Method for detoxification of lipopolysaccharide (LPS) or of lipid A of gram-negative bacteria

The invention lies within the vaccine field and the subject thereof is a method for detoxifying lipopolysaccharide (LPS) or lipid A from Gram-negative bacteria, which can subsequently be used in vaccines as an adjuvant and/or as a vaccine antigen.

LPS is a major constituent of the outer membrane of the wall of Gram-negative bacteria. LPS is toxic to mammals at high dose and, in view of this biological activity, has been called an endotoxin. It is responsible for septic shock, a fatal pathological condition which develops following acute infection with a Gram-negative bacterium.

The structure of LPS consists of a lipid component, called lipid A, covalently bonded to a polysaccharide component.

Lipid A is responsible for the toxicity of LPS. It is strongly hydrophobic and enables the LPS to be anchored in the outer membrane of the wall. Lipid A is composed of a disaccharide structure substituted with fatty acid chains, the number and the composition of the fatty acid chains varies from one species to the other.

The polysaccharide component is constituted of carbohydrate chains which are responsible for the antigenicity. At least 3 major regions can be distinguished in this polysaccharide component:

(i) an inner core consisting of monosaccharides [one or more KDO (2-keto-3-deoxyocrulosonic acid) and one or more heptose (Hep)] which are unvarying within the same bacterial species;

(ii) an outer core bonded to a heptose and consisting of various monosaccharides; and

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

The composition of the polysaccharide component varies from one species to another, and from one serotype (immunotype in meningococcus) to another within the same species.

In a certain number of nonenteric Gram-negative bacteria such as Neisseriae, Bordetellae, Branhamellae, Haemophilus and Moraxellae, the O-specific chain does not exist. The saccharide component of LPS from these bacteria consists only of the oligosaccharide core. Consequently, the LPS from these bacteria is often called lipooligosaccharide (LOS).

LPS is not only toxic, it is also immunogenic. In mammals, anti-LPS antibodies are generated during carrying and infection and can be protective. Thus, the use of LPS has already been envisioned in the prophylaxis of infections caused by Gram-negative bacteria and associated diseases. Furthermore, when it is combined with another antigen of interest, it can also be capable of having an adjuvant effect - i.e. of increasing the immune response of a mammal against this vaccine antigen.

Nevertheless, it is advisable to detoxify it beforehand. To do this, it is not imperative to remove the lipid A in its entirety. Indeed, since the toxicity is more particularly linked to a supramolecular conformation conferred by all the fatty acid chains carried by the disaccharide core of lipid A, it is sufficient, according to one advantageous embodiment, to act on these chains. Detoxification can be obtained according to various approaches: chemical, enzymatic or genetic or else by complexation with a peptide analog of polymyxin B, or alternatively by associating LPS with lipids so as to form complexes such as liposomes. Indeed, LPS or lipid A in liposomes - i.e. associated with the lipid bilayer forming the liposomes - can experience a very substantial decrease in its toxicity. The lipid(s) which are part of the composition of these lipid complexes, la. of these liposomes, may be neutral, cationic and/or anionic lipids. This is described in (i) Petrov et al, Infect. Immun. (1992) 60 (9): 3897, which uses a mixture of neutral lipids, phosphatidylcholine and cholesterol; (ii) Richards et al, Vaccine (1989) 7: 506, which uses a mixture of neutral lipids (dimyristoyl phosphatidylcholine, cholesterol) and anionic lipids (dicetyl phosphate, dimyristoyl phosphatidylglycerol); and (iii) Bennett-Guerrero et al, Infect. Immun. (2000) 68 (11): 6202, which uses a mixture of neutral lipids (dimyristoyl phosphatidylcholine and cholesterol) and anionic lipids (dimyristoyl phosphatidylglycerol); and (iv) Tseng et al, Vet. Immunol. Immunopath. (2009) 131: 285, which uses a particular mixture of cholesterol, stearylamine and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPC) with, in the end, the production of cationic'liposomes.

During comparative studies, it has now been found that cationic liposomes have a greater detoxifying capacity than that of neutral or anionic liposomes. Two tests were used for the purposes of comparison:

The rabbit pyrogen test. This test, the calculations and the reading thereof were carried out according to the European Pharmacopeia guidelines (Edition 6.0, paragraph 2.6.8.).
The LAL (Limulus Amebocyte Lysate) assay carried out according to the European Pharmacopeia guidelines (Edition 6.0, paragraph 2.6.14.).

Consequently, a subject of the present invention is a method for detoxification of lipopolysaccharide (LPS) or of lipid A from a Gram-negative bacterium, according to which the LPS or the lipid A is mixed with a cationic lipid so as to form a complex in which the LPS is associated with the cationic lipid.

Likewise, a subject of the invention is also a complex comprising at least LPS or lipid A from a Gram-negative bacterium and a cationic lipid, in which the LPS or the lipid A is detoxified owing to the complexation thereof with the cationic lipid.
LPS/lipidA

The LPS/lipid A that can be detoxified by means of the method according to the invention may be any LPS/lipid A from Gram-negative bacteria, whether they are enteric or nonenteric, preferably pathogenic. According to one particular aspect, it may be LPS/lipid A from nonenteric bacteria of genera such as Neisseria, Bordetella, Branhamella, Haemophilus and Moraxella. The LPS from these bacteria is also referred to as LOS (lipooligosaccharide) owing to the absence of O-specific polysaccharide. By way of additional example, mention is made of LPS/LOS from the genera or species Klebsiella, Pseudomonas, Burkholderia, Porphyromonas, Franciscella, Yersinia, Enterobacter, Salmonella, Shigella, E. coli; and most particularly the LOS from N. meningitidis.

The N. meningitidis strains are classified as several immunotypes (IT LI to LI3), according to their reactivity with a series of antibodies which recognize varied epitopes on LOS (Achtman et al, 1992, J. Infect. Dis. 165: 53-68). The LOS from these strains is subsequently likewise listed as 13 immunotypes (IT LI to L13). The differences between immunotypes originate from variations in the composition and in the conformation of the oligosaccharide chains. This shows in the table below, derived from table 2 of Braun et al, Vaccine (2004) 22: 898, supplemented with data obtained subsequently and relating to immunotypes L9 (Choudhury et al, Carbohydr. Res. (2008) 343: 2771) and Lll (Mistretta et al, (2008) Poster at the 16th International Pathogenic Neisseria Conference, Rotterdam): n.d.: not determined.

**: when R2 is a glucose residue, R2 is commonly called P chain.

It may be noted, inter alia, that certain LOSs may be sialylated (presence of N-acetylneuraminic acid on the terminal galactose residue (Gal) of the a chain). Thus, immunotypes L3 and L7 differ only by the respective presence/absence of this sialylation. Moreover, most LOSs are substituted with an O-acetyl group on the glucosamine residue (α -GlcNAc or y chain) 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. (above); and Mistretta et al. (above).

The Gaipi-4GlcNAc(31-3Gaipi-4Glc[31-4 carbohydrate unit or lacto-N-neotetraose unit which is present in the a chain of certain N. meningitidis LPS immunotypes constitutes an epitope which can potentially crossreact with human erythrocytes. Thus, with a view to producing a vaccine for use in humans, it is advisable to choose an LPS which does not possess this unit. It may therefore be particularly advantageous to use an LOS of immunotype L8.

Alternatively, it is also possible to envision starting, for example, from a strain of immunotype L2 or L3 in which a gene involved in the biosynthesis of the a chain has been inactivated by mutation, so as to obtain an incomplete LNnT structure. Such mutations are proposed in patent application WO 04/014417. It involves extinguishing, by mutation, the IgtB, IgtE (or IgtH), IgtA or IgtA and IglC genes. Thus, it appears to be possible and advantageous to use an LPS originating from an N. meningitidis strain of immunotype L2 or L3 which is IgtB', IgtE' (or IgtH), IgtA' or IgtA' and IglC.

Generally, it should therefore be understood that an LPS that is of use for the purposes of the present invention can have a structure which is as found naturally or else which has undergone modifications, in particular following mutation.

For the purposes of the present invention, the LPS may be obtained by conventional means: in particular, it can be extracted from a bacterial culture, and then purified according to conventional methods. Many methods of production are described in the literature. By way of example, mention is made, i.a., of Gu & Tsai', 1993, Infect. Immun. 61 (5): 1873; Wu et al, 1987, Anal. Biochem. 160: 281 and US 6,531,131. An LPS preparation can be quantified according to well-known procedures. Assaying of KDO by high performance anion exchange chromatography (HPAEC-PAD) is a method which is most particularly suitable.

With regard to the lipid A, it can be obtained, i.a., by acid hydrolysis of LPS as, for example, described in Gu & Tsai, Infect. Immun. (1993) 61 (5): 1873.

The complex

The complex according to the invention or resulting from the method according to the invention is a complex which is cationic in nature (positively charged). Typically, this can be a cationic liposome.

The term "liposomes" is intended to mean a synthetic entity, preferably a synthetic, vesicle, made up of at least one lipid bilayer membrane (or matrix) enclosing an aqueous compartment. For the purposes of the present invention, the liposomes may be unilamellar (a single bilayer membrane) or multilamellar (several membranes layered like an onion). The lipids constituting the bilayer membrane comprise a nonpolar region which, typically, is made of a chain or chains of fatty acids or of cholesterol, 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, in particular at physiological pH (pH = 7), carry either a negative (anionic lipid) or positive (cationic lipid) net (overall) surface charge, or not carry a net charge (neutral lipid).

The complexes, la. the liposomes, that are of use for the purposes of the present invention may be any type of lipid complexes of cationic type (carrying positive charges), la. cationic liposomes. At least one of the lipids that is part of the composition of these complexes, la. of these liposomes, is a cationic lipid. The cationic lipid(s) may be accompanied by anionic lipids provided that these complexes, la. these liposomes, remain cationic in nature - i.e. the overall charge of the complexes, la. of the liposomes, remains positive.

The cationic lipid

For the purposes of the present invention, the cationic lipid may be:

(i) lipophilic amines or alkylamines such as, for example, dimethyldioctadecyl-ammonium (DDA), trimethyldioctadecylammonium (DTA) or structural homologs of DDA and of DTA [these alkylamines are advantageously used in the form of a salt; mention is made, for example, of dimethyldioctadecylammonium bromide (DDAB)];

(ii) octadecenoyloxy (ethyl-2-heptadecenyl-3-hydroxyethyl)imidazoliniurn (DOTIM) and structural homologs thereof;

(iii) lipospermines such as N- palmitoyl-D-erythrosphingosyl-l- o -carbamoyl-spermine (CCS) and dioctadecylamidoglycylspermine (DOGS, transfectam);

(iv) lipids incorporating an ethylphosphocholine structure, such as 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-.v«-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-5«-glycero-3-ethylphosphocholine, l,2-palmitoyloleoyl-s/'Z-glycero-3-ethylphosphocholine, 1,2-distearoyl-src-glycero-3-ethylphosphocholine (DSPC), l,2-dioleoyl-s/7-glycero-3-ethylphosphocholine (DOEPC or EDOPC or ethyl-DOPC or ethyl PC), and also structural homologs thereof; (v) lipids incorporating a trimethylammonium structure, such as N-(l-[2,3-dioleyloxy]propyl)-N,N,N -trimethylammonium (DOTMA) and structural homologs thereof and those incorporating a trimethylammonium propane structure, such as 1,2-dioleyl-3-trimethylammonium propane (DOTAP) and structural homologs thereof; and also lipids incorporating a dimethyl ammonium structure, such as l,2-dioleyl-3-dimethylammonium propane (DODAP) and structural homologs thereof; and (vi) cationic derivatives of cholesterol, such as 3 β -[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Choi) or other cationic derivatives of cholesterol, such as those described in US patent 5 283 185, and in particular cholesteryl-3(3-carboxamidoethylenetrimethylammonium iodide, cholesteryl-3(3-carboxyamido-ethyleneamine, cholesteryl-3(3-oxysuccinamidoethylenetrimethylammonium iodide and 3P-[N-(polyethyleneimine)carbamoyl]cholesterol.

The term "structural homologs" signifies lipids which have the characteristic structure of the reference lipid while at the same time differing therefrom by virtue of secondary modifications, especially in the nonpolar region, in particular of the number of carbon atoms and of double bonds in 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 (C16:l), octadecanoic or stearic acid (C18:0), cis-9-octadecanoic or oleic acid (C18:1), cis,cis-9,12-octadecadienoic or linoleic acid (C18:2), cis,cis-6,9-octadecadienoic acid (C18:2), all-cis-9,12,15-octadecatrienoic or a-linolenic acid (C1 8:3), all-cis-6,9,12-octadecatrienoic or γ -linolenic acid (C18:3), eicosanoic or arachidic acid (C20:0), cis-9-eicosenoic or gadoleic acid (C20:l), all-cis-8,11,14-eicosatrienoic acid (C20:3), all-cis-5,8,11,14-eicosatetraenoic or arachidonic acid (C20:4), all-cis-5,8,11,14,17-eicosapentaneoic acid (C20:5), docosanoic or behenic acid (C22:0), all-cis-7,10,13,16,19-docosapentaenoic acid (C22:5), all-cis-4.7,10,13,16,19-docosahexaenoic acid (C22:6) and tetracosanoic or lignoceric acid (C24:0).

The characteristic structure of DDAB is:

Generally, the cationic lipid can be advantageously used in combination with a neutral lipid which is often referred to as a co-lipid. According to one advantageous mixing mode, the charged lipid (cationic lipid with or without anionic lipid):neutral lipid molar ratio is between 10:1 and 1:10, advantageously between 4:1 and 1:4, preferably between 3:1 and 1:3, limits included.

With regard to the neutral lipids, mention is made, by way of example, of: (i) cholesterol;
(ii) phosphatidylcholines such as, for example, l,2-diacyl -sn - glycero-3-phosphocholines, e.g. l,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), and also l-acyl-2-acyl- sn - glycero-3-phosphocholines of which the acyl chains are different than one another (mixed acyl chains); and (iii) phosphatidylethanolamines such as, for example, l,2-diacyl- sn -glycero-3-phosphoethanolamines, e.g. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and also l-acy]-2-acyl- sn -glycero-3-phosphoethanol-amine carrying mixed acyl chains.

Thus, according to one 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, in particular in a DC-chol : DOPE molar ratio ranging from 10:1 to 1:10, advantageously from 4:1 to 1:4, preferably from approximately 3:1 to 1:3;

a mixture of EDOPC and cholesterol, in particular in an EDOPCxholesterol molar ratio ranging from 10:1 to 1:10, advantageously from 4:1 to 1:4, preferably from approximately 3:1 to 1:3; and

a mixture of EDOPC and DOPE, in particular in an EDOPC:DOPE molar ratio ranging from 10:1 to 1:10. advantageously from 4:1 to 1:4, preferably from approximately 3:1 to 1:3.

Several techniques known to those skilled in the art make it possible to obtain preparations of liposomes containing LPS ([LPS] liposomes). These various techniques can be more or less suitable depending on the nature and the properties of the LPS, in particular depending on its solubility in an aqueous or organic medium. Those skilled in the art are able to select the method(s) most suitable.

It is, for example, possible to incorporate the LPS into liposomes by preparing a dry lipid film which is subsequently rehydrated with an aqueous solution of LPS, as described by Dijkstra el al. J. Immunol. Methods (1988) 114: 197-205. Alternatively, if the LPS is soluble in the organic solvent used to dissolve the lipids, it is possible to directly prepare an organic solution containing both the LPS and the lipids, followed by drying of this lipid mixture-in the form of a film on the walls of a container and, finally, rehydration of this dry lipid film with an aqueous buffer in order to form liposomes containing LPS. Generally, the reconstitution step in an aqueous medium results in the spontaneous formation of multilamellar vesicles, the size of which is then made homogeneous by gradually decreasing the number of lamellae by extrusion, for example using an extruder, by passing the lipid suspension, under a nitrogen pressure, through polycarbonate membranes with decreasing pore diameters (0.8, 0.4, 0.2 urn).

The LPS can also be incorporated into liposomes via the "dehydration-rehydration" method in which preformed liposomes are mixed with LPS in an aqueous solution, sonicated and freeze-dried, before being taken up in an aqueous buffer. This technique is, for example, used by Petrov et al. Infect. Immun. (1992) 60: 3897-3903.

The LPS can also be incorporated into liposomes by the "detergent dilution" technique in which mixed micelles of LPS/lipids in detergent are greatly diluted in an aqueous buffer so as to reach a detergent concentration which is lower than the critical micellar concentration of the detergent. LPS liposomes then form. This technique is, for example, used by Argita et al, Vaccine (2005) 23: 5091-5098. This method is equivalent to the "detergent dialysis" method described in the examples/experimental data which follow.

According to one advantageous embodiment, in an initial step, a dry lipid film is prepared with all the compounds which are part of the composition of the complexes, La. of the liposomes. The lipid film is then reconstituted in an aqueous medium, in the presence of LPS, for example in a lipid: LPS molar ratio of 100 to 500, advantageously of 100 to 400; preferably of 200 to 300; most particularly preferably of approximately 250. Generally, it is considered that this same molar ratio should not substantially vary at the end of the method of preparing the LPS liposomes.

Generally, the reconstitution step in an aqueous medium results in the spontaneous formation of multilamellar vesicles, the size of which is subsequently made homogeneous by gradually decreasing the number of lamellae by extrusion, for example using an extruder, by passing the lipid suspension, under a nitrogen pressure, through polycarbonate membranes with decreasing pore diameters (0.8, 0.4, 0.2 urn). The extrusion process can also be replaced with another process using a detergent (surfactant) which disperses lipids (see Argita et al (above)). This detergent is subsequently removed by dialysis or by adsorption onto porous polystyrene microbeads with a particular affinity for detergent (BioBeads). When the surfactant is removed from the lipid dispersion, the lipids reorganize in a double layer.

At the end of the complexation, La. of the incorporation of the LPS into liposomes, a mixture consisting of complexes, La. of adhoc liposomes, and of LPS in free form is obtained. Advantageously, the liposomes are then purified in order to be rid of the LPS in free form.

Given the properties of LPS and of lipid A, a complex according to the invention can have a varying use: either as an adjuvant in any vaccine comprising any vaccine antigen; or as a vaccine antigen in a vaccine against Gram-negative bacteria; or as both an adjuvant and a vaccine antigen.

Vaccine/Use as a vaccine antigen

According to another aspect, a subject of the invention is also a vaccine composition (also referred to as vaccine) which comprises an LPS-cationic lipid complex according to the invention; the LPS being present in a sufficient amount for the anti-LPS immune response induced by the vaccine to be capable of protecting an individual against a disease caused by the Gram-negative bacterium.

A vaccine composition according to the invention is in particular of use for treating or preventing an infection with a Gram-negative bacterium which is nonenteric (such as bacteria of the genera Neisseria, Bordetella, Branhamella, Haemophilus and Moraxella); or of the genera Klebsiella, Pseudomonas, Burkholderia, Porphyromonas, Franciscella, Yersinia, Enter obacter, Salmonella, Shigella, Escherichia, e.g. E. coli.

According to a preferred aspect, a vaccine composition according to the invention is in particular of use for treating or preventing an infection caused by N, meningitidis, such as meningitis caused by N. meningitidis, meningococcemia and complications which can derive therefrom, such as purpura fulminans and septic shock; and also arthritis and pericarditis caused by N meningitidis.

It can be produced conventionally. In particular, a therapeutically or prophylactically effective amount of LPS is combined with a carrier or with a diluent.

A vaccine according to the invention may also comprise an adjuvant. According to one advantageous embodiment, the adjuvant may be a lipopolypeptide.

Said lipopolypeptide is typically a polypeptide onto which one or more lipid(s), including in particular fatty acids, isoprenoids and cholesterol, is (are) grafted by covalent bonding, at one or more lipidation site(s).

An advantageous adjuvant is an acylated polypeptide, i.e. a polypeptide bearing at least one, preferably at least two or three acyl groups. Advantageously, the lipopolypeptide is diacylated or triacylated. The acyl group(s) borne by the polypeptide may each independently be a C8 to C22, preferably C12 to C18, most particularly preferably C14 , C16 or C18 , acyl group.

Thus, any acylated, preferably at least diacylated, polypeptide can be used as an adjuvant. It may be of natural or synthetic origin. A lipopolypeptide of natural origin may be that of a prokaryotic or eukaryotic organism.

Proteins are naturally modified with lipids, covalently, by virtue of multiple mechanisms such as, for example, N-myristoylation, S-palmitoylation or prenylation. N-myristoylation consists of the covalent addition of a fatty acid, myristic acid, to a glycine residue in the N-terminal position, via an amide bond. S-palmitoylation consists of the covalent addition of a fatty acid, palmitic acid, to a cysteine residue, via a thioester bond. Prenylation consists of the addition of an isoprenoide, either a C15 famesyl group or a C20 geranylgeranyl group, to a cysteine residue in the C-terminal position, via a thioester bond.

Thus, advantageous lipopolypeptides may be S-, N- and/or O-acylated polypeptides. By way of example, mention is made of lipopolypeptides having one or more of the following characteristics a) to c):

a) the amino acid sequence of the polypeptide comprises at least one cysteine residue which is S-acylated with an acyl group R;

b) the amino acid in the N-terminal position, for example a glycine or cysteine residue, is N-acylated with an acyl group R';

c) the amino acid in the C-terminal position is O-acylated with an acyl group R";
each of the groups R, R" and R'' being independently an acyl group containing from 8 to 22, advantageously from 10 to 20, preferably from 12 to 18, most particularly preferably 14, 15, 16, 17 or 18 carbon atoms.

According to one particularly advantageous embodiment, the lipopolypeptide adjuvant is a prokaryotic lipoprotein, in particular a bacterial lipoprotein, for example from a Gram-negative bacterium. The majority of bacterial lipoproteins are envelope proteins, the latter consisting of two, outer and inner, membranes delimiting in between them a space called the periplasm.

Generally, bacterial envelope lipoproteins are, in their natural environment, proteins anchored to the periplasmic face of one of the two membranes by three fatty acids in the N-terminal position. Two, in diacylglyceryl form, reduce the sulfhydryl (SH) group of the cysteine residue in the N-terminal position (thioether bond). The third acylates the amine group (NH2) of this same residue.

According to one preferred embodiment, the lipopolypeptide in use is a lipoprotein from an enteric or nonenteric, preferably pathogenic, Gram-negative bacterium. According to one particular aspect, it may be a lipoprotein from nonenteric bacteria of genera such as Neisseria, Bordetella, Branhamella, Haemophilus and Moraxella. It may also be a lipoprotein from the genera Klebsiella. Pseudomonas. Burkholderia, Franciscella, Yersinia, Enlerobacter, Salmonella. Shigella and Escherichia, e.g. E. coli.

According to one most particularly preferred embodiment, the LPS and the lipopolypeptide used as an adjuvant both originate from a Gram-negative bacterium, advantageously of the same bacterial species.

Generally, the lipopolypeptide, and more particularly the "polypeptide" component of the latter, comprises one or more T-heJper epitope(s) - i.e. epitopes capable of being recognized by T-helper cells and of activating them. Advantageously, they are T-helper epitopes characteristic of the organism for which the LPS-based vaccine is intended (a mammal, in particular a human) - i.e. epitopes capable of being recognized by the T-helper cells of the recipient organism and of activating them.

By way of indication, the following lipoproteins are mentioned: OspA (outer surface protein A) from Borrelia burgdorferi; GNA1870 (factor H-binding protein) and NMB1969 (NalP or Asp A) from N. meningitidis and lipoprotein D from Haemophilus influenzae or a lipidated fragment of these lipoproteins, in particular an N-terminal fragment.

According to one preferred embodiment, a lipopolypeptide that is of use as an adjuvant may be the human transferrin receptor subunit B (TbpB), which is an outer membrane protein of a certain number of nonenteric Gram-negative bacteria such as Neisserias, Moraxellae, Branhamellae, Bordeiellae and Haemophilus. Alternatively, it may also be a lipidated N-terminal fragment of a TbpB. Most particularly preferably, it is the TbpB from Neisseria meningitidis or a lipidated fragment thereof.

The vaccine composition according to the invention may also comprise one or more additional vaccine antigen(s). Advantageously, they may be chosen from proteins of the bacterial species from which the LPS originates. In connection with this possibility, a vaccine composition according to the invention may also contain a pharmaceutical]y acceptable additional adjuvant other than the lipopolypeptide, for adjuvanting the additional vaccine antigen(s): nevertheless, this option is not the most attractive owing to the complexity thereof.

The amounts of LPS and/or of adjuvant per vaccine dose which are sufficient to achieve the abovementioned aims, and which are effective from an immunogenic, prophylactic or therapeutic point of view, depend on certain parameters that include the individual treated (adult, adolescent, child or infant), the route of administration and the administration frequency.

Thus, the amount of LPS per dose which is sufficient to achieve the abovementioned aims is in particular between 5 and 500 ug, advantageously between 10 and 200 ug, preferably between 20 and 100 ug, entirely preferably between 20 and 80 ug or between 20 and 60 ug, limits included.

When the LPS is adjuvanted with a lipopolypeptide, the amount of the latter per dose may be between 5 and 500 ug. advantageously between 10 and 200 ug, preferably between 20 and 100 ug, entirely preferably between 20 and 80 ug or between 20 and 60 ug. limits included.

In the latter case, the LPS ; dipopolypeptide molar ratio may be from 10"2 to 103, advantageously from 10-1 to 102. preferably from 1 to 50. most particularly preferably from 15 to 30, or approximately 20.

The term "dose" employed above should be understood to denote a volume of vaccine administered to an individual in one go - i.e. at a time T. Conventional doses are of the order of a milliliter, for example 0.5, 1 or 1.5 ml. the definitive choice depending on certain parameters, and in particular on the age and the status of the recipient. An individual can receive a dose divided up into injections at several injection sites on the same day. The dose may be a single dose or, if necessary, the individual may also receive several doses a certain time apart - it being possible for this time apart to be determined by those skilled in the art.

A vaccine composition according to the invention can be administered by any conventional route in use in the prior art, e.g. in the vaccines field, in particular enterally or parenterally. The administration may be carried out as a single dose or as repeated doses a certain time apart. The route of administration varies according to various parameters, for example according to the individual treated (condition, age, etc.).

Finally, a subject of the invention is also:

a method for inducing in a mammal, for example a human, an immune response against a Gram-negative pathogenic bacterium, according to which an immunogenically effective amount of a vaccine according to the invention is administered to the mammal so as to induce an immune response, in particular a protective immune response against the Gram-negative pathogenic bacterium; and a method for prevention and/or treatment of an infection induced by a Gram-negative pathogenic bacterium, according to which a prophylactically or therapeutically effective amount of a vaccine according to the invention is administered to an individual in need of such a treatment.

Experimental data

1. Preparation of the purified LPS Culturing Eight mL of frozen sample of a strain of N. meningitidis serotype A known to express exclusively LPS of immunotype L8 (strain Al) are used to inoculate 800 mL of Mueller-Hinton medium (Merck) supplemented with 4 mL of a solution of glucose at 500 g/L and distributed into Erlenmeyer flasks. The culturing is continued at 36 + 1°C with shaking for approximately 10 hours.

400 mL of a solution of glucose at 500 g/L and 800 mL of a solution of amino acids are added to the preculture. This preparation is used to inoculate a fermenter containing Mueller-Hinton medium, at an OD600nm close to 0.05. The fermentation is continued at 36°C, at pH 6.8, 100 rpm, pO2 30%, under an initial air flow of 0.75 L/min/L of culture.

After approximately 7 hours (OD600nm of approximately 3). Mueller-Hinton medium is added at a rate of 440 g/hour. When the glucose concentration is less than 5 g/L, the fermentation is stopped. The final OD600 nm is commonly between 20 and 40. The cells are harvested by centrifugation and the pellets are frozen at -35°C.

Purification

The pellets are thawed and suspended with 3 volumes of 4.5% (vol./vol.) phenol, with vigorous stirring for 4 hours at approximately 5°C. The LPS is extracted by treatment with phenol.

The bacterial suspension is heated to 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 1 1 000 g. The aqueous phase is removed and stored, while the phenol phase and the interphase are harvested so as to be subjected to a second extraction.

The phenol phase and the interphase are heated to 65°C, and then mixed with a volume of water equivalent to the volume of the aqueous phase previously removed, 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 phenol phase and the interphase are harvested so as to be subjected to a third extraction identical to the second.

The three aqueous phases are dialyzed separately, each against 40 L of water. The dialyzates are then pooled together. One volume of 20 mM Tris. 2 mM MgCl2 is added to 9 volumes of dialyzate. 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 bacterial pellet treated (wet weight). The preparation is stirred at 37°C for approximately 1 hour. The pH is adjusted to 6.8 + 0.2. The preparation is then subjected to filtration through a 0.22 urn membrane, and the filtrate is purified by passing it over a Sephacryl S-300 column (5.0 x 90 cm; Pharmacia™).

The fractions containing the LPS are pooled together and the MgCl2 concentration is raised to 0.5M by adding MgCl2-6H20 powder with stirring.

While continuing the stirring, dehydrated absolute alcohol is added for a final concentration of 55% (vol./vol.). The stirring is continued overnight at 5 + 2°C, and then centrifugation is carried out at 5 000 g for 30 min at 5 + 2°C. The pellets are resuspended with at least 100 mL of 0.5M MgCl2 and then subjected to a second alcoholic precipitation identical to the previous one. The pellets are resuspended with at least 100 mL of 0.5M MgCl2 .

The suspension is subjected to a gel filtration as previously described, The fractions containing the LPS are pooled together, sterilized by filtration (0.8-0.22 μm) and stored at 5 + 26C.

This method of purification makes it possible to obtain approximately 150 mg of LPS L8 per liter of culture.

2. Preparation of LPS liposomes ( i. a., lipids: EDOPC and DOPE)
2.1. Production of [LPS L8] liposomes by detergent dialysis
The LPS L8 liposomes are prepared by detergent dialysis. Briefly, the lipids (EDOPC:DOPE) are made into the form of a lipid film and taken up in 10 mM Tris buffer, and then dispersed in 100 mM octyl- β -D-glucopyranoside (OG; Sigma-Aldrich ref O8001) and filtered sterilely. The LPS L8 in 100 mM OG is added sterilely. The lipid/LPS/OG mixture is then dialyzed against 10 mM Tris in order to remove the OG and to form the liposomes.

Protocol

A lipid preparation in chloroform, of the lipids that will be used to produce the liposomes, is prepared. A dry film is obtained by complete evaporation of the chloroform.
For example, a dry film of 1,2 -dioleoyl- sn -glycero-3-ethy!phosphocholine (EDOPC or efhyl-DOPC) and of l,2-dioleoyl-i77-glycero-3-phosphoethanolamine (DOPE) in an EDOPC:DOPE molar ratio of 3 to 2 is obtained by mixing 12.633 mL of a solution of EDOPC (Avanti Polar Lipids ref 890704) at 20 mg/ml in chloroform and 7.367 mL of a solution of DOPE (Avanti Polar Lipids ref 850725) at 20 mg/ml in chloroform, and evaporating the chloroform until it has completely disappeared.

The dry film is taken up with 30 mL of 10 mM Tris, pH 7.0, so as 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 hour at ambient temperature and then sonicated for 5 min in a bath.

3.333 ml of a sterile 1M solution of OG in 10 mM Tris, pH 7.0, are then added, still with stirring, so as to obtain a clear suspension of lipids at 12 mg/ml, 100 mM OG and 10 mM Tris. The stirring is continued for 1 h at ambient temperature on a platform shaker. Filtration is then carried out sterilely through a Millex HV 0.45 mn filler.

The following preparations are mixed under sterile conditions:

2.005 mL of 10 mM Tris, pH 7.0; 0.223 mL of 100 mM OG in 10 mM Tris; 31.373 mL of the EDOPC:DOPE suspension having a molar ratio of 3:2, at 12 mg/ml in 100 mM OG, 10 mM Tris; and 6.4 mL of a sterile suspension of LPS L8 at 1 mg/ml in 100 mM OG, 10 mM Tris; so as thus to obtain 40 mL of a composition having a lipids:LPS molar ratio of 250 (0.160 mg/mL of LPS L8, 9.412 mg/mL and 100 mM of OG).

After stirring for one hour at ambient temperature, the suspension is transferred sterilely into 4 sterile 10 ml dialysis cassettes. Each cassette is dialyzed.3 times (24 hours -24 hours - 72 hours) against 200 volumes of 10 mM/L Tris, pH 7.0, i.e. 2 L.

The liposomes are recovered under sterile conditions. The increase in volume after dialysis is approximately 30%.

Merthiolate and NaCl are added to this preparation so as to obtain a preparation of liposomes in 10 mM Tris, 150 mM NaCl, pH 7.0, 0.001% merthiolate.
Finally, the preparation is obtained at the following concentrations:
LPS approximately 1 1 0 u.g/ml
lipids approximately 7 mg/ml, of which EDOPC approximately 4.5 mg/ml and DOPE approximately 2.5 mg/ml
lOmM/LTris
150 mM/L NaCl
0.001% merthiolate. The LPS liposomes are stored at +5°C.
2.2. Production of [LPS L8] liposomes by extrusion
[LPS L8] liposomes are prepared with DC-chol or EDOPC in a lipid/LPS molar ratio of 250.

To do this, 129 μ g of LPS and 5.2 mg of DC-chol or 10.4 mg of EDOPC are dissolved in 10 mL of a 4:1 chloroform/methanol mixture. A dry film is prepared by solvent evaporation and additional drying under a strong vacuum. The film is taken up with ultrafiltered water at 50°C using, a vortex. The preparation is passed through an ultrasonic bath, and then subjected to extrusion by membrane filtration (retention threshold: 0.4 μ m ) once; followed by six consecutive membrane filtrations (retention threshold: 0.2 μ m ). Sterilization is carried out by filtration.

3. Evaluation of the detoxification of LPS in liposomes
Three main tests are used: (i) the LAL (Limulus Amebocyte Lysate) assay; (ii) an in vitro assay for release of the cytokines IL6 and TNFa; and (iii) the rabbit pyrogen test.

LAL assay

The LAL assay is a very sensitive colorimetric test for detecting and quantifying endotoxins of Gram-negative bacteria. This test is carried out according to the European Pharmacopeia guidelines (Edition 5.0, paragraph 2.6.14.) using the QCL-1000 kit, ref 50-647 U, from Cambrex-BioWhittaker™ (linear zone of the test: 0.1 to 1 lU/mL) with, as positive control, the E. coli endotoxin, 4 x 103 EU/mL (SigmaTM).

Dilutions (i) of the test samples, (ii) of the standard and (iii) of the positive control are prepared in the respective ranges of 1/10 to 1/105; 0.5 to 0.031 EU/mL; and 1/104 to 1.8 104.

Fifty uL of the dilutions of the samples, of the standard and of the positive control are distributed into the wells of a 96-well ELISA plate. 50 u,L of lysate are added to each of the wells, and then 100 uL of p-nitroaniline. Incubation is carried out for 6 min at 37°C. The reaction is stopped by adding 100 uL of 25% glacial acetic acid (in water). The plate is read by spectrometry at 405 tun.

Calculation of the endotoxin concentration: the average OD value of the "blank" samples is subtracted from all the optical densities (ODs). The linear regression line of the standard range is established (it should be linear from 0.031 EU/mL to 0.5 EU/mL) in order.to calculate the endotoxin concentration (EU/mL) of each sample tested, on the basis of the ODs obtained. These values are then multiplied by the inverse of the corresponding dilutions and the arithmetic mean is calculated.

The detoxification rate is determined as being the LAL value measured on the nonformulated LPS divided by the LAL value measured on the liposome-formulated product, at equivalent LPS concentration.

In vitro cytokine release assay

Human blood collected in sodium heparin (25 000 U/5 mL; Sanofi Aventis) is diluted to 1/5 in AIM-V medium (Invitrogen™). This preparation is distributed into Micronics™ tubes in a proportion of 400 u.L per well. 100 uL of the test substances are added. The tubes are incubated for 24 h at 37°C in a humid atmosphere at 5% CO?.

The tubes are centrifuged for 10 min at 500 g. At least 200 uL of the plasma supernatant are sampled from each tube, and kept frozen at -80°C until the titration is carried out.

The titration of the cytokines is carried out by ELISA using the OptEIA human IL6, IL8 and TNFa kits from Pharmingen™, each of the kits comprising a capture antibody (mouse anti-human cytokine antibody), a detection antibody (biotinylated mouse anti-human cytokine antibody), avidin-peroxidase conjugate and the standards.

The capture antibodies are diluted to 1/250 th in 0.1M carbonate buffer, pH 9.5 (Sigma™). For each test, 100 μ L of the dilution to l/250th are distributed into each well of a 96-well flat-bottom ELISA plate (NUNC Maxisorp 96™). The plates are incubated overnight at 4°C.

The plates are washed in PBS-0.05% Tween 20. 200 uL of PBS-0.05% bovine serum albumin are added per well. Incubation is carried out for 1 h at ambient temperature. The plates are washed in PBS-0.05% Tween 20.

Dilutions of the recombinant cytokines in AIM-V medium are prepared in the following ranges: μ g/mL - 18.75 μ g/mL (IL6); 800 μ g/mL - 12.5 μ g/mL (IL8); and 1000 μ g/mL - 15.87 pg/mL (TNFa). 100 Μ L of each dilution are distributed into the wells in order to establish the standard curve.

The plasmas harvested from blood stimulated with pure LPS are diluted to 1 /25th and 1/125th. Those harvested from blood in contact with LPS liposomes are diluted to l/5th and to l/25th. 100 u.L of each dilution are distributed per well. Incubation is earned out for 2 h at ambient temperature.

The plates are washed in PBS-0.05% Tween 20. The detection antibody and the enzyme are both diluted to 1/250th in PBS containing 10% fetal calf serum. 100 μ 1 of each dilution are added per well. Incubation is carried out for one hour at ambient temperature.
The plates are washed in PBS-0.05% Tween 20. 100 μ l of substrate are distributed per well (solutions A and B of tetramethylbenzidine mixed vol. for vol.). Incubation is carried out for 10 to 30 min at ambient temperature.

The reaction is stopped by adding 100 )^L of 1M phosphoric acid per well. The plates are read at 450 nm.

The standard curves of cytokine concentration as a function of optical density are obtained from a recombinant cytokine dilution range, and the crude results corresponding to the concentrations of the samples are obtained from these standard curves.

The detoxification rate is determined as being the ratio of the concentration of liposome-formulated LPS that induces 50% of maximum release (ED50 expressed in pg/mL) divided by the concentration of nonformulated LPS that induces 50% of maximum cytokine release. The higher the rate, the higher the detoxification. Since the detoxification rate is systematically measured using blood from several independent donors, the results express a mean value.

Rabbit pyrogen test

The rabbit is considered to be the animal having a sensitivity to LPS pyrogenie effects that is equivalent to that observed in humans. The pyrogen test consists in measuring the temperature increase induced by an intravenous injection of a sterile solution of the substances to be analyzed. The test, the reading and the calculations are carried out according to the European Pharmacopeia guidelines (Edition 6.0, paragraph 2.6.8.). A pyrogen effect is recorded when a temperature increase of more than 1.15°C is observed.

4. Immunogenicity study in mice

Mouse immunization

Seven-week-old CD1 female mice (Charles River Lab.) divided up into various groups, receive, subcutaneously, a 200 ul aliquot of the test preparations adjusted in 10 mM Tris, 150 mM NaCl, pH 7.0, to 50 μ g/mL of LPS. Blood samples are taken for analysis,
before each of the two injections. The mice are sacrificed at D35.

Assaying o f anti-LPS antibodies by ELISA (IgG, IgM)

This assay is automated (Staccato automated system, Caliper) according to the following protocol:

The wells of Dynex™ 96-well plates are coated with 1 ug of LPS L8 in IX PBS (phosphate buffered saline) buffer, pH 7.1 + 10 mM MgCl , and the plates are incubated for 2 hours at 37°C and then overnight at 4°C. The plates are blocked by adding, to the •wells, 150 ul of PBS containing 0.05% Tween 20 and 1% (weight/vol.) of powdered skim milk (PBS-Tween-milk). The plates are incubated for 1 h at 37°C.

Serial double dilutions of the test samples are prepared in PBS-0.05% Tween-1% milk.

The plates are incubated for 90 min at 37°C and then washed 3 times with PBS + Tween 20 at 0.05%.

A peroxidase-anti-mouse IgG or -anti-rabbit IgG or peroxidase-anti-mouse IgM or -anti-rabbit IgM conjugate in PBS-Tween-milk is added to the wells and the plates are incubated for 90 min at 37°C. The plates are washed three times. 100 μ l per well of a ready-to-use solution of TMB (3,3',5,5'-tetramefhylbenzidine, substrate for peroxidase) are distributed per well. The plates are incubated in the dark for 20 min at ambient temperature. The reaction is stopped by adding 100 μ 1 of 1M HC1 per well.

The optical density is measured at 450-650 nm with an automatic plate reader (Multiskan Ascent). With there being no standard available, the antibody titers are determined as being the reciprocal dilution giving an optical density of 1.0 on a tendency curve (CodUnit software). The antibody detection threshold is Ologio ELISA -unit. For each titer below this threshold, an arbitrary value of Ologio is assigned.

5. Quantification of LPS and lipids in liposomes

5.1. Assaying of lipids by HPLC-UV

Preparation of the standard range and of the samples to be analyzed

A stock solution containing 1 mg/mL, in chloroform, of each of the EDOPC, DOPC, DOPE, DC-chol and cholesterol lipids is prepared and is subsequently diluted to 1/10th by adding an acetonitrile/water (90/10) mixture. This stock solution is used to prepare the standard range of 2 to 50 μ l/ml by dilution in the acetonitrile/water mixture.

The samples to be analyzed are diluted in acetonitrile/water so as to have a theoretical final concentration of approximately 10 μg/ml.

Analytical conditions

A Zorbax C18 Extend, 3;5 urn, 3 x 150 mm, 80A column (Agilent reference 763954-302) is used, and for the mobile phase, an acetonitrile/water/trifluoroacetic acid (TFA) mixture in the volume proportions 850/150/1 is used. The column is pre-conditioned according to the following process:

flow rate at 0.25 ml/min for 20 minutes (P - 21 bar) - flow rate at 0.5 ml/min for 20 minutes (P = 42 bar)

flow rate at 0.75 ml/min for 20 minutes (P = 60 bar)

flow rate at 1 ml/min for 20 minutes (P = 80 bar)

The measurements are carried out at 60°C, by injecting 10 μ L of the preparation at a mobile-phase flow rate of 1 ml/min. The analytes are delected at OD 200 nm. DC-chol average retention time: 1.6 minutes

Ethyl DOPC average retention time: 7.7 minutes DOPC average retention time: 9.9 minutes DOPE average retention time: 11.5 minutes Choi average retention time: 13.4 minutes 5.2. Assaying of LPS by HPAEC-PAD

The principle of the assay consists in subjecting the LPS to an acid hydrolysis which releases one molecule of KDO per molecule of LPS; then in separating this free KDO from the rest and in quantifying it by high performance ion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

Preparation of the standard range and of the samples to be assayed

The following are prepared in a final volume of 400 uL: a blank and a standard range of KDO of between 42.5 and 1700 ng/mL; which corresponds to an LPS standard range of between 613 and 24 507 ng of LPS/mL. The blank and each of the samples of the range also contain an amount of lipids and/or of detergent substantially equivalent to that present in the samples to be assayed; that is to say, e.g., 0.7 mg/ml of a mixture of EDOPC and of DOPE in a molar ratio of 3:2 together with 0.2 mM octyl glucoside.
The samples to be assayed are prepared in a final volume of 400 uL by dilution, e.g. to 1/10th, of a liposome preparation at a starting theoretical LPS concentration of 100 μ g /mL.

Acid hydrolysis 100 uL of a hydrolysis solution containing 5% acetic acid and glucuronic acid at 20 μ g/mL (compound used as internal standard) prepared extemporaneously are introduced into the standard range + blank samples and into the samples to be assayed.

The hydrolysis is allowed to continue for 1 h at 100°C and is then stopped by centrifugation at 5°C for 5 min.

Extraction of the lipids and. of the detergent 500 u.1 of purified water are added to the previously hydrolyzed solution, followed by 2 mL of a chloroform/methanol (2/1) mixture, and the mixture is vortexed for 30 sec. It is centrifuged at 4500 rpm for 10 min. The aqueous phases are taken, dried at 45°C for 2 hours under a nitrogen stream at 0.5 bar and taken up with 400 μ l of water.

HPAEC-PAD assay

This technique is implemented on an HPAEC system (Dionex™) using the Dionex™ Chromeleon management software for the data acquisition and reprocessing. The chromatography column (Carbopac PA1 4 x 250 mm, Dionex™ reference 035391) is subjected to a temperature of 30°C. The column is equilibrated with an eluting solution (75 mM NaOH, 90 raM NaOAc) and pre-conditioned according to the following scheme:
- flow rate at 0.20 ml/min for 20 minutes (P = 270 psi)
- flow rate at 0.4 ml/min for 20 minutes (P = 540 psi)
- flow rate at 0.6 ml/min for 20 minutes (P = 800 psi)
- flow rate at 0.8 ml/min for 20 minutes (P = 1055 psi) flow rate at 1 ml/min for 20 minutes (P =. 1300 psi).

100 μ l of a sample are injected onto the column at an elution flow rate of 1 mL/min for 22 min.

The amount of KDO present in the sample is determined by integration of the KDO peak of the chromatogram. Since one mole of KDO is released per mole of LPS, it is possible to determine the concentration of LPS present in the initial preparation.

6. Results

6.1. Cationic liposomes have superior LPS-detoxifying property

Three kinds of LPS-containing liposomes were produced according to the extrusion method: (i) liposomes containing a single lipid, the latter being a neutral lipid (DOPC); (ii) liposomes containing a single lipid, the latter being a cationic lipid (EDOPC or DC-chol); and (iii) liposomes containing a cationic lipid and a neutral lipid). These liposomes are described in the following table which also shows the results of the LAL assay and of the rabbit pyrogen test. LPS incorporated into neutral liposomes induces a pyrogenic effect in the rabbit at administered LPS doses which do not produce this effect when the LPS is incorporated into cationic liposomes.



6.2. Study of LPS detoxification as a function of the Iipid:LPS molar ratio, of the liposome composition and/or of the liposome production process

Various kinds of LPS-containing liposomes were produced 'either by extrusion or by detergent dialysis. Their composition is described in the following table. The size of the liposomes produced is analyzed by quasi-elastic light scattering using a Malvern Zetasize'r nano-S apparatus. In all cases, this size is much less than 200 nm.


LPS detoxification is measured at TO and T + 3 months using the LAL and cytokine assay methods. The results are given in the following table:

The detoxification rate assessed by IL6 release is determined as being the ratio of the concentration of liposome-formulated LPS that induces 50% of maximum release (ED50 expressed in pg/mL)/the concentration of nonformulated LPS that induces 50% of maximum release.

In the LAL assay, the detoxification rate is determined as being the LAL value measured with the nonformulated LPS divided by the LAL value measured with the liposome-formulated product, at equivalent LPS concentration.

Even though the detoxification rates seem to follow the LPS concentration in the liposomes, both tests showing inverse tendencies moreover, it is not possible to conclude that there are substantial differences from one concentration to another. The LPS concentration in the liposomes should not have any incidence on the detoxification of said LPS. By contrast, the addition of a co-lipid (cholesterol or DOPE) to the base lipid (EDOPC) seems to promote said detoxification.

The detoxification of LPS in liposomes is also compared to that obtained with the endotoxoid obtained by complexation of purified LPS with the SAEP2-L2 peptide (dimeric, anti-parallel) according to the information given in WO 06/108586. The detoxification is not as high, but still perfectly acceptable, since the SAEP2-L2 peptide detoxifies LPS beyond what is required.

6.3. Study of LPS immunogenicity as a function of the lipid:LPS molar ratio, of the liposome composition and/or of the liposome production process

Mice in groups of 10 were immunized on DO and D21 with 10 ug of LPS by injection of an aliquot of a preparation A-I of LPS in liposomes at 50 fig/ml in 10 mM Tris buffer, 150 mM NaCl, pH 7.0. Negative and positive controls are added to the test. There are two positive controls: purified, non-detoxified LPS from the same batch (10 μ g per injection) as the one used to produce the LPS liposomes and also 10 ug of LPS from this batch in an endotoxoid form - endotoxoid produced according to WO 06/108586.

The amounts of anti-LPS IgG and IgM induced were evaluated by ELISA 35 days after the first injection. The results are given in figures 1 (IgG) and 2 (IgM). In each of these figures, numbers 6 to 14 and 5 respectively correspond to samples A to J described in the tables hereinabove. Sample 1 is a control sample consisting solely of a buffer solution. Sample 3 contains non-detoxified LPS L8. Samples 2 and 4 are the reference samples containing endotoxoid (LPS L8 detoxified by complexation with the SAEP2 L2 peptide, which is a polymyxin B analog).

It is noted that, whatever the LPS formulation mode, the antigenic nature of the detoxified LPS exhibits great homogeneity. Its ability to induce antibodies is entirely comparable to those of non-detoxified LPS and of endotoxoid.

Claims

1. A method for detoxification of lipopolysaccharide (LPS) or of lipid A from a Gram-negative bacterium, according to which the LPS or the lipid A is mixed with a cationic lipid so as to form a complex in which the LPS is associated with the cationic lipid.

2. The method as claimed in claim 1, in which the LPS is the lipooligosaccharide (LOS) from Neisseria meningitidis.

3. The method as claimed in claim 1 or 2, in which the cationic lipid is selected from the group constituted of:

(i) alkylamines;

(ii) octadecenoyloxy(ethyl-2-heptadecenyl-3-hydroxyethyl)imidazolinium (DOTIM) and structural homologues thereof;

(iii) lipospermines;

(iv) lipids incorporating an ethylphosphocholine structure;

(v) lipids incorporating a trimethylairunonium structure or a trimethylammonium propane structure or a dimethylammonium structure; and

(vi) cationic derivatives of cholesterol.

4. The method as claimed in claim 3. in which the cationic lipid is 1,2-dioleyl- sn- glycero-3-ethylphosphocholine (EDOPC) or 3P-[N-(N,.N,-dimethylamino-ethane)carbamoyl] cholesterol (DC-Choi).

5. The method as claimed in one of claims 1 to 4, according to which the LPS or the lipid A is mixed with a cationic lipid and, in addition, a neutral lipid (colipid) so as to form a complex in which the LPS or the lipid A is associated with the cationic lipid and with the neutral lipid.

6. The method as claimed in claim 5, in which the neutral lipid is selected from the group constituted of (i) cholesterol; (ii) phosphatidylcholines; and (iii) phosphatidylethanolamines.

7. The method as claimed in claim 6, in which the neutral lipid is l,2-dioleoyl-.sn- glycero-3-phosphoethanolamine (DOPE).

8. The method as claimed in one of claims 1 to 7, in which the cationic lipid or the cationic lipid and the neutral lipid is (are) in the form of liposomes, the LPS or the lipid A being associated with the liposomes.

9. A complex comprising at least LPS or lipid A from a Gram-negative bacterium and a cationic lipid, in which the LPS or the lipid A is detoxified owing to the complexation thereof with the cationic lipid.

10. The complex as claimed in claim 9, in which the LPS is the lipooligosaccharide from Neisseria meningitidis.

11. The complex as claimed in claim 9 or 10. in which the cationic lipid is selected from the group constituted of:

(i) alkylamines;

(ii) octadecenoyloxy(ethyl-2-heptadecenyl-3-hydroxyethyl)imidazolinium (DOTIM) and structural homologues thereof;

(iii) Iipospennines;

(iv) lipids incorporating an ethylphosphocholine structure;

(v) lipids incorporating a trimethylammonium structure or a trimethylammonium propane structure or a dimethylammonium structure; and

(vi) cationic derivatives of cholesterol.

12. The complex as claimed in claim 9 or 10, in which the cationic lipid is l,2-dioleyl-.sn- -glycero-3-ethylphosphochoIine (EDOPC) or 3(3-[N-(N, N- dimethylaminoethane)carbamoyl] cholesterol (DC-chol).

13. The complex as claimed in one of claims 9 to 12, which comprises, in addition, a neutral lipid.

14. The complex as claimed in claim 13, in which the neutral lipid is selected from the group constituted of (i) cholesterol; (ii) phosphatidylcholines; and (in) phosphatidylethanolamines.

15. The complex as claimed in claim 14, in which the neutral lipid is 1,2-dioleoyl-.w-glycero-3-phosphoethanolamine (DOPE).

16. The complex as claimed in one of claims 9 to 15, in which the cationic lipid or the cationic lipid and the neutral lipid is (are) in the form of a liposome, the LPS or the lipid A being associated with the liposome.

17. The complex as claimed in one of claims 9 to 16, which is a liposome [LPS].

18. The complex as claimed in one of claims 9 to 17, for use as an adjuvant.

19. The complex as claimed in one of claims 9 to 17, for use as a vaccine antigen.

20. A vaccine composition which comprises, as a vaccine antigen, a complex as claimed in one of claims 9 to 19, in combination with an adjuvant which is lipoprotein in nature.