Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/095—Neisseria
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants

Definitions

• the invention is in the field of vaccines against infections due to Neisseria meningitidis and proposes in particular a vaccine composition comprising lipooligosaccharide (LOS) in combination with the subunit B (TbpB) lipid transferrin receptor of a strain of N. meningitidis.
• LOS lipooligosaccharide
• TbpB subunit B
• meningitidis serogroup B This bacterium is responsible for a number of pathologies , including predominantly meningitis and meningococcal disease, but also arthritis and pericarditis. Meningococcal disease can be complicated by purpura fulminons and deadly septic shock.
• meningitis is either of viral origin or of bacterial origin.
• the main causative bacteria are: N. meningitidis and Streptococcus pneumoniae, respectively involved in about 40-50% of cases of bacterial meningitis.
• Haemophilus influenzae also remains an important source of meningitis.
• N. meningitidis meningitis In France, about 600 to 800 cases a year of N. meningitidis meningitis occur. In the United States, the number of cases is about 2,500 to 3,000 per year.
• the N. meningitidis species is subdivided into serogroups according to the nature of the capsule polysaccharides. Although there are a dozen serogroups, 90% of cases of meningitis are attributable to serogroups: A, B, C, Y and Wl 35.
• the capsular polysaccharide of N. meningitidis serogroup B is not or hardly immunogenic in humans, whether in conjugated form or not (Bruge et al, Vaccine (2004) 22: 1087).
• this polysaccharide carries an epitope that can potentially cross-react with human tissues. So, it appears It is highly desirable to seek a vaccine against N. meningitidis-induced meningitis, especially serogroup B other than a capsular polysaccharide vaccine.
• LPS Lipopolysaccharide
• LPS is not only toxic, it is also immunogenic. In mammals, anti-LPS antibodies are generated during carriage and infection and may be protective. Thus, the use of LPS has already been considered in the prophylaxis of infections due to Gram-negative bacteria and associated diseases.
• LPS The structure of LPS consists of a lipid moiety, called lipid A, covalently bound to a polysaccharide moiety.
• Lipid A is responsible for the toxicity of LPS. It is highly hydrophobic and allows to anchor the LPS in the outer membrane of the wall.
• Lipid A is composed of a disaccharide structure substituted with fatty acid chains. The number and composition of fatty acid chains vary from one species to another.
• the polysaccharide moiety consists of carbohydrate chains that are responsible for antigenicity. There are at least 3 major regions in this polysaccharide part:
• an inner core consisting of monosaccharides [one or more KDO (2-keto-3-desoxyoctulosonic acid) and one or more heptose (Hep)] invariant within the same bacterial species;
• an heptose-linked outer core consisting of various monosaccharides;
• LPS LPS
• non-enteric Gram-negative bacteria such as
• LPS lipooligosaccharide
• LPS LPS The structure of LPS varies not only from one species to another, but also within the same species.
• the outer core (or ⁇ chain) is variable depending on the type of oligosaccharide (substituent R1), attached to the glucose residue carried by heptose I.
• lipid A is essentially invariant
• the inner core which is also invariant, of two KDOs (2-keto, 3-desoxy, octulosonic acid) and two heptoses (HepI and HepII)
• heptose II substituted by two KDOs (2-keto, 3-desoxy, octulosonic acid
• HepI and HepII heptoses
• ⁇ -chain consisting of ⁇ -acetylated glucosamine (Glc ⁇ Ac) which may be non-O-acetylated.
• the R2 residue is commonly called ⁇ chain, when R2 is a glucose residue.
• N. meningitidis is classified into several immunotypes (IT L1 to L13), depending on their reactivity with a series of antibodies that recognize various epitopes on LOS (Achtman et al, 1992, J. Infect Dis 165: 53-68).
• the majority of N. meningitidis serogroup B invasive strains are of L3.7 immunotype as evidenced by the reactivity of these strains with a monoclonal antibody called 12.1.9. This monoclonal antibody is capable of recognizing each of the L3, L7 and L9 immunotypes (Gu et al, J.
• LOS can be sialylated (presence of N-acetyl neuraminic acid on the galactose residue (GaI) terminal of the ⁇ chain).
• GaI galactose residue
• the L3 and L7 immunotypes differ only in the respective presence / absence of this sialylation.
• most LOS are substituted with an O-acetyl moiety on the glucosamine residue ( ⁇ -GlcNAc) of the inner core (Wakarchuk et al (1998) Eur J. Biochem 254: 626, Gamian et al. 1992) J.
• Ipu and lo Off-locus genes are Ipu and lo.
• the Ip ⁇ gene encodes a PEA transferase. This enzyme has the ability to attach a phosphoethanolamine (PEA) residue at the O-3 position of heptose II.
• the lo ⁇ gene encodes an LOS O-acetyltransferase that has the ability to O-acetylate the ⁇ chain. It is subject to phase variation.
• the lgt-1 locus has 7 genes: IgtA, IgtB, IgtC, IgtD, IgtE, Igt, and IgtZ each encoding a particular glycosyl transferase.
• IgtA and IgtC are subject to phase variation.
• IgtE and IgtR exhibit allelic variation: the codon determining the amino acid at position 153 codes for either a threonine residue (and in this case the resulting enzyme is a Gal-transferase); either for a methionine residue (and in this case the resulting enzyme is a Glc-transferase).
• the lgt-1 locus is classified into 8 genetic types (Zhu et al., Microbiology (2002) 148: 1833).
• the locus / gt-2 has 2 genes: IgtF and igtK encoding glycosylases.
• the product of the IgtF gene is involved in the construction of the ⁇ chain by allowing the glucose residue to be fixed on heptose I and thus does not interfere with the nature of the immunotype; nor the IgtK gene for that matter.
• the lgt-3 locus has 2 genes: / g / G and Ipt ⁇ .
• the igtG gene encodes an Ices synthetase that has the ability to attach a glucose residue at the O-3 position of IL heptose.
• the Ipt ⁇ gene encodes a PEA transferase that has the ability to attach a phosphoethanolamine (PEA) substituent. position O-6 or O-7 of heptose IL
• PEA phosphoethanolamine
• the lgt-3 locus is classified into 5 genetic types (Wright et al., J. Bact. (Oct. 2004): 6970).
• the carbohydrate motif Gal ⁇ 1-4GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc ⁇ 1-4 or lacto-N-neotetraose motif which is present in the ⁇ chain of certain immunotypes of LOS of JV. meningitidis, is an epitope that can potentially cross-react with human erythrocytes.
• an LOS that does not have this pattern. It may therefore be particularly advantageous to use an LOS derived from immunotype L6 or L8 strains.
• the subject of the invention is a vaccinal pharmaceutical composition against N. meningitidis infections, which comprises:
• a LOS of N. meningitidis consisting in particular of a lipid A, an inner core, an L8-type ⁇ chain, in which the heptose residue II of the inner core (a) bears in position O-3 a phosphoethanolamine (PEA) substituent and does not carry a PEA substituent at the O-6 and O-7 positions; or (b) has a phosphoethanolamine (PEA) substituent at the 0-3 position and the 0-6 or 0-7 position; and
• a vaccine according to the invention comprises: (i) a LOS of N. meningitidis consisting in particular of a lipid A, an inner core, an L8-type ⁇ chain, in which the heptose residue II of the inner core carries in position O-3 a phosphoethanolamine (PEA) substituent and does not carry a PEA substituent at the 0-6 and O-7 positions;
• a LOS of N. meningitidis consisting in particular of a lipid A, an inner core, an L8-type ⁇ chain, in which the heptose residue II of the inner core carries in position O-3 a phosphoethanolamine (PEA) substituent and does not carry a PEA substituent at the 0-6 and O-7 positions;
• PEA phosphoethanolamine
• N. meningitidis consisting in particular of a lipid A, an inner core, an L8-type ⁇ chain, in which the heptose II residue of the inner core bears a phosphoethanolamine (PEA) substituent, in position O-3 and in position O-6 or O-7; and (iii) N. meningitidis TbpB or a lipid fragment thereof.
• PEA phosphoethanolamine
• a composition according to the invention can (i) prevent at least 60%, advantageously at least 70%, preferably at least 80% of infections caused by N. meningitidis, especially serogroup B or (ii) prevent infections due to at least at 60%, advantageously at least 70%, preferably at least 80% of N. meningitidis strains including serogroup B.
• composition according to the invention does not contain OMV (outer membrane vesicle) of N. meningitidis.
• the LOS consisting in particular of a lipid A 5 of an inner core, of an L8 type ⁇ chain, in which the heptose II residue of the inner core carries a phosphoethanolamine (PEA) substituent.
• PPA phosphoethanolamine
• the LOS comes from a strain of serogroup A.
• a vaccine according to the invention by using an LOS consisting in particular of a lipid A, an inner core, an ⁇ chain type L8, in which the Heptose II residue of the inner core carries a phosphoethanolamine (PEA) substituent in the 0-3 position and does not carry a PEA substituent at the O-6 and O-7 positions.
• PDA phosphoethanolamine
• such an LOS originates from an L8 immunotype strain, preferably serogroup A. It is also proposed to obtain such an LOS from a N. meningitidis strain of L6 immunotype modified in such a way. that it expresses an Ip ⁇ gene and that it no longer expresses the Ipt6 and IgtA genes.
• the starting strain that can be used for modification purposes may be strain C708 deposited on March 11, 2008, with the National Collection of Microorganism Culture, 25 rue du Dr Roux 75015 Paris, according to the terms of the Budapest Treaty.
• This strain has the order number CNCM I-3942.
• This strain possesses, inter alia, an active IgtA gene (gene lit "ON”); an igtB gene (non-functional gene); an igtG gene off ("Off”); a truncated Ip ⁇ gene; an active I ⁇ t6 gene; a lofa active gene; and an active msbB gene.
• Strain C708 has a truncated IpB gene. To modify it so that the LOS carries a PEA substituent at position 03, the functionality of the Ipt3 gene can be restored, in particular by homologous recombination making use of a complete Ipf ⁇ gene (full-length).
• this strain is also appropriate to deactivate the iptA and Iptd genes to obtain a strain that no longer expresses these genes.
• the deactivation of these genes may in particular be carried out by total or partial deletion of the IptA and Ipt ⁇ genes or else by intra- genic insertion of an irrelevant sequence, for example of an antibiotic resistance gene.
• an LOS for use in a composition according to the invention has a ⁇ chain which is O-acetylated, at least partially.
• LOS can be obtained by conventional means: in particular, it can be extracted from a bacterial culture; then purified according to conventional methods. Numerous methods of obtaining are described in the literature. For example, La., Westphal & Jann, (1965) Meth. Carbohydr. Chem. 5:83; Gu & Tsai, 1993, Infect. Immun. 61 (5): 1873; Wu et al., 1987, Anal. Biochem. 160: 281 and US 6,531,131. An LOS preparation can be quantified according to well-known procedures. The determination of KDO by high performance anion exchange chromatography (HPAEC-PAD) is a particularly suitable method. Detoxification of LOS
• LOS For incorporation into a vaccine, LOS needs to be detoxified.
• the toxicity of LOS is due to its lipid A. Nevertheless, it is not imperative to remove lipid A in its entirety; nor to modify it for example by mutation (e.g. mutation msbB minus). Indeed, the toxicity being more particularly related to a supramolecular conformation conferred by all the fatty acid chains carried by the disaccharide ring of the lipid, according to an advantageous mode, it is sufficient to act on these chains.
• the detoxification can be obtained according to various approaches: chemical, enzymatic, genetic or even by complexation with a polymixin B analog peptide or by incorporation / formulation into liposomes.
• the level of detoxification of LOS can be assessed according to one of two standard tests:
• the pyrogenic test in rabbits This test, the calculations and their reading and were performed according to the principles set out by the European Pharmacopoeia (Edition 6.0, paragraph 2.6.8.).
• the LAL test (Limulus Amebocyte Lysate) carried out according to the principles stated by the European Pharmacopoeia (Edition 6.0, paragraph 2.6.14.).
• the chemical approach is to treat LOS with a chemical agent.
• the LOS is subjected to a mild acid hydrolysis with acetic acid which removes the lipid A and the or KDOs in branching when the latter (these) is
• the LOS is subjected to a de-O-acylation, preferably primary, the. by treatment with hydrazine which hydrolyzes the esterified primary fatty acid chains of lipid A.
• a de-O-acylation preferably primary
• hydrazine which hydrolyzes the esterified primary fatty acid chains of lipid A.
• the enzymatic approach consists in putting the LOS in the presence of lipases capable of digesting the esterified fatty acid chains of the lipid A.
• lipases are produced by the amoeba Dictyostelium discoideum.
• the amoeba is cultured together (co-culture) and a gram-negative bacterium capable of being phagocytosed by the amoeba, such as N. meningitidis.
• the supernatant is then recovered and the LOS is extracted from the supernatant, which is then free of fatty acid chains.
• acyloxyacyl hydrolase produced by certain human cells (WO 87/07297 Munford R.) or by Salmonella typhimurium (Trent et al 2001 J. Biol Chem 276: 9083-9092, Reynolds et al., 2006 J. Biol Chem 281: 21974-21987) (enzyme encoded by the genes PagL or LpxR in the latter case).
• the genetic approach consists of using an LOS produced by a bacterial strain whose genotype is such that the entity of LOS normally responsible for its toxicity (lipid A and more particularly the lipid tails of lipid A) has a degree of toxicity largely reduced or even nonexistent.
• a bacterial strain can be conveniently obtained by mutation. From a wild strain (that is to say producing a toxic LOS), it is then a question of inactivating by mutation certain genes involved in the biosynthesis of the fatty acid chains or in their attachment on the nucleus. disaccharide lipid A. Thus, it can be expected to inactivate the genes ipxl ⁇ or ipxLl (also called htrBl I J ⁇ trB2) of N.
• meningitidis or their equivalents in other species for example, the equivalent of genes IpxLl and lpxL2 meningococcus are called respectively msbB or ipxM and htrB or ipxL in E. col).
• An inactivating mutation in one of these genes leads to an LOS lacking one or two secondary acyl chains.
• IpxL1 or L2 mutants of N. meningitidis or Haemophilus influenzae are in particular described in patent applications WO 00/26384, US 2004/0171133 and WO 97/019688.
• the endogenous gene ipxA can also be replaced by the homologous gene from E.
• a fourth approach consists in complexing the LOS with a polymixin B analogue peptide, as described, for example, in the patent application WO 06/108586.
• LOS complexed and therefore detoxified is called endotoxoid.
• the polymixin B analogue used in the composition of an endotoxoid useful for the purposes of the present invention may be any peptide capable of detoxifying LOS by simple complexation. Such peptides are described in particular in patents or patent applications US Pat. No. 6,951,652, EP 976,402 and WO 06/06/108586.
• an advantageous peptide may be the peptide of formula (I) NH 2 -A-Cys 1 -B-Cys 2 -C-COOH, in which:
• A is a peptide of 2 to 5, preferably 3 or 4 amino acid residues, wherein at least 2 amino acid residues are independently selected from Lys, HyI (hydroxylysine), Arg and His;
• B is a peptide of 3 to 7, preferably 4 or 5 amino acid residues, which comprises at least two, preferably three amino acid residues selected from Val, Leu, Ile, Phe, Tyr and Trp; and
• this position may be empty or not
• the C position is an empty position.
• peptide of formula (I) are the following peptides: NH 2 -Lys-Thr-Lys-Cysl-Lys-Phe-Leu-Lys-Lys-Cys2-COOH (SAEP2 peptide); NH 2 -Lys-Thr-Lys-Cysl-Lys-Phe-Leu-Leu-Leu-Cys2-COOH (SAEP2-L2 peptide); NH 2 -Lys-Arg-His-Hyl-Cys 1 -Lys-Arg-Ile-Val-Leu-Cys 2 -COOH; NH 2 -LyS-Arg-His-Cysl-Val-Leu-Ile-Trp-Tyr-Phe-Cys2-COOH; NH 2 -Lys-Thr-Lys-Cysl-Lys-Phe-Leu-Leu-Leu-Cys2-COOH
• the peptides of formula (I) may be in the form of a monomer or preferably in the form of dimer, parallel or antiparallel. In general, it is also possible to use a dimeric peptide of formula (II) NH 2 -A-Cys 1 -B-Cys 2 -C-COOH
• a and A ' are independently a peptide of 2 to 5, preferably 3 or 4 amino acid residues, wherein at least 2 amino acid residues are independently selected from Lys, HyI (hydroxylysine), Arg and His;
• B and B ' are independently a peptide of 3 to 7, preferably 4 or 5 amino acid residues, which comprise at least two, preferably three amino acid residues independently selected from Val, Leu, Ile, Phe, Tyr and Trp; and
• C and C are optional (these positions may be empty or not) and are independently an amino acid residue or a peptide of 2 to 3 amino acid residues; provided that the ratio of cationic amino acid / hydrophobic amino acid in the dimer of formula (II) or (III) is 0.4 to 2, preferably 0.5 to 1.2 or 1.5, preferably 0.6 to 1; better from 0.6 to 0.8; for example. 0.75.
• a and A ' are independently a peptide of 2 to 5, preferably 3 or 4 amino acid residues, in which at least one, preferably 2 amino acid residues, are independently selected from Lys.
• HyI, Arg and His those which are not selected from Lys, HyI, Arg and His ("the remaining amino acid residues") being selected from the group of non-amino acid residues. charged, polar or non-polar; preferably Thr, Ser and GIy; most preferably Thr.
• each of them may be a cationic residue; or alternatively, two out of three residues are cationic amino acids, while the remaining residue is selected from the group of unfilled, polar or non-polar amino acid residues; preferably Thr, Ser and GIy; most preferably Thr.
• a and A 'have 4 amino acid residues it is preferred that two or three out of four residues are selected from the cationic amino acid residue groups as defined above, while the remaining residue (s) is ( are) selected from the group of residues of polar or non-polar non-charged amino acid residues as defined above.
• a and A 'have 5 amino acid residues it is preferred that three or four out of five residues are selected from the cationic amino acid residue groups as defined above, while the remaining residue (s) is ( are) selected from the group of residues of polar or non-polar non-charged amino acid residues as defined above.
• B and B ' are independently a peptide of 3 to 7, preferably 4 or 5 amino acid residues, which comprises at least two, preferably three amino acid residues independently selected from Val, Leu, Ile , Phe, Tyr, and Trp; preferably Leu, Ile and Phe; and where appropriate, those which are not selected from Val, Leu, Ile, Phe, Tyr and Trp ("the remaining amino acid residues") being independently selected from the group consisting of Lys, HyI, Arg and His .
• B and B ' may have up to 7 amino acid residues independently selected from Val, Leu, Ile, Phe, Tyr and Trp.
• B and B comprise the sequence - X1 - X2 - X3 -, in which X1 and X2; X2 and X3; or X1, X2 and X3 are independently selected from Val, Leu, Ile, Phe, Tyr and Trp; preferably from Leu, Ile and Phe.
• the sequence - X1 - X2 - X3 - comprises the Phe-Leu motif.
• B and B include: (i) the sequence - X1 - X2 - X3 - wherein: X1 is Lys, HyI, His or Arg, preferably Lys or Arg; preferably Lys; X2 is Phe, Leu, Ile, Tyr, Trp or Val; preferably Phe or Leu; more preferably, Phe; and
• X3 is Phe, Leu, Ile, Tyr, Trp or Val; preferably Phe or Leu; more preferably, Leu; and (ii) where appropriate, the amino acid residues, each being independently selected from the group consisting of Val, Leu, Ile, Phe, Tyr, Trp, Lys, HyI, Arg and His; preferably Val, Leu, Ile, Phe, Tyr and Trp; more preferably Leu, Ile and Phe.
• a and A preferably have at least 3 positively charged amino acid residues.
• the amino acid residues may be any amino acid residue provided that the ratio of cationic amino acid residues to hydrophobic amino acid residues remains within the indicated range.
• they are independently selected from non-charged, polar or non-polar amino acid residues, the latter being preferred.
• the C and C positions are empty positions.
• a preferred class of dimers are of formula (IV) NH 2 -A-Cys 1 -B-Cys 2 -COOH
• a and A ' are preferably the same. The same applies to B and B 'on the one hand; and C and C on the other hand.
• the endotoxoid useful for the purposes of the present invention may advantageously be characterized by a LOS: peptide molar ratio of 1: 1.5 to 1: 0.5, preferably of 1: 1.2 to 1: 0.8, very particularly preferably of 1: 1.1. at 1: 0.9, eg 1: 1.
• LOS detoxified into liposomes When LOS is formulated into liposomes, it does not necessarily appear necessary to detoxify it beforehand. Indeed, LOS in liposomes - that is to say, associated with the lipid bilayer forming liposomes - can see its toxicity decrease very substantially. The extent of this decay, up to a substantial loss, is in part a function of the nature of the components forming the liposome. Thus, when positively charged components (cationic components) are used, the loss of toxicity may be greater than with non-charged (neutral) or anionic components.
• liposomes is meant a synthetic entity, preferably a synthetic vesicle, formed of at least one two-layer lipid membrane (or matrix) closed on an aqueous compartment.
• the liposomes may be unilamellar (a single bilayer membrane) or plurilamellar (several membranes arranged onion).
• the lipids constituting the bilayer membrane comprise a non-polar region which is typically made of fatty acid or cholesterol chain (s), and a polar region, typically made of a phosphate group and / or tertiary or quaternary ammonium salts.
• the polar region may, especially at physiological pH (pH ⁇ 7), be carrying a net surface charge (overall) either negative (anionic lipid) or positive (cationic lipid) or not net charge (neutral lipid).
• liposomes can be any type of liposome; in particular, they may consist of any lipid known for its utility in the manufacture of liposomes.
• the lipid or lipids used in the composition of the liposomes may be neutral, anionic or cationic lipids; the latter being preferred.
• These lipids can be of natural origin (plant or egg extraction products, for example) or synthetic; the latter being preferred.
• the liposomes may also consist of a mixture of these lipids; for example, a cationic or anionic lipid and a neutral lipid, in a mixture.
• the neutral lipid is often referred to as co-lipid.
• the molar lipid (cationic or anionic): neutral lipid molar ratio is between 10: 1 and 1: 10, advantageously between 4: 1 and 1: 4, preferably between 3: 1 to 1: 3, terminals included.
• neutral lipids With regard to neutral lipids, mention is made by way of example: (i) cholesterol; (ii) phosphatidylcholines such as, for example, 1,2-diacyl-5n-glycero-3-phosphocholines eg, 1,2-dioleoyl-yl-glycero-3-phosphocholme (DOPC), and also 1 -acyl-2-acyl-sn-glycero-3-pohosphocholines whose acyl chains are different from each other (mixed acyl chains); and (iii) phosphatidylethanolamines such as 1,2-diacyl- ⁇ -glycero-3-phosphoethanolamines eg, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), as well as 1-acyl -2-acyl-5n-glycero-3-phosphoethanolamine carrying mixed acyl chains.
• phosphatidylcholines such as, for example, 1,2-di
• anionic lipids With regard to the anionic lipids, mention is made by way of example (i) cholesterol hemisuccinate (CHEMS); (ii) phosphatidylserines such as 1,2-diacyl-, s' -glycero-3- [phospho-L-serine] eg, 1H-dioleoyl-src-glycero-S-tphospho-L-serine] ( DOPS), and 1-acyl-2-acyl-s "-glycero-3- [phospho-L-serine] bearing mixed acyl chains; (iii) phosphatidylglycerols such as 1,2-diacyl-5 "-glycero-3- [phosphoro-1- (1-glycerol)] e, 1,2-dioleoyl-5" -glycero-3- [phospho -r ⁇ c- (1-glycerol)] (DOPG), and 1-acyl-2-acyl-sn-gly
• lipophilic amines or alkylamines such as, for example, dimethyldioctadecylammonium (DDA), trimethyldioactadecylammonium (DTA) or the structural homologs of DDA and DTA [these alkylamines are advantageously used in salt form; mention is made, for example, of dimethyldioctadecylammonium bromide (DDAB)];
• DDA dimethyldioctadecylammonium
• DTA trimethyldioactadecylammonium
• DDAB dimethyldioctadecylammonium bromide
• lipospermins such as N ⁇ palmitoyl-D-erythrospingosyl-1-O-carbamoyl spermine (CCS) and dioctadecylamidoglycylspermine (DOGS, Transfectam);
• lipids incorporating an ethylphosphocholine structure such as the cationic derivatives of phospholipids, in particular phosphoric ester derivatives of phosphatidylcholine, for example those described in Patent Application WO 05/049080 and including in particular: 1,2-dimyristoyl , 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-palmitoyl-oleoyl-sn-glycero-3-ethylphosphocholine, 1, 2-distearoyl-sn -glycero-3-ethylphosphocholine (DSPC), 1,2-dioleyl-5 "-glycero-3-ethylphosphocholine (DOEPC or EDOPC or ethyl-DOPC or ethyl PC), as well as their structural counterparts;
• DOEPC or EDOPC or ethyl-DOPC or ethyl PC 1,2-dioleyl-5 "
• lipids incorporating a trimethylammonium structure such as N- (I - [2,3-dioleyloxy] propyl) -N, N, N-trimethylammonium (DOTMA) and its structural homologs and those incorporating a trimethylammonium propane structure, such as 1,2-dioleyl-3-trimethylammonium propane (DOTAP) and its structural counterparts; as well as lipids incorporating a dimethylammonium structure such as 1,2-dioleyl-3-dimethylammonium propane (DODAP) and its structural counterparts; and
• DOTMA trimethylammonium structure
• DODAP 1,2-dioleyl-3-dimethylammonium propane
• cationic derivatives of cholesterol such as 3 ⁇ - [ ⁇ - ( ⁇ ', ⁇ '-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol) or other cationic cholesterol derivatives such as those described in US Pat. 283,185 and especially cholesteryl-3 ⁇ -carboxamidoethylenetri dimethylammonium iodide, the cholesteryl-3 ⁇ -carboxyamidoethylenamine, cholesteryl-3 ⁇ -oxysuccinamidoethylenetetrimethylammonium iodide and 3 ⁇ - [N- (polyethylenimine) carbamoyl] cholesterol.
• structural homologues is meant lipids which possess the characteristic structure of the reference lipid while differentiating therefrom by secondary modifications, especially at the level of the non-polar region, in particular the number of carbon atoms and double bonds of the fatty acid chains.
• fatty acids which are also found in neutral and anionic phospholipids, are, for example, dodecanoic or lauric acid (C12: 0), tetradecanoic or myristic acid (C14: 0), hexadecanoic or palmitic acid.
• C16: 0 cis-9-hexadecanoic or palmitoleic acid (C 16: 1), octadecanoic or stearic acid (C18: 0), cis-9-octadecanoic or oleic acid (C18: 1) cis, cis-9,12-octadecadienoic or linoleic acid (C 18: 2), cis-cis-6,9-octadecadienoic acid (C18: 2), cis-9,12 garlic acid, 15-octadecatrienoic or ⁇ -linolenic acid (C18: 3), cis-6,9,12-octadecatrienoic acid or ⁇ -linolenic acid (Cl 8: 3), eicosanoic acid or arachidic acid (C20: 0), cis-9-eicosenoic or gadoleic acid (C20: 1),
• a mixture of cationic lipid and neutral lipid is used.
• a mixture of DC-chol and DOPE especially in a DC-chol: DOPE molar ratio ranging from 10: 1 to 1: 10, advantageously from 4: 1 to 1: 4, of preferably about 3: 1 to 1: 3
• a mixture of ethyl-DOPC and of cholesterol in particular in a molar ratio ethyl-DOPC: cholesterol ranging from 10: 1 to 1: 10, advantageously from 4: 1 to 1: 4, preferably from about 3: 1 to 1: 3
• a mixture of ethyl-DOPC and DOPE especially in a molar ratio of ethyl-DOPC: DOPE ranging from 10: 1 to 1: 10, advantageously from 4: 1 to 1: 4, preferably from about 3: 1 at 1: 3.
• a dry lipid film is initially produced with all the compounds used in the composition of the liposomes.
• the lipid film is then reconstituted in an aqueous medium, in the presence of LOS, for example in a lipid: LOS molar ratio of from 100 to 500, advantageously from 100 to 400; preferably from 200 to 300; most preferably about 250.
• LOS molar ratio of from 100 to 500, advantageously from 100 to 400; preferably from 200 to 300; most preferably about 250.
• LOS molar ratio of from 100 to 500, advantageously from 100 to 400; preferably from 200 to 300; most preferably about 250.
• it is believed that the same molar ratio should not vary substantially in the final process of preparation of LOS liposomes.
• the reconstitution step in an aqueous medium leads to the spontaneous formation of multi-lamellar vesicles, the size of which is then homogenized by progressive reduction of the number of lamellae by extrusion, for example by means of an extruder by passages. of the lipid suspension under nitrogen pressure, through polycarbonate membranes with increasingly reduced pore diameters (0.8, 0.4, 0.2 ⁇ m). It is also possible to replace the extrusion process with another process using a detergent (surfactant) which disperses the lipids. This detergent is then removed by dialysis or adsorption on porous polystyrene microbeads particularly affine detergent (BioBeads). When the surfactant withdraws from the lipid dispersion, the lipids reorganize into a double layer.
• a detergent surfactant
• the liposomes are then purified in order to get rid of the non-detoxified LOS in free form.
• the LOS is advantageously in the form of a LOS-carrier polypeptide conjugate, especially when it is not in the form of OMVs or liposomes.
• the carrier polypeptide may be any polypeptide, oligopeptide or carrier protein in use in the field of conjugated vaccines; and in particular pertussis, diphtheria or tetanus toxoid, the mutant of the diphtheria toxin called CRM1 97, a bacterial OMP, a bacterial protein complex [for example N. meningitidis POMPC (outer-membrane protein C)], the exotoxin Pseudomonas, Haemophilus influenzae lipoprotein D, pneumolysin Streptococcus pneumoniae, Bordetella pertussis filamentous hemagglutinin, and N. meningitidis human transferrin receptor lipid B-subunit.
• POMPC outer-membrane protein C
• the reactive groups of the LOS involved during the conjugation are those of the inner core or lipid A. It may be, inter alia, the acid function of the KDO or a aldehyde generated following appropriate treatment on the disaccharide of lipid A.
• a phosphatase treatment generates an aldehyde on the structure of the second glucosamine of lipid A N. meningitidis (Brade H. (2002) J. Endotoxin Res 8 (4): 295 Mieszala et al., (2003) Carbohydrate Res 338: 167 and Cox et al., (2005) Vaccine 23 (5): 5054).
• the conjugation method makes use of (i) a bifunctional linker (linker) or (ii) a spacer and a linker.
• the LOS is activated with a bifunctional coupling agent (linker) of formula R1-A-R2 such that the radical R2 reacts with a reactive group of KDO or lipid A in order to obtain an activated LOS; then the activated LOS is conjugated with the polypeptide so that the substituent R1 reacts with a functional group carried by the polypeptide to obtain a conjugate.
• linker bifunctional coupling agent
• the LOS is derived with a spacer of formula R3 - B - R4 so that the radical R3 reacts with a reactive group of KDO or lipid A to obtain a derived LOS; then activating the derivatized LOS with a bifunctional coupling agent (linker) of the formula R 1 - A - R 2 such that the radical R 2 reacts with the radical R 4 to obtain a derivatized and activated LOS; finally, the derivatized and activated LOS is conjugated with the polypeptide such that the R1 radical reacts with a functional group carried by the polypeptide to obtain a conjugate.
• a bifunctional coupling agent linker
• the polypeptide is derived with a spacer of formula R3 - B - R4 so that the radical R4 reacts with a functional group carried by the polypeptide;
• LOS is activated with a bifunctional agent (linker) of formula R1-A-R2 such that R2 is reacted with a reactive moiety of KDO or lipid A to obtain activated LOS; then the activated LOS is conjugated with the derived polypeptide so that the R1 radical of the activated LOS reacts with the R3 radical of the derived polypeptide to obtain a conjugate.
• B may be a carbon chain, preferably carbonyl, alkyl or alkylene, for example C1 to C12.
• R3 and R4 may be independently a thiol or amine group or a residue carrying it, for example a hydrazide group Le., NH 2 -NH-CO-.
• Compounds which can be used as spacers are, for example, of formula NH 2 - B - NH 2, or preferably NH 2 - B - SH and NH 2 - B - S
• cysteamine eg diamines, diaminohexane, adipic acid dihydrazide (ADH), urea and cystamine.
• cysteine eg diamines, diaminohexane, adipic acid dihydrazide (ADH), urea and cystamine.
• ADH adipic acid dihydrazide
• A may be an aromatic or preferably substituted or unsubstituted aliphatic chain, which advantageously comprises from 1 to 12 carbon atoms; preferably 3 to 8 carbon atoms.
• A may be a C2 to C8 alkylene, a phenylene, a C7 to C12 aralkylene, a C2 to C8 alkyl, a phenyl, a C7 to C12 aralkyl, C6 alkanoyloxy or a benzylcarbonyloxy, substituted or unsubstituted.
• Radical R2 is the functional grouping of the linker that creates the link to the derived LOS or LOS.
• R2 is a functional group that can react with a carboxyl, hydroxyl, aldehyde or amine group. If the linker is to react with a carboxyl or aldehyde hydroxyl group, R2 is preferably an amino group or a residue carrying it, for example a hydrazide group Le., NH 2 -NH-CO-.
• R2 is preferably a carboxyl, succinimidyl (eg, N-hydroxy succinimidyl) or sulfosuccinimidyl (eg, N-hydroxy sulfosuccinimidyl) group.
• compounds which can be used as linker can be chosen from adipic acid dihydrazide (ADH); sulfosuccinimidyl-6- (3- [2-pyridyldithio] propionamido) hexanoate (Sulfo-LC-SPDP); succinimidyl-6- (3- [2-pyridyldithio] propionamido) -hexanoate (LC-SPDP); N-succinimidyl-S-acetyl thioacetate (SATA); N-Succinimidyl-3- (2-pyridyl dithio) propionate (SPDP), succinimidyl acetyl thiopiopionate (SATP); succinimidyl-4- (N-maleimido methyl) cyclohexane-1-carboxylate (SMCC); the maleimido benzoyl-N-hydroxy succinimide ester (MBS); N-succin
• the acid function of KDO to derive LOS with cysteamine or cysteine in the presence of EDAC.
• the thiol function thus introduced is reacted with the maleimide function of a homobifunctional linker, such as bis maleimido hexane; or heterobifunctional, such as GMBS.
• the maleimide function thus introduced is then reacted with the thiol functions of the polypeptide.
• the succinimidyl function of the derivatized and activated LOS is reacted with the amino functions of the polypeptide.
• the LOS and the polypeptide may be conjugated to one another in a LOS: polypeptide molar ratio of from 10 "1 to 10 2 , advantageously from 1 to 10 2 , preferably from 1 to 50, most preferably about 20.
• N. meningitidis TbpB The N meningitidis TbpB, as naturally produced by N. meningitidis, is a lipoprotein. Nevertheless, it can advantageously be produced recombinantly in an expression system which makes it possible, in particular, to ensure lipidation of the polypeptide within the body responsible for expression.
• lipidated TbpB is a recombinant TbpB - i.e., recombinantly produced, eg in a heterologous expression system.
• An expression system typically employs an expression cassette and a prokaryotic or eukaryotic (yeast) host cell.
• the expression cassette codes for a precursor of TbpB (also called pro-TbpB).
• This precursor consists of a signal sequence characteristic of a lipoprotein and the sequence of the mature protein having a cysteine residue in the N-terminal position.
• the three amino acids at the C-terminal position of the signal sequence as well as the Cysteine residue at the N-terminal position of the mature sequence constitute the cleavage site (also called lipobox):
• This lipobox typically has the following sequence: Leu-Ser / Ala - Ala / Gly - Cys.
• a typical signal sequence is that of Lpp lipoprotein E.
• the polynucleotide sequence encoding the amino acid sequence of TbpB is fused at 5 'to a suitable signal sequence.
• N. meningitidis TbpB is like any protein, defined by an amino acid sequence. Within the species, this amino acid sequence may exhibit some degree of variability without affecting the biological function of the lipoprotein. We then speak of "allelic variant". At a TbpB of N. meningitidis, correspond a multiplicity of sequences presenting between them a certain degree of identity, each of the sequences coming from a particular strain, one being the allelic variant of the other.
• the present invention is not limited to a lipidated TbpB of wild form. Indeed, it may be not only a wild form, but also a form mutated by addition, substitution or deletion of one or more amino acids.
• a lipid fragment of N. meningitidis TbpB is advantageously the N-terminal lipid fragment of TbpB.
• the "polypeptide" part of the fragment may advantageously comprise one or more T-helper epitopes - that is to say, epitopes capable of being recognized by T helper cells - and activate them.
• Isotype I is expressed in the ST-I clonal complex I and II in the ST-8, ST-18, ST-32, ST-41/44 clonal complexes (Harrison et al., BMC Microbiol 2008, 8). : 66).
• Strains B16B6 (serogroup B) and FAMl 8 (serogroup C) are representatives of isotype I; strains M982, BZ83 and 8680 are representatives of the IL isotype
• the lipidated TbpB is that of a N. meningitidis strain of isotype I or isotype II, preferably isotype II.
• a composition according to the invention comprises the lipidated TbpB of an isotype I strain and an IL isotype strain.
• the lipidated TbpB of a strain of N. meningitidis of isotype I can be that of strain B16B6; and the lipidated TbpB of a N. meningitidis strain of isotype II may be that of strain M982.
• the lipidated and purified TbpB exhibits a certain degree of insolubility in a purely aqueous condition. Consequently, it should be placed in conditions favoring its solubility.
• Those skilled in the art master the techniques for making a lipoprotein soluble. For example, it is possible to use a detergent during the purification of the lipoprotein, to obtain a preparation of a purified lipoprotein soluble in the presence of detergent. The amount of detergent remaining in the final preparation will be controlled so that it is just necessary to maintain the purified lipoprotein in soluble form. Alternatively, it is possible to completely remove the detergent used during purification and then add another product also having the ability to maintain soluble form, the purified lipoprotein.
• the TbpBs can be incorporated with LOS into liposomes or be in a simple mixture with liposomes LOS (LOS formulated into liposomes: this latter embodiment being however preferred.
• liposomes proteoliposomes
• LOS and lipidated TbpB the liposomes useful for this purpose are the same as those previously described for the formulation of LOS alone.
• One way to carry out this formulation is to formulate together in liposomes, LOS and lipidated TbpB, for example by reconstituting a lipid film in an aqueous medium in the presence of LOS and lipidated TbpB, especially in: a lipid: LOS molar ratio from 100 to 500, advantageously from 100 to 400; preferably from 200 to 300; most preferably about 250; and / or a liposomal LOS: TbpB molar ratio of from 10 2 to 10 3 , advantageously from 10 -1 to 10 2 , preferably from 1 to 50, most preferably from 15 to 30, e.g. About 20.
• the liposomes are then purified in order to get rid of LOS in free form.
• the mixture can be used as is for vaccine purposes or the liposomes can be further purified in order to get rid of the free lipid Tbpb.
• the liposomes are completely purified, it is possible to add free lipid Tbpb, especially in a defined amount.
• a vaccine / pharmaceutical composition according to the invention is particularly useful for treating or preventing a N. meningitidis infection, such as N. meningitidis meningitis, meningococcemia and complications that can derive from it such as purpura fulminans and septic shock. ; as well as N. meningitidis arthritis and pericarditis.
• a therapeutically or prophylactically effective amount of the essential components of the vaccine, LOS and TbpB is combined with a pharmaceutically acceptable carrier or diluent.
• a pharmaceutically acceptable carrier or diluent may further comprise a pharmaceutically acceptable adjuvant.
• the (the) LOS is (are) advantageously formulated (s) in liposomes.
• the amounts of LOS and rTbpB per vaccine dose that are immunogenically, prophylactically or therapeutically effective depend on certain parameters including the individual being treated (adult, adolescent, child or infant), the route of administration and its frequency.
• the amount of LOS per dose may be between 5 and 500 ⁇ g, advantageously between 10 and 200 ⁇ g, preferably between 20 and 100 ⁇ g, very preferably between 20 and 80 ⁇ g or between 20 and 60 ⁇ g, terminals included.
• the amount of TbpB lipid per dose may be between 5 and 500 ⁇ g, advantageously between 10 and 200 ⁇ g, preferably between 20 and 100 ⁇ g, most preferably between 20 and 80 ⁇ g or between 20 and 60 ⁇ g, terminals. included.
• the molar ratio LOS: TbpB lipid is 10 " to 10, advantageously 10 " 1 to 10 2 ; preferably from 1 to 50; most preferably from 15 to 30 or about 20.
• the liposized LOS: TbpB molar ratio can be typically 20 or approximately 25, depending on whether the isotype of TbpB is I or IL
• dose should be understood to mean a volume of vaccine administered to an individual at one time - that is, at a time T.
• Conventional doses are in the order of one milliliter, for example 0.5, 1 or 1.5 ml; the final choice depends on certain parameters, in particular the age and status of the recipient.
• An individual may receive a divided dose at multiple injection sites on the same day.
• the dose may be single or if necessary, the individual may also receive several doses at a certain interval time - interval time that can be determined by those skilled in the art.
• a composition according to the invention may be administered by any conventional route used in the field of the art, eg in the field of vaccines, in particular enterally or parenterally.
• the administration may take place in single or repeated doses with a certain interval time.
• the route of administration varies according to various parameters, for example of the individual treated (condition, age, etc.).
• the invention also relates to:
• a method of preventing and / or treating a JV-induced infection meningitidis, according to which a prophylactically or therapeutically effective amount of a composition according to the invention is administered to an individual in need of such treatment.
• Strain C708 is cultured in Brain Heart Infusion (BHI) medium at 37 ° C. under a 10% CO 2 atmosphere.
• BHI Brain Heart Infusion
• the bacterial layer is harvested in liquid BHI medium supplemented with 5 mM MgCl 2 to obtain a bacterial suspension at 10 9 cfu / mL (cfu: colony-forming-unit or colony forming unit).
• the transformation medium is incubated for 30 min at 37 ° C., 10% CO 2 .
• the labeling of the probe is obtained by PCR amplification using the Ready-to-Go PCR Beads kit (GE Healthcare); then the labeled probe is purified on a ProbeQuant G50 Microcolumn column (GE Healthcare).
• the membranes to be hybridized are placed three times in 50 ml of Rapid Hyb buffer (GE Healthcare), 15 min at 65 ° C., with slow stirring, for pre-hybridization.
• the probe, labeled and denatured beforehand for 2 min at 95 ° C., is added to the membranes. Finally, the concentration of the probe is 5 ng / ml. Hybridization is allowed to continue for 2 hours at 65 ° C with slow stirring.
• the membranes are then subjected to successive washes by proceeding as follows:
• the 5 'end of the oligonucleotide is labeled in the following reaction medium (the amounts indicated are those corresponding to the hybridization of an amount of oligonucleotide necessary for the hybridization of 3 membranes in a dish):
• Oligonucleotide free 5'-OH 3 ⁇ l maximum is lO pmoles
• T4 kinase (10 U / ⁇ l) l ⁇ l or 10 U
• reaction medium is incubated for 30 min at 37 ° C. Then the T4 kinase is inactivated by heating for 10 min at 70 ° C.
• the membranes to be hybridized are placed three times in 60 ml of Rapid Hyb buffer (GE Healthcare), 15 min at 48 ° C., with slow stirring, for pre-hybridization.
• the prehybridization buffer is removed and replaced with 50 ml of the following hybridization buffer: 5 X SSC, 5 X Denhardt's solution, 0.5% SDS (wt / vol) and 100 ⁇ g / ml sperm DNA. salmon 10 mg / ml sonicated and denatured for 5 min at 100 ° C.
• the labeled oligonucleotide (10 ⁇ l) is added to the membranes.
• the hybridization is allowed to proceed overnight at a temperature of 5 ° C below the Tm of the oligonucleotide, with slow stirring.
• the membranes are then subjected to successive washings in the following order:
• Strain C708 has a truncated Ipu gene. To modify it so that the LOS carries a PEA substituent at position 03, it is chosen to replace, by homologous recombination, the Ipu gene truncated by the complete Ipu gene (full-length) of the N. meningitidis FAM1 strain. 8 serogroup (strain available in research laboratories, worldwide). The resulting strain will be more conveniently referred to as C708 Ipu FL.
• the pair of primers is the following (couple n ° 1):
• the PCR product on the one hand and the plasmid pUC19 on the other hand were double digested with EcoRI and PstI for 2 hrs at 37 ° C. 10 units of each enzyme per ⁇ g of DNA were used in R ⁇ act2 buffer (Invitrogen). The PCR fragment was then inserted into the linearized pUC19 vector. Ligations were performed in a final volume of 20 ⁇ l with 50 ng of vector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 ⁇ l of 10 mM ATP (Invitrogen) for 16 hours at 16 ° C. The ligase was then inactivated by heating for 10 min at 65 ° C.
• the vector thus obtained was transferred by the electroporation technique into a strain of E. coli. coli XLl blue MRF resistant to kanamycin and made electro competent.
• the parameters monitored for electroporation are as follows: Capacitance: 500 ⁇ FD; Resistance: 200 ohms; Voltage: 1700 volts.
• the selection of the transformed clones was done by spreading on LB ampicillin plates
• plasmid pM1222 100 ⁇ g / ml. Authentication of 10 of the 50 positive clones was performed by PCR amplification. 100% of the clones had the expected profile. Finally, the gene lpt2 in a plasmid of one of these clones (plasmid pM1222) was verified by sequencing.
• the bacteria were plated on 17 140 mm petri dishes at a theoretical concentration of 30,000 cfu per dish; or 510,000 cfu (colonies-forming-units), and then placed overnight at 37 ° C. The dishes were then placed for 30 minutes at + 4 ° C.
• the number of control plates made it possible to estimate the number of cfu at 27,000 per box for the mutant.
• the selection of the positive clones was done by hybridization of the DNA fixed on the membranes with a DNA probe labeled with 33 P dCTP corresponding to the truncated part of the Ipt3 gene, thus present only in the recombinant clones.
• the pair of primers is the following (couple n ° 2):
• thermocycler program is as follows:
• part of the area taken around the positive clones was kept in freezing medium (M199 medium, 20% fetal calf serum, 10% glycerol) and the other part was used to PCR authentication.
• freezing medium M199 medium, 20% fetal calf serum, 10% glycerol
• each of the samples was first taken up with 80 .mu.l of BHI broth, so as to spread 30 .mu.l of this suspension, in mini-tablecloth on a BHI box.
• thermocycler program is as follows:
• the next step was to isolate a pure clone.
• one of the heterogeneous positive clones was spread in isolated cfu and several of these cfu (40) were analyzed by PCR, with the pairs of primers 1 or 2.
• Each cfu was resuspended in 100 ⁇ l of nuclease-free water, 30 ⁇ l were deposited on a BHI dish and the remaining 70 ⁇ l were lysed for 5 min at 95 ° C. and the supernatant, which serves as a template for the PCR reaction. , was taken after centrifugation.
• the PCRs were performed with Platinum® Taq High Fidelity (Invitrogen) as already described for the amplification of the lpt1 probe.
• the hybridization temperature was 54 ° C. After the reaction, 1/10 of the PCR products were deposited on agarose gel for verification. Five of the 40 clones were found to be pure clones.
• the mini sheet pure clones was resumed in freezing medium, aliquoted in 100 .mu.l and stored at -70 0 C. The purity and identity of this frozen material were validated. 3. Construction of a strain of N. meningitidis expressing an LOS whose ⁇ chain is that of an L6 L6 immunotype and comprising only in the O3 position of the heptose II residue (hep II) of the inner core, a phosphoethanolamine substituent ( PEA)
• the strain of N. meningitidis C708 (Lpt2) FL obtained as previously described is used as a starting strain.
• the objective is to inactivate the Ipt ⁇ gene of this strain by deletion of a central part of the gene.
• the pair of primers is the following (couple n ° 3):
• thermocycler program is as follows:
• the PCR product on the one hand and the plasmid pUC19 on the other hand were double digested with EcoRI and PstI for 2 hrs at 37 ° C. 10 units of each enzyme per ⁇ g of DNA were used in R ⁇ act2 buffer (Invitrogen).
• the PCR fragment was then inserted into the linearized pUC19 vector.
• Ligations were performed in a final volume of 20 ⁇ l with 50 ng of vector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 ⁇ l of 10 mM ATP (Invitrogen) for 16 hours at 16 ° C.
• the ligase was then inactivated by heating for 10 min at 65 ° C.
• the vector thus obtained was transferred by the electroporation technique into a strain of E. coli. coli XLl blue MRF resistant to kanamycin and made electro competent.
• the parameters monitored for electroporation are as follows: Capacitance: 500 ⁇ FD; Resistance: 200 ohms; Voltage: 1700 volts.
• the selection of transformed clones was made by plating on LB ampicillin 100 ⁇ g / ml dishes.
• the authentication of the positive clones was carried out by Ndel enzymatic digestion after extraction of the DNA by miniprep. Of 20 clones analyzed, 6 had the expected profile.
• the recombinant plasmid of the selected clone was named pM1223.
• thermocycler program is as follows:
• the PCR product was digested with BamHI at the rate of 10 U of enzyme per ⁇ g of DNA. Once digested, it was purified by electroelution followed by phenol-chloroform extraction.
• the ligation of the vector on itself was performed in a final volume of 20 .mu.l with 0.5 U of T4 DNA ligase (Invitrogen) and 1 .mu.l of 10 mM ATP (Invitrogen) for 16 hours at 16.degree.
• the ligase was then inactivated by heating for 10 min at 65 ° C.
• the last step was to transfer the vector thus ligated into an Escherichia coli strain as described for pM1222.
• Authentication of positive clones was carried out by Ndel-PstI enzymatic digestion after extraction of the DNA by miniprep. Of the 4 clones analyzed, 100% have the expected profile.
• the recombinant plasmid of the selected positive clone was renamed pM1224 and this clone was stored in glycerol at -70 ° C. The presence in the plasmid pM1224 of a Igt ⁇ gene deleted from its central part was verified by sequencing.
• strain C708 was done following the technique described in section A.1.1.
• the bacteria were plated on 16 140 mm petri dishes at a theoretical concentration of 50,000 cfu per dish; that is 800 000 cfu. The dishes were placed overnight at 37 ° C and then placed 30 min at + 4 ° C.
• the recombination event that is to say the replacement of the Ipt ⁇ FL gene by the Ipt ⁇ TR gene, was detected after membrane transfer of clones and hybridization with a labeled probe according to the methods described in sections A.1.2. . and A.1.4.
• Clones transferred to membranes are subjected to lysis and washing steps.
• the DNA is fixed on the membranes by placing the latter for 2 hours at 80 ° C.
• the samples were first taken up in 80 .mu.l of BHI broth, so as to spread, in a mini layer on a BHI box, 30 .mu.l of each suspension.
• the remaining volume was centrifuged for 5 min at 6000 rpm, and then the pellet was taken up in 50 ⁇ l of nuclease-free water.
• the seeds were lysed for 5 min at 95 ° C and the supernatant, which serves as template for the PCR reaction, was removed after centrifugation.
• PCR amplification was performed with Platinum® Taq High Fidelity (Invitrogen) and the following pair of primers
• thermocycler program is as follows:
• Each cfu was resuspended in 50 ⁇ l of nuclease-free water, 20 ⁇ l were deposited on a BHI dish and the remaining 30 ⁇ l were lysed for 5 min at 95 ° C. and the supernatant, which serves as a template for the PCR reaction. , was taken after centrifugation.
• the PCRs were performed with Platinum® Taq High Fidelity (Invitrogen) as already described for the screening of positive spot samples.
• the mini sheet pure positive clone was resumed in freezing medium, aliquoted in 100 .mu.l and stored at -7O 0 C. The purity and identity of this frozen material were validated.
• the N. meningitidis strain C708 lpt2) FL Ipt ⁇ TR obtained as previously described in section A.3 is used as a starting strain.
• the objective is to inactivate the igtA gene of this strain by deletion of a central part of the gene.
• PCR amplification polymerase chain reaction
• meningitidis MC58 serogroup B NMB 1929 gene
• the pair of primers is as follows:
• the PCR product obtained in 4.1. on the one hand and plasmid pUC19 on the other hand were double digested with EcoRI and PstI for 2 hours at 37 ° C. Ten units of each enzyme per ⁇ g of DNA were used in REact2 buffer (Invitrogen). The PCR fragment was then inserted into the linearized pUC19 vector. The ligations were carried out in a final volume of 20 ⁇ l with 50 ng of vector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 ⁇ l of 10 mM ATP (Invitrogen) for 16 hours at 16 ° C. The ligase was then inactivated by heating for 10 min at 65 ° C.
• the vector thus obtained was transferred by the electroporation technique into the E. coli strain. coli XL1 blue MRF resistant to kanamycin and made electro-competent.
• the parameters monitored for electroporation are as follows: Capacitance: 500 ⁇ FD; Resistance: 200 ohms; Voltage: 1700 volts.
• the selection of transformed clones was made by plating on LB ampicillin 100 ⁇ g / ml dishes.
• the authentication of positive clones was performed by enzymatic digestion after extraction of the DNA by miniprep. 1/5 of the clones analyzed showed the expected enzyme digestion profile.
• PCR amplification polymerase chain reaction of the erythromycin resistance gene (erm)
• the PCR amplification of the erythromycin (erm) cassette from pMGC10 was done with primers to integrate the BamHI-XbaI restriction sites.
• the pair of primers used is the following:
• thermocycler program was as follows: Initial denaturation: 95 0 C for 2 mn 30 cycles of:
• the last step was to transfer the vector into the strain of E. coli XLl blue MRF resistant to kanamycin and made electro competent.
• the authentication of positive clones was performed by enzymatic digestion after extraction of the DNA by miniprep. 4.5. Construction of plasmid pUC19 IgtA :: erm
• PCR product Erm on the one hand and the plasmid pUC19 IgtA TR resulting from the reverse PCR on the other hand were each subjected to double digestion with BamHI and XbaI under the following conditions:
• the digestion products were then completely deposited on a 0.8% agarose gel, and after migration, the bands were cut to be electroeluted (that of the plasmid is 3.2 kbs).
• the linearized plasmid and the digested PCR product were ligated together as previously described.
• the product of the ligation was used to transform, as previously described, E. coli strain XL1 Blue MRF resistant to kanamycin and rendered electro-competent.
• the recombinant clones were analyzed by enzymatic digestion. 4/11 clones analyzed showed the expected enzyme digestion profile.
• the expression strains are the E. coli strains BL21 respectively containing the plasmid pTG9219 / pTG9216
• These plasmids comprise in particular a kanamycin selection and the polynucleotide encoding the rTbpB of N. meningitidis strain M982 (pTG9219) or B16B6 (pTG9216) (the sequences are as described in patent EP 586,266), fused to the signal sequence RIpB (Real lipoprotein B) from E. coli and placed under the control of the promoter arabinose ( ⁇ raB).
• This preculture is used to seed a fermentor containing TGM16 medium (yeast extract 9g / L, K 2 SO 4 0.795 g / L, K 2 HPO 4 3.15 g / L, NaCl 0.75 g / L, CaCl 2 , 2H 2 O 0.005 g / L, FeCl 3 , 6H 2 O 0.021 g / L, MgSO 4 JH 2 O 0.69 g / L, casein acid hydrolyzate without salt 37.5 g / L, casein acid hydrolyzate without salt 37.5 g / L, casein acid hydrolyzate without salt 37.5 g / L, casein acid hydrolyzate without salt 37.5 g / L, casein acid hydrolyzate without salt 37.5 g / L, casein acid hydrolyzate without salt 37.5 g / L, casein acid hydrolyzate without salt 37.5 g / L, casein acid hydrolyzate without salt 37.5 g / L)
• the culture is continued at 37 ° C. with stirring, at a pressure of 100 mbar and under an air supply of 1 L / min / L of culture, readjusting the concentration of glycerol at 20 g / L over time ( eg at OD 60O of 15 + 2).
• the concentration of glycerol at 20 g / L over time (eg at OD 60O of 15 + 2).
• the OD 6 oo is between 21 and 27
• the expression of rTbpB is induced by the addition of arabinose to obtain a final concentration of 10 g / l.
• the culture is stopped while cooling to around 10 ° C.
• the bacterial pellets are recovered by centrifugation and stored in the cold. 1.2. Purification
• a bacterial pellet equivalent to one liter of culture (about 72 g of seeds, wet weight) is thawed at a temperature of 20 ° C. +/- 5 ° C.
• the thawed (or partially thawed) germs are resuspended by 800 ml. room temperature solution of 50 mM Tris HCl, 5 mM EDTA, pH 8.0.
• 9 protease inhibitor pellets are immediately added (7 pellets of Complete Mini, EDTA free, ROCHE ref 11836170001 + two pellets of Complete, EDTA free, ROCHE ref 11836170001).
• a second extraction is carried out: homogenization with Turrax in 800 ml of 50 mM Tris-HCl buffer, 5 mM EDTA pH 8.0 and stirring for 30 min. MgCl 2 (8 ml of a molar solution) is added. Incubation is continued for 10 minutes. The suspension is centrifuged 15,000 g for 1 hr 30. Bacterial Lyse
• the pellet is resuspended with 1400 ml of 50 mM Tris HCl supplemented with 4 pellets of protease inhibitor and 8 ⁇ l of Benzonase.
• the solution is homogenized with Turrax 15 seconds. Lysis is carried out at + 4 ° C. for 30 minutes thanks to the addition of 14 ml (10 mg / ml final) of lysozyme at 100 mg / ml in 25 mM Na acetate, 50% glycerol.
• the suspension is centrifuged at 30,000 g for 30 minutes (pellet C2 containing the protein, vs supernatant S2 containing the rTbpB contaminants).
• the pellet containing the membranes can be frozen at this stage.
• the C2 lysis pellet is taken up in 50 mM Tris HCl (1100 ml). After homogenization, (Turrax 15 seconds), it is washed for one hour at + 4 ° C. As before, centrifugation at 30,000 g for 30 minutes is used, the pellet (C3, vs supernatant S3) is frozen at -45 ° C.
• the 50 mM Tris HCl buffer makes it possible to eliminate a small amount of protein (supernatant S3) and solubilizes only a very small amount of rTbpB.
• the C3 pellet is taken up in 50 mM Tris HCl buffer, 8 M urea pH 8.0 (800 ml).
• This buffer makes it possible to eliminate a part of the contaminating proteins without solubilizing the membranes containing the rTbpB.
• the solution is then stirred for one hour at +4 ° C.
• centrifugation is carried out at 30,000 g for 30 minutes, which makes it possible to obtain a pellet of membranes that can be frozen.
• the supernatant S4 (about 845 ml) is deposited at a rate of 6 ml / minute.
• the direct eluate (part that does not cling to the column during sample deposition) contains the protein of interest rTbpB.
• This eluate (1150 ml) is removed, then dialyzed at + 4 ° C. (for 6 days) against 6 liters of 50 mM Tris-HCl buffer, 2 M urea, 1% eluent, pH 7.5 in order to lower to 1 mM. EDTA concentration and eliminate NaCl.
• a K50 column of 490 ml of fresh Q Sepharose Fast Flow gel is equilibrated in 50 mM Tris HCl buffer, 2 M urea, 1% Elugent pH 7.5.
• the dialyzed solution (1080 ml) is deposited on the column (flow rate 6 ml / minute); then 5 saline elution steps in this same buffer are performed: 20 mM, 50 mM, 100 mM, 250 mM and 1 M NaCl (working rate 6 ml / minute).
• the rTbpB protein is eluted from the column at two salt concentrations (50 mM and 100 mM).
• the 50 mM elution fraction is the fraction of interest, the rTbpB protein being the purest and most important (2.6 times more protein than in the 100 mM NaCl fraction).
• the pH of the fraction corresponding to the 50 mM NaCl elution peak is lowered with magnetic stirring to pH 5.5 by addition of 1.7 N acetic acid.
• the solution (860 ml) is placed in dialysis against 5 liters of 10 mM sodium acetate buffer, 1 M urea, 0.2% eluent, pH 5.5 (24 hrs at + 40 ° C.) then against 4 liters of 10 mM sodium acetate buffer, 1 M urea, 0.2 eluent %, pH 5.5 (17 hrs at + 4 ° C).
• a K50 column of 100 ml SP Sepharose Fast Flow gel (Ge Healthcare, ref 17-0729-01) is equilibrated in 10 mM sodium acetate buffer, 1 M urea, 0.2% eluent, pH 5.5.
• the dialyzed protein solution (850 ml) is deposited on the column (flow rate 6 ml / minute). Then, five saline elution levels are carried out: 50 mM, 100 mM, 250 mM, 500 mM and 1 M NaCl, in buffer cited above.
• the rTbpB protein is eluted exclusively in the 250 mM NaCl fraction and the low molecular weight contaminants are essentially removed in the direct eluate (40%). About 35 mg of purified rTbpB M982 are thus obtained and a little less for rTbpB B16B6.
• the fractions corresponding to the 250 mM elution peak of the SPI column are combined (volume 274 ml).
• the pH of the solution is raised to pH 7.3 by adding, with stirring, approximately 800 ⁇ l of 0.5 N NaOH.
• the solution is placed in dialysis at + 40 ° C. (Spectra Por 1: cut-off point 6-8000 D) against two baths of 10 liters of PBS Elugent 0.2% pH 7.1 (66 hrs and 22 hrs).
• the dialysate is concentrated to a volume of 21.1 ml by diafiltration frontal concentration on 30 kD Amicon membrane in PBS (ref PBTK06510).
• the solution is then filtered aseptically onto a Durapore Millex 0.22 ⁇ m filter (Ref Millipore SLGV 033RS).
• the purified rTbpB protein batch obtained is frozen at -80 ° C.
• the protein concentration is 1642 ⁇ g / ml. 1.3. Preparation of the rTbpB for injection
• the rTbpB solution obtained in section 1.2. contains 2 mg / mL of Elugent TM.
• the amount of Bio-Beads TM to be used is determined based on the amount of Elugent TM to be removed.
• OD O oo nm After about 7 hrs (OD O oo nm of about 3) was added Mueller-Hinton medium at 440 g / hr. When the glucose concentration is less than 5 g / L the fermentation is stopped. OD O oo nm finish is commonly between 20 and 40. Cells were harvested by centrifugation and pellets frozen at -35 ° C.
• the pellets are thawed and suspended with 3 volumes of phenol 4.5% (vol./vol.) Shaking vigorously for 4 hours at about 5 ° C. LOS is extracted by phenol treatment.
• the bacterial suspension is heated at 65 ° C. and then mixed vol./vol. with 90% phenol with vigorous stirring for 50-70 min at 65 ° C.
• the suspension is then cooled to ambient temperature and then centrifuged for 20 min at 11,000 g.
• the aqueous phase is removed and stored while the phenolic phase and the interphase are harvested for a second extraction.
• the phenolic phase and the interphase are heated at 65 ° C. and then mixed with a volume of water equivalent to that of the aqueous phase previously removed, while stirring vigorously for 50-70 min at 65 ° C.
• the suspension is then cooled to room temperature. room temperature and then centrifuged for 20 min at 11000 g.
• the aqueous phase is removed and preserved while the phenolic phase and the interphase are harvested to be subjected to a third extraction identical to the second.
• the three aqueous phases are dialysed separately against 40 L of water each. Then the dialysates are gathered together. To 9 volumes of dialysate is added a volume of 20 mM Tris, 2 mM MgCl 2 . The pH is adjusted to 8.0 ⁇ 0.2 with 4N sodium hydroxide. Two hundred and fifty international units of DNAse are added per gram of pellet. The pH is adjusted to 6.8 + 0.2. The preparation is placed at 37 ° C for about 2 hours with magnetic stirring; then subjected to 0.22 ⁇ m membrane filtration. The filtrate was purified by passage through a column of Sephacryl S-300 (5.0 x 90 cm; Pharmacia TM). The LOS-containing fractions are pooled together and the MgCl 2 concentration is raised to 0.5M by adding MgCl 2 , 6H 2 O powder with stirring.
• the suspension is subjected to gel filtration as previously described.
• the fractions containing the LOS are pooled together and sterilized by filtration (0.8-0.22 ⁇ m) and stored at 5 ⁇ 2 ° C.
• LOS liposomes are prepared by detergent dialysis.
• the lipids EOPC: DOPE
• the lipids are put into lipid film form and taken up in 10 mM Tris buffer, then dispersed in the presence of 100 mM octyl ⁇ -D-glucopyranoside (OG) (Sigma-Aldrich ref O8001) and sterile filtered.
• OG octyl ⁇ -D-glucopyranoside
• LOS in 100mM OG is added sterilely.
• the lipid / LOS / OG mixture is then dialyzed against 10 mM Tris buffer to remove the OG and form the liposomes. Protocol
• a lipid preparation of chloroform lipids that will be used for the manufacture of liposomes A dry film is obtained by complete evaporation of the chloroform.
• Dry film of 1,2 dioleyl-OT-glycero-3-ethylphosphochorine (EDOPC or ethyl-DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a molar ratio EDOPC: DOPE of 3 to 2 is obtained by mixing 12.633 mL of a solution of EDOPC (Avanti Polar Lipids ref 890704) to 20 mg / ml in chloroform and 7.367 mL of a solution of DOPE (Avanti Polar Lipids ref 850725) to 20 mg / ml in chloroform and evaporating the chloroform until complete disappearance.
• EDOPC 1,2 dioleyl-OT-glycero-3-ethylphosphochorine
• DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
• the dry film is taken up in 30 ml of 10 mM Tris buffer pH 7.0 to obtain a suspension containing 13.333 mg of lipids / ml (8.42 mg / ml of EDOPC and 4.91 mg / ml of DOPE). The suspension is stirred for 1 hr at room temperature and then sonicated for 5 min in a bath.
• a composition is prepared under sterile conditions by bringing together LOS and lipids in a lipid: LOS molar ratio of 250 (0.160 mg / ml of LOS, 9.412 mg / ml of lipids and 100 mM of OG). 40 ml of such a composition results from the mixture of the following preparations:
• the suspension After stirring for one hour at room temperature, the suspension is transferred sterilely into 4 sterile 10 ml dialysis cassettes. Each cassette is dialyzed 3 times (24 hrs - 24 hrs - 72 hrs) against 200 volumes of 10 mM Tris pH 7.0 or 2 L. The liposomes are recovered under sterile conditions. The volume increase after dialysis is approximately 30%.
• merthiolate and NaCl are added to obtain a liposome preparation in 10 mM Tris, 150 mM NaCl, pH 7.0, 0.001% Merthiolate which contains in fine approximately 110 ⁇ g / ml of LOS and 7 mg / ml of lipids. of which approximately 4.5 mg / ml EDOPC and approximately 2.5 mg / ml DOPE (theoretical concentrations).
• the liposomes are adjusted to the required concentration of LOS (especially for immunogenicity tests) in 10 mM Tris 150 mM NaCl pH 7.4. The concentration of merthiolate is maintained at 0.001%. 4. Preparation of a mixture liposomes [LOS] + rTbpB
• RTbpB was mixed in PBS (section B.1.3.) With liposomes [LOS] (section B.3.) In a ratio weight: weight rTbpB: LOS equal to 1. Then 10 mM Tris buffer was added, NaCl 150 mM, pH 7.4 to obtain a preparation in which each of the components (rTbpB and LOS) is concentrated at 80 ⁇ g / ml. The concentration of merthiolate is maintained at 0.001%.
• the rabbits of each group receive in a volume of 0.5 ml divided into 2 concomitant intramuscular injections in the legs, at OJ, J21 and J42: Group A: 40 ⁇ g of liposomes [LOS ⁇ chain L8, PEA-3, PEA-6] and 40 ⁇ g rTbpB M982, in 10 mM Tris buffer, 150 mM NaCl pH 7.4; Group B: 40 ⁇ g of liposomes [LOS ⁇ L8 chain, PEA-3, PEA-6] and 40 ⁇ g rTbpB
• Group C 40 ⁇ g of liposomes [LOS ⁇ L8 chain, PEA-3] and 40 ⁇ g rTbpB M982, in 10 mM Tris buffer 150 mM NaCl pH 7.4;
• Group D 40 ⁇ g of liposomes [LOS ⁇ L8 chain, PEA-3, PEA-6] in 10 mM Tris buffer 150 mM NaCl pH 7.4;
• Group E 40 ⁇ g rTbpB M982 and 40 ⁇ g of liposomes without LOS in 10 mM Tris buffer 150 mM NaCl, 0.5% Tween pH 7.0;
• Group F 40 ⁇ g rTbpB B 16B6 and 40 ⁇ g of liposomes without LOS in Tris buffer 10 mM NaCl 150 mM, 0.5% Tween pH 7.0;
• Group G 40 ⁇ g of liposomes [LOS ⁇ L8 chain, PEA-3] in 10 mM Tris buffer 150 mM NaCl pH 7.4;
• Group H Tris buffer 10 mM NaCl 150 mM pH 7.4
• the IgGs were purified by affinity chromatography using the HiTrap rProtein A FF column (GE Healthcare / Amersham Biosciences) according to the supplier's recommendations.
• the bactericidal activity of the purified IgGs was tested against the strains mentioned in Table II below.
• a culture of JV strains is carried out. meningitidis in BHI medium supplemented or not for 2 h 30 with Desferal 50 ⁇ M (iron chelating agent in free form, which allows the expression of TbpB).
• Bacteria + inactivated rabbit baby complement + test serum (serum control).
• the bactericidal titer is expressed as being the reverse of the dilution giving 50% of bacterial death as compared with the complement control.
• Bacterium test 34 strains of N. meningitidis were tested in cross-bacterial bacteria. Their names appear in Table II below.
• the bactericidal activity of the purified IgGs is expressed in "fold increase".
• the "fold increase” is the bactericidal titre ratio of the purified IgGs of the interest group: bactericidal titre of the corresponding negative control group.
• the fold increase seroconversion rate measures the increase in the bactericidal titre. It is considered that bactericidal activity is significant when a factor x 8 is observed between the interest group and the corresponding negative control group ("fold increase" greater than or equal to x 8).
• the purified IgGs from the negative control immunization group show no bactericidal activity against any of the strains ("fold increase" less than x4).
• Purified IgGs from immunization groups D and G show cross-bacterial disease of less interest. Only the bacterial results expressed in "fold increase” obtained with the purified IgGs of groups A, B, C, E and F are presented in Table II below. Table III presents the bactericidal results expressed as "fold increase”. fold increase, purified IgGs from group A, against 22 strains cultured in the presence / absence of Desferal.
• Table IV shows, for various vaccine compositions, the percentage of protection deduced from the cross-bacterial study including 34 strains cultivated in the presence of Desferal.
• Table II (i)
• L6 means an LOS having an ⁇ chain of type L6
• L8 means an LOS having an ⁇ chain of type L8
• PEA-3 means that LOS has a single PEA substituent at position 3 of heptose II
• PEA-3, -6 means that LOS has a PEA substituent at the 3-position and a PEA substituent at the 6-position of IL heptose

Applications Claiming Priority (3)

Publications (1)

Family

ID=42738824

Family Applications (1)

Country Status (7)

Families Citing this family (3)

Family Cites Families (4)

• 2010
  • 2010-05-12 WO PCT/FR2010/000368 patent/WO2010130899A1/fr active Application Filing
  • 2010-05-12 AU AU2010247255A patent/AU2010247255A1/en not_active Abandoned
  • 2010-05-12 NZ NZ597008A patent/NZ597008A/xx not_active IP Right Cessation
  • 2010-05-12 CA CA2762033A patent/CA2762033A1/fr not_active Abandoned
  • 2010-05-12 US US13/319,868 patent/US20120156281A1/en not_active Abandoned
  • 2010-05-12 EP EP10723645A patent/EP2429576A1/de not_active Withdrawn
• 2011
  • 2011-12-13 ZA ZA2011/09172A patent/ZA201109172B/en unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events