EP2429576A1 - Meningococcal vaccine based on lipooligosaccharide (los) and neisseria meningitidis protein - Google Patents

Abstract

The invention relates to a vaccine against N. meningitidis infections, comprising (i) an N. meningitidis LOS especially consisting of a lipid A, an inner core, and an L8-type a chain in which the heptose II residue of the inner core (a) carries a phosphoethanolamine (PEA) substituent in position O-3, and does not carry a PEA substituent in positions O-6 and O-7, or (b) carries a phosphoethanolamine (PEA) substituent in position O-3 and in position O-6 or O-7; and (ii) the lipidated sub-unit B (TbpB) of the receptor of the human transferrine of an N. meningitidis strain or a lipid fragment of said TbpB.

Description

 Meningococcal Lipooligosaccharide (LOS) Vaccine and Neisseria meninsitidis Protein

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.

In the field of vaccines, one of the major challenges of the coming years will include the marketing of a vaccine intended to prevent all infections N. 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.

In general, meningitis is either of viral origin or of bacterial origin. In developed countries, the main causative bacteria are: N. meningitidis and Streptococcus pneumoniae, respectively involved in about 40-50% of cases of bacterial meningitis. In developing countries Haemophilus influenzae also remains an important source of 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.

Effective capsular polysaccharide vaccines are available to prevent N. meningitidis meningitis in serogroups A, C, Y and Wl 35. These polysaccharides are as low as or not immunogenic in children under 2 years do not induce immune memory. However, these disadvantages can be overcome by conjugating these polysaccharides to a carrier protein.

On the other hand, 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). On the other hand, 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.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of the Gram-negative bacterial wall. LPS is toxic in high doses for mammals and in terms of this biological activity, has been called endotoxin. It is responsible for septic shock, a fatal pathology that develops following an acute infection with a Gram-negative bacterium.

Nevertheless, 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.

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:

(i) 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; (ii) an heptose-linked outer core consisting of various monosaccharides; and

(iii) an O-specific outer chain consisting of a sequence of repeating units - repetitive units themselves composed of one or more different monosaccharides.

From one species to another, the structure of LPS is variable. For example, in a number of non-enteric Gram-negative bacteria such as

Neisseriae, Bordetellae, Branhamellas, Haemophilus and Moraxellae, the O-chain specific does not exist. The saccharide portion of LPS of these bacteria consists only of the oligosaccharide core. Therefore, the LPS of these bacteria is often referred to as lipooligosaccharide (LOS).

The structure of LPS varies not only from one species to another, but also within the same species.

Thus, not all strains of N. meningitidis have an LOS of the same structure, although all meningococcal LOS share the basic structure schematized in the following structural formula:

KDO α chain R1 - A β chain

GlcΝAc γ chain

The outer core (or α chain) is variable depending on the type of oligosaccharide (substituent R1), attached to the glucose residue carried by heptose I.

Whereas 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), carries various substitutions to level (i) of heptose II (substituents R2 and R3); and (ii) γ-chain, consisting of Ν-acetylated glucosamine (GlcΝAc) which may be non-O-acetylated. In the literature, the R2 residue is commonly called β chain, when R2 is a glucose residue.

Strains of N. meningitidis are 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. Clin Microbiol (1992) 30: 2047, Moran et al, Infect Immun (1994) 62 (12): 5290 and Scholten et al., J. Med Microbiol (1994) 41: 236). The LOS classification follows that of the strains. The differences between immunotypes arise from variations in the composition and conformation of the oligosaccharide chains. This is shown in the table below, derived from Table 2 of Braun et al, Vaccine (2004) 22: 898, supplemented by later data on L9 immunotypes (Choudhury et al., Carbohydr Res (2008) 343). : 2771) and LI l (Mistretta et al, (2008) Poster at the 16th International Pathogenic Neisseria Conference, Rotterdam):

Table I:

nd: not determined.

Among other things, it can be noted that some LOS can be sialylated (presence of N-acetyl neuraminic acid on the galactose residue (GaI) terminal of the α chain). Thus, the L3 and L7 immunotypes differ only in the respective presence / absence of this sialylation. Furthermore, most LOS are substituted with an O-acetyl moiety on the glucosamine residue (α-GlcNAc) of the inner core (Wakarchuk et al (1998) Eur J. Biochem 254: 626, Gamian et al. 1992) J. Biol Chem 267: 922, Kogan et al (1997) Carbohydr Res 298: 191, Di Fabio et al (1990) Can J. Chem 68: 1029, Michon et al (1990). J. Biol Chem 275: 9716, Choudhury et al (supra) and Mistretta et al (supra).

Other variations in the structure of LOS are due to various genetic factors including: (i) the presence / absence of certain genes involved in the LOS biosynthetic pathway and the possible associations of the genes with each other; (ii) the phase variation to which certain genes are subject;

(iii) homologous recombination [some genes with conserved regions (IgtB, IgtE and igtΑ) and other genes being hybrids (igtZ is the hybrid of igtA and IgtB genes), rearrangements can occur]; and (iv) mutations.

The genes involved in the LOS biosynthetic pathway (with the exception of two) are divided into three loci (lgt-1, lgt-2 and lgt-3). The description of these genes and their function is given below, illustrated schematically in Figure 1 which shows the structure of the LOS of N. meningitidis, the different sites where the variability is expressed as well as the intervention levels. of genes.

Off-locus genes are Ipu and lo. The Ipύ 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 loύ gene encodes an LOS O-acetyltransferase that has the ability to O-acetylate the γ 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. Among these genes, 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 α 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 Iptβ. 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 Iptβ gene encodes a PEA transferase that has the ability to attach a phosphoethanolamine (PEA) substituent. position O-6 or O-7 of heptose IL The igtG gene is subject to phase variation. When it is "On" and accompanied by a functional Ipβ gene, the attachment of the glucose residue is always at the expense of the PEA (whose attachment is mediated by Ipύ).

The lgt-3 locus is classified into 5 genetic types (Wright et al., J. Bact. (Oct. 2004): 6970). The carbohydrate motif Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-4 or lacto-N-neotetraose motif which is present in the α chain of certain immunotypes of LOS of JV. meningitidis, is an epitope that can potentially cross-react with human erythrocytes. Thus, in order to make a vaccine for humans, one should choose 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.

Alternatively, it is also possible to start from, for example, an L2 or L3 immunotype strain in which a gene intervening in the α chain biosynthesis has been inactivated by mutation, so as to obtain an incomplete LNnT structure. Such mutations are proposed in patent application WO 04/014417. LOS of mutated strains that result, possess an L6-type α chain and a phosphoethanolamine (PEA) substituent at the O-3 position of IL heptose

It has now been found that the LOS of an L8 immunotype strain (immunogenic L8 L8), in association with the lipid subunit B (TbpB) of the human transferrin receptor of a strain of N. meningitidis, could induce an improved bactericidal activity compared to that induced by LOS (in association with the same TbpB) possessing an L6-type α chain, and a phosphoethanolamine (PEA) substituent at the O-3 position of heptose II.

This is why the subject of the invention is a vaccinal pharmaceutical composition against N. meningitidis infections, which comprises:

(i) a LOS of N. meningitidis consisting in particular of a lipid A, an inner core, an L8-type α 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

(ii) the lipidated subunit B (TbpB) of the human transferrin receptor of a N. meningitidis strain or a lipid fragment of this TbpB. In a particular aspect, 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 α 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;

(ii) a LOS of N. meningitidis consisting in particular of a lipid A, an inner core, an L8-type α 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.

In the rest of the text, the term "or a lipid fragment of the latter" no longer appears, for the sake of simplification. Nevertheless it remains implied whenever the term "TbpB lipid" or "TbpB" appears.

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.

Advantageously, a composition according to the invention does not contain OMV (outer membrane vesicle) of N. meningitidis.

*****

Lélos

For use in a composition according to the invention, the LOS consisting in particular of a lipid A ₅ of an inner core, of an L8 type α chain, in which the heptose II residue of the inner core carries a phosphoethanolamine (PEA) substituent. ), at the O-3 position and at the O-6 or O-7 position, may be that of an atypical L8 immunotype strain such as the Al strain (Zhu, Klutch & Tsai, FEMS Microbiology Letters (2001) 203: 173 as well as Gu, Tsai & Karpas, J. Clin Microbiol (Aug. 1992) 30 (8): 2047). According to an advantageous mode, the LOS comes from a strain of serogroup A. It is also possible to manufacture a vaccine according to the invention, by using an LOS consisting in particular of a lipid A, an inner core, an α 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. Typically, 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 Ipύ 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 Ipβ gene; an active Iρt6 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 Ipfα gene (full-length). When this strain is used in the method according to the invention, it 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 Iptβ genes or else by intra- genic insertion of an irrelevant sequence, for example of an antibiotic resistance gene.

The N. meningitidis serogroup against which a priority vaccine is imperative is serogroup B (vaccines against other prevalent serogroups A, C, Y and Wl 35 are already available). However, the purification of LOS from a serogroup B strain can lead to a product containing residual amounts of capsule polysaccharide, undesirable in the vaccine. In order to overcome these difficulties, it has now been found that an adhoc LOS derived from a serogroup A strain can meet the vaccination requirements against serogroup B. Advantageously, an LOS for use in a composition according to the invention , has a γ chain which is O-acetylated, at least partially. For purposes of the present invention, 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

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.).

Chemical detoxification

The chemical approach is to treat LOS with a chemical agent. In a particular embodiment, 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

(are) present in the structure of the LOS. Such a treatment is for example described in Gu & Tsai Infect. Immun. (1993) 61: 1873. According to an alternative and preferred mode, 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. Such a treatment is for example described in USP 6,531,131, Gupta et al., Infect. Immun. (1992) 60 (8): 3201 and Gu et al, Infect. Immun. (1996) 64 (10): 4047.

Detoxification by enzymatic route

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. Such lipases are produced by the amoeba Dictyostelium discoideum. According to a particularly advantageous mode, 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. It may also be an 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).

Genetic detoxification

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. Such 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 ipxlΛ or ipxLl (also called htrBl I JιtrB2) 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. In N. meningitidis the endogenous gene ipxA can also be replaced by the homologous gene from E. coli or Pseudomonas aeruginosa. The fatty acid chains are modified, resulting in a less toxic lipid A (Steeghs et al., Cell Microbiol (2002) 4 (9): 599). The genetic approach is preferred when LOS is purified as OMVs.

LOS detoxified by complexation with an analog peptide of B-polymer

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.

Thus, 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

It is optional (this position may be empty or not) and is an amino acid residue or a peptide consisting of 2 to 3 amino acid residues; with the proviso that the ratio of cationic amino acid / hydrophobic amino acid in the peptide of formula I is from 0.4 to 2, advantageously from 0.5 to 1.2 or 1.5, preferably from 0.6 to 1; better from 0.6 to 0.8; for example 0.75.

Preferably in the peptide of formula (I) the C position is an empty position.

Particular examples of the 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; and NH ₂ -Hyl-Arg-His-Lys-Cysl-Phe-Tyr-Trp-Val-Ile-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

I I

NH ₂ -A '-Cys1-B'-Cys2-C'-C00H wherein the two Cysl residues are linked together by a disulfide bridge and the two Cys2 residues are linked together by a disulfide bridge; or of formula (III)

NH ₂ -A-Cysl-B-Cys 2-C-COOH

I I

COOH-C'-Cys2-B'-Cys1-A'-NH ₂ in which the Cys1 residues are bonded to the Cys2 residues by peptide interchain disulfide bridges; formulas (II) and (III) in which:

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. Advantageously, 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; and where appropriate, 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.

When A and A 'have 3 amino acid residues, 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.

When 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.

When 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.

Advantageously, 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 . As can easily be understood, B and B 'may have up to 7 amino acid residues independently selected from Val, Leu, Ile, Phe, Tyr and Trp.

Advantageously, 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. In a preferred embodiment, the sequence - X1 - X2 - X3 - comprises the Phe-Leu motif. Particular embodiments of 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.

When B and B 'have more than 4 non-polar amino acid residues, A and A preferably have at least 3 positively charged amino acid residues. In C and C, 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. Advantageously, they are independently selected from non-charged, polar or non-polar amino acid residues, the latter being preferred. However, in a preferred manner, the C and C positions are empty positions.

Therefore, a preferred class of dimers are of formula (IV) NH ₂ -A-Cys 1 -B-Cys 2 -COOH

I I

NH ₂ -A'-Cysl-B'-Cys 2 -COOH wherein the two Cysl residues are linked together by a disulfide bridge and the two Cys2 residues are linked together by a disulfide bridge; or of formula (V)

NH ₂ -A-Cysl-B-Cys 2 -COOH

IIOCOC-Cys2-B'-Cys1-A'-NH ₂ in which the Cys1 residues are linked to the Cys2 residues by interchain peptide disulfide bridges; formulas (IV) and (V) wherein A, A ', B and B' are as described above; provided that the ratio of cationic amino acid / hydrophobic amino acid in the dimer of formula (IV) or (V) 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.

In formulas (II) to (V), 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. A dimer of formula (II) to (V), in which A and A '; B and B '; and C and C are two to two identical, is referred to as a homologous dimer.

For the latter, mention is made by way of example, the parallel and antiparallel dimers formed from peptide S AEP2-L2: NH ₂ -Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2- COOH

I I

NH ₂ -Lys-Thr-Lys-Cysl-Lys-Phe-Leu-Leu-Leu-Cys2-COOH; and

NH ₂ -Lys-Thr-Lys-Cys 1 -Lys-Phe-Leu-Leu-Leu-Cys2-COOH HOOC-Cys2-Leu-Leu-Leu-Phe-Lys-Cys1-Lys-Thr-Lys-NH ₂ .

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.

By "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. For the purpose of the present invention, 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. Depending on its composition, 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). For LOS detoxification purposes, 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. In the latter two cases, the neutral lipid is often referred to as co-lipid. According to an advantageous mixing mode, 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.

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-α-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.

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 -rαc- (1-glycerol)] (DOPG), and 1-acyl-2-acyl-sn-glycero-3- [phospho-rac- (1-glycerol)] bearing mixed acyl chains; (iv) phosphatidic acids such as 1,2-diacyl-sn-glycero-3-phosphate eg, 1,2-dioleoyl-5'-glycero-3-phosphate (DOPA), and 1-acyl-2 -acyl-5'-glycero-3-phosphate bearing mixed acyl chains; and (v) phosphatidylinositols such as 1,2-diacyl-sn-glycero-3- (phosphoinositol) eg, 1,2-dioleoyl-5n-glycero-3- (phosphoinositol) (DOPI) and 1-acyl-2-acylsulfonyl-3- (phosphoinositol) bearing mixed acyl chains. With regard to cationic lipids, mention is made by way of example:

(i) 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)];

(ii) roctadecenoyloxy (ethyl-2-heptadecenyl-3-hydroxyethyl) imidazolinium (DOTIM) and its structural counterparts;

(iii) lipospermins such as N ~ palmitoyl-D-erythrospingosyl-1-O-carbamoyl spermine (CCS) and dioctadecylamidoglycylspermine (DOGS, Transfectam);

(iv) 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;

(v) 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

(vi) cationic derivatives of cholesterol such as 3β- [Ν- (Ν ', Ν'-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol) or other cationic cholesterol derivatives such as those described in US Pat. 283,185 and especially cholesteryl-3β-carboxamidoethylenetri dimethylammonium iodide, the cholesteryl-3β-carboxyamidoethylenamine, cholesteryl-3β-oxysuccinamidoethylenetetrimethylammonium iodide and 3β- [N- (polyethylenimine) carbamoyl] cholesterol.

By "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.

These 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 α-linolenic acid (C18: 3), cis-6,9,12-octadecatrienoic acid or γ-linolenic acid (Cl 8: 3), eicosanoic acid or arachidic acid (C20: 0), cis-9-eicosenoic or gadoleic acid (C20: 1), cis-8,11,14-eicosatrienoic acid (C20: 3), cis-5,8, 11,14-eicosatetraenoic acid or arachidonic acid (C20: 4), cis-5,8, 11,14,17-eicosapentaneoic acid (C20: 5), docosanoic or behenic acid (C22: 0), cis-7,10,13,16,19-docosapentaenoic acid (C22: 5), cis-4,7,10,13,16,19-docosahexaenoic acid (C22: 6) and tetracosanoic acid or lignoceric (C24 .O).

In a particular embodiment, a mixture of cationic lipid and neutral lipid is used. By way of example, mention is made of: 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; and 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. According to an advantageous method of preparation, 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. In general, it is believed that the same molar ratio should not vary substantially in the final process of preparation of LOS liposomes.

In general, 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 μm). 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.

At the end of the incorporation of LOS into liposomes, it is commonly possible to obtain a mixture consisting of adhoc liposomes and LOS free form. Advantageously, the liposomes are then purified in order to get rid of the non-detoxified LOS in free form.

Conjugation of LOS

In a vaccine according to the invention, 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.

Many methods of conjugation exist in the technical field. Some are listed for example in patent applications EP 941 738 and WO 98/31393. In general, 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. For example, 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).

Advantageously, the conjugation method makes use of (i) a bifunctional linker (linker) or (ii) a spacer and a linker.

For example, in the first case, 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. For example, in the second case, 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.

In the second case, it is also possible to proceed as follows: 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.

In the formula of the spacer, 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

- S - B '- NH ₂ . As a particular example, mention may be made of: cysteamine, cysteine, eg diamines, diaminohexane, adipic acid dihydrazide (ADH), urea and cystamine.

In the formula of the linker, 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. For example, 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. Thus, 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-. If the linker is to react with an amino group, R2 is preferably a carboxyl, succinimidyl (eg, N-hydroxy succinimidyl) or sulfosuccinimidyl (eg, N-hydroxy sulfosuccinimidyl) group.

Thus, 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-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB); succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB); bromoacetic acid -N-hydroxy succinimide (BANS) ester; dithiobis (succinimidyl propionate) (DTSSP); the H- (γ-maleimido butyryloxy) succinimide ester (GMBS); succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate; N-succinimidyl-4- (4-maleimidophenyl) butyrate; N- [β-maleimidocaproic acid] hydrazide (BMCH); N-succinimidyl-4-maleimidobutyrate; and N-succinimidyl-3-maleimido benzoate. For example, it is proposed to use the acid function of KDO to derive LOS with DHA in the presence of a carbodiimide [eg, 3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride (EDAC)]. The aminated function thus introduced is then reacted with the carboxyl functions of the polypeptide, in the presence of EDAC, after protecting the amino functions of the latter (Wu et al (2005) Vaccine 23: 5177) or having transformed them into acidic functions. (Succinylation of the protein, Pavliakova et al, Infect Immun (1999) 67 (10): 5526).

Alternatively, it is proposed to use the acid function of KDO to derive LOS with cysteamine or cysteine in the presence of EDAC. Then 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. In the first case, the maleimide function thus introduced is then reacted with the thiol functions of the polypeptide. In the second case, the succinimidyl function of the derivatized and activated LOS is reacted with the amino functions of the polypeptide.

Depending on the chosen conjugation mode, 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. In a preferred embodiment, 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. coli: Met - Lys - AIa - Thr - Lys - Leu - Val - Leu - GIy - Ala - Val - Ile - Leu - GIy - Ser - Thr - Leu - Leu - AIa - GIy. Thus, in the expression cassette, 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.

Thus, it will be readily understood that the present invention is not limited to the use of a particular TbpB, defined by a particular amino acid sequence. Any reference to an amino acid sequence is for illustrative, non-limiting.

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.

For use in the present invention, 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. Advantageously, these are T-helper epitopes characteristic of the organism to which the LOS-based vaccine is intended (a mammal, in particular a human) - that is to say of epitopes capable of being recognized by T helper cells of the recipient organism and to activate them.

The open reading frame (ORF) encoding the TbpB (tbpB) of several strains of N. meningitidis has already been identified by its sequence; and the amino acid sequence of the corresponding protein deduced. Thus, the tbpB and TbpB sequences of N. meningitidis strains of serogroup B, M982 and B16B6, are disclosed in patent application EP 586 266. Those strains of meningococcal MC58 (serogroup B), Z2491

(Serogroup A) and FAMl 8 (Serogroup C) are respectively disclosed in Tettelin et al., Science, March 2000, 287: 1809 or WO 00/66791; Parkhill et al, Nature (March 2000) 404: 502; and Bentley et al, PLoS Genet., 3, e23 (2007).

The identification of these latter sequences having been made within the framework of the complete sequencing of the genomes, a sequence number was assigned to them. Thus, in Tettelin et al (supra) or WO 00/66791, the tpbB I TbpB sequences of the MC58 strain are designated under the reference NMB 0460. In the remainder of the text, the proteins of N. meningitidis can be designated without this refers in a limiting manner to the sequences of strain MC58.

In N. meningitidis, two large families of TbpB have been documented to date: isotype I characterized by a 1.8 kb tbpB gene and isotype II characterized by a 2.1 kb tbpB gene (EP 560 969 and EP 586 266). ). 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

For use in the present invention, the lipidated TbpB is that of a N. meningitidis strain of isotype I or isotype II, preferably isotype II. According to one particular embodiment, 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. Because of its lipid tail, it is expected that, in purified form, 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.

When LOS is formulated into liposomes, 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.

When liposomes (proteoliposomes) are combined with 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.

At the end of the liposome incorporation of LOS and lipidated TbpB, it is commonly possible to obtain a mixture consisting of liposomes adhoc (proteoliposomes),

LOS and / or TbpB lipidated in free form. Advantageously, the liposomes are then purified in order to get rid of LOS in free form. Once the free LOS has been removed, 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. Once 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.

It can be manufactured conventionally. In particular, 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. Advantageously, it may further comprise a pharmaceutically acceptable adjuvant.

For use in a composition according to the invention, 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.

Thus, the amount of LOS per dose may be between 5 and 500 μg, advantageously between 10 and 200 μg, preferably between 20 and 100 μg, very preferably between 20 and 80 μg or between 20 and 60 μg, terminals included. The amount of TbpB lipid per dose may be between 5 and 500 μg, advantageously between 10 and 200 μg, preferably between 20 and 100 μg, most preferably between 20 and 80 μg or between 20 and 60 μg, terminals. included.

In the vaccine according to the invention, 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. By way of example, it is indicated that the liposized LOS: TbpB molar ratio can be typically 20 or approximately 25, depending on whether the isotype of TbpB is I or IL

The term "dose" used above 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.). Finally the invention also relates to:

A method for inducing in a mammal, for example a human, an immune response against N. meningitidis, wherein the mammal is administered an immunogenically effective amount of a composition of the invention to induce immune response, particularly a protective immune response to N. meningitidis; and

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.

EXPERIMENTAL DATA

A Experimental data for strains derived from JV. meningitidis C708

1. Material & Methods 1.1 Transformation of strain C708

Strain C708 is cultured in Brain Heart Infusion (BHI) medium at 37 ° C. under a 10% CO ₂ atmosphere. 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). To 900 μl of the liquid medium BHI + MgCl ₂ 5 mM, 10 μg of DNA necessary for the transformation (linearized plasmid) are added; then 100 μl of the bacterial suspension (10 ⁸ germs). The transformation medium is incubated for 30 min at 37 ° C., 10% CO ₂ .

Five hundred μL of this preparation (ie approximately 5.10 ⁷ cfu) are used to inoculate 4.5 mL of 5 mM BHI + MgCl ₂ . The bacteria are allowed to regenerate for 2 hours at 37 ° C, 10% CO ₂ . Then, from this suspension, dilutions of 5 mM BHI + MgCl _{2 are carried out} . 300 μl of a dilution containing approximately 30,000 cfu are spread on a 140 mm BHI agar plate. The dishes are placed at 37 ° C, 10% CO ₂ for at least 20 hrs.

1.2. Blot of Transformant Colonies The colonies are transferred to Hybond-XL 137 mm membrane (GE Healthcare, #RPN 137S). The membrane-deposited seeds are lysed in denaturation buffer (0.5 M NaOH, 1.5 M NaCl). The membranes are washed in neutralization buffer (0.5 M Tris, 1.5 M NaCl, pH 7.5); transferred to 2X SSC medium; then dried. The DNA is fixed by incubation for 2 hrs at 80 ° C. 1.3. Detection of transformants by hybridization with a ³³ P dCTP-labeled probe

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:

In low stringency buffer (2 X SSC, 0.1% SDS (wt / vol) 15 min at room temperature with slow stirring;

Medium-stringency buffer (1 X SSC, 0.1% SDS (weight / vol) 20 min at 65 ° C. with slow stirring, and In low stringency buffer (0.1 X SSC, 0.1% SDS (weight / vol) 45 min at 65 ° C with slow stirring.

Once dried, the membranes are revealed by autoradiography (Biomax MR film).

1.4. Detection of transformants by hybridization with a [γ ³² P] ATP labeled oligonucleotide

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 μl maximum is lO pmoles

Phosphorylation buffer 10 X l μl or 1 X

[γ- ³² P] ATP 10 mCi / ml 5 μl or 50 μCi

T4 kinase (10 U / μl) l μl or 10 U

H ₂ O qsp 10 μl

The 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 μg / ml sperm DNA. salmon 10 mg / ml sonicated and denatured for 5 min at 100 ^° C.

The labeled oligonucleotide (10 μl) 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:

In low stringency buffer (2 X SSC, 0.1% SDS (weight / vol) 5 min at

Tm of the oligonucleotide - 5 ° C, with slow stirring;

In medium-stringency buffer (1 X SSC, 0.1% SDS (weight / vol) 15 min at Tm of the oligonucleotide -5 ° C, with slow stirring, and in low-stringency buffer (0.1 X SSC, 0.1% SDS (weight / vol) 10 min to

Tm of the oligonucleotide - 5 ° C, with slow stirring. Once dried, the membranes are revealed by autoradiography (Biomax MR film).

2. Construction of a strain of N. meningitidis expressing an LOS whose α chain is that of an L6 immunotype L6 and comprising in each of the O3 and O6 positions of the heptose II (hep II) residue of the inner core, a Phosphoethanolamine Substitute (PEA)

The N. meningitidis strain C708 of serogroup A and immunotype L6, which has the following characteristics among others, is used as the starting strain:

An active igtA gene (gene turned ON);

An igtB gene - (non-functional gene); - an igtG gene extinguished ("OFF");

A truncated Ipu gene;

An active Iptβ gene; and

An active gene.

Strain C708 was filed 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 bears the order number CNCM 1-3942.

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.

2.1. PCR amplification (polymerase chain reaction) of the complete Ipt3 gene (FL = full-length) of N. meningitidis strain FAM18 One hundred ng of genomic DNA of strain FAMl 8 was used for amplification with Platinum® Taq DNA polymerase High Fidelity (Invitrogen, # 11304-011).

The pair of primers is the following (couple n ° 1):

CG GAATTC GCC GTC TCA ATG AAA AAA TCC CTT TTC GTT CTC (Tm = 55.9 ° C); and AA CTGCAG TCA TTG CGG ATA AAC ATA TTC (Tm = 57.1 ^° C). (The EcoRI and PsI sites are respectively underlined). For amplification the following mixture has been realized:

Component Volume Final Concentration

Buffer 1 OX High Fidelity PCR 5 μl IX

Mixing 10 mM dNTP 1 μl 0.2 mM each MgSO ₄ 50 mM 2 μl 2 mM

Mix of primers (10 μM each) 1 μl 0.2 μM each

Genomic DNA x μl 100 ng

Platinum® Taq High Fidelity 0.2 μl 1.0 unit Nuclease free water for 50 μl final Does not apply The thermal cycler program is as follows:

Initial denaturation: 94 ⁰ C for 30 seconds

30 cycles of: Denaturation: 94 ° C for 30 seconds Hybridization: 55 ° C for 30 seconds Extension: 68 ° C for 1 minute / kb of PCR product. After the reaction, 1/10 of the PCR product was deposited on agarose gel for verification. 2.2. Construction of a transformation vector

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 μg of DNA were used in RΕact2 buffer (Invitrogen). The PCR fragment was then inserted into the linearized pUC19 vector. Ligations were performed in a final volume of 20 μl with 50 ng of vector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 μl 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 μFD; Resistance: 200 ohms; Voltage: 1700 volts.

The selection of the transformed clones was done by spreading on LB ampicillin plates

100 μg / 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.

2.3. Transformation of strain C708 and detection of the homologous recombination event 2.3.1. Transformation

40 μg of pM1222 were digested with 400 U of EcoRI and 10 μg were used to transform the C708 strain according to the method described in section A.1.1.

After transformation, 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.

After 24 hours at 37 ° C, the number of control plates made it possible to estimate the number of cfu at 27,000 per box for the mutant.

2.3.2. Selection of clones The recombination event, that is to say the replacement of the lpt2 TR gene (truncated) by the Ipt3 FL gene (complete), was detected after transfer of hybridization membrane clones and hybridization with a hybridization membrane. probe labeled with ³ P dCTP according to the methods described in sections A.1.2. and A.1.3.

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.

Preparation of the probe

In order to obtain the 270 bp lpt2 probe, 10 ng of plasmid pUC / t were used for PCR amplification with Platinum® Taq High Fidelity DNA polymerase (Invitrogen, # 11304-011).

The pair of primers is the following (couple n ° 2):

CGC CGA ATA CTT CTT TAT GAG GC (Tm = 60.6 ° C); and

CTC GCC AAA GAG CAG GGC (Tm - 60.5 ^° C).

For amplification, the following mixture was made: Components Volume Final concentration 10X High Fidelity PCR buffer 5 μl IX

Mixing 10 mM dNTP 1 μl 0.2 mM each

MgSO ₄ 50 mM 2 μl 2 mM

Mix of primers (10 μM each) 1 μl 0.2 μM of each plasmid pUC x μl 100 ng

Platinum® Taq High Fidelity 0.2 μl 1.0 unit Nuclease free water for 50 μl final Does not apply

The thermocycler program is as follows:

Initial denaturation: 94 ^° C. for 30 seconds 30 cycles of: Denaturation: 94 ° C. for 30 seconds

Hybridization: 55 ° C for 30 seconds Extension: 68 ⁰ C for 45 seconds.

After the reaction, 1/10 of the PCR products were deposited on agarose gel to ensure the specificity of plasmid, and then the PCR fragment was purified using the QIAquick PCR Purification Kit (Qiagen, # 28104).

Hybridization and revelation

The probe labeling, hybridization, washing and revelation steps were performed as described in section A.l.3.

About 460,000 cfu were tested. After exposure with BioMax MR films, the autoradiographs revealed 5 positive spots (C708 containing an Ipβ FL gene) each on a different membrane.

Screening and authentication of positive clones

After identification on the Petri dish, 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.

To do this, 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.

The remaining volume was centrifuged for 5 min at 6000 rpm, and then the pellet was taken up in 50 μl 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. For each sample corresponding to a positive spot and the controls, a PCR amplification with the pair of primers used to amplify the C708 iptZ probe of 270 bp (pair # 2) was performed with the Expand Long Kit. PCR template (Roche) as described below. Components Volume Final Concentration

1OX ELT PCR buffer 5 μl IX

Mix of dNTPs (10 mM each) 2 μl 0.4 mM each

Mix of primers (10 μM each) 1.5 μl 0.3 μM each

DNA template 40 μl Not applicable Polymerase ELT 0.75 μl 3.75 units

Nuclease free water for 50 μl Not applicable

The thermocycler program is as follows:

Initial denaturation: 94 ⁰ C for 2 min

10 cycles of: Denaturation: 94 ° C for 10 seconds Hybridization: 54 ° C for 30 seconds

Extension: 68 ⁰ C for 45 seconds

20 cycles of: Denaturation: 94 ^° C. for 15 seconds

Hybridization: 54 ° C for 30 seconds

Extension: 68 ° C for 45 seconds + 20 sec / cycle Final elongation: 68 ° C for 7 min

After the reaction, 1/10 of the PCR products were deposited on agarose gel for verification. Four of the 5 clones had the expected profile. The frequency of obtaining a true positive clone was 1 / 115,000 raw tested.

The next step was to isolate a pure clone. For this, 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 μl of nuclease-free water, 30 μl were deposited on a BHI dish and the remaining 70 μl 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 α 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 Iptβ gene of this strain by deletion of a central part of the gene.

3.1. PCR amplification (polymerase chain reaction) of the full-length Ipt6 gene of N. meningitidis strain Z2491 of serogroup A (NMA 0408 gene) One hundred ng of genomic DNA of strain Z2491 (strain available in the research laboratories, globally) were used for amplification with Platinum® Taq DNA polymerase High Fidelity (Invitrogen, # 11304-011).

The pair of primers is the following (couple n ° 3):

CG GAATTC GCC GTC TCA A GGT TGC CTA TGT TT CCT GTT TT G (Tm = 59.7 ⁰ C); and

AA CTGCAG CTA ACG GGC AAT TT CAA AAC GTC (Tm = 59.3 ° C). (are respectively underlined the EcoRI and PstI sites).

For amplification the following mixture has been realized:

Components Volume Final concentration Buffer 10X High Fidelity PCR 5 μl IX

Mixing 10 mM dNTP 1 μl 0.2 mM each

MgSO ₄ 50 mM 2 μl 2 mM

Mix of primers (10 μM each) 1 μl 0.2 μM each

Genomic DNA x μl 100 ng Platinum® Taq High Fidelity 0.2 μl 1.0 unit

Nuclease free water for 50 μl final Not applicable The thermocycler program is as follows:

Initial denaturation: 94 ° C for 30 seconds

30 cycles of: Denaturation: 94 ⁰ C for 30 seconds Hybridization: 55 ⁰ C for 30 seconds Extension: 68 ° C for 1 minute / kb of PCR product.

After the reaction, 1/10 of the PCR product was deposited on agarose gel for verification.

3.2. Construction of the vector pM1223 (pUC19 Iptβ FL)

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 μg of DNA were used in RΕact2 buffer (Invitrogen).

The PCR fragment was then inserted into the linearized pUC19 vector. Ligations were performed in a final volume of 20 μl with 50 ng of vector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 μl 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 μFD; Resistance: 200 ohms; Voltage: 1700 volts.

The selection of transformed clones was made by plating on LB ampicillin 100 μg / ml dishes. The authentication of the positive clones (presence of an Iptβ FL gene) 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.

3.3. Deletion of the central part of the Iptβ gene originating from the strain Z2491 Construction of a transformation vector

With the Εxpand Long Template PCR kit (Roche), reverse PCR was performed from plasmid pM1223 using the following pair of primers (pair no. 4): CG GGATCC CGA CAT CAC GAA CGC CGC (Tm = 60.5); and CG GGATCC CGC CGC TTA ACT ACG ACA TC (Tm ≈ 59.4); (The BamRi sites are underlined). This makes it possible to reamplify the plasmid by deleting the part that one wishes to remove (808 bp).

For amplification the following mixture was realized: Components Volume Final Concentration

Buffer 1 OX ELT PCR 5 μl IX

Mix of dNTPs (10 mM each) 2 μl 0.4 mM each Primer Mix (10 μM each) 1.5 μl 0.3 μM each DNA Matrix (pM1223) 40 μl Not applicable

Polymerase ELT 0.75 μl 3.75 units

Nuclease free water for 50 μl Not applicable

The thermocycler program is as follows:

Initial denaturation: 94 ⁰ C for 2 min

10 cycles of: Denaturation: 94 ⁰ C for 10 seconds Hybridization: 54 ° C for 30 seconds Extension: 68 ⁰ C for 3 min

20 cycles of: Denaturation: 94 ° C for 15 seconds Hybridization: 54 ° C for 30 seconds Extension: 68 ° C for 3 min + 20 sec / cycle

Final elongation: 68 ⁰ C during 7 min

After the reaction, 1/10 of the PCR products were deposited on agarose gel.

After purification on a QiaQuick column, the PCR product was digested with BamHI at the rate of 10 U of enzyme per μg 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 Igtδ gene deleted from its central part was verified by sequencing.

3.4. Transformation of the C708 lpt2 FL strain and detection of the homologous recombination event

Transformation

Ten μg of plasmid pM1224 were linearized with EcoRI at a rate of 10 units of enzyme per μg of plasmid to be digested in the appropriate buffer for 2 hours at 37 ° C.

The transformation into strain C708 was done following the technique described in section A.1.1.

After transformation, 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.

Selection of clones: Preparation of the probe, hybridization and revelation The recombination event was detected after colony blotting and hybridization with a γ ³² P dATP-labeled oligonucleotide.

The recombination event, that is to say the replacement of the Iptβ FL gene by the Iptβ 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 selection of positive clones was by hybridization of the DNA fixed on Hybond N + membranes with a radioactive oligonucleotide whose sequence is overlapping on both recombinant ends. This is the following oligonucleotide: GTC GAT ATG GGG ATC GCG CTT AAC G (Tm = 69.5 ° C).

About 840,000 cfu were tested. After exposure with BioMax MR films, the autoradiographs revealed 16 positive spots (C708 containing an Iptβ TR gene) distributed over 9 different membranes.

Screening and authentication of positive clones For each of the 16 positive spots, after identification on the petri dish, part of the area taken around the positive clones was kept in freezing medium (Ml 99, SVF 20%, glycerol 10%) and the other part was removed. has been used for PCR authentication.

To do this, 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 μl 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.

For each sample corresponding to a positive spot, PCR amplification was performed with Platinum® Taq High Fidelity (Invitrogen) and the following pair of primers

(couple n ° 5)

CGC ACT GGC GGA ATT GGG (TM ≈ 60.5 ⁰ C); and

CCCATTTCTTCC TGA CGG AC (Tm≈ 59.4 ^° C.).

For amplification, the following mixture was made: Components Volume Final concentration

Buffer 1 OX High Fidelity PCR 5 μl IX

Mixing 10 mM dNTP 1 μl 0.2 mM each

MgSO4 50 mM 2 μl 2 mM

Mix of primers (10 μM each) 1 μl 0.2 μM of each DNA template x μl 100 ng

Platinum® Taq High Fidelity 0.2 μl 1.0 unit

Nuclease free water for 50 μl final Not applicable

The thermocycler program is as follows:

Initial denaturation: 94 ⁰ C for 1 min

30 cycles of: Denaturation: 94 ⁰ C for 30 seconds Hybridization: 55 ° C for 30 seconds Extension: 68 ⁰ C for 50 seconds.

After the reaction, 1/10 of the PCR product was deposited on agarose gel for verification. Two out of 16 candidates were found to be true positives: either a frequency of obtaining a true positive clone for 425,000 cfu tested. The next step was to isolate a pure clone. For this, one of the 2 heterogeneous positive clones was spread in isolated cfu and several of these cfu (24) were analyzed by PCR, with the pair of primers No. 5.

Each cfu was resuspended in 50 μl of nuclease-free water, 20 μl were deposited on a BHI dish and the remaining 30 μl 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.

After the reaction, 1/5 of the PCR products were deposited on agarose gel for verification. Only one in 24 clones proved to be a true positive.

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.

4. Construction of a strain of N. meningitidis expressing an LOS whose α chain is that of an L8 L8 immunotype and comprising only in the O3 position of the heptose II residue (hep II) of the inner core, a phosphoethanolamine substituent ( PEA)

The N. meningitidis strain C708 lpt2) FL Iptβ 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. 4.1. PCR amplification (polymerase chain reaction) of the complete lgt A gene (full-length) of the iV strain. meningitidis MC58 serogroup B (NMB 1929 gene)

One hundred ng of genomic DNA of the MC58 strain (strain available in research laboratories, globally) was used for amplification with Platinum® Taq High Fidelity DNA polymerase (Invitrogen, # 11304-011).

The pair of primers is as follows:

GAATTC GCC GTCTCAAATG CCGTCT GAA GCC TTCAG (Tm = 59.4 ° C); and

AA CTGCAG AAC GGT TT TCA GCA ATC GGT GC (Tm = 60.6 ° C). (are respectively underlined the EcoRI and Pstl sites). For amplification the following mixture has been realized:

Components Volume Final Concentration

Buffer 1 OX High Fidelity PCR 5 μl IX

Mixing 10 mM dNTP 1 μl 0.2 mM each MgSO ₄ 50 mM 2 μl 2 mM

Mix of primers (10 μM each) 1 μl 0.2 μM each

Genomic DNA x μl 100 ng

Platinum® Taq High Fidelity 0.2 μl 1.0 unit Nuclease free water for 50 μl final Does not apply The thermal cycler program is as follows:

Initial denaturation: 94 ° C for 30 seconds

30 cycles of: Denaturation: 94 ⁰ C for 30 seconds Hybridization: 55 ° C for 30 seconds Extension: 68 ° C for 1 minute / kb of PCR product. After the reaction, 1/10 of the PCR product was deposited on agarose gel for verification. 4.2. Construction of the pUC19 lgtA FL vector

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 μg 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 μl with 50 ng of vector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 μl 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 μFD; Resistance: 200 ohms; Voltage: 1700 volts.

The selection of transformed clones was made by plating on LB ampicillin 100 μg / ml dishes. The authentication of positive clones (presence of a lgtA FL gene) was performed by enzymatic digestion after extraction of the DNA by miniprep. 1/5 of the clones analyzed showed the expected enzyme digestion profile. 4.3. 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:

CG GGATCC GGA AGG CCC GAG CGC AGA AGT (Tm: 65.7 ^° C); and GC TCTAGA CAA CTT ACT TCT GAC AAC CGG GAT (Tm: 61 ° C)

For amplification, the following mixture has been produced:

Components Volume Final concentration Buffer PfU turbo 1 OX 5 μl IX

10 mM dNTP mixture 0.4 μl 0.2 mM each

Mix of primers (10 μM each)) 11 μμl 0.2 pMGCIO x μl 10 ng

Pfu turbo (Stratagene) 1 μl 2.5 units Nuclease free water to 50 μl Not applicable

The thermocycler program was as follows: Initial denaturation: 95 ⁰ C for 2 mn 30 cycles of:

Denature: 95 ⁰ C for 30 seconds Anneal: 55 ⁰ C for 30 seconds

Extend: 72 ⁰ C for 1 minute Final elongation: 72 ⁰ C for 10 mn

After the reaction 1/10 PCR products are deposited on agarose gel.

4.4. Construction of the plasmid pUC19 IgtA TR (deletion of the central part of the IgtA gene by inverse PCR)

With the Expand Long Template PCR Kit (Roche), reverse PCR was performed from plasmid pUC19 IgtA FL, obtained in A.4.2., For the dual purpose of removing the central part of the gene and creating two sites of restriction at the ends (BamHI and XbaI). The following pair of primers was used: CG GGATCC GCCAAT TCA TCC AGC CCGATG (Tm = 61.8 ° C); and CG TCTAGA CCC GGT TCG ACA GCC TTG (Tm = 60.5 ^° C); This makes it possible to reamplify the plasmid by deleting the part that one wishes to remove.

For amplification, the following mixture was made: Volume Components Final Concentration

Buffer 1 OX ELT PCR 5 μl IX Mix of dNTPs (10 mM each) 2 μl 0.4 mM each

Primer mix (10 μM each) 1.5 μL 0.3 μM each DNA template (10 ng pUC19 IgtA FL) Not applicable ELT Polymerase 0.75 μl 3.75 units

Nuclease free water at 50 μl Not applicable The thermal cycler program is as follows:

Initial denaturation: 94 ° C for 2 min

10 cycles of: Denaturation: 94 ⁰ C for 10 seconds

Hybridization: 55 ° C for 30 seconds

Extension: 68 ° C for 1 min per kb 20 cycles of: Denaturation: 94 ° C for 15 seconds

Hybridization: 55 ° C for 30 seconds

Extension: 68 ° C for 1 min per kb + 20 sec / cycle

Final elongation: 68 ⁰ C during 7 min

After the reaction, 1/10 of the PCR products were deposited on agarose gel for size verification (3.2.kbs). The PCR product was purified on QiaQuick column.

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 (igtA deleted from its central part) was performed by enzymatic digestion after extraction of the DNA by miniprep. 4.5. Construction of plasmid pUC19 IgtA :: erm

The 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:

2 μg of DNA were in contact with 20 units of XbaI in buffer 2 (InVitrogen) under 60 μl for 2 hours at 37 ° C. Xbal was then inactivated for 10 minutes at 65 ° C. We then 7 μl of 1M NaCl, 20 units of BamHI and 1 μl of buffer 2 were added. The digestion was continued for 2 hrs at 37 ° C.

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).

After purification, 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.

4.6. Transformation of the C708 Ipt3 FL Ipt6 TR strain and detection of the homologous recombination event

Ten μg of plasmid pUC19 igtA :: erm were linearized with EcoRI at a rate of 10 units of enzyme per μg of plasmid to be digested in the appropriate buffer for 2 hours at

37 ° C.

The transformation in the C708 Ipύ FL Iptβ TR strain was made following the technique described in section A.1.1.

After transformation, 1.24 10 bacteria were plated on BHI + erythromycin medium at 2 μg / mL. Incubation is continued overnight at 37 ° C. The observed transformation frequency is 1 / 2.5 10 ⁶ .

B. Experimental Data Relating to Vaccine Compositions 1. Preparation of lipidated rTbpB For the sake of linguistic simplification, simply indicate below, rTbpB or TbpB.

1.1. Production

Strains expressing lipidated TbpB M982 or B16B6

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 (αraB). Culture

Three freezes of the strain of E. coli BL21 / pTG9219 or BL21 / pTG9216 (1 ml each) are used to seed 3 liters of medium LB (Luria Broth) distributed erlens. Incubation is continued for 15 to 18 hours at 37 ° C.

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) supplemented with glycerol 20 g / L, with 10% (vol./vol.).

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). When 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. After one hour of induction, 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

Extraction of membranes containing rTbpB LOS extraction

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). Part of the germs lysing spontaneously, 4 .mu.l of benzonase (1 IU activity DΝAse / ml final Merck ref K32475095) are also added. The incubation is continued at + ^{40 °} C. for 45 minutes with magnetic stirring after homogenization with Turrax (15 sec).

Then 4 ml of 1 M MgCl ₂ was added to be at 5 mM final. The magnetic stirring is increased to 10 minutes. Centrifugation at 15,000 g for 45 minutes allows the pellet (Cl pellet vs supernatant Sl) containing the rTbpB protein to be harvested.

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 μl 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.

Washing Membrane Fragments 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. After homogenization (without Turrax), the solution is then stirred for one hour at +4 ^° C. As before, 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.

Membrane solubilization The thawed membrane pellet is solubilized with 780 ml of 50 mM Tris HCl buffer 6 mM EDTA 2 M urea Elugent 4% at pH 7.5. The presence of the 4% detergent and the 2 M urea makes it possible to carry out the solubilization of the pellet. The solution is stirred at + 4 ° C overnight (16h minimum). Centrifugation at 30,000 g (1 hour at + 4 ° C) of the solution leaves only a small pellet (C4) containing some impurities. Supernatant S4 containing the rTbpB protein is recovered for deposit on a first cation exchange column (QS I).

Purification by anion exchange chromatography on Q Sepharose at pH 7.5

Two successive chromatographies are carried out, the product of the first chromatography is collected and then deposited after a dialysis step on a second chromatography column which uses different conditions (absence of EDTA).

I ^{e you} chromatography in the presence of EDTA (Chromatography QS I)

A 600 ml column (K50, diameter 20 cm2) of Q Sepharose Fast Flow gel (ref. 17- 0510-01 GE Healthcare) is mounted, packed in equilibration buffer. Tris HCl 50 mM EDTA 6 mM, Urea 2 M, Elugent 1% at pH 7.5 at a flow rate of 8 ml / minute.

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.

2 ^έme chromatography (QSII), EDTA

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).

Purification by cation exchange chromatography on SP Sepharose (SPI) at pH

5.5

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.

Dialysis and concentration of SPI product (250 mM fractions)

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 μl 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).

Then the concentrate obtained is dialyzed against 2 liters of PBS Elugent 0.2% pH 7.1 (Slide A Lyser ref 66810: cutoff threshold 10 kD).

The solution is then filtered aseptically onto a Durapore Millex 0.22 μm filter (Ref Millipore SLGV 033RS). The purified rTbpB protein batch obtained is frozen at -80 ^° C. The protein concentration is 1642 μg / ml. 1.3. Preparation of the rTbpB for injection

The solution of rTbpB obtained in section B.1.2. is adsorbed on Bio-Beads ™ SM-2 to remove excess Elugent ™ detergent (alkyl glucoside surfactant) that may destabilize LOS liposomes. Activation of Bio-Beads ™

About 2.5 mL of methanol is added to 500 mg of Bio-Beads ™ and intermittently homogenized for 15 minutes. at room temperature. After a settling time, the supernatant is removed. This washing operation is repeated twice.

About 5 ml of ultrafiltered sterile water are then added and the mixture is intermittently homogenized for 15 minutes. at room temperature. After a settling time, the supernatant is removed. This washing operation is repeated twice.

About 5 mL of PBS is then added and the mixture is intermittently homogenized for 15 minutes. at room temperature. It is stored at 5 ^° C. and used the same day.

Finally, the weight of Bio-Beads ™ increased by a factor R (equal to about 1.2). Removal of the detergent by adsorption on Bio-Beads ™

The rTbpB solution obtained in section 1.2. contains 2 mg / mL of Elugent ™. The amount of Bio-Beads ™ to be used is determined based on the amount of Elugent ™ to be removed.

For one mL of the rTbpB solution obtained in section B.1.2., 29 X R mg of activated Bio-Beads ™ is added. Stir vigorously for one hour at room temperature. Then the maximum amount of liquid is recovered, to which the final 0.001% merthiolate is added. Everything is done in sterile condition.

2. Preparation of the purified LOS

Culture Eight mL of N. meningitidis strain C708, serogroup A, Ipt3 FL Ipt6 TR igtA: erm previously obtained in A.4.6. or Al strain N. meningitidis serogroup A known to express exclusively LOS of immunotype L8 and whose LOS carries 2 PEAs, one in position 3, the other in position 6 of heptose II, used to seed 800 mL of Mueller Hinton (Merck) medium supplemented with 4 niL of a glucose solution at 500 g / L and distributed in erlens. The culture is continued with stirring at 36 ± 1 ° C for about 10 hours.

400 ml of a 500 g / L glucose solution and 800 ml of an amino acid solution are added to the preculture. This preparation is used to seed a fermentor containing Mueller-Hinton medium, at a _{OD 6} oo _{m n} close to 0.05. The fermentation is continued at 36 ° C, pH 6.8, 100 rpm, ₂ pθ 30% under an initial air flow of 0.75 L / min / L of culture.

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.

Purification (adapted method from Westphal & Jann, (1965) Meth Carbohydr Chem 5: 83)

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 μm membrane filtration. The filtrate was purified by passage through a column of Sephacryl S-300 (5.0 x 90 cm; Pharmacia ™). 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.

While continuing the stirring, dehydrated absolute alcohol is added for a final concentration of 55% (vol / vol). Stirring is continued overnight at 5 ± 20 ^° C. and then centrifuged at 5.000 g for 30 minutes at 5 ± 2 ° C. The pellets are resuspended with at least 100 ml of 0.5 M MgCl ₂ and then subjected to a second alcohol precipitation identical to the previous one. The pellets are resuspended with at least 100 mL of 0.5 M MgCl ₂ .

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 μm) and stored at 5 ± 2 ° C.

This purification process makes it possible to obtain approximately 150 mg of LOS per liter of culture. 3. Preparation of liposomes [LOS] by detergent dialysis 3.1. Preparation of liposomes

LOS liposomes are prepared by detergent dialysis. In brief, the lipids (EDOPC: DOPE) are put into lipid film form and taken up in 10 mM Tris buffer, then dispersed in the presence of 100 mM octyl β-D-glucopyranoside (OG) (Sigma-Aldrich ref O8001) and sterile filtered. 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. 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.

3.333 ml of a sterile solution of octyl β-D-glucopyranoside (OG) (Sigma-Aldrich ref O8001) 1 M in 10 mM Tris buffer pH 7.0 are then added with stirring to obtain a clear suspension of lipids at 12 ° C. mg / ml, 100 mM OG, 10 mM Tris buffer. Stirring is continued for 1 hour at room temperature on a stirring table. Then sterile filtered on Millex HV 0.45 microns.

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:

2.005 mL of 10 mM Tris buffer pH 7.0; 0.223 mL of 100 mM OL in 10 mM Tris; 31.373 ml of the EDOPC: DOPE suspension of molar ratio 3: 2 to 12 mg / ml in 100 mM OG Tris 10 mM; and 6.4 mL of a sterile 1 mg / mL LOS suspension in 100mM OG Tris 10mM.

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%.

To this preparation, 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 μg / 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).

LOS liposomes are stored at + 5 ° C. 3.2. Preparation of injectables

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 μg / ml. The concentration of merthiolate is maintained at 0.001%.

5. Immunogenicity study in rabbits

The various formulations tested were manufactured as described in one of the previous sections.

5.1. Immunizations of Rabbits Twenty four 7-week-old NZ KBL (Charles River Lab.) Rabbits are divided into four test groups of four (groups A to D) and four groups of two (groups E to H).

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 μg of liposomes [LOS α chain L8, PEA-3, PEA-6] and 40 μg rTbpB M982, in 10 mM Tris buffer, 150 mM NaCl pH 7.4; Group B: 40 μg of liposomes [LOS α L8 chain, PEA-3, PEA-6] and 40 μg rTbpB

B16B6, in Tris buffer 10 mM NaCl 150 mM pH 7.4;

Group C: 40 μg of liposomes [LOS α L8 chain, PEA-3] and 40 μg rTbpB M982, in 10 mM Tris buffer 150 mM NaCl pH 7.4;

Group D: 40 μg of liposomes [LOS α L8 chain, PEA-3, PEA-6] in 10 mM Tris buffer 150 mM NaCl pH 7.4; Group E: 40 μg rTbpB M982 and 40 μg of liposomes without LOS in 10 mM Tris buffer 150 mM NaCl, 0.5% Tween pH 7.0; Group F: 40 μg rTbpB B 16B6 and 40 μg of liposomes without LOS in Tris buffer 10 mM NaCl 150 mM, 0.5% Tween pH 7.0; Group G: 40 μg of liposomes [LOS α 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

Animal blood is collected for analysis at OJ, J42 (before the third injection) and at J56.

5.2. Measurement of bactericidal activity of purified IgG against JV strains. heterologous meningitidis at strain C708

From the pool of sera, the IgGs were purified by affinity chromatography using the HiTrap rProtein A FF column (GE Healthcare / Amersham Biosciences) according to the supplier's recommendations.

From the purified IgGs, serial dilutions of Reason 2 in PBS Dulbecco's gelatin containing calcium and magnesium ions are carried out. The dilutions are made in 96-well plate for a final volume of 50 μL per well.

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 μM (iron chelating agent in free form, which allows the expression of TbpB).

Twenty five μL of the exponential phase culture (4.10 ³ CFU / mL) and 25 μL of 1 / 1.5 rabbit baby supplement are added to each well. The plate is incubated for one hour at 37 ° C., with stirring.

Fifty μL of the mixture of each well is then deposited on bioMérieux Mueller-Hinton agar plates and incubated overnight at 37 ° C. under 10% CO ₂ . We count the number of clones. The witnesses are three in number:

Bacteria + baby rabbit supplement, serum-free to test (complement control);

Bacteria + inactivated rabbit baby supplement, without serum to be tested (control "germs"); and

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.

5.3. Results and discussion

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. Thus, 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).

As expected, 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 (no adjuvantation of LOS with TbpB) 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)

'Jl

9 \

Table II (i)

IU

Table II (in)

20

In Table II above:

IT means "immunotype"

L6 means an LOS having an α chain of type L6

L8 means an LOS having an α 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

Table III

Table IV

Claims

claims

1. A vaccine against N. meningitidis infections, which includes:

(iii) a LOS of N. meningitidis consisting in particular of a lipid A, an inner core, an L8-type α chain, in which the heptose II residue 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 O-3 position and the O-6 or 0-7 position; and (iv) the lipidated subunit B (TbpB) of the human transferrin receptor of a N. meningitidis strain or a lipid fragment of this TbpB.

A vaccine according to claim 1, which comprises:

(iv) a LOS of N. meningitidis consisting in particular of a lipid A, an inner core, an L8-type α chain, in which the heptose II residue of the inner core carries a phosphoethanolamine substituent at the O-3 position;

(PEA) and does not carry a PEA substituent at positions O-6 and O-7;

(v) a LOS of N. meningitidis consisting in particular of a lipid A, an inner core, an L8-type α chain, in which the heptose II residue of the inner core bears a phosphoethanolamine (PEA) substituent, in position

0-3 and in position 0-6 or O-7; and (vi) N. meningitidis TbpB or a lipid fragment thereof.

A vaccine according to claim 1 or 2, wherein the TbpB is TbpB of a strain of N. meningitidis of IL isotype

4. A vaccine according to claim 3, wherein the TbpB of a N. meningitidis strain of isotype II is the TbpB of N. meningitidis M982 strain.

5. A vaccine according to claim 3 or 4 which further comprises TbpB of a N. meningitidis strain of isotype I.

A vaccine according to claim 5, wherein the TbpB of the N. meningitidis strain of isotype I is the TbpB of N. meningitidis strain B16B6.

7. A vaccine according to one of claims 1 to 6, wherein the LOS is formulated into liposomes (LOS liposomes).

8. A vaccine according to one of claims 1 to 7, wherein the LOS and the TbpB (s) are formulated together into liposomes.

9. A vaccine according to one of claims 1 to 7, wherein the LOS is formulated into liposomes and wherein the TbpB (s) are mixed with LOS liposomes.

10. A vaccine according to one of claims 1 to 9, which does not contain N. meningitidis outer membrane vesicle (OMV).