulations. All mice were housed under specific-pathogen- free conditions at the PST “Animal eries” animal facility (Universite de Tours, France) and had access to food and water ad libitum. All experiments complied with the French government’s ethical and animal experiment regulations (APAFIS#201604071220401.V2-4885 and 2016111512369894 V3 - 7590).

[0102] At the day of experiment, mice were treated with 10 pg of recombinant flagellin (FlagellinA 174-400) 24h before infection with 5x105 or 8x105 CFU / mouse of PAMDR by the nasal route. One-hour post-infection, mice were treated by intraperitoneal injection (i.p.) with 15 mg / kg of gentamicin purchased from Sigma Aldrich (Sant-Quentin Fallavier, France). The choice of gentamicin dose was based on previous experiments in vivo showing that a single dose of 15 mg / kg i.p. was the minimum able to decrease PAMDR bacterial load (in mice infected with 105 CFU / mouse). Sixteen hours after challenge with PAMDR, mice were sacrificed and airways were washed four times with 0.5 mL of Phosphate Buffered Saline (PBS) for bronchioalveolar lavage (BAL) collection. After centrifugation at 400g for 5 min, BAL fluids were stored at -80°C for subsequent measurement of inflammatory mediators and pellets were recovered in PBS 2% Fetal Bovine Serum (FBS). The pellet was resuspended and filtered, and erythrocytes were discarded using a red blood cell lysis buffer. Leukocytes were counted and analyzed by flow cytometry. After BAL recovery, lungs were perfused with 10 mL PBS injected into the heart. The lungs were photographed for inflammation assessment based on lung morphology and color. The right lungs were homogenized with 1 mL of PBS using the Gentle MACSTM Octo Dissociator (Miltenyi Biotech) and 100 pL of lung suspension was plated for bacterial count. 1.5 Analysis of synergy between flagellin and gentamicin

[0103] The effect of treatments (standalone Flagellin, standalone GNT or combination Flagellin+GNT) against PAMDR was quantified as the percentage of bacterial growth (%growth), corresponding to the ratio of the mean bacterial load in the lungs of infected and treated mice to that of infected and untreated mice (control). For example, the effect of treatment A was calculated as follows: % growth [A] = (mean CFU[A] / mean CFU[control]) x 100. The predicted additive effect (or predicted % growth) of a combination treatment was calculated as described previously (Planer et al., 2014). Briefly, the predicted % growth of a treatment combining compounds A and B is the product of the experimentally defined %growth values for each standalone treatment (predicted % growth [A+B] = % growth [A] x % growth [B]). If the experimental % growth for the combination treatment is lower or higher than the predicted %growth, then the two drugs are synergistic or antagonistic, respectively. When the experimental and predicted % growth values are identical, the two drugs’ effects are additive.

[0104] 1.6 Flow cytometry analysis

[0105] Bronchioalveolar lavage from all mice was dispensed into round bottomed 96-well plates and were centrifuged at 500 g at 4°C for 5 min. Samples were further stained using specific antibodies and appropriate isotype controls. To identify total leukocytes, neutrophils, total dendritic cells and alveolar macrophages, cells were stained with a lineage cocktail containing anti-CD45 (APCVio770 -XX), anti-CDl lb (PE - clone MI / 70, BD cat.no. 553311), anti-CDl lc (PEVio770 - (clone N418, Bio cat.no. 117318), anti-Ly6G (APC - XX), anti- SiglecF (VioBlue - XX), anti-MHCII (PerCP - XX), and LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, cat.no. L34966). Flow cytometry data were acquired on a MACSQuant® Analyzer and analyses were performed using the VenturiOne software according to the gating strategy described in Supplementary Figure 2.

[0106] 1.7 ELISA

[0107] BAL were centrifuged 5 min at 500 g and supernatants were stored at -80°C. DuoSet ELISA (Mouse IL-6, IL-ip, TNF-a and GM-CSF) were performed according to the manufacturer’s (R&D Systems) instructions.

[0108] 1.8 Stati sti cal analy si s

[0109] Sample size estimation was based on previous animal studies conducted by our group using P. aeruginosa-infection mice model. There was no randomization or blinding procedure. Statistical analyses were performed using GraphPad Prism. Data are reported as mean ± SEM, with n = the number of individual animals. Statistical values, including the number of replicates (nr) and the statistical test used, can be found in the figure legends. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001..

[0110] Results

[0111] The combination of preventive flagellin and the antibiotic gentamicin displays synergistic therapeutic activity against a MDR strain of P. aeruginosa

[0112] Short intro on the MDR strain of PA (PAMDR) and refer to Supplementary Figure 1?

[0113] To evaluate the impact of preventive intranasal flagellin in combination with the antibiotic gentamicin against PAMDR, we compared mice that received both prophylactic flagellin (24 hours before infection) and GNT 1-hour post-infection (“Flagellin+GNT group”) with groups of mice that received either standalone prophylactic flagellin (“Flagellin group”), or mice only treated with GNT (“GNT group”), or infected non-treated mice (“Untreated group”). We also considered animals that were neither treated nor infected as healthy (“Healthy group”) (see Figure 1A). We assessed bacterial load and inflammatory status in the BAL fluids of these mice 16 hours post-infection (i.e. 40 hours after flagellin administration).

[0114] As expected, infected untreated mice presented a high bacterial load in the lungs 16 hours after PAMDRchallenge (4.5xl06CFU ± 1.7xl06) (see Figure IB). Although standalone GNT was not able to significantly decrease the bacterial load of infected mice, prophylactic administration of flagellin significantly reduced PAMDRCFU (p<0.05). Interestingly, when mice received the combination of prophylactic flagellin and GNT 1-hour post-infection, they exhibited much lower bacterial CFU (a 400-fold reduction compared to infected non-treated mice; p<0.001). This result suggests a significant therapeutic advantage for the combination treatment compared to standalone flagellin or GNT treatments.

[0115] When two drugs are administered in combination, their effect may be simply additive, or they may work in synergy to improve the host's condition. Hence, the interaction between two drugs is classified as synergistic when the total effect of the two drugs combined is greater than that predicted by their individual potencies. Using the equation described in the materials and methods, the expected percentage of bacterial growth when combining flagellin and GNT should be 9.4% (55% growth for GNT x 17% growth for flagellin = 9.4%). However, Flagellin+GNT combination therapy led to only 0.2% of PAMDRgrowth compared to the control (infected non-treated mice). Since the real percentage of growth is lower than the theoretical predicted value, we can conclude that there is a synergistic effect between the two drugs.

[0116] Preventive intranasal administration of flagellin decreases proinflammatory cytokines and cell infiltration but increases neutrophils and dendritic cells in the BAL of PAMDR-infected mice Using the same experimental design as described above, we examined the inflammatory status of mice. We analyzed the appearance of their lungs (data not shown), immune cell populations (Figure 2A), and the secretion of proinflammatory cytokines in BAL (Figure 2B), at 16 hours post-infection, for all experimental groups.

[0117] The lungs appaerence shows the difference between representative lungs of a mouse from each group. As anticipated, mice infected with PAMDRbut not treated with either flagellin or antibiotic, exhibited severe redness compared with healthy lungs, suggesting local inflammation. Mice that only received GNT 1-hour post-infection experienced similar lung redness. On the other hand, animals pre-treated with flagellin before infection exhibited a clear reduction in redness, which was even more pronounced in animals that received the combination therapy of flagellin and GNT.

[0118] The total number of cells and leukocytes (CD45+) in the BAL was analyzed by flow cytometry using the gating strategy described in Supplementary Figure 2. also compared between groups (Figure 2A.1 and 2A.2, respectively). We showed that animals treated with preventive instillation of flagellin (in combination or not with GNT) presented significantly fewer cells in the BAL at 16 hours post-infection, compared to infected non-treated or those only treated with GNT (p<0.05 for group Flagellin vs. group Untreated and p<0.001 for group Flagellin+GNT vs. group GNT). Interestingly, among the cells present in the BAL, significantly more were CD45+ immune cells for the animals that received preventive flagellin (p<0.01 for group Flagellin vs. group Untreated and p<0.001 for group Flagellin+GNT vs. group GNT).

[0119] According to the study of Fougeron et al. (2015), the number of neutrophils and total DCs in the lungs of mice remain elevated for approximately 72 hours after being challenged nasally with flagellin. In this context, we investigated both immune cell populations (neutrophils identified as CD45+CD1 lb+Ly6G+and total DCs as CD45+CDl lc+SiglecF' MHCTI ) in the BAL (Figure 2A.3 and 2A.4, respectively). As expected, P. aeruginosa infection induced neutrophil recruitment to the airways. The mucosal delivery of flagellin (in combination or not with GNT) induced an even higher increase in the number of neutrophils (p<0.01 for group Flagellin vs. group Untreated and p<0.001 for group Flagellin+GNT vs. group GNT). Flagellin instillation also caused an increase in total DCs (p<0.01 for group Flagellin vs. group Untreated and p<0.001 for group Flagellin +GNT vs. group GNT). Regarding AM (identified as CD45+CDl lc+SiglecF+; Figure 2A.5), infection with PMDRdecreased the number of this cell population in BAL (a 5-fold reduction compared to the noninfected group) and treatment with the antibiotic GNT was not enough to revert such drop. Pre- treatment with flagellin (in combination or not with GNT) preserved the level of AM (p<0.01 for group Flagellin vs. group Untreated and p<0.001 for group Flagellin+GNT vs. group GNT).

[0120] Sixteen hours after challenge with PAMDR, infected non-treated animals presented high levels of IL-6, Tumor Necrosis Factor alpha (TNF-a), GM-CSF and IL-ip 16h post-infection (Figure 2B). Whereas GNT -treated mice also presented high proinflammatory cytokines, mice that received flagellin (in combination or not with GNT) showed a significant reduction for all tested cytokines (p<0.01 or p<0.001). Thus, flagellin pretreatment reduced the expression of proinflammatory cytokines triggered by PAMDRinfection. The combination of flagellin and GNT fully restores the level of pro-inflammatory mediators released from the lungs of PAMDR-infected mice to a homeostatic basal level.

[0121] Combination of prophylactic flagellin and therapeutic gentamicin protects mice against a lethal dose of a MDR strain of P. aeruginosa

[0122] We assessed the efficacy of the Flagellin+GNT combination therapy in protecting mice from a lethal challenge with PAMDR. To this end, we monitored mice mortality over several days following infection and treatment (Figure 3A). As anticipated for a virulent MDR strain of PA, most infected non-treated mice succumbed to the infection within 24 hours (Figure 3B). Standalone GNT was unable to protect mice from the lethal challenge. Remarkably, preventive nasal administration of flagellin alone was sufficient to substantially reduce mortality rates, even in the absence of antibiotics (80% survival for group “Flagellin” vs. 20% survival for group “GNT”, p<0.001). Moreover, the combination of prophylactic flagellin with therapeutic GNT resulted in a remarkable 100% increase in mice survival rates compared to non-treated, infected animals (p<0.001).

[0123] Discussion

[0124] In the present study we used an in vivo mouse model to investigate the effect of prophylactic nasal administration of flagellin in combination, or not, with the antibiotic GNT against a MDR strain of P. aeruginosa (PAMDR). We demonstrated that (i) a single prophylactic immunization of flagellin was sufficient to decrease bacterial load in the lungs and, when combined with GNT Ih post-infection, this reduction was even more accentuated, which suggests a synergistic effect between the two agents; (ii) pre-treatment with flagellin, in combination or not with GNT, reduced inflammation but increased the number of neutrophils, total DCs and maintained AM levels in the BAL, 16h post-infection.; and (iii) combination of prophylactic flagellin and GNT Ih post-infection led to 100% survival, contrary to the control groups. Several studies have shown that nasal administration of flagellin can reduce bacterial load in an animal infection model (Kinnebrew et al., 2010; Munoz et al., 2010; Yu et al., 2010; Kone et al. , 2020). For example, the study from Munoz N., et al. demonstrated that respiratory treatment with flagellin leads to S. pneumoniae clearance in the lungs and promotes survival of infected mice. They attributed such results to increased neutrophil infiltration into the airways as a result of strong chemokine secretion by flagellin-stimulated ECs. Our findings are consistent with such studies. We observed that animals pre-treated with flagellin 24 hours before infection, receiving or not GNT 1-hour after challenge, presented significantly less PAMDRbacterial load and higher neutrophil counts in the BAL, compared with infected nontreated mice or only treated with GNT. Neutrophils play a critical role in combating infections through their antimicrobial functions. Particularly in the context of P. aeruginosa, it has been demonstrated that early neutrophil influx to the respiratory airways is essential for the clearance of the pathogen (Koh et al., 2009; Jarchum etal., 2011; Mij ares et al. , 2011). Accordingly, we hypothesize that, the rise in neutrophil counts in flagellin-treated mice could play a role in the decrease of P. aeruginosa CFU in those same mice. The secretion of antimicrobial compounds by ECs (Kinnebrew et al., 2010 and Yu et al., 2010) may also contribute to the reduction of PAMDR. TO fully comprehend the effect of antimicrobial effectors in our model, additional experiments should be carried out.

[0125] While standalone flagellin was effective in clearing PAMDR, our study showed that the combination of prophylactic flagellin and therapeutic GNT confers an additional advantage. Mice treated with this combination showed an even greater reduction in lung bacterial load compared to those treated with flagellin alone, suggesting a synergy between the antibiotic and flagellin. Since we did not find any significant difference in immune cell populations, particularly neutrophils, between mice treated with flagellin alone and those treated with Flagellin+GNT, the observed additional advantage in the combination group may be attributed to the direct antibiotic's impact, despite GNT's limited efficacy against PAMDRwhen used alone. We hypothesize that the prophylactic nasal administration of flagellin may have contributed not only to the recruitment of phagocytes to the site of infection but also to the secretion of multiple antimicrobial peptides, which in turn lead to the elimination of, at least, some of the bacteria present in the respiratory tract. When GNT was administered, Ih after infection, less viable bacteria in the flagellin-regulated lung mucosa enabled the antibiotic to efficiently clear most of the remaining bacteria.

[0126] The mechanisms behind GNT resistance are still unidentified in the / Jaeruginosa strain used in this study. However, regardless of the bacterial resistance mechanism(s) that our strain may employ against GNT, the impact that flagellin-stimulated immune mediators may have had in bacterial fitness and / or bacterial tissue distribution may also have facilitated the action of the antibiotic, resulting in synergy between the two molecules. Such results strongly highlight the benefits of using flagellin as a host-directed immunomodulator in combination with an antibiotic.

[0127] Thus, our work is reminiscent of previous studies showing that a host-directed immunomodulator can contribute to the clearance of a pathogen, facilitating the action of an antibiotic (Kaiser et al., 2014; Porte et al., 2015; Matarazzo et al., 2019; Jiang et al, 2020). Secoeudesma sesquiterpenes lactone A (SESLA) is an anti-inflammatory molecule proved to modulate NFKB and PI3K / Akt signaling pathways (Jiang et al, 2020). When in simultaneous combination with the antibiotic meropenem, it promotes bacterial clearance and survival in mice infected with carbapenem-resistant Klebsiella pneumoniae. Recently, a similar strategy was found to be efficacious in the context of intestinal infections with Salmonella Typhimurium (Kaiser et al., 2014). Indeed, the combination of systemic administration of ciprofloxacin and a TLR4 or TLR9 agonist was able to further reduce the number of antibiotic- tolerant Salmonella in the gut and in the associated draining lymph nodes, compared with standalone treatments. Another study also showed that the simultaneous combination of flagellin and the antibiotic amoxicillin, administered 12 hours post-infection, can provide additional protection by effectively eliminating a resistant strain of S. pneumoniae (Matarazzo et al. , 2019).

[0128] A host-directed immunomodulator, when paired with an antibiotic, should also promote a balanced inflammatory response and confer long-lasting immunity (Zumla et al., 2016; Kaufmann et al., 2018). In our study, immunization with flagellin 24-hours before challenge, either alone or in combination with GNT, effectively reduced excessive local inflammation caused by PAMDRinfection. Mice receiving the combination of flagellin and GNT exhibited even lower tissue redness, a decrease in total cell infiltration in the BAL, and a return of proinflammatory cytokine levels to steady state by 16 hours post-infection. These findings are in alignment with a recent investigation conducted by Lopez-G lvez R. et al (2021). Using airway epithelial cells and a pig model, their research demonstrates that flagellin serves as an immunostimulant molecule capable of eliciting a transient proinflammatory reaction. This proinflammatory stimulus plays a pivotal role initiating an efficient host immune response towards a forthcoming pathogen. However, because flagellin’ s proinflammatory stimulus is short-lived and the molecule itself is also rapidly degraded within the airway mucosa, a few hours after its administration there is no longer inflammation driven by flagellin. They also show that, particularly in the context of P. aeruginosa infection, a first prophylactic stimulus with flagellin is enough to attenuate / ’. c / era / «osc / -triggered inflammatory response. Although the detailed mechanism is still unclear, Lopez-Galvez R. et al (2021) suggest that epigenetic changes or trained immunity caused by flagellin (also present in P. aeruginosa flagellum) may also contribute to restrain inflammation upon P. aeruginosa challenge (Netea et al. , 2016; Bigot et al., 2020). For example, the research of Bigot and colleagues demonstrated that bronchial epithelial cells pre-exposed with flagellin modify their inflammatory response to a second stimulus due to epigenetic regulation (Bigot et al, 2020). The observed decreased inflammatory response could also be related to TLR5 signalling desensitization due to a decrease in receptor availability or tachyphylaxis, as observed when repeated doses of TLR ligands are administered to mice (Alfaro et al., 2014). In addition, it has been shown that flagellin treatment increased concentration of TNFAIP3. This key negative regulator of the innate immune response, can decrease the signalling of both TLR4 and TLR5 pathways, thereby modifying the ability of airway epithelial cells to respond to a second stimulation by P. aeruginosa (Ashall et al., 2009; Caballero et al., 2017).

[0129] Our findings also demonstrate that preventive administration of flagellin can enhance the preservation of AM, which were diminished following P. aeruginosa infection. Such apparent reduction has also been previously reported in the context of Influenza and SARS- Cov-2 infection (Ghoneim, Thomas and McCullers, 2013; Winkler et al, 2020). While we have yet to fully elucidate the precise mechanisms underlying this decrease in our PAMDRinfection model, our data strongly suggests that nasal flagellin administration effectively mitigates AM reduction. The advantages of preserving AM in the context of respiratory infections are vast (Allard, Panariti and Martin, 2018). These cells are the first to encounter pathogens in the alveoli. After an antigen exposure, AM become activated and not only are able to internalize and kill pathogens but also produce several proinflammatory cytokines and chemokines that recruit neutrophils to the site of infection, helping with clearance. Thus, flagellin-dependent AM preservation may contribute to the reduction of P. aeruginosa infections.

[0130] It has been shown that total DCs, which include monocyte derived-DCs and conventional DCs are increased in the airway mucosa after stimulus with flagellin. Although we did not discriminate DC subtypes in the current study, we also observed an increase in these immune cell population in animals that were nasally challenged with flagellin, 24h before infection, (receiving or not GNT Ih post-infection). These results may indicate possible long- lasting immunity driven by prophylactic nasal flagellin against a MDR strain of P. aeruginosa, although further studies would have to be conducted to confirm such hypothesis. Finally, the beneficial synergy between flagellin and gentamicin was evidenced in a survival study. The combination between flagellin and GNT protected all infected mice, which was not observed in any other group. The observed survival outcome may be attributed to the synergistic effects of flagellin and the restoration of GNT effectiveness, involving several mechanisms: (i) flagellin enhances the influx of neutrophils into the lungs, (ii) these cells aid in bacterial clearance, (iii) the reduced bacterial burden increases the antibiotic-to-bacteria ratio, thereby restoring the efficacy of GNT, and (iv) flagellin's prophylactic effect mitigates excessive inflammation in the lungs, ultimately contributing to improved host survival. Such results highlight the advantageous synergy between the two molecules and underline the importance of investigating such combination further. Moreover, our work extends the current knowledge about the beneficial effect of flagellin as host-directed immunomodulator to MDR strains of P. aeruginosa, which present a significant challenge for patients with chronic respiratory conditions and limited treatment options. Although more research is required to confirm the feasibility of prophylactic flagellin administration in patients, this study provides a basis for a novel dual therapy against P. aeruginosa in the context of increasing antimicrobial resistance.

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Claims

CLAIMS:

1. A combination of (i) a flagellin polypeptide and (ii) an antibiotic, for the simultaneous or sequential use in the treatment of bacterial infections due to Pseudomonas aeruginosa drug resistance strain.

2. The combination for use according to claim 1 wherein the flagellin polypeptide comprises: a) a N-terminal peptide having at least 90% amino acid identity with the amino acid sequence starting from the amino acid residue located at position 1 of SEQ ID NO:3 and ending at an amino acid residue selected from the group consisting of any one of the amino acid residues located at positions 99 to 173 of SEQ ID NO:3 ; and b) a C-terminal peptide having at least 90% amino acid identity with the amino acid sequence starting at an amino acid residue selected from the group consisting of any one of the amino acid residues located at positions 401 to 406 of SEQ ID NO:3 and ending at the amino acid residue located at position 494 of SEQ ID NO:3 , wherein : the said N-terminal peptide is directly linked to the said C-terminal peptide, or the said N- terminal peptide and the said C-terminal peptide are indirectly linked, one to the other, through a spacer chain.

3. The combination for use according to claim 2 wherein said N- terminal and C-terminal peptides consist of the amino acid sequences 1-173 and 401-494 of SEQ ID NO:3, respectively.

4. The combination for use according to claim 2 or 3 wherein said N-terminal peptide and the said C-terminal peptide are indirectly linked, one to the other, through an intermediate spacer chain consisting of a NH2-GIy-AIa-AIa-GIy-COOH (SEQ ID NO:4) peptide sequence5. The combination for use according to claim 4 wherein the flagellin polypeptide is the polypeptide having the amino acid sequence of SEQ ID N°5.

6. The combination for use according to claim 1 to 5 wherein the antibiotic is selected from the list consisting of P-lactams, aminoglycosides, antibiotic agents which inhibit acid nucleic synthesis comprising aminocoumarines and quinolones, antibiotics which inhibit protein synthesis comprising amphenicols, tetracyclins macrolides and ketolides,antibiotics which inhibit folate metabolism comprising sulfonamides and sulfanilamides, antibiotic agents comprising cyclic lipopeptides, glycylcyclines, oxazolidinones and lipiarmycins.

7. The combination for use according to claim 6 wherein the aminoglycoside antibiotic is gentamycin.

8. The combination for use according to claim 1 to7, wherein the flagellin polypeptide is preferably administered by the intranasal route.

9. A flagellin polypeptide as defined according to claim 1 to 5, for use in a method for enhancing sensitivity to an antibiotic of a patient suffering from bacterial infection due to Pseudomonas aeruginosa drug resistance strain.

10. The flagellin polypeptide for use according to claim 9 wherein the antibiotic is selected from the list consisting of 0-lactams, aminoglycosides, antibiotic agents which inhibit acid nucleic synthesis comprising aminocoumarines and quinolones, antibiotics which inhibit protein synthesis comprising amphenicols, tetracyclins macrolides and ketolides, antibiotics which inhibit folate metabolism comprising sulfonamides and sulfanilamides, antibiotics agent comprising cyclic lipopeptides, glycylcyclines, oxazolidinones and lipiarmycins.

11. The flagellin polypeptide for use according to claim 9 wherein the aminoglycoside antibiotic is gentamycin.

12. A pharmaceutical composition comprising: i. a flagellin polypeptide (as defined according to claim 1 to 5), ii. an antibiotic agent (as defined as defined according to claim 1 to 5); and iii. a pharmaceutically acceptable carrier. for use in the prevention or the treatment of antibiotic-resistant bacterial infection due to Pseudomonas aeruginosa antibiotic-resistant strain in a patient in need thereof13. The pharmaceutical composition for use according to claim 9, for use in a method of treating or preventing a lung infectious disease in a subject.

14. The pharmaceutical composition for use according to claim 12 or 13 wherein the flagellin polypeptide is administered by the intranasal route.

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