CN100354297C - Adjunct Meningococcal Composition - Google Patents

Abstract

The combination of CpG oligonucleotides and polymer microparticles is a very effective adjuvant for neisserial antigens. The invention therefore provides a composition comprising: a neisserial antigen; (b) a CpG oligonucleotide; (c) biodegradable polymer microparticles.

Description

Adjunct Meningococcal Composition

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

technical field

The invention relates to vaccines, more particularly vaccines against Neisseria meningitidis.

background field

The genome sequences of Neisseria meningitidis (meningococcus) serogroups A [1] and B [2,3] have been reported. Serogroup B sequences were studied to identify vaccine antigens [eg refs 4 to 9] and candidate antigens were manipulated to improve heterologous expression [refs 10 to 12].

Antigens generally require co-administration of adjuvants to improve their immunogenicity in vaccines [13]. Freund's adjuvant has been used for serogroup B meningococci [9] and the licensed vaccine against serogroup C, Menjugate ^TM , uses aluminum hydroxide [14]. The use of oligonucleotide adjuvants containing CpG motifs has been reported to enhance the bactericidal activity of Neisseria antigens [15].

It is an object of the invention to provide further and improved adjuvants for Neisseria antigens.

disclosure of invention

The combination of CpG oligonucleotides and polymer particles has been found to be an extremely effective adjuvant for Neisserial antigens, with the combination producing much better results than either component alone. The invention thus provides a composition comprising: (a) Neisseria antigens; (b) CpG oligonucleotides; (c) biodegradable polymer particles.

Neisserial antigen

Neisserial antigens may be protein antigens, nucleic acids encoding protein antigens, or carbohydrate antigens. The antigen preferably elicits a bactericidal or protective immune response (eg, antibody response) in the recipient mammal.

Antigens may be derived from any Neisserial strain, including N. gonorrhoeae (N. gonorrhoeae), N. lactamica (N. lactamica), and N. meningitidis. Neisseria meningitidis antigens are preferred and may be from any serogroup. When the antigen is from serogroup B, protein antigens are preferred; when from serogroups A, C, W135 or Y, carbohydrate antigens are preferred. When carbohydrate antigens are used, these are generally derived from bacterial capsular polysaccharides (eg oligosaccharides, eg obtained by hydrolysis), which are usually conjugated to a carrier protein (eg CRM ₁₉₇ ).

Preferred protein antigens derived from serogroup B Neisseria meningitidis are:

any one of the proteins described in references 4, 5, 6, 7, 8 or 9 (in particular the 446 even-numbered SEQIDs shown in reference 4 (i.e. 2, 4, 6, ..., 890, 892), 45 even SEQ IDs (i.e. 2, 4, 6, ..., 88, 90) shown in reference 5 and 1674 even SEQ IDs 2-3020, even SEQ IDs 3040-3114 and all SEQ IDs 3115-3241);

A protein comprising an immunogenic fragment of one or more proteins of any one of references 4, 5, 6, 7, 8 or 9.

A protein comprising a sequence having sequence identity (preferably greater than 50%, such as 60%, 70%, 80%, 90%, 95%, 99% or higher).

A protein as described in any one of references 10, 11 or 12.

A protein comprising a sequence having sequence identity (preferably greater than 50%, such as 60%, 70%, 80%, 90%, 95%, 99% or higher).

A particularly preferred protein antigen from serogroup B Neisseria meningitidis is protein '287'. This protein can be used in wild-type form [such as GenBank accession number gi:7228690; the polymorphic arrangement of 287 is shown in Figures 5 & 15 of Reference 8], but derivatives of the wild-type protein can be used. For example, proteins with 50% or greater sequence identity (eg, 60%, 70%, 80%, 90%, 95%, 99% or greater) to gi:7228690 may be used. Proteins containing truncations or protein deletion variants can be used, as described in refs 10 to 12 for N-terminally truncated forms (specifically 'ΔG287', in which the protein N- ends are deleted). Fusion proteins containing sequences such as 287 can be used. All these 287 forms, more particularly those that retain the immunogenicity of the wild-type 287 protein, fit within the meaning of '287' as used herein.

Another particularly preferred protein antigen from serogroup B Neisseria meningitidis is protein '961', also known as 'NadA' [16]. This protein can be used in wild-type form [eg GenBank accession number gi:7227256; the allele of 961 is disclosed in ref. 17], but derivatives of the wild-type protein can be used. For example, proteins with 50% or greater sequence identity (eg, 60%, 70%, 80%, 90%, 95%, 99% or greater) to gi:7227256 may be used. Proteins containing truncations or protein deletion variants may be used, as described in refs 10 to 12 (in particular '961c', which lacks a C-terminal membrane anchor). Fusion proteins containing sequences such as 961 can be used. All these 961 forms, especially those that retain the immunogenicity of the wild-type 961 protein, fit within the meaning of '961' as used herein.

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 hybrid proteins as described in references 10 to 12, especially hybrid proteins comprising one or more of: 287 protein, 953 protein, 963 protein and/or 741 protein.

Protein antigens may be derived from any N. meningitidis strain. Preferably used antigens are from strains 2996, MC58, 95N477 and 394/98.

In addition to strain variants, single or multiple conservative amino acid substitutions may be made according to the invention to alter the immunogenicity of the antigen used.

In addition to or in place of protein antigens, nucleic acids encoding protein antigens may be included in compositions of the invention. Once administered to a mammalian recipient, the nucleic acid is expressed in vivo and produces protein antigens. Such nucleic acid immunization is well known [eg, references 18 to 23, etc.]. The nucleic acid is usually a DNA plasmid.

A preferred carbohydrate antigen derived from serogroup C N. meningitidis is the oligosaccharide conjugate used in Menjugate ^™ [24, 25], which comprises 12 to 22 monosaccharide units from the serogroup C capsular polysaccharide.

A preferred carbohydrate antigen derived from serogroup A is an oligosaccharide in which one or more hydroxyl groups on the constituent monosaccharide units are replaced by blocking groups [26].

Further oligosaccharide antigens obtained from serogroups A, W135 and Y are disclosed in ref. 27.

Compositions of the invention may include more than one Neisserial antigen. When carbohydrates from N. meningitidis serogroups A and C are both included, it is preferred that the MenA sugar:MenC sugar ratio (w/w) is greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1). 1, 10:1 or higher)

The inventive composition is preferably an immunogenic composition or vaccine. Such compositions include an immunologically effective amount of the antigen. "Immunologically effective amount" refers to an amount of antigen contained in a composition of the invention administered to an individual (single dose or a series of fractions) effective to raise a therapeutic or prophylactic immune response. This amount will vary depending on the health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (e.g., non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the desired Degree of protection, formulation of the vaccine, assessment of the medical condition by the attending physician, and other relevant factors. The amount can be within a relatively wide range, which range can be determined by routine experimentation. Antigens are usually present at concentrations of at least 1 μg/ml each.

Dosed therapy may be a single dose or a multi-dose arrangement (eg including booster doses).

CpG oligonucleotides

CpG oligonucleotides are known to be used as vaccine adjuvants [eg ref. 28] and they induce strong Th1 immune responses. They are used as parenteral and mucosal adjuvants [29].

A CpG oligonucleotide used according to the invention is a nucleic acid comprising at least one CpG dinucleotide, ie a cytosine nucleotide followed by a guanosine nucleotide. An oligonucleotide may contain multiple CpG dinucleotides.

The CG sequence in the oligonucleotide can be flanked by two purines at the 5' and two pyrimidines at the 3', ie RRCGYY.

Cytosine nucleotides in CpG oligonucleotides may be methylated, but preferably they should be unmethylated.

Cytosine and guanosine nucleotides are preferably deoxyribonucleotides and the nucleic acid is preferably DNA. To increase nuclease resistance, oligonucleotides may include modified backbones, such as phosphorothioate backbones. Instead of using DNA, it is possible to use PNA (peptide nucleic acid). In addition, oligonucleotides may include substitutions of sugar moieties and nitrogenous base moieties.

The oligonucleotide preferably comprises between about 6 and about 100 nucleotides, more preferably between about 8 and about 50 nucleotides, most preferably between about 10 and about 40 nucleotides.

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

Examples of CpG oligonucleotide adjuvants are found in references 30 to 55.

biodegradable polymeric microparticles

Biodegradable polymer particles are known for use as vaccine adjuvants [eg ref. 56]. They are used as parenteral and mucosal adjuvants [29].

In addition to being biodegradable, the polymers used to make the microparticles are generally sterilizable and nontoxic (biocompatible). Suitable biodegradable polymers are commercially available, including polymers derived from polyhydroxybutyrate; polycaprolactone; polyorthoesters; polyanhydrides; poly(hydroxybutyrate); ). Preferred polymers are formed from one or more poly(alpha-hydroxy acids) such as poly(L-lactide), poly(D,L-lactide), D,L-lactide and glycolide Copolymers (such as poly(D,L-lactide-co-glycolide) or copolymers of D,L-lactide and caprolactone. Preferably formed from poly(D,L-lactide-co- Glycolide) ('PLG') microparticles.

These polymers come in a variety of molecular weights and the appropriate molecular weight for a particular antigen can be readily determined. For poly(L-lactide), a suitable molecular weight corresponds to about 2,000 to 250,000. For PLG, suitable molecular weights range generally from about 10,000 to 200,000, preferably from about 15,000 to about 150,000, most preferably from about 50,000 to 100,000.

For PLG microparticles, a variety of lactide:glycolide ratios can be used, and the choice of ratio will depend largely in part on the co-administered antigen and the desired rate of degradation. For example, a 50:50 PLG polymer containing 50% D,L-lactide and 50% glycolide rapidly resorbs the copolymer while 75:25 PLG degrades more slowly due to the increased lactide content, 85: 15, 90:10 or even slower. The appropriate ratio of lactide:glycolide is readily determined on the basis of the nature of the antigen and the disease in question. In addition, mixtures of microparticles with varying ratios of lactide:glycolide were used in the formulation to obtain the desired release kinetics for a specific antigen and to provide primary and secondary immune responses. The rate of degradation of the microparticles of the invention can also be controlled by factors such as polymer molecular weight and crystallinity.

As used herein, the term 'microparticle' refers to particles having a diameter of about 100 nm to about 150 μm, more preferably about 200 nm to about 30 μm in diameter, most preferably about 500 nm to about 10 μm in diameter. Preferred particle diameters allow parenteral administration without occlusion of needles or capillaries. Particle size is readily determined by techniques well known in the art, such as photon correlation spectroscopy, laser diffractometry and/or scanning electron microscopy. The term 'microparticle' includes within its scope 'nanoparticles' [57]. Preferably the microparticles are microspheres, although flake-like particles may also be used [58].

Microparticles can be prepared by any number of methods known in the art [eg ref. 59]. For example, double emulsion/solvent evaporation techniques [eg refs 60 & 61] can be used to form microparticles. These techniques involve the formation of primary emulsions consisting of droplets of polymer solution containing the antigen (if the antigen is to be embedded in microparticles), followed by mixing with a continuous aqueous phase containing particle stabilizers/surfactants.

More specifically, water-in-oil-in-water (w-o-w) solvent evaporation systems can be used to form microparticles, as described in refs 62, 63 and 64. In this technique, specific polymers are combined with organic solvents such as ethyl acetate, dimethyl chloride (also known as dichloromethane and dichloromethane), acetonitrile, acetone, chloroform, etc. The polymers are provided in about 2-15% solutions, organic solvents. An approximately equal amount of antigen solution (eg in water) is added and the polymer/antigen solution is emulsified eg with a homogenizer. The emulsion is then combined with a bulky aqueous solution of an emulsion stabilizer, such as polyvinyl alcohol (PVA) and polyvinylpyrrolidone. Emulsion stabilizers are usually provided in about 2-15% solution, more usually in about 4-10% solution. The mixture is then homogenized to produce a stable w/o/w double emulsion. The organic solvent was then evaporated.

Formulation parameters can be manipulated to produce small (<5 μm) and large (>30 μm) microparticles [eg 63, 65]. For example, reducing agitation produces larger particles, as does increasing the volume of the dispersed phase. Small particles are produced by high concentrations of PVA with low aqueous phase volumes.

Microparticles can also be formed by spray-drying and agglomeration [e.g. refs 66, 67 &68]; air-suspension coating techniques such as pan coating and Worcester coating [69, 70]; ion gelation [71] .

Prior to use of the microparticles, the antigen content is generally determined so that an appropriate amount of microparticles can be delivered to a subject to elicit an appropriate immune response.

The antigen content of the microparticles can be determined according to methods known in the art, such as splitting the microparticles and extracting the entrapped antigen. For example microparticles can be dissolved in dimethyl chloride and proteins extracted into distilled water [eg refs 72, 73, 74]. Alternatively, the microparticles can be dispersed in 0.1M NaOH containing 5% (w/v) SDS. The samples are stirred, centrifuged and the supernatant analyzed by an appropriate assay.

Antigen and/or CpG oligonucleotides can be localized within or on the microparticles. Entrapment is generally accomplished by the presence of antigen/oligonucleotides during microparticle formation, while surface adsorption is accomplished by adding antigen/oligonucleotides to preformed microparticles.

One method of adsorbing antigens/oligonucleotides onto preparative microparticles is as follows. The microparticles are rehydrated and dispersed into an essentially monomeric suspension of the microparticles, using dialyzable anionic or cationic detergents. Useful detergents include, but are not limited to, any of the various N-methylglucamides (known as MEGAs), such as heptanoyl-N-methylglucamide (MEGA-7), octanoyl-N-methylglucamide Glucamide (MEGA-8), Nonanoyl-N-methylglucamide (MEGA-9) and Decanoyl-N-methylglucamide (MEGA-10); Cholic acid; Sodium cholate; Deoxy Cholic acid; Sodium deoxycholate; Taurocholic acid; Sodium taurocholate; Taurodeoxycholic acid; Sodium taurodeoxycholate; 3-3[(3-cholaminopropyl)dimethylamino]-1 -propane-sulfonates (CHAPs); N-octyl glucoside, 3-3[(3-cholaminopropyl)dimethylamino]-2-hydroxy-1-propane-sulfonate (CHAPSO); N -Dodecyl-N,N-dimethyl-3-amino-1-propane-sulfonate (ZWITTERGENT 3-12); N,N-bis-(3-D-glucosaminopropyl)- Deoxycholamide (DEOXY-BIGCHAP); Sucrose Monolaurate; Glycocholic Acid/Sodium Glycocholate; Laurosarcosine (Sodium Salt); Glyodeoxycholic Acid/Sodium Glycocholate ; Sodium dodecylsulfonate (SDS); Cetyltrimethylammonium bromide (CTAB); Dodecyltrimethylammonium bromide; Cetyltrimethylammonium bromide; Bromide Tetradecyltrimethylammonium; Benzyldodecyldimethylammonium bromide; Benzylhexadecyldimethylammonium chloride; Benzyltetradecyldimethylammonium bromide. The above detergents are commercially available. Various cationic lipids known in the art can also be used as detergents [76, 77].

The particle/detergent mixture is then physically ground, eg, with a porcelain mortar and pestle, until a smooth slurry is formed. An appropriate aqueous buffer such as phosphate-buffered saline (PBS) or Tris-buffered saline is then added, and the resulting mixture is sonicated or homogenized until the microparticles are well suspended. Antigen/oligonucleotides are then added to the microparticle suspension and the system is dialyzed to remove detergent. Preferably the polymer particle and detergent system allow the antigen/oligonucleotide to be adsorbed to the surface of the particle while still remaining active. The resulting surface-adsorbed antigen/oligonucleotide-containing microparticles can be washed free of unbound antigen/oligonucleotide and stored as a suspension in a suitable buffer formulation, or lyophilized with a suitable excipient, as further described below.

Antigen/CpG/microparticle combination

Various physical relationships are possible between the three essential components of the inventive composition. This is because the microparticles have an internal volume and a surface, either of which can be used to localize CpG oligonucleotides and/or antigens.

Thus the antigen can be embedded within the microparticles, it can be adsorbed to the microparticles, or it can simply be mixed with the microparticles without entrapment or adsorption. Adsorption is preferred.

Similarly, CpG oligonucleotides can be embedded within the microparticles, it can be adsorbed to the microparticles, or it can simply be mixed with the microparticles. Adsorption can be accomplished with detergents such as CTAB.

Both the CpG oligonucleotide and the antibody have the same physical relationship to the microparticles, or they can be different. Also CpG oligonucleotide and antigen can be adsorbed to the same microparticle or CpG oligonucleotide and antigen can be adsorbed to different microparticles. All possible combinations are included in the invention:

CpG oligonucleotides embedding Adsorption mix anti embedding yes yes yes Adsorption yes yes yes mix yes yes yes

The composition of the invention may comprise a mixture of the above, eg some microparticles in the composition entrap the antigen and some particles adsorb the antigen.

pharmaceutical composition

For pharmaceutical use, inventive compositions generally include a pharmaceutically acceptable carrier. This leads to the inventive pharmaceutical composition. A pharmaceutically acceptable carrier can be any substance which does not itself induce antibodies deleterious to the patient receiving the composition and which can be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactivated virus particles. Such vectors are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present in such vehicles. Liposomes are suitable carriers. A full discussion of drug carriers is in reference 78.

The compositions of the invention can be prepared in a variety of forms. For example, the compositions can be prepared as injectable liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The composition may be prepared for topical administration, eg, as an ointment, cream or powder. The composition may be prepared for oral administration, eg as a tablet or capsule or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration eg as an inhaler, using a fine powder or spray. The composition can be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration, eg as drops, spray or powder [eg 79].

Pharmaceutical compositions are preferably sterile. It is preferably pyrogen free. It is preferably buffered, eg between pH 6 and pH 8, typically around pH 7.

Pharmaceutical compositions can 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

Compositions of the invention may be used therapeutically (ie, to treat an existing Neisserial infection) or prophylactically (ie, to prevent future Neisserial infections).

The invention provides compositions of the invention for use as a medicament.

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

The invention also provides methods for treating a mammal suffering from Neisserial infection and/or disease comprising administering to the patient a pharmaceutical composition of the invention.

The invention also provides methods for protecting a mammal from Neisserial infection and/or disease comprising administering a pharmaceutical composition of the invention to the mammal.

The invention also provides the use of (a) Neisserial antigens, (b) CpG oligonucleotides and (c) biodegradable polymer particles in the manufacture of a medicament to prevent or treat Neisserial disease and/or Infect.

The mammal is preferably a human. A human can be an adult, or preferably a child. The inventive composition is especially useful for immunizing children and adolescents.

The uses and methods of the invention are particularly useful for treating/protecting against Neisseria meningitidis infection. The uses and methods are particularly useful in the prevention/treatment of disease, including bacterial meningitis.

The efficacy of the treatment can be tested by monitoring Neisserial infection following administration of the inventive composition. The efficacy of prophylactic treatment can be tested by monitoring the anti-Nisserial immune response following administration of the composition.

The compositions of the invention are generally administered directly to the patient. Direct delivery can be accomplished by parenteral (eg, subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial), rectal, oral, vaginal, topical, transdermal, ophthalmic, nasal, aural, or pulmonary administration. Injection and intranasal administration are preferred.

Dosage therapy can be a single dose schedule or a multiple dose schedule.

further ingredients

In addition to CpG oligonucleotides and polymer particles, compositions of the invention may include adjuvants. Preferred further adjuvants include, but are not limited to: (A) Aluminum compounds (such as aluminum hydroxide, aluminum phosphate, aluminum hydrogen phosphate, hydroxides, orthophosphates, sulfates, etc. [see for example chapters 8 & 9 of ref. 13 ]), or a mixture of different aluminum compounds, the compound taking any suitable form (e.g. gel, crystalline, amorphous, etc.), preferably adsorbed; (B) MF59 (5% squalene, 0.5% Tween 80 and 0.5% Span 85, formulated as submicron particles with microfluidizers) [see Chapter 10 of 13, see also Ref. 80]; (C) liposomes [see Chapters 13 and 14 of Ref. 13]; (D) ISCOMs [see Chapter 23 of ref. 13] can be used without additional detergents [81]; (E) SAF with 10 squalene, 0.4% Tween 80 and 5% pluronic-blocking polymer L121 and thr- MDP, microfluidized into submicron emulsions or vortexed to produce larger particle size emulsions [see Chapter 12 of ref. 13]; (F) Ribi ^TM Adjuvant System (RAS) (Ribi Immunochem) with 20% squalene , 0.2% Tween 80 and one or more bacterial cell wall components, the cell wall components are from monophosphoryl lipid A (MPL), trehalose dimycolic acid (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox ^TM ); (G) saponin adjuvants such as QuilA or QS21 [see chapter 12 of ref. 13], also known as Stimulon ^TM [82]; (H) chitosan [eg 83]; (I ) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (J) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferon (such as interferon-γ), macrophage colony-stimulating factor, tumor necrosis factor, etc. [see Chapter 27&28 of reference 13]; (K) monophosphoryl lipid ( M) polyoxyethylene ether or polyoxyethylene ester [85]; (N) polyoxyethylene sorbitan ester surfactant combined with octoxynol [86] or polyoxyethylene alkyl ether or ester surfactant combined with at least An additional nonionic surfactant such as octoxynol [87]; (O) particles of metal salts [88]; (P) saponins and oil-in-water emulsions [89]; (Q) saponin Glycoside (QS21) + 3d MPL + IL-12 (optional + sterol) [90]; (R) E. coli heat-labile enterotoxin (“LT”) or its detoxified mutants, such as K63 or R72 mutants [as in Chapter 5 of ref. 91]; (S) cholera toxin ("CT") or a detoxified mutant thereof [as in Chapter 5 of ref. 91]; (T) double-stranded RNA and (U) other substances , as an immunostimulant to enhance the efficacy of the composition [eg Chapter 7 of ref. 13]. Aluminum (especially aluminum phosphate and/or aluminum hydroxide) and MF59 are further preferred as adjuvants for parenteral immunizations. Mutant toxins are preferred mucosal adjuvants.

Muramyl peptides include N-acetyl-muramoyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-demethylmuramoyl-L-alanyl-D-isoglutamine Glutamine (nor-MDP), N-acetylmuramoyl-L-alanyl-D-isoglutamyl--L-alanine-2(1'-2'-dipalmitoyl-sn -glycerol-3-hydroxyphosphoryloxy)-ethylamine MTP-PE) and the like.

In addition to Neisserial antigens, the invention may further comprise antigenic components. Antigens that may be included in compositions of the invention include:

Antigens from Helicobacter pylori, such as CagA [92 to 95], VacA [96, 96], NAP [98, 99, 100], HopX [eg 101], HopY [eg 101], and/or urease .

Outer membrane vesicle (OMV) preparations from N. meningitidis serogroup B, as shown in refs 102, 103, 104, 105 et al.

Carbohydrate antigens from Streptococcus pneumoniae [eg, 106, 107, 108].

Antigens from hepatitis A virus, such as inactivated virus [eg 109, 110].

Antigens from HBV, such as surface and/or core antigens [eg 110, 111].

Antigens from hepatitis C virus [eg 112].

Antigens from Bordetella pertussis (Bordetella pertussis), such as pertussis holotoxin (PT) and filamentous hemagglutinin (FHA) from Bordetella pertussis, optionally also with pertactin ) and/or combinations of agglutinogens 2 and 3 [eg refs 113 & 114].

Diphtheria antigens such as diphtheria toxoid [eg chapter 3 of ref. 115], eg CRM ₁₉₇ mutant [eg 116].

Tetanus antigen, such as tetanus toxoid [eg, chapter 4 of ref. 115].

Carbohydrate antigen from Haemophilus influenzae B [eg 23].

Antigens from Chlamydia pneumoniae [eg 117, 118, 119, 120, 121, 122, 123].

Antigens from Chlamydia trachomatis [eg 124].

Antigens from Porphyromonas gingivalis [eg 125].

Polio antigens [eg 126, 127] eg IPV or OPV.

Rabies antigen [eg 128], for example lyophilized inactivated virus [eg 129, RabAvert ^™ ].

Measles, mumps and/or rubella antigens [eg Chapters 9, 10 & 11 of ref. 115].

Antigens from influenza viruses [eg, chapter 19 of ref. 115], such as hemagglutinin and/or neuraminidase surface proteins.

Antigens from paramyxoviruses, such as respiratory syncytial virus (RSV [130, 131] and/or parainfluenza virus (PIV3 [132]).

Antigens from Moraxella catarrhalis [eg 133].

Antigens from Streptococcus agalactiae (group B Streptococcus) [eg, 134, 135].

Antigens from Streptococcus pyogenes (group A Streptococcus) [eg, 135, 136, 137].

Antigens from Staphylococcus aureus [eg 138].

Antigens from Bacillus anthracis [eg 139, 140, 141].

Antigens from viruses of the Flaviviridae family (genus Flaviviruses), such as from yellow fever virus, Japanese encephalitis virus, 4 serotypes of dengue virus, tick-borne encephalitis virus, West Nile virus.

Pestivirus antigens, eg from classical swine fever virus, bovine virus diarrhea virus and/or border disease virus.

Parvovirus antigens, such as from parvovirus B19.

Prion protein (eg, CJD prion protein).

Amyloid proteins such as beta peptides [142].

Cancer antigens, as listed in Table 1 of reference 143 or Tables 3 & 4 of reference 144.

The composition may comprise one or more of these further antigens.

Toxic protein antigens can be detoxified if desired (eg, by chemical and/or genetic methods to detoxify pertussis toxin).

When diphtheria antigens are included in the composition, tetanus antigens and pertussis toxin are also preferably included. Similarly, when tetanus antigens are included, diphtheria antigens and pertussis toxin are also preferably included. Similarly, when pertussis toxin is included, diphtheria and tetanus antigens are also preferably included.

The antigen is preferably adsorbed to the aluminum salt.

The antigens in the composition are usually present at a concentration of at least 1 μg/ml each. Generally, the concentration of a particular antigen is sufficient to elicit an immune response against that antigen.

Instead of using protein antigens in the compositions of the invention, nucleic acids encoding the antigens may be used. Thus the protein component of the inventive composition may be replaced by a nucleic acid (preferably DNA, eg in the form of a plasmid) encoding the protein.

definition

The term "comprising" means "comprising" and "consisting of". For example, the composition "comprising" X may completely consist of X or may include other components such as X+Y.

Percent sequence identity between two amino acid sequences refers to the percentage of amino acids that, when aligned, compare the two sequences to be the same. This alignment and percent homology or sequence identity can be determined using software programs known in the art, for example as described in section 7.7.18 of ref. 145 . The preferred permutations were determined by the Smith-Waterman homology search algorithm with an affine gap search, a gap opening penalty of 12 and a gap extension penalty of 2, and a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is taught in ref. 146.

mode of invention

Parenteral sensitization and mucosal boosting with Neisseria meningitidis serogroup B antigens

Reference 6 discloses a protein from N. meningitidis serogroup B called '287'. References 10 to 12 disclose methods to improve their expression. One approach involves deleting the N-terminus of the protein and including 6 repeated glycine residues. This protein is called 'ΔG287'.

Mice were sensitized and boosted with MenBΔG287 antigen (20 μg/dose) from strain 2996 made for intramuscular (IMU) administration by adsorption to PLG microparticles, with or without CpG oligonucleotides (also adsorbed to particles). Another formulation for intranasal (IN) administration uses LT-K63 adjuvant. Mice received 3 IM doses or 2 IM followed by 2 IN doses (doses: day 0; day 28; day 84; optionally day 98).

Group preparation way dosage Antibody GMT 2 weeks later dose 2 Dose 3 dose 4 1 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 4 PLG/287+PLG/CpG IM 1, 2 34,891 15,167 16,556 287+LT-K63 IN 3,4

Thus, inclusion of CpG oligonucleotides increased antibody titers against intramuscularly administered MenB protein 287 (comparison groups 1 & 2). Antibody titers could be increased by replacing the 3rd intramuscular dose with 2 intranasal doses (comparison groups 1 & 3). CpG increases were also seen with the intramuscular/intranasal regimen (comparison groups 3 & 4).

Comparing adjuvants for MenB protein 287

ΔG287 was formulated with various adjuvants and administered to mice. Sera from mice were evaluated with a bactericidal antibody (BCA) assay and titered as follows:

Adjuvant After BCA-2 After BCA-3 Freund's adjuvant 2048 8192 aluminum <4 256 Aluminum + CpG oligonucleotides 256 4097 MF59 <4 <4 CpG oligonucleotides <4 128 PLG particles (adsorption) 8 1024 PLG particles (adsorption) + CpG 2048   16384

CpG oligonucleotides are therefore only moderately effective as adjuvants, almost comparable to aluminum. PLG microparticles were more effective than aluminum and CpG, but not as effective as Freund's adjuvant. In striking contrast, however, the adjuvantity of the CpG and PLG mixture was comparable to that of Freund's adjuvant after the second immunization period, and surpassed that of Freund's adjuvant after the third immunization.

Enhancement of PLG adjuvantity with CpG was also found in a separate study (02-0279):

Adjuvant After GMT2 3 after GMT MF59 6967 13417 PLG particles (adsorption) 7070 11367 PLG particles (adsorption) + CpG 15099 26833

The effect of adsorption on adjuvant

The effect of adsorption on adjuvant properties was studied. Protein ΔG287 was adsorbed to PLG microparticles with DSS surfactant or SDS or simply mixed with the particles. Immunizations were performed on days 0, 21 and 35 and titers were assessed on days 35 and 49. The result is as follows:

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

The adjuvant properties of the mixture of CpG and microparticles for ΔG287 are therefore optimal when antigen is adsorbed to the microparticles.

Reference 6 revealed a protein from N. meningitidis serogroup B called '961' (now also called 'NadA' [16, 17]). References 10 to 12 disclose methods for improving NadA expression. One approach involves deleting the C-terminus of the protein to remove its membrane anchor (ie, amino acids 341-405 of strain 2996) as well as naturally removing its leader peptide. This protein is called '961c'. As described above for 287, the effect of adsorption on the adjuvant properties of PLG was studied when CpG was co-administered:

preparation BCA Antibody titers 2 weeks after dose 3 961 adsorbed on PLG (SDS) 2048 20661 961+PLG (no adsorption) 256 1706 287 adsorbed on PLG 4096 63057 Adsorbed 287+soluble 961 on PLG 4096 287:86052; 961:1924 287 adsorbed on PLG+961 adsorbed on PLG 8192 287:107142; 961:11717

287 (no adsorption) + 961 (no adsorption) + 'blank' PLG 1024 287:1266; 961:145 287(adsorption)+961(adsorption)+‘blank’ PLG 8092 287:78176; 961:20876

Thus for ΔG287, the adjuvantity of CpG and the microparticle mixture used for 961c is optimal when the antigen is adsorbed to the microparticles. This is also true for the antigen itself and when the antigen is bound to ΔG287.

Thus, for ΔG287 and 961c alone and in combination, the adjuvant properties of the mixture of CpG and PLG were optimal when the antigen was adsorbed onto the PLG microparticles.

PLG, CpG, Al and MF59

Different combinations of PLG, CpG, Al and MF59 were tested for the protein ΔG287 expressed as a His-tagged product. Serum bactericidal titers after 3 immunizations were as follows:

Adjuvant Titer aluminum 2048 Aluminum+CpG 32768 MF59 8192 MF59+CpG 32768 PLG (antigen adsorbed to PLG) 1024 PLG+CpG (both antigen and CpG are adsorbed to PLG) 4096 PLG+MF59 (antigen adsorbed to PLG) 2048 PLG+MF59+CpG (antigen adsorbed to PLG) 8192 Complete Freund's Adjuvant 32768 PLG+complete Freund's adjuvant (antigen adsorbed to PLG) 2048

A similar test was carried out and the results were as follows:

Adjuvant Titer PLG (antigen adsorbed to PLG) 1024 PLG+CpG (antigen adsorbed to PLG)   16384 PLG+CpG (both antigen and CpG are adsorbed to PLG)   16384 PLG+Al (antigen adsorbed to PLG) 1024 PLG+Al+CpG (antigen adsorbed to PLG)   16384 PLG+Al+CpG (both antigen and CpG are adsorbed to PLG) 8192

PLG+MF59 (antigen adsorbed to PLG) 4096 PLG+MF59+CpG (antigen adsorbed to PLG)   16384 Aluminum (antigen adsorbed to aluminum) 256 CpG 128 Aluminum+CpG 1024 Aluminum+CpG+PLG (antigen adsorbed to aluminum; CpG adsorbed to PLG) 4096 CpG+PLG (CpG adsorbed to PLG; antigen not adsorbed) 64

Thus MF59 and aluminum may further enhance the efficacy of the CpG/PLG mixture, adsorption of CpG to PLG microparticles is not necessary for adjuvantity, but again antigen adsorption to microparticles is seen to be optimal.

antigen mixture

The effect of adsorption on adjuvantity was investigated for the proteins ΔG287 and 961c alone and in combination. Antibody titers after 3 doses were as follows:

preparation Antibody GMT anti 287 961 961 adsorbed on CpG+PLG - 20661 CpG+961+PLG (no adsorption) - 1706 287 adsorbed on CpG+961+PLG 86052 1924 961 adsorbed on CpG+PLG+287 adsorbed on PLG   107142   11717 287 adsorbed on CpG+PLG 63057 - 287 and 961 co-adsorbed on CpG+PLG 57306 6251 961+PLG adsorbed on CpG+PLG 287+PLG adsorbed on PLG 78176 20876 287+961+PLG (no antigen adsorption) 1266 145

Thus for ΔG287, the adjuvantity of CpG and microparticle mixture for protein 961c is optimal when the antigen is adsorbed to the microparticles.

For proteins ΔG287 and 961c, the combination of adjuvant and PLG microparticles was further tested. CpG is soluble or adsorbed to PLG microparticles. The result is as follows:

Formulation + PLG particles BCA GMT resistance

287 961 287 (adsorption on PLG)+961 (adsorption on PLG) 256 5719 2412 287 (adsorption on PLG)+961 (adsorption on PLG)+CpG 512   17553 8627 287 (adsorption on PLG) + 961 (adsorption on PLG) + CpG (adsorption on PLG) 1024   16906 6720 287 (adsorption on PLG)+961 (adsorption on PLG)+MF59 64 4636 3969 287 (adsorption on PLG)+961 (adsorption on PLG)+MF59+CpG 2048   23642 48446

Similar work was performed on groups of 10 CD-1 mice using 20 μg per PLG-adsorbed antigen per IM dose (0, 21 and 35 days). When CpG was present, 10 μg per dose was given. ELISA titers (GMT) were calculated as reciprocal serum dilutions at _OD450nm 0.5, sera tested for both antigens. Serum bactericidal activity titers (SBA) were calculated as reciprocal serum dilutions that killed 50% of the target bacteria, and the activity of serum against 2996 strain and against heterologous strain MC58 was tested. Titers at 49 days (2 weeks after the third dose) are as follows:

287 961 additional adjuvant GMT SBA 287 961 2996 M58 x - - 8375 - 512 <4 x - soluble CpG 33736 - 1024 128 x PLG-adsorbed CpG 32058 - 1024 64 - x - - 3818 nd nd - x soluble CpG - 14149 2048 <4 - x PLG-adsorbed CpG - 18526 2048 <4 x x - 13557 2476 nd nd x x soluble CpG 21664 6557 8192 64 x x PLG-adsorbed CpG 27259 7510 2048 128 x x Soluble CpG+MF59 27981 26826 2048 256 Control: soluble 287 with CFA 37889 - 1024 <32

Control: soluble 961 with CFA - 50453 4096 <4 Control: soluble 287 and 961 with CFA 16781 27069 512 ＜32

Nd not detected

Reference 12 discloses a combination of three proteins including among them 5 different N. meningitidis antigens: (1) 961c ₂₉₉₆ ; (2) ΔG287 _NZ -953 ₂₉₉₆ ; (3) 936 ₂₉₉₆ -ΔG741 _MC58 . Antigen mixtures in ref. 12 were tested with aluminum hydroxide adjuvant. According to the invention, the mixture of antigens is assisted by adsorption to biodegradable polymer particles plus oligonucleotides. After the third dose the titers were as follows:

immunity ELISA GMT SBA (anti-7 strains) 961 287 741 953 2996 MC58 BZ133 394/98 NGH38 F6124 44/76 (1) 961 on aluminum 12346 - - - 4096 ＜4 ＜4 ＜4 ＜4 64 ＜4 (2) 287-953 on aluminum - 6415 - 585 1024 1024 256 1024 4096 256 1024 (2) 936-741 on aluminum - - 10625 - ＜4 32678 16384 1024 128 16384 32768 (1), (2) and (3) on aluminum 42302 18206 33881 4549 8192 32768 32768 2048 4096 32768 65536 (1) 961 on aluminum 14185 - - - 2048 4 ＜4 ＜4 16 256 ＜4 (2) 287-953 on aluminum - 43515 - 478 2048 128 2048 2048 8192 4096 128 (2) 936-741 on Aluminum - - 16150 - ＜4 32768 16384 1024 512 8192 262144 (1), (2) and (3) on aluminum 6735 24304 13801 1214 4096 65536 32768 2048 4096 32768 65536 (1), (2) and (3)+CpG on aluminum 10896 40697 26966 2301 8192 262144 65536 4096 8192 32768 262144

Compared to the aluminum adjuvant used in ref. 12, the PLG+CpG mixture produced lower total antibody titers (except for protein 287), but importantly gave higher bactericidal titers against a broad range of strains. Thus the inventive adjuvant confers antibody production favorably towards bactericidal antibodies despite lower absolute titers.

It is to be understood that the invention is described by way of example only and that modifications may be made within the scope and spirit of the invention.

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Claims (12)

1. An immunogenic composition, characterized in that, said composition comprises (a) Neisserial antigen; (b) CpG oligonucleotide; (c) biodegradable poly(D, L- lactide-co-glycolide) polymer particles. 2. The composition of claim 1, wherein the Neisserial antigen is a protein antigen. 3. The composition of claim 2, wherein the Neisseria meningitidis protein comprised by the Neisserial antigen is selected from the group consisting of NadA protein; 287 protein; 741 protein; 953 protein. 4. A composition according to any one of the preceding claims, wherein the CpG oligonucleotide comprises 6 to 100 deoxyribonucleotides. 5. Composition according to any one of the preceding claims, characterized in that the Neisserial antigen is embedded within the microparticles. 6. Composition according to any one of the preceding claims, characterized in that the Neisserial antigen is adsorbed to the microparticles. 7. A composition according to any one of the preceding claims, wherein the CpG oligonucleotides are embedded within the microparticles. 8. A composition according to any one of the preceding claims, wherein the CpG oligonucleotides are adsorbed to the microparticles. 9. The composition of claim 1, wherein the composition comprises MF59 adjuvant. 10. The composition of claim 1, comprising a silver salt adjuvant. 11. The composition of any one of the preceding claims, further comprising a pharmaceutically acceptable carrier. 12. Use of the composition of claim 1 for the manufacture of a medicament for preventing or treating Neisserial infection in mammals.