RU2360699C2 - Compositions of meningococcal vaccines with adjuvants - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there is offered immunogenic composition containing antigen Neisseria containing protein of serogroup B Neisseria meningitides chosen from the group including protein NadA, protein 287, protein 741, protein 953; CpG-oligonucleotide; and also biologically degradable microparticles representing copolymer D, L of lactide with glycolipid PLG. Besides, there is offered method of intensifying the antibody response, mammal protection and application of the composition for drug preparation.

EFFECT: invention can be used for making the vaccines.

15 cl, 11 tbl

Description

All documents cited here are incorporated by reference in their entirety.

FIELD OF TECHNOLOGY

This invention relates to vaccines, and more particularly to vaccines against Neisseria meningitidis.

BACKGROUND OF THE INVENTION

Genomic sequences for Neisseria meningitidis (meningococcus) serogroups A (1) and B (2, 3) have been described. The sequence for serogroup B was studied in relation to the identification of vaccine antigens (for example, references 4 to 9), and candidate antigens were manipulated to improve heterologous expression (references 10 to 12).

Antigens usually require coadministration of adjuvants to enhance their immunogenicity in vaccines (13). Freund's adjuvant was used for serogroup B meningococci (9), and aluminum hydroxide was used in the licensed Menjugate ^TM vaccine against serogroup C (14). An increase in the bactericidal activity of Neisseria antigens was also reported with the use of oligonucleotide adjuvants containing CpG motifs (15).

The object of this invention is to obtain additional and improved adjuvants for Neisseria antigens.

DESCRIPTION OF THE INVENTION

It was found that the combination of CpG oligonucleotides and polymer microparticles is an extremely effective adjuvant for Neisseria antigens, and the combination gives significantly better results than any of the individual components. The present invention therefore provides a composition consisting of: (a) Neisseria antigen; (b) a CpG oligonucleotide; and (c) biodegradable polymer microparticles.

Neisseria antigen

The Neisseria antigen may be a protein antigen, a nucleic acid encoding a protein antigen, or a saccharide antigen. The antigen preferably elicits a bactericidal or protective immune response (e.g., a humoral immune response) in a recipient mammal.

The antigen can be derived from any species of Neisseria, including N. gonorrhoeae, N. lactamica, and N. meningitidis. The N.meningitidis antigen is preferred and may be from any serogroup. When the antigen comes from serogroup B, it is preferable to use a protein antigen; when it comes from serogroups A, C, W135 or Y, it is preferable to use a saccharide antigen. When saccharide antigens are used, they are usually derived from capsular polysaccharides of bacteria (e.g., oligosaccharides, such as oligosaccharides obtained by hydrolysis), and they will usually be conjugated to carrier proteins (e.g., CRM ₁₉₇ ).

Preferred protein antigens derived from N.meningitidis serogroup B are:

- a protein described in any of the sources 4, 5, 6, 7, 8 or 9 (in particular, 446 even SEQ IDs (i.e. 2, 4, 6, ..., 890, 892) described in source 4, 45 even SEQ IDs (i.e. 2, 4, 6, ..., 88, 90) described in source 5, and 1674 even SEQ IDs 2-3020, even SEQ ID 3040-3114 and all SEQ ID 3115-3241, described in source 6);

- a protein containing an immunogenic fragment of one or more proteins described in any of the sources 4, 5, 6, 7, 8 or 9;

- a protein containing a sequence having sequence identity (preferably more than 50%, for example 60%, 70%, 80%, 90%, 95%, 99% or more) with one or more proteins described in any of the sources 4, 5 6, 7, 8 or 9;

- the protein described in any of the sources 10, 11 or 12;

- a protein containing a sequence having sequence identity (preferably more than 50%, for example 60%, 70%, 80%, 90%, 95%, 99% or more) with one or more proteins described in any of the sources 10, 11 or 12.

A particularly preferred protein antigen from N. meningitidis serogroup B is the '287' protein. This protein can be used in the wild-type form (for example, GenBank entry gi: 7228690; alignment of polymorphic forms 287 are shown in Figs. 5 and 15 from source 8), but derivatives of the wild-type protein can also be used. For example, proteins having 50% or more sequence identity (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) can be used with gi: 7228690. You can use variants of this protein - proteins with shortening or deletion, such as truncated at the N-end of the form described in sources 10 through 12 (in particular, ΔG287, in which the N-end of the protein up to and including six repeating glycine residues deleted). You can use fusion proteins containing such sequences 287. All of these forms 287, and more specifically those that retain the immunogenicity of wild-type 287 proteins, are included in the value '287', as used here.

Another particularly preferred protein antigen from N.meningitidis serogroup B is the protein '961', also known as 'NadA' (16). This protein can be used in the wild-type form (for example, GenBank entry gi: 7227256; alleles 961 are described in source 17), but derivatives of the wild-type protein can be used. For example, proteins having 50% or more sequence identity (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) with gi: 7227256 can be used. You can use variants of this protein - proteins with truncation or deletion, such as the proteins described in sources 10 through 12 ('961 s', in particular, which does not have a C-terminal membrane anchor). Fusion proteins containing such 961 sequences can be used. All of these forms of 961, and especially those that retain the immunogenicity of wild-type 961 proteins, are included in the value '961' or 'NadA', which is used here.

Other preferred protein antigens are protein '741' and protein 'ORF46.1', and proteins 'ORF1', 'ORF4', 'ORF25', 'ORF40', 'ORF83', 'NMB1343', '230', '233' , '292', '594', '687', '736', '907', '919', '936', '953' and '983'. Other preferred protein antigens are fusion proteins described in Sources 10 through 12, especially proteins comprising one or more of: protein 287, protein 953, protein 936 and / or protein 741.

Protein antigens can be obtained from any strain of N.meningitidis. Antigens from strains 2996, MC58, 95N477 and 394/98 are preferably used.

In addition to strain variants, single or multiple substitutions of conservative amino acids can be made with a change in the immunogenicity of antigens used in accordance with this invention.

In addition to or instead of protein antigens, nucleic acids encoding a protein antigen may be included in the compositions of this invention. Nucleic acids will be expressed in vivo when administered to a mammalian recipient, and a protein antigen will be produced. Such immunization with nucleic acids is well known (e.g., sources 18 to 23, etc.). Nucleic acid will usually be plasmid DNA.

A preferred saccharide antigen derived from serogroup C N.meningitidis is the oligosaccharide conjugate used in Menjugate ^™ (24, 25), which contains 12 to 22 monosaccharide units from the capsular polysaccharide of serogroup C.

A preferred saccharide antigen derived from serogroup A is an oligosaccharide in which one or more of the hydroxyl groups in the composite monosaccharide elements are replaced by a blocking group (26).

Additional oligosaccharide antigens from serogroups A, W135 and Y are described in source 27.

The composition of this invention may include more than one Neisseria antigen. When saccharides from both serogroups A and C of N.meningitidis are included, it is preferable that the ratio (weight / weight) of MenA saccharide: MenC saccharide be greater than 1 (e.g.

2: 1, 3: 1, 4: 1, 5: 1, 10: 1 or more).

The composition of the present invention is preferably an immunogenic composition or vaccine. Such compositions include an immunologically effective amount of antigen. By “immunologically effective amount” is meant that the administration to an individual of a composition of the invention containing such an amount of antigen (either in a single dose or as part of a series) is effective for generating a therapeutic or prophylactic immune response. This amount varies depending on the state of health and physical condition of the individual to be treated, the age, taxonomic group of the individual to be treated (for example, a non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the composition of the vaccine, the judgment of the attending physician about the medical situation, and other significant factors. This amount may correspond to a relatively wide range, which can be determined by routine testing. Antigens are usually present in a concentration of at least 1 μg / ml each.

The dosage regimen for treatment may consist of a single dose or multiple doses (for example, including booster doses).

CpG oligonucleotide

It is known that CpG oligonucleotides are used as adjuvants in vaccines (e.g., source 28), and they elicit a strong Th1 immune response. They are suitable as adjuvants for parenteral administration and for application to mucous membranes (29).

The CpG oligonucleotide used in accordance with this invention is a nucleic acid that includes at least one CG dinucleotide, i.e. a cytosine nucleotide followed by a guanosine nucleotide. An oligonucleotide may contain multiple CG dinucleotides.

The CG sequence in the oligonucleotide can be flanked by two purines on the 5'-side and two pyrimidines on the 3'-side, i.e. RRCGYY.

The cytosine nucleotides in the CpG oligonucleotide may be methylated, but preferably they should be unmethylated.

The cytosine and guanosine nucleotides are preferably deoxynucleotides, and the nucleic acid is preferably DNA. To increase resistance to nucleases, the oligonucleotide may contain a modified skeletal chain, such as a phosphorothioate skeletal chain. As an alternative to using DNA, PNA (peptide-nucleic acid) can be used. In addition, oligonucleotides may include substitutions of sugar groups and nitrogen bases.

The oligonucleotide preferably consists of from 6 to about 100 nucleotides, more preferably from about 8 to about 50 nucleotides, most preferably from about 10 to about 40 nucleotides.

Oligonucleotides containing at least one CG dinucleotide can be conveniently prepared using conventional oligonucleotide synthesis.

Examples of CpG oligonucleotide adjuvants are in sources 30 to 55.

Biodegradable Polymer Microparticles

Biodegradable polymer microparticles are known to be used as vaccine adjuvants (e.g., Source 56). They are suitable as adjuvants for parenteral administration and for application to mucous membranes.

In addition to being biodegradable, the polymer used to make microparticles should usually be sterilizable and non-toxic (biocompatible). Suitable biodegradable polymers can be easily purchased, and they include polymers that are derived from polyhydroxybutyric acid; polycaprolactone; polyorthoester; polyanhydride; polyhydroxybutyrate and poly-α-hydroxy acids. Preferred polymers are derived from one or more of poly-α-hydroxy acids, for example poly-L-lactide, poly-D, L-lactide, copolymers of D, L-lactide and glycolide (such as random copolymer D, L-lactide with glycolide) or a copolymer of D, L-lactide or caprolactone. Microparticles obtained from random copolymer D, L-lactide with glycolide (PLG) are preferred.

There are polymers with different molecular weights, and a suitable molecular weight for a given antigen can be easily determined. For poly-L-lactide, a suitable molecular weight will be on the order of from about 2000 to 250,000. For PLG, a suitable molecular weight will usually be in the range of from about 10,000 to about 200,000, preferably from about 15,000 to about 150,000, and most preferably from about 50,000 to about 100,000.

For PLG microparticles, lactide: glycolide can be used in different ratios, and the ratio is mostly a matter of choice, depending in part on the antigen introduced along with them and on the degree of their destruction. For example, a 50:50 PLG containing 50% D, L-lactide, and 50% glycolide will produce a rapidly absorbable copolymer, while a 75:25 PLG decomposes more slowly, and 85:15 and 90:10 even more slowly due to high content of lactide component. A suitable lactide: glycolide ratio is readily determined based on the nature of the antigen and disease in question. In addition, mixtures of microparticles with different ratios of lactide: glycolide will be used in preparations in order to achieve the desired release kinetics for a given antigen, and to obtain both a primary and secondary immune response. The decomposition rate of the microparticles of the present invention can also be controlled by factors such as the molecular weight of the polymer and the crystallinity of the polymer.

The term "microparticle", as used here, refers to a particle with a diameter of from about 100 nm to about 150 microns, more preferably a diameter of from about 200 nm to about 30 microns, and most preferably a diameter of from about 500 nm to about 10 microns. Preferably, the microparticles will be of a diameter that allows for parenteral administration without clogging needles and capillaries. The microparticle size is readily determined by methods well known in the art, such as photon correlation spectroscopy, laser diffractometry, and / or scanning electron microscopy. The term “microparticle” includes “nanoparticles” (57) within their boundaries. Preferred microparticles are microspheres, although lamellar particles can also be used (58).

Microparticles can be obtained using any of several methods well known in the art (e.g., source 59). For example, to obtain microparticles, you can use the methods of double evaporation of the emulsion / solvent (for example, sources 60 and 61). These techniques include the formation of a primary emulsion consisting of droplets of a polymer solution containing antigen (if the antigen should be contained in a microparticle), which is then mixed with a continuous aqueous phase containing a particle stabilizer / surfactant.

More specifically, a water-in-oil-in-water (w / m / v) solvent evaporation system can be used to prepare microparticles as described in Sources 62, 63 and 64. In this technique, a particular polymer is combined with an organic solvent such like ethyl acetate, dimethyl chloride (also called methylene chloride and dichloromethane), acetonitrile, acetone, chloroform and the like. This polymer will be present in an approximately 2-15% solution in an organic solvent. A approximately equal amount of antigen solution (for example, in water) is added and the polymer / antigen solution is emulsified using, for example, a homogenizer. The emulsion is then combined with a large volume of an aqueous emulsion stabilizer solution, such as polyvinyl alcohol (PVA) or polyvinylpyrrolidone. The emulsion stabilizer is usually presented in about 2-15% solution, more typically in about 4-10% solution. The mixture is then homogenized to give a stable double emulsion (w / m / v). The organic solvents are then evaporated.

The parameters of the drug can be manipulated to provide the possibility of obtaining small (<5 μm) and large (> 30 μm) microparticles (for example, 63, 65). For example, reduced mixing results in larger microparticles, resulting in an increase in the volume of the internal phase. Fine particles are produced using small volumes of the aqueous phase using high concentrations of PVA.

Microparticles can also be formed using spray drying and coacervation (for example, sources 66, 67 and 68); suspended layer techniques, such as drying in a suspended layer and a Wurster layer (69, 70), ion gelation (71).

Before using microparticles, the antigen content is usually determined so that an appropriate amount of microparticles can be administered to the subject to obtain an adequate immune response.

The antigen content in the microparticles can be determined by methods known to those skilled in the art, such as the destruction of microparticles and extraction of the antigen contained therein. For example, microparticles can be dissolved in dimethyl chloride and the protein extracted into distilled water (e.g., sources 72, 73, 74). Alternatively, the microparticles can be dispersed in 0.1 M NaOH containing 5% (w / v) SDS. The sample is shaken, centrifuged, and a quantitative analysis of the supernatant for antigen is carried out using an appropriate analysis method (75).

Antigen and / or CpG oligonucleotides can be incorporated into microparticles. Capture will usually be accomplished by ensuring the presence of antigen / oligonucleotide during the formation of microparticles, while adsorption on the surface is achieved by adding antigen / oligonucleotide to preformed microparticles.

One of the methods for adsorption of antigen / oligonucleotide on previously obtained microparticles is the following. Water is added to the microparticles and dispersed to a substantially monomeric suspension of microparticles using dialyzable anionic and cationic detergents. Suitable detergents include, but are not limited to, any of a variety of N-methylglucamides (known as MEGA) such as heptanoyl-N-methylglucamide (MEGA-7), octanoyl-N-methylglucamide (MEGA-8), nonanoyl-N-methylglucamide (MEGA-9) and decanoyl-N-methylglucamide (MEGA-10); cholic acid; sodium cholate; deoxycholic acid; sodium deoxycholate; taurocholic acid; sodium taurocholate; taurodeoxycholic acid; sodium taurodeoxycholate; 3 - ((3-cholamidopropyl) dimethylammonio) -1-propanesulfonate (CHAPS); N-octyl glucoside; 3 - ((3-cholamidopropyl) dimethylammonio) -2-hydroxy-1-propanesulfonate (CHAPSO); N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (ZWITTERGENT 3-12); N, N-BIS- (3-D-gluconamidopropyl) -deoxycholamide (DEOXYBIGCHAP); sucrose monolaurate; glycocholic acid / sodium glycocholate; laurosarcosine (sodium salt); glycodeoxycholic acid / sodium glycodeoxycholate; sodium dodecyl sulfate (SDS; SDS); and hexadecyltrimethylammonium bromide (CTAB); dodecyltrimethylammonium bromide; hexadecyltrimethylammonium bromide; tetradecyltrimethylammonium bromide; benzyldimethyldodecylammonium bromide; benzyldimethylhexadecylammonium chloride; benzyldimethyltetradecylammonium bromide. The above detergents are available for purchase. Various cationic lipids known in the art can also be used as detergents (76, 77).

The microparticle / detergent mixture is then physically triturated, for example using a ceramic mortar and pestle, until a uniform, thick suspension is formed. A suitable aqueous buffer is then added, such as phosphate buffered saline (PBS) or Tris buffered saline, and the resulting mixture is sonicated or homogenized until the microparticles are completely suspended. An antigen / oligonucleotide is then added to the microparticle suspension and the system is dialyzed to remove detergent. The microparticles of the polymer and the detergent system are preferably selected so that the antigen / oligonucleotide will be adsorbed on the surface of the microparticles, while maintaining activity. The resulting microparticles containing antigen / oligonucleotide adsorbed on the surface can be washed from the unbound antigen / oligonucleotide and stored as a suspension in an appropriate buffer composition or lyophilized with suitable excipients, which are described below.

Antigen / CpG / Microparticle Combination

Various physical relationships are possible between the three main components of the compositions of this invention. These relationships arise due to the fact that the microparticles have an internal volume and surface, both of which can be used to locate the CpG oligonucleotide and / or antigen.

Thus, this antigen can be enclosed within the microparticles, it can be adsorbed on the microparticles, or it can be in a simple mixture with microparticles without inclusion or adsorption. Adsorption is preferred.

Similarly, a CpG oligonucleotide can be incorporated into microparticles, it can be adsorbed on microparticles, or it can be in a simple mixture with microparticles. Adsorption can be achieved using detergents such as CTAB.

CpG oligonucleotide and antigen, both can interact with both microparticles and with each other, or the interaction may be different. Also, the CpG oligonucleotide and antigen can be adsorbed on the same microparticles or the CpG oligonucleotide and antigen can be adsorbed on different microparticles. All possible combinations are encompassed by this invention:

The compositions of this invention may include the mixtures described above, for example, some microparticles in the composition have an antigen contained therein, and some have an adsorbed antigen.

Pharmaceutical Compositions

For pharmaceutical use, the compositions of this invention will typically include a pharmaceutically acceptable carrier. This gives the pharmaceutical composition of the present invention.

A pharmaceutically acceptable carrier may be any substance that does not itself produce antibodies that are harmful to the patient receiving the composition and that can be administered without excessive toxicity. Suitable carriers may be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactivated viral particles. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers may include liquids such as water, saline, glycerin and ethanol. Excipients, such as wetting or emulsifying agents, pH buffering agents and the like, may also be present in such carriers. Suitable carriers are liposomes. A full discussion of pharmaceutical carriers can be found in source 78.

The compositions of this invention can be obtained in various forms. For example, the compositions can be made in the form of injectable forms, or solutions, or suspensions in liquids. Solid forms suitable for preparing solutions or suspensions in liquid carriers prior to injection may also be made. A topical composition may be prepared, for example, ointment, cream or powder. A composition for oral administration can be made, for example, in the form of a tablet or capsule, or syrup (optionally with flavoring agents). This composition can be made for pulmonary administration, for example, in the form of an inhaler using a finely divided powder or aerosol. This composition can be made in the form of a suppository or pessary. A composition may be formulated for nasal administration, in the ears or eyes, for example, in the form of drops, in the form of an aerosol, or in the form of a powder (for example, 79).

The pharmaceutical composition is preferably sterile. Preferably it is pyrogen-free. It is preferably buffered, for example with a pH of 6 and a pH of 8, usually around pH 7.

The pharmaceutical composition may be lyophilized.

The invention also provides a delivery device comprising a pharmaceutical composition of the invention. The device may be, for example, a syringe.

Medical treatment and use

The compositions of this invention can be used therapeutically (i.e., for the treatment of an existing infection caused by neysseries) or prophylactically (i.e. to prevent future infection caused by neysseries).

The present invention provides a composition of the present invention for use as a therapeutic agent.

The invention also provides a method for increasing an antibody response in a mammal, comprising administering a pharmaceutical composition of the invention to a mammal. The antibody response is preferably an IgA or IgG response, and preferably it is bactericidal.

The invention also provides a method of treating a mammal suffering from a neisseria infection and / or disease, comprising administering to a patient a pharmaceutical composition of the invention.

The present invention also provides a method of protecting a mammal from neisseria-induced infections and / or diseases, comprising administering to the mammal a pharmaceutical composition of the invention.

The invention also provides the use of (a) a neisseria antigen, (b) a CpG oligonucleotide and (c) biodegradable polymer microparticles in the manufacture of a medicament for the prevention or treatment of a disease and / or infection in a mammal.

The mammal is preferably a human. The person may be an adult or preferably a child. The compositions of this invention are particularly useful for immunizing children and adolescents.

The use and methods of this invention are particularly suitable for the treatment / protection against infections caused by N.meningitidis. Application and methods are especially useful for the prevention / treatment of diseases, including bacterial meningitis.

The effectiveness of therapeutic treatment can be tested by monitoring the infection caused by Neisseria after administration of the composition of the present invention. The effectiveness of prophylactic treatment can be tested by monitoring the immune response to Neisseria after administration of this composition.

The compositions of this invention will usually be administered directly to the patient. Direct delivery can be achieved by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly or into the interstitial space of the tissue) or by rectal, oral, vaginal, topical, transdermal, into the eyes, nasal, in the ears or pulmonary. Injections and intranasal administration are preferred.

Dosage during treatment can be represented by a single dose regimen or a multiple dose regimen.

Additional components

The compositions of this invention may include adjuvants in addition to CpG oligonucleotides and polymer microparticles. Preferred additional adjuvants include, but are not limited to: (A) aluminum compounds (e.g. aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, oxyhydroxide, orthophosphate, sulfate, etc. (e.g. see chapters 8 and 9 of source 13)), or mixtures of different aluminum compounds, the compounds taking any suitable form (for example, gel, crystalline, amorphous, etc.), and adsorption is preferred; (B) MF59 (5% squalene, 0.5% tween 80, and 0.5% spen 85, made as submicron particles using a microfluidizer) (see chapter 10 of 13; see also source 80) ; (C) liposomes (see chapters 13 and 14 of source 13); (D) ISCOM (see chapter 23 of source 13), which may not contain additional detergent (81); (E) SAF containing 10% squalene, 0.4% tween 80, 5% pluronium block polymer L121 and thr-MDP, or micro-fluidized to a submicron emulsion or shaken to form an emulsion with coarse particles (see chapter 12 of source 13 ); (F) adjuvant Ribi ™ system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% tween 80 and one or more of the components of the bacterial cell wall from the group consisting of monophosphoryl lipid A (MPL), trehalose dimicolate ( TDM) and a cell wall framework (CWS), preferably MPL + CWS (Detox ™); (G) saponin adjuvants such as QuilA or QS21 (see chapter 22 of source 13), also known as Stimulon ™ (82); (H) chitosan (e.g., 83); (I) Freund's complete adjuvant (PAF; CFA) and Freund's incomplete adjuvant (NAF; IFA); (J) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon -g), macrophage colony stimulating factor, tumor necrosis factor, etc. (see chapters 27 and 28 of source 13); (K) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (e.g. chapter 21 of source 13); (L) combinations of 3dMPL with, for example, QS21 and / or oil-in-water emulsions (84); (M) polyoxyethylene ether or polyoxyethylene ester (85); (N) surfactant (PVA) - polyoxyethylene sorbitan ester in combination with octoxynol (86) or PVA - polyoxyethylene alkyl ether or ester in combination with at least one additional non-ionic PVA such as octoxynol (87); (N) metal salt particles (88); (O) saponin and an oil-in-water emulsion (89); (P) saponin (e.g. QS21) + 3dMPL + IL-12 (optional + sterol) (90); (Q) E. coli heat labile enterotoxin (“LT”) or its detoxified mutants, such as K63 or R72 mutants (for example, chapter 5 of source 91); (R) cholera toxin (“HT”) or its detoxified mutants (for example, chapter 5 of source 91); (S) double-stranded RNA; and (T) other substances that act as immunostimulating agents to increase the effectiveness of the composition (for example, see chapter 7 of source 13). Alum (especially aluminum phosphate and / or hydroxide) and MF59 are preferred adjuvants for parenteral immunization. Mutant toxins are preferred mucosal adjuvants.

Muramyl peptides include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylnormuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L -alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine MTP-PE), etc.

In addition to the Neisseria antigen (s), the composition may also include additional antigenic components. Antigens that may be included in the composition of this invention include:

- antigens from Helicobacter pylori, such as CagA (c 92-95), VacA (96, 97), NAP (98, 99, 100), HopX (e.g. 101), HopY (e.g. 101) and / or urease;

- the preparation of the outer membrane vesicles (OMV) from N.meningitidis serogroup B, such as described in source 102, 103, 104, 105, etc .;

- saccharide antigen from Streptococcus pneumoniae (e.g. 106, 107, 108);

- an antigen from hepatitis A virus, such as an inactivated virus (e.g. 109, 110);

- an antigen from hepatitis B virus, such as surface and / or nuclear antigens (for example, 110, 111);

- antigen from hepatitis C virus (for example, 112);

- an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and microvill hemagglutinin (FHA) from B. pertussis, optionally also in combination with pertactin and / or agglutinogens 2 and 3 (for example, sources 113 and 114);

- diphtheria antigen, such as diphtheria toxoid (for example, chapter 3 from source 115), for example CRM ₁₉₇ mutant (for example, 116);

- tetanus antigen, such as tetanus toxoid (for example, chapter 4 of source 115);

- saccharide antigen from Haemophilus influenzae B (e.g. 23);

- an antigen from Chlamidya pneumoniae (for example, 117, 118, 119, 120, 121, 122, 123);

- antigen from Chlamidya trachomatis (e.g. 124);

- antigen from Porphyromonas gingivalis (e.g. 125);

- polyoantigen (s) (e.g. 126, 127), such as IPV or OPV;

- rabies antigen (s) (e.g. 128), such as lyophilized inactivated virus (e.g. 129, RabAvert ™);

- antigen (s) of measles, mumps and / or rubella (for example, chapters 9, 10 and 11 from source 115);

- antigen (s) from influenza virus (for example, chapter 19 from source 115), such as hemagglutinin and / or neuraminidase surface proteins;

- antigen (s) from paramyxoviruses, such as respiratory syncytial virus (RSV (130, 131)) and / or parainfluenza virus (PIV3 (132));

- antigen from Moraxella catarrhalis (e.g. 133);

- antigen from Streptococcus agalactiae (group B streptococci) (e.g. 134, 135);

- antigen from Streptococcus pyogenes (group A streptococci) (e.g. 135, 136, 137);

- antigen from Staphylococcus aureus (e.g. 138);

- antigen from Bacillus anthracis (e.g. 139, 140, 141);

- an antigen from a virus of the family Flaviviridae (genus Flavivirus), such as from yellow fever virus, Japanese encephalitis virus, four serotypes of Dengue viruses, tick-borne encephalitis virus, West Nile fever virus;

a pestivirus antigen, such as from classic swine fever virus, cattle diarrhea virus and / or borderline disease virus;

- parvovirus antigen, for example, from parvovirus B19;

- prion protein (for example, prion protein CJD);

an amyloid protein, such as a betapeptide (142);

- a cancer antigen, such as the antigens listed in table 1 of source 143 or in tables 3 and 4 of source 144.

The composition may include one or more of these additional antigens.

Antigens, which are proteins of toxins, can be detoxified when necessary (for example, detoxification of pertussis toxin with chemical and / or genetic means (114)).

When a diphtheria antigen is included in the composition, it is also preferable to include tetanus antigen and pertussis antigens. Similarly, when a tetanus antigen is included, it is preferable to also include diphtheria antigen and pertussis antigens. Similarly, when pertussis antigen is included, it is preferable to also include diphtheria antigen and tetanus antigen.

Antigens are preferably adsorbed onto aluminum salts.

Antigens in the composition will usually be present at a concentration of at least 1 μg / ml each. Basically, the concentration of any given antigen will be sufficient to obtain an immune response against this antigen.

As an alternative to using protein antigens in the composition of this invention, nucleic acids encoding these antigens can be used. The protein components of the compositions of this invention can thus be replaced by nucleic acids (preferably DNA, for example, in the form of plasmids) that encode proteins.

Definitions

The term “comprising” means “comprising” as well as “consisting of”, for example, a composition “comprising” X may consist solely of X or may include something additional, for example X + Y.

Mention of the percentage of sequence identity between two amino acid sequences means, when aligned, the percentage of amino acids that are the same when comparing two sequences. This alignment and percent homology or sequence identity can be determined using software known in the art, for example, the programs described in section 7.7.18 of source 145. The preferred alignment is determined by the Smith-Waterman homology search algorithm using affinity-range search with deletion an open interval of 12, and with a lengthening of the removal interval of 2, the BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is indicated in source 146.

MODES FOR CARRYING OUT THE INVENTION

Parenteral priming and mucosal secondary immunization with serogroup B antigen Neisseria meningitidis

Source 6 describes a protein from N.meningitidis serogroup B, called '287'. Sources 10 through 12 describe ways to improve its expression. One pathway involves deletion of the N-terminus of the protein before and including six repeating glycine residues. This protein is called “DG287".

Mice were primirovanny and re-immunized with MenB DG287 antigen (20 μg / dose) from strain 2996, prepared in the form for intramuscular (IM) administration by adsorption on PLG microparticles, with or without CpG oligonucleotide (also adsorbed on microparticles). An adjuvant LT-K63 was used as an additional preparation for intranasal (IN) administration. Mice received either 3 doses of IM or 2 doses of IM, then 2 doses of IN (doses were administered on: day 0; 28 day; 84 day and, optionally, 98 day).

Table 1 Group A drug Way Dose GMT antibodies after 2 weeks dose 2 dose 3 dose 4 one PLG / 287 IM 1,2,3 10,729 2,853 - 2 PLG / 287 + PLG / CpG IM 1,2,3 15,673 4,163 - 3 PLG / 287 IM 1,2 9,064 7,948 9,412 287 + LT-K63 IN 3.4 four PLG / 287 + PLG / CpG IM 1,2 34,891 15,167 16,556 287 + LT-K63 IN 3.4

Thus, the inclusion of a CpG oligonucleotide increased antibody titers against the intramuscularly introduced protein MenB 287 (compared groups 1 and 2). The titers could be increased by replacing the third intramuscular injection with two intranasal doses (compared groups 1 and 3). CpG amplification was also observed in the intramuscular / intranasal regimen (compared groups 3 and 4).

Comparison of adjuvants for MenB-287 protein

DG287 was formulated with various adjuvants and administered to mice. Serum from mice was evaluated using a bactericidal antibody assay (BCA), and the titers were as follows:

table 2 Adjuvant BCA, after 2 BCA, after 3 Freund's adjuvant 2048 8192 Alum <4 256 Alum + CpG Oligonucleotide 256 4096 Mf59 <4 <4 CpG oligonucleotide <4 128 PLG microparticles (adsorbed) 8 1024 PLG microparticles (adsorbed) + CpG 2048 16384

The CpG oligonucleotide was thus only moderately effective as an adjuvant, almost comparable to alum. Microparticles from PLG were more effective than alum and CpG, but not as effective as Freund's adjuvant. In noticeable contrast, however, the activation mixture of CpG and PLG was equal to Freund's adjuvant in the stage after the second immunization and was superior to Freund's adjuvant after the third immunization.

An increase in the adjuvant PLG using CpG was also observed in a separate study (02-0279):

Table 3 Adjuvant GMT after 2 GMT after 3 Mf59 6967 13417 PLG microparticles (adsorbed) 7070 11367 PLG microparticles (adsorbed) + CpG 15099 26833

The effect of adsorption on adjuvant

The effect of adsorption on adjuvance was investigated. Protein DG287 was either adsorbed onto PLG microparticles using a DSS or SDS surfactant, or simply mixed with particles. Immunization was performed on days 0, 21, and 35, and titers were evaluated on days 35 and 49. The results were as follows:

Table 4 A drug BCA Antibody titer after 2 weeks Dose 2 Dose 3 CpG + 287 adsorbed on PLG (DSS) 4096 45817 67921 CpG + 287 adsorbed on PLG (SDS) 4096 39730 29911 CpG + 287 + PLG (no adsorption) <16 62 1065 DSS + 287 adsorbed on alum <16 1209 1249 CpG + 287 adsorbed on alum 1024 4054 12236 287 adsorbed on alum 128 646 2454

The adjuvant mixtures of CpG and microparticles for DG287 are thus optimal when the antigen is adsorbed onto the microparticles.

Source 6 describes a protein from N. meningitidis of serogroup B, called “961” (currently known as “NadA” (16, 17)). Sources 10 and 12 describe ways to improve NadA expression. One of the ways involves the deletion of the C-terminus of the protein with the removal of its membrane anchor (i.e., the removal of amino acids 351-405 for strain 2996), as well as the natural removal of its leader peptide. This protein is called "961 s." The effect of adsorption on the adjuvant PLG when co-administered with CpG was studied for 961 s, as described above for 287:

Table 5 A drug BCA Antibody titer 2 weeks after 3 doses 961 adsorbed on PLG (SDS) 2048 20661 961 + PLG (without adsorption) 256 1706 287 adsorbed on PLG 4096 63057 287 adsorbed on PLG + soluble 961 4096 287: 86052; 961: 1924 287 adsorbed on PLG + 961 adsorbed on PLG 8192 287: 107142; 961: 11717 287 (not adsorbed) +961 (not adsorbed) + “pure” PLG 1024 287: 1266; 961: 145 287 (adsorbed) +961 (adsorbed) + “pure” PLG 8192 287: 78176; 961: 20876

Therefore, with regard to DG287, the adjuvance of mixtures of CpG and microparticles for 961c is optimal when the antigen is adsorbed on the microparticles. This is true for antigen per se and antigen when combined with DG287.

Therefore, with respect to DG287 and 961c, separately and in combination, the best adjuvant for CpG and PLG mixtures is observed when antigens are adsorbed on PLG microparticles.

PLG, CpG, Alum and MF59

Different combinations of PLG, CpG and alum were tested for the DG287 protein expressed as a His-labeled product. Serum bactericidal titers after three immunizations were as follows:

Table 6 Adjuvant Titer Alum 2048 Alum + CpG 32768 Mf59 8192 MF59 + CpG 32768 PLG (antigen adsorbed on PLG) 1024 PLG + CpG (antigen and CpG, both adsorbed on PLG) 4096 PLG + MF59 (antigen adsorbed on PLG) 2048 PLG + MF59 + CpG (antigen adsorbed on PLG) 8192 Freund's complete adjuvant 32768 PLG + Freund's complete adjuvant (antigen adsorbed onto PLG) 2048

Carried out the same experiments and the results were as follows:

Table 7 Adjuvant Titer PLG (antigen adsorbed on PLG) 1024 PLG + CpG (antigen adsorbed on PLG) 16384 PLG + CpG (antigen and CpG, both adsorbed on PLG) 16384 PLG + alum (antigen adsorbed on PLG) 1024 PLG + alum + CpG (antigen adsorbed on PLG) 16384 PLG + alum + CpG (antigen and CpG, both adsorbed on PLG) 8192 PLG + MF59 (antigen adsorbed onto PLG) 4096 PLG + MF59 + CpG (antigen adsorbed on PLG) 16384 Alum (antigen adsorbed on alum) 256 Cpg 128 Alum + CpG 1024 Alum + CpG + PLG (antigen adsorbed on alum; CpG adsorbed on PLG) 4096 CpG + PLG (CpG adsorbed on PLG; antigen not adsorbed) 64

Thus, MF59 and alum can further enhance the effectiveness of CpG / PLG mixtures; adsorption of CpG on microparticles from PLG is not necessary for adjuvantation, but antigen adsorption on microparticles was again observed to be optimal.

Mixtures of antigens

The effect of adsorption on adjuvance has been studied for proteins DG287 and 961 s, separately or in combination. Antibody titers after three administrations were as follows:

Table 8 A drug Antibodies GMT to 287 961 CpG + 961 adsorbed on PLG - 20661 CpG + 961 + PLG (without adsorption) - 1706 CpG + 961 + 287 adsorbed on PLG 86052 1924 CpG + 961 adsorbed on PLG, +287 adsorbed on PLG 107142 11717 CpG + 287 adsorbed on PLG 63057 - CpG + 287 and 961 co-adsorbed on PLG 57306 6251 CpG + 961 adsorbed on PLG, +287 adsorbed on PLG + PLG 78176 20876 287 + 961 + PLG (without antigen adsorption) 1266 145

Therefore, with regard to DG287, the adjuvant mixtures of CpG and microparticles for protein 961c is optimal when the antigen is adsorbed on microparticles.

Additional combinations of adjuvants with microparticles from PLG were tested for proteins DG287 and 961c. CpG was either dissolved or adsorbed on PLG microparticles. The results were as follows:

Table 9 The drug + PLG microparticles BCA GMT to 287 961 287 (adsorbed on PLG) +961 (adsorbed on PLG) 256 5719 2412 287 (adsorbed on PLG) +961 (adsorbed on PLG) + CpG 512 17553 8627 287 (adsorbed on PLG) +961 (adsorbed on PLG) + CpG (adsorbed on PLG) 1024 16906 6720 287 (adsorbed on PLG) +961 (adsorbed on PLG) + MF59 64 4636 3969 287 (adsorbed on PLG) +961 (adsorbed on PLG) + MF59 + CpG 2048 23642 48446

Similar work was performed on groups of 10 CD-1 mice using 20 μg of PLG-adsorbed antigen per IM dose (days 0, 21 and 35). When CpG was present, it was given at 10 μg per dose. Typhoid titers (GMT) were calculated as the reciprocal of the serum dilution giving an OD of _{450 nm} 0.5, and the sera were tested for both antigens. Serum bactericidal activity titers (ALS) were calculated as the inverse of the dilution of serum killing 50% of the target bacteria, and the sera were tested for activity against strain 2996 and against MC58, a heterologous strain. The titers on day 49 (2 weeks after the third dose) were as follows:

Table 10 287 961 Additional adjuvant GMT BASS 287 961 2996 MS58 X - - 8375 - 512 <4 X - Dissolved CpG 33736 - 1024 128 X - Adsorbed on PLG CpG 32058 - 1024 64 - X - - 3818 but but - X Dissolved CpG - 14149 2048 <4 - X Adsorbed on PLG CpG - 18526 2048 <4 X X - 13557 2476 but but X X Dissolved CpG 21664 6557 8192 64 X X Adsorbed on PLG CpG 27259 7510 2048 128 X X Dissolved CpG + MF59 27981 26826 2048 256 Control: dissolved 287 with CFA 37889 - 1024 <32 Control: dissolved 961 with CFA - 50453 4096 <4 Control: dissolved 287 and 961 with CFA 1678 27069 512 <32

Source 12 describes a combination of three proteins that include five different N.meningitidis antigens: (1) 961c ₂₉₉₆ ; (2) DG287 _NZ -953 ₂₉₉₆ and (3) 936 ₂₉₉₆ -DG741 _MS58 . A mixture of antigens was tested at source 12 using aluminum hydroxide as an adjuvant. In accordance with this invention, the stimulating activity of a mixture of antigens is enhanced by adsorption on biodegradable polymer particles plus a CpG oligonucleotide. The titers after the third dose were as follows:

Table 11 Immunization TIFA GMT ALS (versus seven strains) 961 287 741 953 2996 MS58 BZ133 394/98 NGH38 F6124 44/76 (1) 961 in alum 12346 - - - 4096 <4 <4 <4 <4 64 <4 (2) 287-953 on alum - 6415 - 585 1024 1024 256 1024 4096 256 1024 (3) 936-741 on alum - - 10625 - <4 32768 16384 1024 128 16384 32768 (1), (2) and (3) on alum 42302 18206 33881 4549 8192 32768 32768 2048 4096 32768 65536 (1) 961 per PLG 14185 - - - 2048 four <4 <4 16 256 <4 (2) 287-953 on PLG - 43515 - 478 2048 128 2048 2048 8192 4096 128 (3) 936-741 on PLG - - 16150 - <4 32768 16384 1024 512 8192 262144 (1), (2) and (3) on the PLG 6735 24304 13801 1214 4096 65536 32768 2048 4096 32768 65536 (1), (2) and (3) on PLG + CpG 10896 40697 26966 2301 8192 262144 65536 4096 8192 32768 262144

Compared to the aluminum adjuvant used in source 12, the PLG + CpG mixture leads to lower total antibody titers (except for protein 287), but it is important that it gives higher bactericidal titers against a wide range of strains. Despite the fact that the absolute titers are lower, the adjuvant of the present invention, therefore, mainly increases the production of bactericidal antibodies.

It will be understood that the invention has been described by way of example only, and modifications can be made while remaining within the scope and spirit of the invention.

Claims (15)

1. An immunogenic composition comprising:
(a) a Neisseria antigen containing a protein of the serogroup B Neisseria meningitides selected from the group consisting of:
a) a NadA protein or sequence that is more than 80% homologous to the NadA protein;
b) protein 287 or a sequence that is more than 80% homologous to protein 287;
c) protein 741 with the sequence:
1 VNRTAFCCLS LTTALILTAC SSGGGGVAAD IGAGLADALT APLDHKDKGL
51 QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ
101 IEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI
151 GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI
201 EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA
251 QEVAGSAEVK TVNGIRHIGL AAKQ
or a sequence that is more than 80% homologous to protein 741;
d) a protein with the sequence:
1 MKKIIFAALA AAAISTASAA TYKVDEYHAN ARFAIDHFNT STNVGGFYGL
51 TGSVEFDQAK RDGKIDITIP IANLQSGSQH FTDHLKSADI FDAAQYPDIR
101 FVSTKFNFNG KKLVSVDGNL TMHGKTAPVK LKAEKFNCYQ SPMEKTEVCG
151 GDFSTTIDRT KWGMDYLVNV GMTKSVRIDI QIEAAKQ
or a sequence that is more than 80% homologous to protein 953;
(b) a CpG oligonucleotide; and
(c) biodegradable polymer microparticles, which are a copolymer of D, L lactide with glycolide (PLG). 2. The composition according to claim 1, in which the CpG oligonucleotide contains from about 6 to about 100 deoxyribonucleotides. 3. The composition according to claim 1, in which the biodegradable polymer microparticles contain poly-α-hydroxy acids. 4. The composition according to claim 1, in which the Neisseria antigen is enclosed inside the microparticles. 5. The composition according to claim 1, in which the Neisseria antigen is adsorbed on microparticles. 6. The composition according to claim 1, in which the CpG oligonucleotide is enclosed inside the microparticles. 7. The composition according to claim 1, in which the CpG oligonucleotide is adsorbed on microparticles. 8. The composition according to claim 1, additionally containing an adjuvant. 9. The composition of claim 8, containing the adjuvant MF59. 10. The composition of claim 8, containing the adjuvant is an aluminum salt. 11. The composition according to claim 1, containing at least one additional antigen is not Neisseria. 12. The composition according to claim 1, additionally containing a pharmaceutically acceptable carrier. 13. A method of increasing the humoral response in a mammal, comprising administering to the mammal a composition according to any one of claims 1-12. 14. A method of protecting a mammal from infection and / or disease caused by Neisseria, comprising administering to the patient a composition according to any one of claims 1-12. 15. The use of a composition according to claim 1, comprising (a) a Neisseria antigen comprising a NadA protein, 287, 741, 953 of a Neisseria meningitides serogroup B; (b) a CpG oligonucleotide; and (c) biodegradable polymer microparticles, which are a copolymer of D, L lactide with glycolide (PLG) for the manufacture of a medicament for the prophylaxis or treatment of a disease and / or infection in a mammal.