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WO2025172452A1 - Combinaison pour le traitement d'une infection bactérienne due à une souche résistante aux antibiotiques de pseudomonas aeruginosa - Google Patents

Combinaison pour le traitement d'une infection bactérienne due à une souche résistante aux antibiotiques de pseudomonas aeruginosa

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Publication number
WO2025172452A1
WO2025172452A1 PCT/EP2025/053880 EP2025053880W WO2025172452A1 WO 2025172452 A1 WO2025172452 A1 WO 2025172452A1 EP 2025053880 W EP2025053880 W EP 2025053880W WO 2025172452 A1 WO2025172452 A1 WO 2025172452A1
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WO
WIPO (PCT)
Prior art keywords
flagellin
antibiotic
combination
amino acid
gnt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/053880
Other languages
English (en)
Inventor
Virginie Herve
Adeline CEZARD
Mustapha SI-TAHAR
Jean-Claude Sirard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Lille
Universite de Tours
Institut Pasteur
Universite de Lille
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Lille
Universite de Tours
Institut Pasteur
Universite de Lille
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut Pasteur de Lille, Institut National de la Sante et de la Recherche Medicale INSERM, Centre Hospitalier Universitaire de Lille , Universite de Tours, Institut Pasteur, Universite de Lille filed Critical Centre National de la Recherche Scientifique CNRS
Publication of WO2025172452A1 publication Critical patent/WO2025172452A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose

Definitions

  • 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.
  • the present invention meets this need.
  • PA Pseudomonas aeruginosa
  • CF cystic fibrosis
  • Multidrug-resistant (MDR) strains of PA are even harder to eradicate and cause higher mortality rates (Matos et al., 2018; Ikuta et al., 2022).
  • WHO World Health Organization
  • MDR strains of PA in a list of pathogens requiring urgent attention for the development of new therapeutic strategies (Tacconelli etal., 2018).
  • 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).
  • flagellinAi74-4oo 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).
  • TLR5 Toll-like receptor 5
  • PRR pattern recognition receptor
  • ECs epithelial cells
  • AM alveolar macrophages
  • eDCs conventional dendritic cells
  • 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).
  • NFKB transcription factor nuclear factor-kappa B
  • MAPK Mitogen-activated protein
  • 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:
  • 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.
  • 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.
  • 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.
  • P-lactamase-resistant penicylin derivatives Methicillin, Nafcillin, Oxacillin, Cioxacillin, Dicloxacillin, Flucloxacillin.
  • Aminopenicillins Ampicillin, Amoxicillin, Pivampicillin, Hetacillin, Bacampicillin, Metampicillin, Talampicillin, Epicillin.
  • Example of Carboxypenicillins Carbenicillin, Ticarcillin, Temocillin.
  • P-lactamase inhibitors penicylin derivatives Clavulanic acid, Sulbactam, Tazobactam.
  • 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.
  • 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.
  • Antibiotic agents which block DNA gyrase (topoisomerase specific to bacteria) : aminocoumarines, and quinolones.
  • Antibiotic agents which block the bacterial RNA polymerase rifampicine.
  • Antibiotic agents which block the formation of the peptide bond amphenicols (examples: chloramphenicol, thiamphenicol azidamfenicol and florfenicol)
  • 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
  • Sulfonamides also called sulphonamides, sulfa drugs or sulpha drugs (examples: Sulfamethoxazole) and sulfanilamides.
  • cyclic lipopeptides such as daptomycin
  • glycylcyclines such as tigecycline
  • oxazolidinones such as linezolid
  • lipiarmycins such as fidaxomicin
  • 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.
  • MDR multidrug-resistant
  • 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 .
  • flagellin 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.
  • 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.
  • TLR5 flagellin or a fragment thereof that retains the ability to bind and activate TLR5.
  • 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.
  • a TLR5 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.
  • 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.
  • 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.
  • the flagellin polypeptide of the present invention comprises the domains of flagellin involved in TLR5 signaling.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • said N- terminal and C-terminal peptides consist of the amino acid sequences 1-173 and 401-494 of SEQ ID NO:3, respectively.
  • said N- terminal and C-terminal peptides consist of the amino acid sequences 1-160 and 406-494 of SEQ ID NO:3 , respectively.
  • said N- terminal and C-terminal peptides consist of the amino acid sequences 1-137 and 406-494 of SEQ ID NO:3 , respectively.
  • 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.
  • the asparagine amino acid residue located at position 488 of SEQ ID NO: 3 is replaced by a serine.
  • the flagellin polypeptide as above described comprises an additional methionine residue at the N-terminal end (regarding flagellin polypeptide of SEQ ID N°3).
  • 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).
  • 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.
  • FLAMOD modified recombinant flagellin
  • the flagellin polypeptide of the present invention is produced by any method well known in the art.
  • 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.
  • 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.
  • 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.
  • S. D. Shine-Dalgarno
  • 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.
  • eubacteria such as Gram-negative or Grampositive organisms
  • 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.
  • Strain SIN41 of Salmonella Typhimurium 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.
  • the host cell secretes minimal amounts of proteolytic enzymes.
  • 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.
  • 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.
  • 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.
  • 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.).
  • 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.
  • 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).
  • the flagellin polypeptide is administered between 24 and 48 hours before antibiotic agent administration.
  • 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).
  • 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).
  • 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.
  • the flagellin polypetide of the invention is preferably administered by intranasal route and the antibiotic agent intravenous /intraperitoneal/intramuscular route.
  • medicaments according to the invention comprise a pharmaceutically- acceptable carrier.
  • a pharmaceutically- acceptable carrier e.g., a pharmaceutically-acceptable styrene foam, a pharmaceutically-acceptable styrene foam, a pharmaceutically-acceptable styrene foam, a pharmaceutically-acceptable styrene foam, a pharmaceutically-acceptable styrene foam, a pharmaceutically-acceptable s, a pharmaceutically- acceptable carrier.
  • suitable carriers 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.
  • 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.
  • the lung disease is a lung infectious disease (i.e. lung bacterial infection).
  • lung infectious disease 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.
  • a microbial agent e.g. common cold
  • bacterial infectious diseases such as Legionnaire's disease (Legionella), tuberculosis, infections by E. coll, Staphylococci, , Pseudomonas, Streptococci Hemophilus influenzae, Klebsiella pneumoniae, ....
  • the lung infectious disease is a lung infection due to Pseudomonas aeruginosa antibiotic-resistant strain
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 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”).
  • mice 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..
  • FIG. 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 5xlO 5 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 (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.
  • FIG. 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.
  • 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.
  • the custom-designed flagellin primarily used in the present study 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.
  • MIC minimal inhibitory
  • CSLI Clinical and Laboratory Standards Institute
  • 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.
  • a plate reader TECAN Infinite 200, Lyon, France
  • mice 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).
  • 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.
  • flagellinA 174-400 recombinant flagellin
  • 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).
  • mice 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.
  • PBS Phosphate Buffered Saline
  • FBS Fetal Bovine Serum
  • 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.
  • 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.
  • DuoSet ELISA Mae IL-6, IL-ip, TNF-a and GM-CSF were performed according to the manufacturer’s (R&D Systems) instructions.
  • preventive flagellin displays synergistic therapeutic activity against a MDR strain of P. aeruginosa
  • 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”).
  • Flagellin group mice only treated with GNT
  • Untreated group mice only treated with GNT
  • Healthy group mice that were neither treated nor infected as healthy
  • infected untreated mice presented a high bacterial load in the lungs 16 hours after PA MDR challenge (4.5xl0 6 CFU ⁇ 1.7xl0 6 ) (see Figure IB).
  • prophylactic administration of flagellin significantly reduced PA MDR CFU (p ⁇ 0.05).
  • 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.
  • 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).
  • animals treated with preventive instillation of flagellin 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).
  • 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.
  • 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.
  • Secoeudesma sesquiterpenes lactone A (SESLA) is an anti-inflammatory molecule proved to modulate NFKB and PI3K/Akt signaling pathways (Jiang et al, 2020).
  • SESLA Secoeudesma sesquiterpenes lactone A
  • 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).
  • immunization with flagellin 24-hours before challenge either alone or in combination with GNT, effectively reduced excessive local inflammation caused by PA MDR infection.
  • 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.
  • 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.
  • 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.
  • flagellin a first prophylactic stimulus with flagellin is enough to attenuate/’. c/era / «osc/-triggered inflammatory response.
  • 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).
  • 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).
  • AM 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.
  • 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.

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Abstract

La présente invention concerne une combinaison de (i) un polypeptide de flagelline et (ii) un antibiotique, pour une utilisation simultanée ou séquentielle dans le traitement d'infections bactériennes dues à une souche résistante aux antibiotiques de Pseudomonas aeruginosa. La présente invention concerne en outre un polypeptide de flagelline, destiné à être utilisé dans une méthode d'amélioration de la sensibilité à un antibiotique chez un patient souffrant d'une infection bactérienne, due à une souche résistante aux antibiotiques de Pseudomonas aeruginosa. En effet, à l'aide d'un modèle de souris in vivo, les inventeurs montrent que, tandis que la flagelline prophylactique seule atténue une infection provoquée par une souche multirésistante (MDR) de PA (PAMDR), son association avec un antibiotique tel que la gentamicine (GNT) conduit à une forte diminution de la charge bactérienne dans le poumon et à une réduction significative de l'infiltration cellulaire et des cytokines inflammatoires. De plus, des souris recevant de la flagelline en combinaison avec GNT ont présenté un taux de survie de 100 %, un résultat que GNT seul n'était pas en mesure de produire contre PAMDR.
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