WO2009106085A1 - Compositions vaccinales comprenant des antigènes saccharidiques - Google Patents

Compositions vaccinales comprenant des antigènes saccharidiques Download PDF

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WO2009106085A1
WO2009106085A1 PCT/DK2009/050047 DK2009050047W WO2009106085A1 WO 2009106085 A1 WO2009106085 A1 WO 2009106085A1 DK 2009050047 W DK2009050047 W DK 2009050047W WO 2009106085 A1 WO2009106085 A1 WO 2009106085A1
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vaccine composition
composition according
group
carrier protein
clinical condition
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PCT/DK2009/050047
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English (en)
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Åsa SCHIÖTT
Nikolai Søren Kirkby
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Nordic Vaccine A/S
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins

Definitions

  • Vaccine compositions comprising saccharide antigens
  • Prokaryotes are surrounded by cell walls usually comprising saccharide moieties. In addition, many prokaryotes are encapsulated by polysaccharide.
  • immunological responses against a prokaryote may be specific to particular saccharide moieties displayed on the prokaryote surface, for example to the polysaccharide capsule of a given prokaryote.
  • Pathogenic bacteria commonly produce a thick mucous-like layer of polysaccharides. Examples of polysaccharide encapsulated bacteria includes Haemophilus influenza type B, Streptococcus pneumoniae, Neisseria meningitides, Group B streptococcus and Salmonella typhi.
  • Vaccines protecting against bacterial infections may in some instances comprise saccharide antigens.
  • one major drawback of the current vaccine is the low efficiency, in particular vaccines comprising free saccharide antigen in general are non efficient in young children below the age of 2.
  • vaccines have been prepared wherein saccharide antigens are covalently linked to a protein carrier. Children below the age of 2 also respond to these kinds of vaccines
  • Saccharide antigens are in general T-independent antigens and cannot be processed or presented on MHC molecules to interact with T-cells. They can however stimulate the immune system though an alternate mechanism.
  • multivalent vaccine comprising polysaccharide-protein conjugates.
  • PRP Haemophilus influenzae type b polysaccharide
  • TT tetanus toxoid
  • Examples of currently used vaccines comprising saccharide antigen includes:
  • Prevenar comprises capsule polysaccharides from 7 different pneumococcal serotypes covalently bound to CRMi 97 protein (2 to 4 ⁇ g polysaccharide from each serotype per dosage).
  • the vaccine may be given also to young children, is however, only effective against pneumococcus of the 7 serotypes comprised therein.
  • Pneumax (Sanofi Pasteur) comprises 23 different pneumococcal serotypes (25 ⁇ g polysaccahride from each serotype per dosage).
  • the vaccine is not useful for children below the age of 2.
  • the vaccine is only useful against pneumococcus of one of the specific 23 serotypes.
  • NeisVac-C (Baxter) comprises Neisseria meningitides serogroup C.
  • the vaccine may be given to infants older than 2 months, however it is only effective against serogroup C.
  • ISCOMs are structurally well defined complexes of saponin and cholesterol.
  • the major ISCOM constituents are quillaja saponins and cholesterol.
  • the procedure for preparation of ISCOM comprises solubilization of amphipathic polypeptides in preferably nonionic detergents, addition of Quillaja saponins, cholesterol, and possibly also phosphatidylcholine.
  • ISCOM particles are formed on removal of the detergent.
  • compositions for transdermal delivery may comprise an immunogen, an occlusion vehicle and a delivery system in the form of a Posintro or an ISCOM.
  • the current vaccines comprising saccharide antigen are in need of improvement, in particular in relation to efficiency.
  • said vaccines may comprise only very small amounts of saccharide antigen.
  • the present invention in one aspect provides a vaccine composition
  • a vaccine composition comprising
  • an immune stimulating complex comprising a. at least one saponin; and b. at least one sterol; and c. at least one contacting group capable of binding a nucleic acid, wherein said contacting group may be covalently attached to said at least one saponin or to said at least one sterol or to at least one lipophilic moiety, said complex being associated with at least one carrier protein (herein designated carrier protein A) and ii) at least one saccharide antigen attached to a carrier protein (herein designated carrier protein B); and iii) at least one aluminium containing adjuvant.
  • carrier protein A carrier protein
  • carrier protein B herein designated carrier protein B
  • the saccahride antigen attached to carrier protein B is adhered to said aluminium containing adjuvant.
  • the immune stimulating complex further comprises a lipophilic moiety.
  • the present invention provides a vaccine composition comprising i) an immune stimulating complex comprising a. at least one saponin; and b. at least one sterol; and c.
  • At least one contacting group capable of binding a nucleic acid, wherein said contacting group may be covalently attached to said at least one saponin or to said at least one sterol or to at least one lipophilic moiety, and said complex being associated with at least one carrier protein (herein designated carrier protein A) and ii) at least one saccharide antigen attached to a carrier protein (herein designated carrier protein B); and iii) at least one aluminium containing adjuvant, said composition being capable of inducing an immune response to said carrier protein A and said at least one saccharide antigen.
  • carrier protein A carrier protein
  • carrier protein B carrier protein
  • aluminium containing adjuvant said composition being capable of inducing an immune response to said carrier protein A and said at least one saccharide antigen.
  • the immune stimulating complex comprises a lipophilic moiety.
  • the invention furthermore relates to uses of such vaccines in treatment of infections, preferably in the prophylactic treatment of infections.
  • Figure 5 illustrates preferred sterols comprising a contacting group which may be contained in the immune stimulating complexes according to the invention.
  • the listed compounds are ChoTB and ChoSC as described by Leventis, R. et.al. (1989) Biochim Biophys Acta 1023, 124; DC-Choi and TC-Chol as described by Avanti Lipids and further characterised herein; Lipid 67 (also known as GL-67), as described by Lee, E. R. et.al.
  • BGTC is an acronym for 3-beta[N',N'- diguanidioethyl-aminoethane)carbamoyl]cholesterol
  • BGSC is an acronym for 3- beta[4N-(1 N,8N-diguanidino spermidine)-carbamoyl]cholesterol.
  • FIG. 6 Mice were immunized with HBsAg combined with different adjuvant systems. Twelve days after the second immunization cells from the draining lymph nodes were cultured in vitro with HBsAg or medium only as control.
  • mice were immunized with HBsAg combined with different adjuvant systems. Twelve days after the second immunization cells from the draining lymph nodes were cultured in vitro with HBsAg or medium only as control. After 5 days incubation in vitro the cells were harvested and analyzed on a flow cytometer. The total amount of proliferating cells were gated and counted. The HBsAg induced number of cells was obtained by subtracting the number of positive cells incubated with HBsAg with the number of positive cells incubated with medium only. HBsAg induced proliferating cell number with mean values ⁇ SD from 5-6 individual animals in each group except PBS control group constituted of 3 animals taken from two individual experiments are shown. Statistical analysis, shown in the graph, was done by comparing different groups with the group receiving HBsAg+PBS.
  • FIG. 8 HBsAg induced CD4+ and CD8+ T-cells. Mice were immunized with HBsAg combined with different adjuvant systems. Twelve days after the second immunization cells from the draining lymph nodes were cultured in vitro with HBsAg or medium only as control.
  • HBsAg induced number of either a. antigen specific CD4+ T-cells or b. antigen specific CD8+
  • T-cells was obtained by subtracting the number of positive cells incubated with HBsAg with the number of positive cells incubated with medium only.
  • HBsAg induced CD4+ or CD8+ T-cell numbers with mean values ⁇ SD from 5-6 individual animals in each group except the PBS control group constituted of 3 animals. Induced cell numbers from two individual experiments are shown. Statistical analysis, shown in the graph, was done by comparing different groups with the group receiving
  • FIG. 9 HBsAg induced cellular responses measured by DTH-assay. Mice were immunized twice and one week after the last immunization all mice were immunized s.c. in both back footpads. Before injection the footpad thickness was measured. In one footpad PBS was injected and in the other footpad HBsAg. 24 hours later the footpad thickness was measured. The mean increase in mm of the footpad ⁇ SD, from 4 individual animals in each group, was recorded for each adjuvant.
  • mice were immunized with HBsAg combined with different adjuvant systems. Twelve days after the second immunization cells from the spleen were cultured with HBsAg coated filter plates for detection of anti-HBsAg producing B-cells. HBsAg induced antibody secreting B-cells/ million spleen cells from 5-6 individual animals in each group except the PBS control group constituted of 3 animals. Data from two individual experiments are shown. Statistical analysis, shown in the graph, was done by comparing different groups with the group receiving HBsAg+PBS.
  • FIG. 1 HBsAg induced total IgG titers in guinea pigs. Guinea pigs were immunized twice with HBsAg with different adjuvant systems. 4 weeks after the last immunization serum was extracted and total IgG titer was measured. Mean values ⁇ standard deviation from 10 individual animals/group are shown. Hepatitis B specific IgG antibody titers were calculated from interpolation of the linear part of the titration curves as the dilution of serum at 50% saturation at a Iog10 scale for the dilution. All values were correlated to an internal standard. Statistical analysis, shown in the graph, was done by comparing different groups with the group receiving Twinrix.
  • mice were immunized twice with HBsAg with different adjuvant systems. 12 days after the last immunization serum was extracted and total IgG titer was measured. Mean values ⁇ SD from 6 individual animals in each group are shown. Hepatitis B specific IgG antibody titers were calculated from interpolation of the linear part of the titration curves as the dilution of serum at 50% saturation at a log 10 scale for the dilution. All values were correlated to an internal standard. Statistical analysis, shown in the graph, was done by comparing different groups with the group receiving HBsAg+PBS.
  • FIG. 13 HBsAg induced total IgGI titers in mice. Mice were immunized twice with HBsAg with different adjuvant systems. 12 days after the last immunization serum was extracted and total IgGI titer titer was measured.
  • FIG. 14 HBsAg induced total lgG2a titers in mice. Mice were immunized twice with HBsAg with different adjuvant systems. 12 days after the last immunization serum was extracted and total lgG2a titer was measured. Mean values ⁇ SD from 6 individual animals in each group are shown. Hepatitis B specific lgG2a antibody titers were calculated from interpolation of the linear part of the titration curves as the dilution of serum at 50% saturation at a Iog10 scale for the dilution. All values were correlated to an internal standard. Statistical analysis, shown in the graph, was done by comparing different groups with the group receiving HBsAg+PBS.
  • FIG. 15 Mice were immunized with HBsAg combined with different adjuvant systems. 12 days after the second immunization cells from the draining lymph nodes were cultured in vitro with HBsAg or medium only as control. After 5 days incubation supernatant was taken and analyzed on the IFN-gamma contents using the Luminex detection system. Data from 3-6 individual animals/group and two individual experiments is shown. Statistical analysis, shown in the graph, was done by comparing different groups with the group receiving HBsAg+PBS, the Posintro group gave a p- value of 0.0566.
  • FIG. 16 Mice were immunized with HBsAg combined with different adjuvant systems. 12 days after the second immunization cells from the draining lymph nodes were cultured in vitro with HBsAg or medium only as control. After 5 days incubation supernatant was taken and analyzed on the IL-3 contents using the Luminex detection system. Data from 3-6 individual animals/group and two individual experiments is shown. Statistical analysis, shown in the graph, was done by comparing different groups with the group receiving HBsAg+PBS., the Posintro group gave a p-value of 0.0566.
  • FIG. 1 Mice were immunized with HBsAg combined with different adjuvant systems. 12 days after the second immunization cells from the draining lymph nodes were cultured in vitro with HBsAg or medium only as control. After 5 days incubation supernatant was taken and analyzed on IL-5 contents using the Luminex detection system. Data from 3-6 individual animals/group and two individual experiments is shown. Statistical analysis, shown in the graph, was done by comparing different groups with the group receiving HBsAg+PBS, the Posintro group gave a p-value of 0.0566.
  • NMRI mice were immunized parenteral with Prevenar ® together with HBsAg combined with PosintroTM or the commercial Hepatitis vaccines Twinrix ® (with Al as adjuvant) or Fendrix ® (with Al and MPL as adjuvant).
  • the vaccine compositions according to the present invention comprise an immune stimulating complex.
  • the immune stimulating complex according to the invention comprises a. at least one saponin and b. at least one sterol and c. optionally a lipophilic moiety
  • said complex is an ISCOM.
  • the immune stimulating complexes according to the present invention may in one preferred embodiment adopt a micro-particle structure in the form of a cage-like matrix similar to, or preferably identical to, that known as an ISCOM.
  • An ISCOM adopts a cage-like structure with icosahedral symmetry.
  • the immune stimulating complexes of the invention have a cage-like structure with icosahedral symmetry having a diameter of 30 to 40 nanometers and are composed of 12 nanometer ring-like subunits.
  • Beside ISCOM structures, the interaction between sterols and saponins have been reported to result in a variety of different structural entities, including entities such as e.g.
  • immune stimulating complexes may adopt any of the aforementioned structures.
  • the molar ratio between saponins and sterols in the immune stimulating complexes according to the present invention is preferably from less than 1000:1 to preferably more than 1 :1000.
  • Preferred ratios are about 100:1 , for example about 80:1 , such as about 60:1 , for example about 50:1 , such as about 40:1 , for example about 30:1 , such as about 25:1 , for example about 20:1 , such as about 18:1 , for example about 16:1 , such as about 14:1 , for example about 12:1 , such as about 10:1 , for example about 9:1 , such as about 8:1 , for example about 7:1 , such as about 6:1 , for example about 5:1 , such as about 4:1 , for example about 3:1 , such as about 2:1 , for example about 1.9:1 , such as about 1 .8:1 , for example about 1 .7:1 , such as about 1.6:1 , for
  • the immune stimulating complex is a complex comprising i) at least one saponin and ii) at least one sterol and iii) at least one contacting group capable of binding a nucleic acid,
  • said contacting group may be covalently attached to said at least one saponin or to said at least one sterol or to said at least one lipophilic moiety.
  • the contacting group may be any group which is capable of contacting and binding a nucleic acid via electrostatic or hydrophobic interactions.
  • the immune stimulating complex is thus preferably capable of binding a nucleic acid.
  • the immune stimulating complex is also capable of binding other molecules or complexes.
  • the immune stimulating complex is in general capable of binding proteins, such as carrier protein A.
  • the immune stimulating complex may preferably also be capable of binding saccharides, such as mono-, oligo-, or polysaccharides.
  • said immunostimulating complex is a Posintro.
  • the terms "Posintro” and “PosintroTM” as used herein cover an immune stimulating complex comprising i) at least one saponin and ii) at least one sterol and iii) at least one cationic moiety
  • said cationic moiety may be covalently attached to said at least one saponin or to said at least one sterol or to said at least one lipophilic moiety.
  • the immune stimulating complex comprises a lipophilic moiety.
  • the Posintro of the present invention is capable of binding DNA and is in general further also capable of binding other molecules or complexes.
  • the Posintro may in particular be capable of binding proteins, such as carrier protein A.
  • the Posintro may also be capable of binding saccharides, such as mono-, oligo-, or poly-saccharides.
  • the immune stimulating complex may be any of the complexes comprising saponin and sterol described in US20031 18635, for example the immune stimulating complex may be a Posintro, which is a common name for the saponin and sterol containing complexes containing cationic moiety described in US20031 18635, and in a most preferred embodiment of the present invention, the immunostimulating complex is any complex as described and claimed in US20031 18635. US20031 18635 is incorporated herein in its entirety by reference.
  • the sterol of the complex may be any sterol capable of forming a complex with a saponin, for example the sterol may any of the sterols described in US20031 18635, thus the sterol may be a first sterol or a second sterol as described in US20031 18635.
  • the saponin may be any saponin capable of forming a complex with a sterol, for example the saponin may be any of the saponins described in US20031 18635, thus the saponin may for example be a first saponins or a second saponin as described in US20031 18635.
  • Sterols such as first and second type sterols and saponins, such as first and second type saponins according to the present invention may be as described in
  • the sterol may be any sterol comprising the characteristic skeleton structure of gonane as depicted below. Said gonane may be substituted independently at any of the carbons of the gonane skeleton with any suitable substituent (see below). All sterols of the invention comprises the characteristic molecular structure composed of 17 carbon atoms arranged in four rings conventionally denoted by the letters A, B, C, and D and bonded to hydrogen atoms and suitable substituents.
  • This skeleton structure (1 ) is named gonane when all C atoms are fully substituted with hydrogen atoms, i.e. one hydrogen atom bond to each of the carbon atoms no. 5, 8, 9, 10, 13, and 14 and two hydrogen atoms bound to each of the remaining carbon atoms no. 1 , 2, 3, 4, 6, 7, 1 1 , 12, 15, 16, and 17.
  • This structure is often referred to as the steroid nucleus.
  • Substituents or additional bonds may be introduced at any of the 17 carbon atoms in the structure (1 ), and more than one substituent or additional bond may be introduced at a single carbon atom, in particular at the carbon atoms no. 1 , 2, 3, 4, 6, 7, 1 1 , 12, 15, 16, and 17.
  • Suitable substituents may for example be hydroxyl (-OH); un-branched alkyls, such as methyl, ethyl, propyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or longer alkyls; branched alkyls; branched or un-branched acyclic alkenes; branched or un-branched partly cyclic or entirely cyclic alkenes; alkynes; or sulphur, for instance in the form of a thiol-group (-SH), for example the substituents are selected from the group consisting of (-OH), C 1- - I 0 linear or branched alkyl, sulphur, amides and any of the aforementioned substituted with an amino group, which may be primary, secondary, tertiary or quaternary, preferably quaternary.
  • carbon atom no. 3 may preferably be substituted with a thiol group (-SH), such as is the case in e.g. thiocholesterol, or more preferably carbon atom no. 3 may be substituted with a hydroxyl group (-OH), such as is the case in e.g. cholesterol and lanosterol.
  • the C-17 carbon may be substituted with an alkyl chain, such as a C 1- - I 0 linear or branched alkyl.
  • any stereocenters, such as a chiral carbon atom, of the optionally substituted structure (1 ) may be of any suitable configuration.
  • the skilled artisan will know how to isolate steroids from plants and animals, and optionally prepare derivatives by chemical and/or enzymatic treatment of natural steroids. The skilled artisan will also know how to synthesize synthetic steroids from simpler compounds, or natural precursors or parts thereof, and optionally prepare derivatives of such compounds by chemical and/or enzymatic treatment of steroids thus obtained.
  • At least one sterol of the complex is selected from the group of sterols consisting of cholesterols, lanosterols, lumisterols, stigmasterols, sitosterols, mycosterols, ergosterols, and thiocholesterols.
  • at least one sterol is cholesterol.
  • the complex comprises at least two different sterols, wherein the first sterol preferably is any of the above-mentioned sterols.
  • the second sterol preferably is covalently linked to a cationic moiety.
  • the second sterol is, comprises, or essentially consists of 3 ⁇ -[N-(Dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol) and/or N-(trimethylammonioethane)-carbamoylcholesterol (TC-cholesterol).
  • one preferred sterol according to the invention is a sterol, and any cationic derivative thereof, in particular cationic derivatives obtained by linking a cationic moiety (such as any of the cationic moieties described herein above) to e.g. an OH-group of the sterol, including an OH-group located at position 3 of the steroid skeleton, including the OH-group of cholesterol located at position 3 (C3, or 3-OH).
  • preferred sterols according to the present invention are cholesterol (CAS (Chemical Abstract) accession no. 57-88-5) and any cationic derivative thereof, in particular cationic derivatives obtained by linking a cationic moiety, to the OH-group located at position 3 of the steroid skeleton (C3, or 3-OH).
  • Saponins according to the present invention are compounds comprising a saccharide part linked by means of a glycosidic bond to one of i) a triterpene aglycone part, ii) a steroid aglycone part , and iii) a steroid alkaloid aglycone part.
  • Saponin as used herein denotes either a substantially purified saponin, or one or more saponins comprised in a crude composition or a composition obtained by predetermined purification means.
  • the saponin is a substantially purified saponin.
  • the saponins pertaining to the present invention may be naturally occurring or synthetic, or they may be made by chemical synthesis, or enzymatic synthesis involving one or more enzyme catalysed steps, either in vitro or in vivo.
  • At least one saponin is selected from the group consisting of saponins comprising a triterpene glycoside, saponins comprising a steroid glycoside, and saponins comprising a steroid alkaloid glycoside. More preferably, the saponin comprises or essentially consists of a triterpene glycosid comprising an aglycone compound and a saccharide compound. In a preferred embodiment the saponin comprises or essentially consists of a triterpene glycosid isolated from a Quillaja plant species. More preferably, the saponin is isolated from a Quillaja saponaria species.
  • the saponin is isolated from Quillaja saponaria Molina or Quillaja saponaria Officinalis.
  • the saponin is Quillajabark Araloside A (Quil A).
  • said Quil A comprises saponins such as QA-7, QA-17, QA-18 and QA-21 also denoted QS-7, QS-17, QS-18 and QS-21 , respectively.
  • the saponin comprises or even essentially consists of QS-21 . By “essentially consists of” is meant that no other saponin is detectable in the complex.
  • the immune stimulating complexes according to the invention may optionally comprise a lipophilic moiety.
  • the lipophilic moiety may be any compound with a LogP value, which is higher than 1 , preferably higher than 2, for example higher than 3.
  • Preferred lipophilic moieties are a naturally-occurring, synthetic or semi-synthetic (modified natural) compounds which is generally amphipathic. Lipids typically comprise a hydrophilic component and a hydrophobic component.
  • semi-synthetic (modified natural) denotes a natural compound that has been chemically modified in some fashion.
  • the lipophilic moieties may serve the purpose of facilitating complex formation while at the same time acting as a "docking" group for contacting groups, such as cationic moieties.
  • the lipophilic moiety may be an amphipathic compound whose molecules contain both polar and non-polar domains.
  • Surfactants are examples of amphipathic compounds. Surfactants typically possess a non-polar portion that is often an alkyl, aryl or terpene structure. In addition, a surfactant possesses a polar portion that can be anionic, cationic, amphoteric or non-ionic. Examples of anionic groups are carboxylate, phosphate, sulfonate and sulfate.
  • Non-ionic domains are amine salts and quaternary ammonium salts.
  • Amphoteric surfactants possess both an anionic and a cationic domain.
  • Non-ionic domains are typically derivatives of a fatty acid carboxy group and include saccharide and polyoxyethylene derivatives.
  • a lipophilic moiety can also comprise two or more compounds possessing non-polar domains, wherein each of the compounds has been bonded to a linking group, which, in turn, is covalently attached to a component of the immune stimulating complex, including any saponin component and/or any sterol component comprised in said complex, including any residues of said sterol component and any residues of said saponin component, including any aglycone part and/or any saccharide part.
  • phospholipids such as phosphatidylcholine, phosphatidylethanolamine, triglycerides, fatty acids, and hydrophobic amino acids residues including membrane spanning hydrophobic amino acid segments.
  • the immune stimulating complexes to be used with the present invention comprises i) a saponin derived from a Quil A fraction as described by WO 92/06710, which is incorporated herein by reference, ii) cholesterol and iii) phosphatidylcholine or phosphatidylethanolamine.
  • lipophilic moieties capable of being used in connection with the present invention are lipids other than sterols, for example fats or fat resembling substances such as e.g. triglycerides or mixed triglycerides containing fatty acids with up to 50 carbon acids such as saturated fatty acids having for example 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 and 30 carbon atoms e.g.
  • burytic acid caprole acid, caprylic acid, captic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid; unsaturated fatty acids with up to 30 carbon atoms, such as hexadecene acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid; hydroxy-fatty acids such as 9,10- dihydroxy stearic acid; unsaturated hydroxy fatty acids such as castor oil; branched fatty acids such as glycerol ethers; waxes i.e.
  • esters between higher fatty acids and monohydric alcohols phospholipides such as derivatives of glycerol phosphates such as derivatives of phosphatidic acids i.e. lecithin, cephalin, inositol phosphatides, spingosine derivatives with 14, 15, 16, 17, 18, 19 and 20 carbon atoms; glycolipids; isoprenoids; sulpholipids; and carotenoids.
  • lipophilic moieties capable of forming part of the complexes according to the present invention are lipids covalently linked to a cationic moeity.
  • examples of such lipids are phosphatidyl ethanolamine, phospatidyl choline, glycero-3- ethylphosphatidyl choline and fatty acyl esters thereof, di- and trimethyl ammonium propane, di- and tri-ethylammonium propane and fatty acyl esters thereof.
  • Suitable lipophilic moieties which may be comprised in the complexes of the invention may also be any of the lipophilic moieties described on pp. 66-74 of WO 02/080981 .
  • ISCOMS or ISCOM-like structures
  • such ISCOMS or structures may be prepared e.g. essentially as described in US20031 18635, see e.g. pages 4-6 (of the original document as filed) and example 2.
  • ISCOM-like complexes may also be prepared essentially as described in US Patent 5679354, such as in examples 1 to 5.
  • the Posintro has a zeta-potential which is not too negative. Accordingly, the Posintro preferably has a zeta-potential which is less negative or more positive than about -50 mV, such as a zeta-potential of about -45 mV, for example a zeta-potential of about -40 mV, such as a zeta-potential of about -37 mV, for example a zeta-potential of about -35 mV, such as a zeta-potential of about -32 mV, for example a zeta-potential of about -30 mVsuch as a zeta-potential of about -29 mV, for example a zeta-potential of about -28 mV, such as a zeta-potential of about -27 mV, for example a zeta-potential of about -26 mV,
  • Preferred Posintro to be used with the present invention may thus have a zeta-potential of from about -50 mV to about -40 mV, such as from about -40 mV to about -35 mV, for example of from about -35 mV to about -30 mV, such as from about -30 mV to about -25 mV, for example of from about -25 mV to about -20 mV, such as from about -20 mV to about -15 mV, for example of from about -15 m V to about -10 m V, such as from about -10 mV to about -5 mV, for example of from about -5 mV to about 0 mV, such as from about 0 mV to about 5 mV, for example of from about 5 mV to about 10 mV, such as from about 10 mV to about 15 mV, for example of from about 15 mV to about 20 mV, such as from about 20 mV to
  • Posintros to be used with the present invention comprise i) any given saponin(s) of interest, ii) cholesterol, and iii) any given second sterol of interest, preferably a cholesterol derivative as listed in Fig.
  • Posintros have a zeta-potential which is at least about 5 mV less negative or more positive than said reference complexes, such as a zeta-potential which is at least about 10 mV less negative or more positive than said reference complexes, for example a zeta-potential which is at least about 15 mV less negative or more positive than said reference complexes, such as a zeta-potential which is at least about 20 mV less negative or more positive than said reference complexes, for example a zeta-potential which is at least about 25 mV less negative or more positive than said reference complexes, such as a zeta-potential which is at least about 30 mV less negative or more positive than said reference complexes, for example a zeta-potential which is at least about 35 mV less negative or more positive than said reference complexes, such as a zeta-potential which is at least about 40 mV
  • the values of the zeta-potentials are preferably determined as described in Example 4 of WO 02/080981.
  • the aluminium adjuvant comprises at least one anionic group
  • the adjuvant is AI(OH) 3
  • the immune stimulating complex is a Posintro.
  • the Posintro may be any of the Posintros described above.
  • the vaccine compositions according to the present invention in general comprises at least two carrier proteins, herein designated carrier protein A and carrier protein B, respectively. It is also comprised within the invention that the vaccine composition may comprise more than two carrier proteins, such as more than one kind of carrier protein A and more than one kind of carrier protein B. Preferably, however, the vaccine composition comprises only one kind of carrier protein A and one kind of carrier protein B.
  • the vaccine compositions according to the present invention comprises only carrier protein A.
  • Carrier protein A is associated with the immune stimulating complex according to the invention, whereas carrier protein B is associated with at least one saccharide antigen.
  • carrier protein A is associated with the immune stimulating complex by means of hydrophobic or electrostatic interaction, a combination of these or covalently bound.
  • carrier protein A is not covalently linked to any saccharide antigen.
  • carrier protein B is associated with at least one saccharide antigen, preferably by means of a covalent bond. Frequently, carrier protein B will be covalently linked to at least one saccharide antigen via a linker. It is furthermore preferred that carrier protein B is not directly associated with the immune stimulating complex.
  • Carrier protein B can thus be linked to the saccharide antigen by means of a linker.
  • the linker may for example be a labile linker such as e.g. a linker of the type disclosed in WO 97/49425.
  • the linker may also be the type of linker disclosed in Shafer et al., 2000, Vaccine 18:1273-1281 or Lees et al., 1996, Vaccine 14(3):190-198, such as a hexanediamine or adipic dihydrazide.
  • carrier protein A and carrier protein B are similar proteins.
  • carrier protein A and carrier protein B may be derived from the same protein, they may have similar sequences or even identical sequences. It is however preferred that carrier protein A is different to carrier protein B.
  • the function of a carrier protein can for example be to increase the molecular weight of the immunogenic determinant or the antigenic determinant in order to increase the activity or immunogenicity of the determinant, to confer stability to the determinant, to increase the biological activity of the determinant, or to increase its serum half-life.
  • an immune response may be induced by carrier protein A.
  • Carrier protein A and carrier protein B may independently be any suitable carrier protein known to the skilled person.
  • carrier protein A and carrier protein B may independently be selected from the group consisting of bacterially derived proteins, virally derived proteins, lipopeptides, members of the polyhistidine triad family of proteins and fusions thereof, keyhole limpet hemocyanin, PPD (purified protein derivative of Tuberculin), pneumococcal pneumolysin (PLY), including detoxified PLY for example dPLY-GMBS or dPLY-formol, OMPC (meningococcal outer membrane protein - usually extracted from N. meningitidis serogroup), PorB (from N.
  • PD Haemophilus influenzae protein D
  • heat shock protein Plasmodium flaciparum pfg27, lactate dehydrogenase peptide, gp120 of HIV, pertussis proteins, cytokines, lymphokines, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens, such as N19 protein, pneumococcal surface protein, iron uptake proteins, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin, ovalbumin, immunoglobulins and hormones, such as insulin and immune stimulating peptides such as P2 and/ or P30 from Tetanus Toxin or the synthethic PADRE epitope (Pan DR Epitope).
  • pathogen derived antigens such as N19 protein, pneumococcal surface protein, iron uptake proteins, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin, oval
  • carrier protein A and carrier protein B may independently be selected from the carrier proteins described in WO2007/07171 1 , in particular from the carrier proteins described therein on p. 8, 1. 18 to p. 1 1 , 1. 28 or from the proteins mentioned in US patent application 2006/0051361 , sections 43-47 or in WO2005/105141 p. 5, I. 18-36.
  • Bacterially derived proteins may be any protein, which is naturally produced by a bacterium or fragments and variants thereof, wherein fragments comprises at least 10, such as at least 20, for example at least 30 consecutive amino acids of the naturally produced bacterial protein and wherein variants are proteins sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity with a naturally produced bacterial protein.
  • Bacterially derived proteins are preferably bacterial toxins or variants of bacterial toxins, for example such variants which are not or are less toxic to mammals, such as human beings. Variants of bacterial toxins, which are inactivated either by genetic mutation, chemical treatment or by conjugation are also referred to as toxoids herein.
  • Bacterial toxins may for example be tetanus toxin and/or diphtheria toxin and/or Pseudomonas aeruginosa exotoxin A or toxin A or B of C. difficile.
  • Variants of bacterial toxins useful as carrier proteins within the scope of the present invention includes bacterial toxoids, such as tetanus toxoid, fragment C of tetanus toxoid, diphtheria toxoid (such as diphtheria toxoid as described in US patent 4,496,538, for example in Example III) , pertussis toxoid, bacterial cytolysins or pneumolysin.
  • CRM197 (SEQ ID NO:2) is a form of the diphtheria toxin with little or no toxicity.
  • the CRM197 protein has the same molecular weight as the diphtheria toxin but differs from it by a single base change in the structural gene. This leads to a glycine to glutamine change of amino acid at position 52 which makes fragment A unable to bind NAD and therefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem. 46; 69-94, Rappuoli Applied and Environmental Microbiology Sept 1983 p560-564).
  • Virally derived protein may be any protein, which is naturally encoded by a viral genome or fragments and variants thereof, wherein fragments comprises at least 10, such as at least 20, for example at least 30 consecutive amino acids of the natural viral protein and wherein variants are proteins sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity with a natural viral protein.
  • Preferred virally derived proteins are proteins derived from Hepatitis type B, Polio, Morbilli paramyxovirus, Parotis, Rubella, and human papillom virus.
  • HBsAg Hepatitis type B surface antigen
  • the polyhistidine triad family of proteins comprises PhtA, PhtB, PhtD or PhtE proteins that have an amino acid sequence sharing 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with a sequence disclosed in WO 00/37105 or WO 00/39299 (e g. with ammo acid sequence 1 -838 or 21 -838 of SEQ ID NO 4 of WO 00/37105 for PhtD).
  • a histidine triad motif is the portion of polypeptide that has the sequence HxxHxH where H is histidine and x is an amino acid other than histidine.
  • carrier protein A and/or carrier protein B comprises one or more histidine triad motifs. 299).
  • carrier protein A and/or carrier protein B may be a lipopeptide, in particular carrier protein A may be a lipopeptide.
  • the lipopeptide may be any lipopeptide, such as a synthetic lipopeptide, such as any of the synthetic lipopeptides described in US patent application 2007/0160631.
  • Suitable lipopeptide preferably comprises at least one lysine moiety and a lipid moiety linked to the peptide via the N- terminal amino group or via the ⁇ -amino group of lysine.
  • Preferred synthetic lipopeptides includes lipopeptides of formula Vl of US patent application 2007/016063 p. 8-9, sections 108-1 15. More preferred synthetic lipopeptides includes the lipopeptides disclosed in figures 1 and 2 of US patent application 2007/016063.
  • the carrier protein A and/or carrier protein B and in particular carrier protein A may be HBsAg or functional equivalents thereof.
  • Functional equivalents of HBsAg are polypeptides sharing at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with HBsAg of SEQ ID NO: 1.
  • carrier protein A and carrier protein B may independently be selected from the group consisting of tetanus toxoid, diphtheria toxoid, diphtheria toxin mutant CRM197, any of the above mentioned lipopeptides and HBsAg and any of the functional equivalents of HBsAg described above.
  • carrier protein A is HBsAg, any of the functional equivalents of HBsAg described above or any of the above mentioned lipopeptides, more preferably HBsAg.
  • Carrier protein B preferably is CRM197.
  • the protein carrier For immunization of humans, the protein carrier must be a physiologically acceptable carrier acceptable to humans and safe. However, tetanus toxoid and/or diptheria toxoid are suitable carriers in one embodiment of the invention.
  • Carrier protein A and carrier protein B may be prepared by any suitable means, for example they may be purified from a natural source by conventional techniques or they may be recombinantly produced. Methods for preparation of recombinant proteins are well known to the skilled person and are for example described in Molecular Cloning: A Laboratory Manual (Third Edition) by Sambrook and Russell. Briefly, a nucleic acid is introduced into a desirable host cell, said nucleic acid comprising a first nucleic acid sequence encoding the carrier protein is operably linked to a second nucleic acid sequence directing expression of the first nucleic acid sequence in said host cell. Carrier protein expressed by the host cell may then be purified by conventional means.
  • the carrier protein such as carrier protein A is HBsAg
  • said HBsAg may be purified from plasma or more preferably recombinantlyt manufactured.
  • the carrier protein, such as carrier protein B is CRM197 then CRM197 is preferably recombinantly produced.
  • the vaccine compositions according to the invention comprise one aluminium containing adjuvant.
  • An aluminium containing adjuvant according to the present invention is any compound capable of modifying an immune response to a particular antigen, preferably augmenting an immune response to a particular antigen when co-administered with said antigen to a mammal, wherein the compound comprises aluminium.
  • Modification of the immune response means augmentation, intensification, or broadening the specificity of either or both antibody and cellular immune responses.
  • the aluminium containing adjuvant has a rigid structure, for example in the form of a grid or a crystal. More preferably, said grid structure has an overall negative charge.
  • the saccharide antigen attached to carrier protein A is capable of being adsorped onto said aluminium containing adjuvant. It is also preferred that said immune stimulating complex is capable of being adsorped onto said aluminium containing adjuvant.
  • the vaccine composition comprises an aluminium containing adjuvant to which immune stimulating complex and saccharide antigen/carrier protein B is adsorbed.
  • the aluminium containing adjuvant may comprise aluminium in any form such as aluminium in alum or aluminium in the form of any other salt.
  • the aluminium containing adjuvant comprises Al 3+ ions.
  • the corresponding anion may be any suitable anion, for example sulphate, hydroxide, halogens or phosphate.
  • the aluminium containing adjuvant may be selected from the group consisting of AIK(SO 4 ) 2 , AINa(SO 4 ) 2 , AINH 4 (SO 4 ), AI(PO 4 ) and AI(OH) 3 .
  • the aluminium containing adjuvant is aluminium phosphate or aluminium hydroxide, such as AI(PO 4 ) or AI(OH) 3 .
  • the immune stimulating complex is a Posintro, such as any of the Posintros described herein above.
  • the vaccine compositions may comprise further adjuvants.
  • adjuvant is any substance whose admixture with an administered antigen modifies the immune response. Modification of the immune response means augmentation, intensification, or broadening the specificity of either or both of antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses such as the induction of tolerance.
  • the further adjuvant could for example be of mineral, bacterial, plant, synthetic or host origin or they could be oil in water emulsions.
  • emulsion lipopolysaccharides, lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides, wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes, Titermax, Lipid A derivatives, Heat Shock Proteins, HSP derivatives, LPS derivatives, synthetic peptide matrixes, GMDP, IL-1 , IL-2, IL-5, IL-10, IL-12, IL-15 and lipopeptides.
  • FCA Freund's Complete Adjuvant
  • Merck Adjuvant 65 polynucleotides, wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes
  • the saccharide antigen according to the present invention is in general oligosaccharides comprising or preferably consisting of associated saccharide monomers.
  • oligosaccharides have a low number of repeat units (typically 5- 30 repeat units) and are typically hydrolysed polysaccharides, such as polysaccharides purified from for example a bacterial capsule, which have been hydrolysed.
  • the saccharide antigen is a polysaccharide purified from a bacterial capsule of a fragment thereof.
  • Polysaccharides from bacterial capsules may be purified according to conventional methods, for example as described in Example I of US patent 4,496,538.
  • many capsular bacterial polysaccharides may be commercially available.
  • bacterial polysaccharide may be directly used as saccharide antigens, however, preferably the bacterial polysaccharides are fragmented into shorter oligosaccharides, preferably by hydrolysis.
  • the saccharide antigen is preferably an oligosaccharide.
  • Hydrolysis may be performed by a number of methods known to the skilled person, for example by treatment with hydrogen peroxide, for example as described in international patent application WO2005/105141 , p. 5, 1. 5-17. The skilled person will be able to adapt that description of WO2005/105141 to other polysaccharides.
  • the vaccine compositions may comprise more than one different saccharide antigen, such as in the range of 2 to 200, for example in the range of 2 to 150, such as in the range of 2 to 100, for example in the range of 2 to 75, such as in the range of 2 to 50, for example in the range of 2 to 30, such as in the range of 5 to 30 for example in the range of 10 to 30, such as in the range of 15 to 30, for example at least 7, such as at least 10, for example at least 15, such as at least 20, for example at least 23, such as at least 30, for example at least 40, such as at least 50, for example in the range of 20 to 90, such as in the range of 10 to 90 different saccharide antigens.
  • saccharide antigen such as in the range of 2 to 200, for example in the range of 2 to 150, such as in the range of 2 to 100, for example in the range of 2 to 75, such as in the range of 2 to 50, for example in the range of 2 to 30, such as in the range of 5 to 30 for example in the range of 10 to
  • the different saccharide antigens may be derived from different microbial organisms, such as from different bacteria, for example from bacteria of different order, such as from bacteria of different familiy, such as from bacteria of different genus. It is however also possible that all or at least some of the different saccharide antigens are derived from bacteria of a specific species, preferably from different serotypes of bacteria of a specific species.
  • the saccharide antigen(s) to be comprised within the vaccines of the present invention are preferably derived from a microbial organism (such as a microbial organism, which is a pathogen), more preferably from a microbial organism (preferably microbial organisms, which are pathogens) selected from the group consisting of amoebae and bacteria, even more preferably from a bacteria, preferably a bacteria, which is a pathogen.
  • a microbial organism such as a microbial organism, which is a pathogen
  • a microbial organism preferably microbial organisms, which are pathogens
  • saccharide is isolated from said microbial organism or that said saccharide is identical to saccharides isolated from said microbial organism.
  • the term “derived from” is not intended to include saccharides isolated from microbial organisms, which have been subsequently been modified by for example substitution or otherwise.
  • the saccharide antigen may be covalently linked to carrier protein B.
  • saccharides derived from a given microbial organism may also be fragments of saccharides obtainable by purification from bacteria, such as encapsulated bacteria, wherein said fragment preferably is an oligosaccharide comprising at least 5, such as at least 8 sugar residues. The fragments may be obtained by fragmentation, for example by hydrolysis.
  • Capsular polysaccharides of encapsulated bacteria such as Streptococcus pneumoniae comprise repeating oligosaccharide units which may in general contain up to 8 sugar residues.
  • a saccharide antigen may be a full length polysaccharide, however in others it may be one oligosaccharide unit, or a shorter than native length saccharide chain of repeating oligosaccharide units.
  • the saccharide antigen is an oligosaccharide comprising at least one repeating unit, and thus for example if the saccharide antigen is derived from Streptococcus pneumoniae, the saccharide antigen is an oligosaccharide preferably comprising at least 8 sugar residues.
  • the size of the saccharide antigen may be any suitable size, such as an average size molecular weight; Mw of above 8OkDa, for example above 10OkDa, such as above 20OkDa, for example above 30OkDa, such as above 40OkDa, for example above 50OkDa, such as above 100OkDa, for example in the range of 200 and 2000 KDa.
  • the saccharide antigen may preferably be derived from a bacteria, which carries polysaccharides on the surface, more preferably, the saccharide antigen may be derived from an encapsulated bacteria.
  • encapsulated bacteria covers any bacterium which have an outer covering (capsule) made of polysaccharide.
  • the vaccine compositions according to the invention comprises more than one different saccharide antigen, such as in the range of 2 to 200, for example in the range og 2 to 150, such as in the range of 2 to 100, for example in the range of 2 to 75, such as in the range of 2 to 50, for example in the range of 2 to 30, such as in the range of 5 to 30 for example in the range of 10 to 30, such as in the range of 15 to 30, for example at least 7, such as at least 10, for example at least 13, such as at least 15, for example at least 20, such as at least 23, for example at least 30, such as at least 40, for example at least 50, such as in the range of 20 to 90, for example in the range of 10 to 90 different saccharide antigens, then it is preferred that at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, yet more preferably at least 90%, even more preferably at least 95%, yet more preferably essentially all, even more preferably all saccharide antigens are derived from en
  • the saccharide antigen(s) may preferably be derived from one or more selected from the group consisting of haemophilus influenza type B, streptococcus pneumoniae, neisseria meningitides, group B streptococcus and Salmonella enterica.
  • the saccharide antigen(s) may be derived from Haemophilus influenzae, which may also be referred to as Pfeiffers bacillus or Bacillus influenzae, preferably the saccharide antigen may be derived from H. influenzae selected from the group consisting of type a, b, c, d, e, and f, preferably from H. influenzae type b.
  • H. influenzae selected from the group consisting of type a, b, c, d, e, and f, preferably from H. influenzae type b.
  • One non- limiting example of a saccharide antigen from H. influenzae is the PRP polysaccharide, which is a heteropolymer of ribitol, ribose and phosphate.
  • a vaccine composition comprising one or more saccharide antigens derived from H.
  • influenzae may in particular be useful for reducing the risk of infection by H. influenzae.
  • a vaccine composition comprising one or more saccharide antigens derived from H. influenzae may in particular be useful for reducing the risk of an individual encountering one or more of bacteremia, acute bacterial meningitis, bacterial meningitis, cellulutis, osteopmyelitis, epiglottitis or joint infections, preferably bacteremia or bacterial meningitis.
  • the saccharide antigen may also be derived from Neisseria meningitides also referred to as meningococcus.
  • the saccharide antigen may be derived from meningococcus of any of the serotypes A, B, C, H, I, K, L, 29E, W135, X, Y and/or Z, preferably from serotypes A, C, W135 and/or Y.
  • a vaccine composition comprising one or more saccharide antigens derived from Neisseria meningitides may in particular be useful for reducing the risk of infection by Neisseria meningitides.
  • a vaccine composition comprising one or more saccharide antigens derived from Neisseria meningitides may in particular be useful for reducing the risk of an individual encountering one or more of bacterial meningitis and meningococcal septicaemia.
  • the saccharide antigen(s) may also be derived from Group B Streptococcus, which may also be referred to as GBS or Streptococcus agalactiae.
  • a vaccine composition comprising one or more saccharide antigens derived from Group B Streptococcus may in particular be useful for reducing the risk of infection by Group B Streptococcus.
  • a vaccine composition comprising one or more saccharide antigens derived from Group B Streptococcus may in particular be useful for reducing the risk of an individual encountering one or more of mastitis, such as bovine mastitis (if the individual is bovine) and perinatal GBS disease.
  • the saccharide antigen(s) may also be derived from Salmonella enterica, for example the saccharide antigen may be derived from Salmonella enterica of any serotype, for example selected from the saccharide antigen may be derived from the Serovar Typhi or serovar typhimurium, such as typhimorium LT2.
  • a saccharide antigen from Salmonella Typhi is the Vi polysaccharide.
  • a vaccine composition comprising one or more saccharide antigens derived from Salmonella enterica may in particular be useful for reducing the risk of infection by Salmonella enterica.
  • a vaccine composition comprising one or more saccharide antigens derived from Salmonella enterica may in particular be useful for reducing the risk of an individual encountering one or more of typhoid fever (in particular if the saccahride antigen is derived from serovar Typhi) or gastroenteritis (in particular if the saccharide antigen is derived from serovar typhimurium).
  • the vaccine compositions comprises at least one antigen derived from pneumococcus, such as at least 2, for example at least 5, such as at least 7, for example at least 13, such as in the range of 5 to 100, for example in the range of 7 to 91 , such as in the range of 10 to 91 , for example in the range of 13 to 91 , such as in the range of 20 to 91 different saccharide antigens derived from pneumococcus, preferably from different serotypes of pneumococcus.
  • at least one antigen derived from pneumococcus such as at least 2, for example at least 5, such as at least 7, for example at least 13, such as in the range of 5 to 100, for example in the range of 7 to 91 , such as in the range of 10 to 91 , for example in the range of 13 to 91 , such as in the range of 20 to 91 different saccharide antigens derived from pneumococcus, preferably from different serotypes of pneumococcus.
  • the saccharide antigen may be derived from any serotype of pneumococcus, such as from one or more selected from the group consisting of serotypes 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 1 1 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, for example one or more selected from the group consisting of serotypes 1 , 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 1 1A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, 33F, such as one or more selected fro the group consisting of serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, for example one or more selected from the group consisting of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.
  • Streptococcus pneumoniae and presently at least 91 different serotypes are known.
  • the serotypes differ in their polysaccharide capsule and thus they carry different saccharide antigens.
  • Capsular polysaccharides of Streptococcus pneumoniae comprise repeating oligosaccharide units which may in general contain up to 8 sugar residues.
  • oligosaccharide units for the key Streptococcus pneumoniae serotypes see JONES, Christopher. Vaccines based on the cell surface carbohydrates of pathogenic bacteria. An. Acad. Bras. Cienc, June 2005, vol.77, no.2, p.293-324. ISSN 0001 -3765.
  • a vaccine composition comprising one or more saccharide antigens derived from pneumococcus may in particular be useful for reducing the risk of infection by pneumococcus.
  • a vaccine composition comprising one or more saccharide antigens derived from pneumococcus may in particular be useful for reducing the risk of an individual encountering one or more of pneumonia, invasive pneumococcal disease, bacteremia, chronic obstructive pulmonary disease, acute sinusitis, otitis media, meningitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, pharyngitis sepsis and brain abscess, more preferably one or more of pneumonia, otitis media and meningitis.
  • the vaccine composition is a vaccine composition
  • a vaccine composition comprising a. an immune stimulating complex comprising i. at least one saponin, and ii. at least one sterol, and iii. at least one contacting group capable of binding a nucleic acid, wherein said contacting group may be covalently attached to said at least one saponin or to said at least one sterol or to at least one lipophilic moiety, and iv. optionally a lipophilic moiety said complex being associated with at least one carrier protein designated carrier protein A; and b. at least one saccharide antigen attached to a carrier protein designated carrier protein B; and c. at least one aluminium containing adjuvant
  • the vaccine composition is a vaccine composition
  • a vaccine composition comprising a. an immune stimulating complex comprising i. at least one saponin, and ii. at least one sterol, and iii. at least one contacting group capable of binding a nucleic acid, wherein said contacting group may be covalently attached to said at least one saponin or to said at least one sterol or to at least one lipophilic moiety, and said complex being associated with at least one carrier protein designated carrier protein A; and b. at least one saccharide antigen (which for example may be any of the saccharide antigens described herein above in this section) attached to a carrier protein designated carrier protein B; and c. at least one aluminium containing adjuvant.
  • the immune stimulating complex further comprises a lipophilic moiety.
  • the vaccine composition is a vaccine composition
  • a vaccine composition comprising a. an immune stimulating complex comprising i. at least one saponin, and ii. at least one sterol, and iii. at least one contacting group capable of binding a nucleic acid, wherein said contacting group may be covalently attached to said at least one saponin or to said at least one sterol or to at least one lipophilic moiety, and said complex being associated with at least one carrier protein designated carrier protein A; and b. one or more different Streptococcus pneumoniae saccharide antigens attached to a carrier protein designated carrier protein B; and c. at least one aluminium containing adjuvant, wherein each dosage unit of said vaccine composition comprises at the most 1 ⁇ g of each of said Streptococcus pneumoniae saccharide antigens.
  • the immune stimulating complex further comprises a lipophilic moiety.
  • the vaccine composition is a vaccine composition
  • a vaccine composition comprising a. an immune stimulating complex comprising i. at least one saponin, and ii. at least one sterol, and iii. at least one contacting group capable of binding a nucleic acid, wherein said contacting group may be covalently attached to said at least one saponin or to said at least one sterol or to at least one lipophilic moiety, and said complex being associated with at least one carrier protein designated carrier protein A; and b. one or more different Neisseria miningitidis saccharide antigens attached to a carrier protein designated carrier protein B; and c. at least one aluminium containing adjuvant.
  • the immune stimulating complex further comprises a lipophilic moiety.
  • the vaccine composition is a vaccine composition
  • a vaccine composition comprising a. an immune stimulating complex comprising i. at least one saponin, and ii. at least one sterol, and iii. at least one contacting group capable of binding a nucleic acid, wherein said contacting group may be covalently attached to said at least one saponin or to said at least one sterol or to at least one lipophilic moiety, and said complex being associated with at least one carrier protein designated carrier protein A; b. at least one saccharide antigen, and c. at least one aluminium containing adjuvant.
  • the immune stimulating complex further comprises a lipophilic moiety.
  • the at least one saccharide antigen may for example be any of the saccharide antigen described herein above in this sections, preferably one or more different Neisseria miningitidis saccharide antigens and/or one or more different Streptococcus pneumoniae saccharide antigens.
  • the vaccine compositions according to the invention in general comprises at least one saccharide antigen, which may be any of the saccharide antigens described in the section "Saccharide Antigen” herein above, attached to a carrier protein B, which may be any of the carrier proteins described herein above in the section "Carrier Protein”.
  • said saccharide antigen is covalently bound to said carrier protein B, optionally through a linker.
  • saccharide antigen covalently bound to carrier protein B is also referred to as "saccharide antigen/carrier protein conjugate”.
  • the saccharide antigen/carrier protein conjugate is adhered to an aluminium containing adjuvant.
  • adherence of the saccharide antigen/carrier protein conjugate to the aluminium containing adjuvant is by non-covalent interactions, such as electrostatic interactions.
  • the saccharide antigen/carrier protein conjugate may be prepared by any suitable method known to the skilled person.
  • some saccharide antigens comprise reactive groups, for example amine, carboxyl or aldehyde groups which may conveniently be reacted with a reactive group of the carrier protein B for form a covalent linkage.
  • carrier protein B preferably comprises one or more reactive groups, preferably selected from the group consisting of primary amine groups (for example at the N-terminus or in the side chains of the amino acids arginine or lysine), carboxyl groups (for example at the C-terminus or in the side chains of aspartate or glutamate) and amide groups with primary amines (for example in the side chains of aspargine or glutamine).
  • the saccahride antigen antigen may be coupled to the carrier protein B via a linker.
  • saccharide antigens require activation before they can be conveniently coupled to carrier protein B.
  • One suitable method for activating saccahride antigens for coupling to carrier protein B is cyanylation for example with CNBr or 1 -cyano-4- dimethylaminopyridinium tetraluoroborate (CDAP).
  • the saccharide antigen may optionally be coupled to a linker.
  • Suitable linkers for linking a saccharide antigen to carrier protein B includes for example ADH, B-propionamido, nitrophenyl-ethylamine, haloalkyl halides, glycisudic linkages, 6-amino-caproic acid, hexanediamine or adipic dihydrazide, preferably the linker may be hexane diamine or adipic dihyrazide.
  • saccharide antigen/carrier protein B conjugate examples include Shafer et al., 2000, Vaccine 18:1273-1281 or Lees et al., 1996, Vaccine 14(3):190-198.
  • Other suitable methods are described in WO2007 07171 1 and references therein, in particular the methods described on p. 14, I. 32 to p. 18, 1. 35 may be used for preparing saccahride antigen/carrier protein B conjugates according to the present invention.
  • Yet other suitable methods are described in US patent 4,496,538, in particular in Examples II, III and IV. The skilled person will be able to adapt these methods to a given saccharide antigen/carrier protein B.
  • Carrier protein B may also be linked to the saccharide antigen by means of a labile linker. Suitable methods for preparing conjugates of saccharide antigen linked to carrier protein B via a labile linker are disclosed in WO 97/49425.
  • the ratio of carrier protein B to saccharide antigen is between 1 :5 and 5:1 ; e.g. between 1 :0.5-4:1 , 1 :1 -3.5:1 , 1.2:1 -3:1 , 1.5:1 -2.5:1 ; e.g. between 1 :2 and 2.5:1 ; 1 :1 and 2:1 (w/w).
  • the majority of the conjugates, for example 6, 7, 8, 9 or more of the conjugates have a ratio of carrier protein B to saccharide antigen that is greater than 1 :1 , for example 1 .1 :1 , 1 .2:1 , 1 .3:1 , 1.4:1 , 1.5:1 or 1 .6:1.
  • the vaccine compositions according to the present invention may comprise more than one different kind of carrier protein B and more than one kind of saccharide antigen. If so, it is possible that different kinds of saccharide antigen are attached to different carrier protein B.
  • all saccharide antigens of a specific kind may be attached to only one kind of carrier protein B and each kind of carrier protein B may be attached to only this kind of saccharide antigen or each kind of carrier protein B may be attached to different kinds of saccharide antigens.
  • each kind of carrier protein B is only attached to one kind of saccharide antigen, and each kind of saccharide antigen may be attached only to this kind of carrier protein B or each kind of saccharide antigen may be attached to different kinds of carrier protein B.
  • the vaccine composition comprises only one kind of carrier protein B and more than one kind of saccharide antigen in which case all different kinds of saccharide antigen preferably are attached to, preferably covalently bound to this particular kind of carrier protein B
  • Some useful saccharide antigen optionally linked to carrier protein and optionally adhered to adjuvant are also commercially available.
  • Useful commercially available saccharide antigen compositions include for example MeningoVax A+C from Sanofi Pasteur MSD, Prevenar from Wyeth and pneumovax from Sanofi Pasteur MSD.
  • the present invention relates to vaccine compositions comprising i) an immune stimulating complex comprising at least one saponin and at least one sterol associated with at least one carrier protein A and ii) at least one saccharide antigen attached to carrier protein B adhered to an aluminium containing adjuvant.
  • these vaccine compositions are employed for generation of a protective immune response recognising the saccharide antigen comprised within the vaccine composition.
  • the vaccine compositions are in general used for prophylactic treatment of an individual before said individual actually encounters a clinical condition.
  • the vaccine composition comprises more than one saccharide antigen it is contemplated that the vaccine compositions are employed for generation of a protective immune response recognising all of the saccharide antigens comprised within the vaccine composition.
  • the vaccine compositions of the invention may be employed for generation of an immune response, preferably a protective immune response against carrier protein A or carrier protein B, preferably against carrier protein A.
  • the vaccine compositions can also be employed for generation of an immune response, preferably a protective immune response against both carrier protein A and the saccharide antigen.
  • protein A is derived from a pathogen against which an immune response is desirable.
  • protein A is preferably a bacterially derived or a virally derived protein, such as any of the bacterially derived or virally derived proteins described herein above in the section "Carrier Protein”.
  • prophylactic treatment means administration of the vaccine composition to an individual which does not yet suffer from the clinical condition against which treatment is directed and that said administration inhibits or at least reduces the risk of said individual encountering said clinical condition.
  • a vaccine composition comprising a given saccharide antigen according to the present invention when administered to an individual (preferably a human being) reduces the risk that said individual encounters a clinical condition caused by infection of a microorganism carrying said saccharide antigen on the surfaced is reduced by at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, yet more preferably at least 90%, such as at least 95%, for example at least 98%.
  • the vaccine compositions of the invention may in some instances also be used in an ameliorating or curative treatment of a clinical condition.
  • the vaccine composition is administered to an individual already suffering from said clinical condition.
  • the clinical condition to be treated, preferably prophylactically treated is preferably infection by one or more microbial organism(s) (preferably encapsulated bacteria, in particular said microbial organism(s) from which the saccharide antigens comprised in the given vaccine composition are derived.
  • microbial organism(s) preferably encapsulated bacteria, in particular said microbial organism(s) from which the saccharide antigens comprised in the given vaccine composition are derived.
  • the vaccine composition comprises one or more saccharide antigen(s) derived from Steptococcus pneumonia
  • the clinical condition to be treated is infection by Streptpcoccus pneumonia.
  • the vaccine composition comprises one or more saccharide antigen(s) derived from Steptococcus pneumonia and one or more saccharide antigens derived from Haemophilus influenzae
  • the clinical condition to be treated is infection by Streptpcoccus pneumonia and/or Haemophilus influenzae.
  • the clinical condition is preferably one or more selected from the group consisting of bacteremia, acute bacterial meningitis, bacterial meningitis, cellulutis, osteopmyelitis, epiglottitis or joint infections meningitis and meningococcal septicaemia mastitis, such as bovine mastitis (if the individual is bovine) and perinatal GBS disease, typhoid fever, gastroenteritis, pneumonia, invasive pneumococcal disease, bacteremia, chronic obstructive pulmonary disease, acute sinusitis, otitis media, meningitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, pharyngitis sepsis and brain abscess.
  • bacteremia acute bacterial meningitis, bacterial meningitis, cellulutis, osteopmyelitis, epiglottitis or joint infections meningitis and meningococcal
  • the vaccine composition can also be used to treat the combination of a viral and a bacterial infection.
  • the vaccine composition may comprise one or more saccharide antigen(s) and at least one carrier protein A which is capable of inducing an immune response.
  • Carrier Protein A is a protein derived from Hepatitis type B, such as HBsAg
  • the clinical condition is preferably hepatitis.
  • the composition in addition also comprises one or more saccharide antigens, then the vaccine composition is useful for treating (preferably prophylactically treating) hepatitis as well as one or more additional clinical conditions.
  • the clinical conditions are preferably one or more selected from the group consisting of bacteremia, acute bacterial meningitis, bacterial meningitis, cellulutis, osteopmyelitis, epiglottitis or joint infections meningitis and meningococcal septicaemia mastitis, such as bovine mastitis (if the individual is bovine) and perinatal GBS disease, typhoid fever, gastroenteritis, pneumonia, invasive pneumococcal disease, bacteremia, chronic obstructive pulmonary disease, acute sinusitis, otitis media, meningitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, pharyngitis sepsis, brain abscess, polio, tetanus, hepatitis B, and diphtheria.
  • Administration and formulation are preferably one or more selected from the group consisting of bacteremia, acute bacterial mening
  • the individual to receive the vaccine composition according to the present invention may be any individual in need thereof, preferably a mammal in need thereof, more preferably a human being in need thereof.
  • the vaccine composition comprises one or more saccharide antigens derived from Group B Streptococcus
  • the individual may preferably be a human being or the individual may be bovine.
  • one or more saccharide antigens are derived from one or more encapsulated bacteria selected from the group consisting of Neisseria meningitides, H. influenzae, Salmonella enterica, and Streptococcus pneumoniae then the individual is preferably a human being.
  • the vaccine composition is administered prophylactically (see above) and in this embodiment the individual in need thereof, may be any individual at risk of encountering the given clinical condition against which the vaccine composition is directed.
  • the individual may thus be any human being and in this embodiment the individual will frequently be a young human being, such as a child, for example a child below the age of 10, such as below the age of 5, for example below the age of 2.
  • One advantage of the vaccine compositions of the present invention is that they are capable of inducing a protective immune response even when administered to young children with an immature immune system, for example to children below the age of 2.
  • the vaccine compositions may be administered in any suitable manner.
  • the vaccine compositions may be administered enterally, parenterally, transdermal ⁇ , orally, by inhalation and/or over a mucosal membrane.
  • the vaccine compositions are administered parenterally.
  • parenteral routes of administration include injections and infusions, e.g. intravenous, intraarterial, intramuscular, intracardial, subcutaneous, intraosseous, intradermal, intraperitonal or intrethecal.
  • a preferred route of parenteral administration of the vaccine comporision is by subcutaneous injection.
  • a suitable administration form for parenteral administration is a solution or dispersion.
  • the pharmaceutical composition comprises an isotonic agent. In particular when the pharmaceutical composition is prepared for administration by injection or infusion it is often desirable that an isotonic agent is added.
  • composition may comprise at least one pharmaceutically acceptable additive which is an isotonic agent.
  • the pharmaceutical composition may be isotonic, hypotonic or hypertonic. However it is often preferred that a pharmaceutical composition for infusion or injection is essentially isotonic, when it is administrated. Hence, for storage the pharmaceutical composition may preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it may be diluted to become an isotonic solution prior to administration.
  • the isotonic agent may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate.
  • ionic isotonic agents include but are not limited to NaCI, CaCI 2 , KCI and MgCI 2
  • non-ionic isotonic agents include but are not limited to mannitol, sorbitol and glycerol.
  • At least one pharmaceutically acceptable additive is a buffer.
  • the composition comprises a buffer, which is capable of buffering a solution to a pH in the range of 4 to 10, such as 5 to 9, for example 6 to 8.
  • the pharmaceutical composition may comprise no buffer at all or only micromolar amounts of buffer.
  • the buffer may for example be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffer.
  • TRIS buffer is known under various other names for example tromethamine including tromethamine USP, THAM, Trizma, Trisamine, Tris amino and trometamol.
  • TRIS covers all the aforementioned designations.
  • the buffer may furthermore for example be selected from USP compatible buffers for parenteral use, in particular, when the pharmaceutical formulation is for parenteral use.
  • the buffer may be selected from the group consisting of monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic, dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric, polybasic acids such as citric and phosphoric and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.
  • monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic
  • dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric
  • polybasic acids such as citric and phosphoric and bases such as ammonia, diethanolamine, glycine, triethanol
  • the vaccine composition may also comprise antioxidants and/or reducing agents for example acetone sodium bisulfite, ascorbate, bisulfite sodium, butylated hydroxy anisole, butylated hydroxy toluene, cystein/cysteinate HCL, dithionite sodium, gentisic acid, gentisic acid ethanolamine, glutamate monosodium, formaldehyde sulfoxylate sodium, metabisulfite potassium, metabisulfite sodium, monothioglycerol, propyl gallate, sulfite sodium and thioglycolate sodium.
  • antioxidants and/or reducing agents for example acetone sodium bisulfite, ascorbate, bisulfite sodium, butylated hydroxy anisole, butylated hydroxy toluene, cystein/cysteinate HCL, dithionite sodium, gentisic acid, gentisic acid ethanolamine, glutamate monosodium, formaldehyde sulf
  • the vaccine composition is administered transdermally.
  • useful administration forms for transdermal administration include ointment, gel, cream, gel-like cream, paste, liquid, lotion, aerosol, spray, liniment, plaster, poultice, foam, bath admixture, a patch and a bandage.
  • the vaccine composition is to be administered transdermally, it is preferably in the form of a patch.
  • Ointments, lotions, creams and the like may be prepared as described in Remington "The science and practice of pharmacy", chapter 44 pages 845-851 , 20 th edition.
  • Patches for transdermal vaccines may be prepared by any conventional methods, for example as described in EP1384403 or WO2004/030696.
  • patches for transdermal vaccines are prepared essentially as described in Examples 4 to 13 of WO2004/03069 except that the transdermal patches should comprise the vaccine composition according to the present invention.
  • the skilled person will readily be able to make the required adaptations.
  • the vaccine composition may be administered in the form of an aerosol and/or spray dosage form which can be prepared, for example, by filling an aerosol container with above-mentioned solution for example, together with an injection agent such as liquefied petroleum gas.
  • a poultice can be prepared by adding the above mentioned vaccine composition to an ointment base formed from a partially neutralized polyacrylic acid, sodium polyacrylate, and the like.
  • the vaccine composition may be administered in the form of a tablet, capsule, drop, liquid mixture or powder. Tablet, capsules, and drops may be swallowed or chewed. Oral administration may result in uptake via the mucosa in the mouth, such as buccal or sublingual uptake, and/or in uptake via the gastro-intestinal route, such as uptake over the mucosa of the intestines.
  • pH adjuster for example, lactic acid, citric acid, phosphoric acid and the like can be mentioned for adjusting to a lower pH range, and sodium hydroxide, potassium hydroxide, sodium lactate, sodium citrate, monoethanolamine and diisopropanolamine and the like can be mentioned for adjusting to a higher pH range.
  • carboxyvinyl polymer which is a water-soluble polymer, can also achieve a lower pH.
  • Buffers such as acetate buffer, a phosphate buffer, a citrate buffer, a succinate buffer or TRIS may also be included for pH adjustment.
  • ascorbic acid for example, ascorbic acid, dibutylhydroxytoluene, sodium thiosulfate, sodium thioglycolate, sodium thiomalate, erythorbic acid, sodium erythorbate, sodium pyrosulfite, benzoic acid, sodium benzoate, sodium alginate, sodium caprylate, L- arginine, L-cysteine, dl-. alpha. -tocopherol, tocopherol acetate, propyl gallate, disodium edetate and the like can be mentioned.
  • benzethonium chloride for example, benzalkonium chloride, methylparaben, ethylparaben, propylparaben, chlorobutanol, benzyl alcohol, thimerosal and the like can be mentioned.
  • the vaccine compositions of the present invention may also be administered over a mucosal membrane.
  • the vaccine composition may be applied to any mucous membrane including the conjunctiva, nasopharynx, orthopharnyx, vagina, colon, urethra, urinary bladder, lung, large (rectal) and small (enteral) intestine.
  • compositions of the present invention can be formulated in an aqueous solution buffered to a pH of between 3.0 and 8.0, most preferably pH 5.0-5.4, by means of a pharmaceutically acceptible buffer system.
  • a pharmaceutically acceptable buffering system capable of maintaining the pH in the preferred ranges can be used in the practice of this invention.
  • a typical buffer will be, for example, an acetate buffer, a phosphate buffer, a citrate buffer, a succinate buffer, or the like.
  • concentration of buffer typically range from between 0.005 and 0.1 molar, most preferably about 0.02 molar.
  • the container e.g. nasal applicator or eye drop bottle, may contain sufficient composition for a single nasal or ocular dosing or for several sequential dosages.
  • the vaccine compositions of the present invention may be formulated for sustained release.
  • one or more of the immune stimulating complex, carrier protein, saccharide antigen and/or aluminium containing adjuvant may be combined with a silicone elastomer that releases the saccharide antigen over a long period of time.
  • the silicone elastomer can also comprise albumin. See U.S. Pat. No. 4,985,253, the contents of which are fully incorporated by reference herein.
  • the release rate of the antigen from the silicone elastomer can be controlled by incorporation of a water soluble or fat soluble mixing agent or cosolvent (e.g., polyethylene glycol 400, polysorbate 80, sodium alginate, L-alanine, sodium chloride, polydimethylsiloxane) into the silicone elastomer. Any other additive can also be incorporated into the silicone elastomer for the purpose of accelerating the release rate.
  • a water soluble or fat soluble mixing agent or cosolvent e.g., polyethylene glycol 400, polysorbate 80, sodium alginate, L-alanine, sodium chloride, polydimethylsiloxane
  • the vaccine composition according to the invention may be formulated into unit dosage forms, wherein each unit, for example a sealed container, comprises one dosage of the vaccine composition.
  • the vaccine compositions may be administered once; however, more often the vaccine compositions are administered more than once, such as more than 2 times, for example more than 5 times, such as more than 10, for example more than 15, such as more than 20, for example more than 30 times, such as more than 50 times, for example more than 100 times.
  • the vaccine compositions are administered in the range of 2 to 10 times, more preferably in the range of 2 to 5 times, such as twice.
  • the time period between two individual administrations is typically in the range of 1 week to 5 years, such as in the range of 1 month to 2 year, for example in the range of 1 month to 1 year, such as in the range of 2 months to 7 months. If the vaccine composition is administered more than twice, then the time period between the individual administrations may each individually be any of the aforementioned. Thus by way of example the interval between the first and second administration may be around 2 months and the interval between the second and the third administration may be around 7 months.
  • the immune stimulating complex is of the ISCOM type.
  • the immune stimulating complex is an ISCOM.
  • the immune stimulating complex is a Posintro.
  • the immune stimulating complex is in general associated with carrier protein A.
  • the immune stimulating complex associated with carrier protein A is administered in a vaccine composition that comprises a pharmaceutically effective concentration of said complex.
  • the vaccine compositions comprise a concentration from 0.01% to 10%, such as from 0.05% to 8%, for example from 0.1% to 7%, such as from 0.25% to 6%, for example from 0.5% to 5%, such as from 0.6% to 4%, for example from 0.7% to 2%, such as from 0.75% to
  • the immune stimulating complex may be administered in a vaccine composition that comprises from 0.01 % to 0.05%, such as from 0.05% to 0.1 %, for example from 0.1 % to 0.25%, such as from 0.25% to 0.5%, for example from 0.5% to 0.75%, such as from 0.75% to 1.0%, for example from 1 ,0% to 1.25%, such as from 1.25% to 1.5%, for example from 1.5% to 2.0% w/w of said complex.
  • the amount of saccharide antigen administered per dosage is dependent on the individual to whom the vaccine composition is administered. In general in the range of 0.01 to 100 ⁇ g, preferably in the range of 0.01 to 50 ⁇ g, more preferably in the range of 0.1 to 10 ⁇ g, for example in the range of 0.1 to 1 ⁇ g of each saccharide antigen comprised within a vaccine composition is administered per dosage.
  • the vaccine composition comprises more than one saccharide antigen
  • the composition may comprise the same or similar amount of each saccharide antigen preferably as determined by weight, but it is also comprised within the invention that the vaccine composition comprises different amounts of the different saccharide antigens.
  • each dosage unit of the vaccine composition preferably comprises at the most 1 ⁇ g, preferably at the most 0.5 ⁇ g, more preferably at the most 0.4 ⁇ g, such as at the most 0.3 ⁇ g, for example at the most 0.2 ⁇ g, such as at the most 0.1 ⁇ g of each of said Streptococcus pneumoniae saccharide antigens.
  • each dosage unit of said vaccine composition preferably comprises in the range of 0.01 to 1 ⁇ g, such as in the range of 0.05 to 1 ⁇ g, for example in the range of 0.1 to 1 ⁇ g, such as in the range of 0.05 to 1 ⁇ g, for example in the range of 0.1 to 1 ⁇ g, such as in the range of 0.05 to 0.8 ⁇ g, for example in the range of 0.05 to 0.6 ⁇ g, such as in the range of 0.05 to 0.4 ⁇ g, for example in the range of 0.05 to 0.2 ⁇ g, such as in the range of 0.1 to 0.8 ⁇ g, for example in the range of 0.1 to 0.6 ⁇ g, such as in the range of 0.1 to 0.5 ⁇ g, for example in the range of 0.1 to 0.4 ⁇ g, such as in the range of 0.1 to 0.3 ⁇ g, for example in the range of 0.1 to 0.2 ⁇ g of each of said Streptococcus pneumoniae saccharide antigens.
  • the composition may comprise the same or similar amount of each saccharide antigen preferably as determined by weight, but it is also comprised within the invention that the vaccine composition comprises different amounts of the different saccharide antigens.
  • Each dosage unit of the vaccine composition preferably comprises in the range of a total of 0.1 to 20 ⁇ g, such as in the range of 0.1 to 16 ⁇ g, for example in the range of 1 to 10 ⁇ g, such as in the range of 1 to 16 ⁇ g Streptococcus pneumoniae saccharide antigens. Examples
  • a stock solution consisting of 1 ,17 mq/ml Hepatitis B surface Antigen, 200 mq/ml Mega-10, 200 mg/ml SDS, and 400 ⁇ M DTT is mixed with 200 mg/ml Mega -10, 20 mg/ml DC-CH in 200 mg/ml Mega -10, 20 mg/ml CH in 200 mg/ml Mega -10, 20 mg/ml POPC in 200 mg/ml Mega -10, 17,5 mg/ml Quil A, and PBS. Everything except Quil A and PBS is mixed and stirred at 37 ° C. After 30 minutes Quil A is added and the formulation is injected into a Slide-a-Lyzer and dialysed according to the following scheme:
  • the formulation is withdrawn from the Slide-a-Lyzer, and the volume is estimated by weight of the formulation. There is an expected loss of 25% of both saponin and protein.
  • the PosintroTM is an ISCOM (immune stimulatory complex) with the unique ability to incorporate the antigen of interest.
  • the surface Hepatitis B antigen is incorporated into the PosintroTM, referred to as PosintroTM-HBsAg here after.
  • the pneumococcal vaccine Prevenar (which includes 7 streptococcus pneumoniae types conjugated to a carrier, CRM197 carrier protein mutated difteri toxin and absorbed to Alum hydroxide) is used in high and low dose and the low dose is combined with the PosintroTM-HBsAg.
  • mice NMRI strain
  • the mice are immunized twice, with three weeks interval, parenteral (subcutaneous) in the neck with only Prevenar or a mixture of Prevenar and PosintroTM-HBsAg according to below table.
  • Blood were taken before priming, just before boosting and 3 weeks after boosting. Serum is extracted from the blood.
  • Results Figure 1 shows that the anti-19F IgG titer is significantly higher in mice immunised with Prevenar+PosintroTM-HBsAG, than in mice immunised with Prevenar only., even when a high dosis of Prevenar was administrered.
  • the MAX-OD value of anti-19F is also significantly higher (see figure 3).
  • figure 2 shows that the anti-23F IgG titer is significantly higher in mice immunised with Prevenar+PosintroTM-HBsAG, than in mice immunised with prevanar only. In fact, anti-23F IgG was not detectable in mice immunised with Prevenar only. The MAX-OD value of anti-23F is also significantly higher (see figure 4).
  • the Prevenar is a commercial pneumococcal vaccine including 7 streptococcus pneumoniae types, 19F and 23F being two of them.
  • the addition of PosintroTM-HBsAg to Prevenar increases both the anti-19F and anti-23F IgG titer and the Max-OD values to anti-19F and anti-23F.
  • NeisVac-C ® is a commercial meningococcal vaccine including serotype C.
  • NeisVac-C ® includes tetanus toxoid as carrierprotein, absorbed to alumininum hydroxide.
  • NeisVac-C ® is combined, at different doses, with the experimental adjuvant system PosintroTM-HBsAg.
  • NMRI mice were immunized subcutaneouslv with NeisVac-C ® together with HBsAg incorporated in PosintroTM.
  • the NeisVac-C ® were administrated at two different dose levels; 40% and 4% NeisVac-C, corresponding to 40% and 4% respectively of normal human dose.
  • Total IgG, IgGI , and lgG2 titers were measured after 2 and 3 immunizations.
  • the combined vaccine Posintro-HBsAg and NeisVac-C ® induced a strong enhanced effect of the immune response against polysaccharides serotype C in NeisVac-C ® after 2 immunizations and preferably the isotype lgG2a.
  • lgG2a is known as the primary isotype mediating optimal complement fixation and opsonisation leading to bacterial clearance.
  • mice were immunized with HBsAg combined with different adjuvant systems. Twelve days after the second immunization cells from the draining lymph nodes were cultured in vitro with HBsAg or medium only as control.
  • Lymph node cells were washed twice in PBS and incubated together with 0.5 ⁇ M CFSE (Invitrogen), diluted in PBS, for 5 minutes at RT. The reaction was stopped with 0.5 % BSA (Sigma-Aldrich) in PBS. The cells were diluted in R10 culture medium. Lymph node cells (10 5 cells/well) were plated in flat bottom 96-well plates and cultured for 5 days with 10 ⁇ g/ml HBsAg or only R5 medium. Five replicate cultures were performed for each assay. After the incubation time, the five replicate wells were pooled and the cell number was counted using the Trucount tubes (Becton Dickinson).
  • the percentage blasted cells were multiplied with the total cell number both for cells incubated with HBsAg and cells incubated with medium only.
  • the HBsAg induced number of proliferating cells was obtained by subtracting the number of positive cells incubated with medium only from the number of positive cells incubated with HBsAg.
  • a CFSE labelling assay was performed. Mice were immunized intradermal ⁇ and cells from draining lymph nodes were stained with CFSE and incubated in vitro together with HBsAg to detect proliferating cells. After 5 days incubation the proliferating CD4 + and CD8 + T-cells were separated by surface antigen staining and analyzed on a flow cytometer (Fig. 8a and b). Approximately 5.000 CD4 + or CD8 + cells were analyzed in each sample.
  • Percentage CFSE low CD4 h ⁇ gh or CD8 h ⁇ gh positive cells were multiplied with the total cell number, both for cells incubated with HBsAg and cells incubated with medium only.
  • the HBsAg induced number of CD4 + and CD8 + cells was obtained by subtracting the number of positive cells incubated with medium only from the number of positive cells incubated with HBsAg.
  • mice were immunized intradermal ⁇ in the base of the tail with 4 ⁇ g HBsAg (the commercial vaccines antigen dose was also set to 4 ⁇ g) in 100 ⁇ l saline (the same dilution was performed as for immunizations) followed by a boost dose with the same amount 3 weeks later.
  • HBsAg the commercial vaccines antigen dose was also set to 4 ⁇ g
  • a boost dose with the same amount 3 weeks later.
  • the Posintro TM adjuvant system induced activated CD4 + antigen specific T-cells to a significantly higher degree than the commercial Aluminium oxide hydrate and aluminium phosphate adjuvant system (Twinrix ® ) (with a p-value of 0.01 17) and higher, but not significant, induction of CD8+ antigen specific T-cells compare to Twinrix ® .
  • the other commercial vaccine Fendrix ® induced almost the same level of both CD4 and CD8 positive T-cells as Posintro TM and with significantly higher CD4 + antigen specific T- cells than the PBS control group. Results from the delayed type hypersensitivity test are shown in figure 9. The thickness of the footpad was estimated for each adjuvant.
  • the Posintro TM adjuvant system induced stronger DTH than the commercial vaccine Fendrix ® and as strong DTH as the commercial vaccine Twinrix ® .
  • the induction of antigen specific B-cells was detected using ELISPOT. Mice were immunized intradermal ⁇ and the spleen cells were incubated on HBsAg coated membrane plate. The spots detected were antigen specific B-cells producing antibodies binding to the HBsAg coated membrane.
  • the ELISPOT technique was used to measure the number of Hepatitis B specific antibody secreting B-cells. Briefly a PVDF membrane micro 96-well-plate (Pierce, Rockford, IL) was coated with 1 ⁇ g/well of HBsAg diluted in a carbonate coating buffer (0.1 M Na 2 CO 3 and 0.1 M NaHCO 3 , pH 9.6) over night at 4 Q C. Tetanus Toxoid, 1 ⁇ g/well (SSI, Copenhagen, Denmark), was used as a negative control and medium only was used as background control. The wells were washed in washing buffer (Pierce) and a blocking buffer (Pierce) was added for 1 h at 37 Q C.
  • a carbonate coating buffer 0.1 M Na 2 CO 3 and 0.1 M NaHCO 3 , pH 9.6
  • Tetanus Toxoid 1 ⁇ g/well (SSI, Copenhagen, Denmark) was used as a negative control and medium only was used as background control.
  • the wells were
  • the blocking buffer was washed off and a single cell suspension from the spleen from each individual animal was added in an amount of 10 6 cells/well diluted in R10 culture medium, with duplicates for each animal.
  • the plate was wrapped in aluminium foil and incubated in an incubator at 37 Q C for 3 hours.
  • the plate was washed 6 times in washing buffer and was shaken between each washing step.
  • Biotinylated anti-mouse IgG (DakoCytomation, Glostrup, Denmark) was added and incubated for 1 hour at 37 Q C.
  • the wells were washed and Streptavidine-alkaline phosphate (Pierce) was added for 30 minutes at room temperature.
  • the numbers of positive spots were subtracted by the spots induced by an irrelevant antigen, tetanus toxoid (Fig. 4).
  • the Posintro TM adjuvant system induced a higher number of positive cells with less variation between the animals, i.e. a more robust response of antigen specific B-cells, than both the commercial Hepatitis B vaccines Twinrix ® and Fendrix ® .
  • the adjuvant system tested were FIA, Twinrix ® (aluminium oxide hydrate and aluminium phosphate), Fendrix ® (aluminium phosphate +MPL), QS21 (purified saponin fraction), the MF59 (oil-in-water emulsion) and PosintroTM.
  • Antibody levels both total IgG titer and the isotypes IgGI and lgG2a antibody titer, were assessed in serum and measured by ELISA. Serum was obtained from the blood by centrifugation at 2160 g for 10 minutes. Total anti-HBsAg mouse IgG antibody levels and total anti-HBsAg Guinea Pig IgG antibody levels and mouse isotype IgGI and lgG2a antibody level were measured by coating microtiter plates (96 well, MaxiSorb, Nunc, Roskilde, DK) with 50 ⁇ l of 0.4 ⁇ g/ml HBsAg (serum Institute of India) in carbonate buffer (5OmM, pH 9.6).
  • the plates Prior to use, the plates were washed three times with PBS containing 1% Triton X-100 and blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS. Serum was added in a series of 5 fold dilutions ranging from 5 fold dilutions to 390625 fold dilutions and incubated for 1 hour at 37 Q C.
  • BSA bovine serum albumin
  • the plate was washed and alkaline phosphatase labelled Goat-anti-mouse IgG, IgGI or lgG2a (Southern biotech, Birmingham, AL) or alkaline phosphates labelled Goat-anti-Guinea Pig IgG (Southern biotech) diluted 1 :4000 in PBS containing 1 % (w/v) BSA was added and incubated for 1 hour at 37 Q C.
  • the plate was washed and p-NPP (Kem-En-Tec Diagnostics, Copenhagen, Denmark) was added as substrate for 30 minutes at RT and the reaction was stopped by 1 M NaOH.
  • the absorbance was read at 405 nm using a Vmax kinetic Microplate Reader (Molecular Devices Corporation, CA).
  • Hepatitis B specific antibody titers were calculated from interpolation of the linear part of the titration curves as the dilution of serum at 50% saturation at a Iog10 scale for the dilution. All values were correlated to an internal standard.
  • the Posintro TM adjuvant system induced the highest average IgG antibody titer compared to the other adjuvant systems, but only significantly (p ⁇ 0.05) higher than Twinrix ® , Fendrix ® and QS21 (Fig 1 1 ).
  • the Posintro TM adjuvant system induced the highest total IgG antibody titer (Fig. 12).
  • IgGI a Th2 induced isotype
  • lgG2a a Th1 induced isotype
  • the Posintro TM was the only adjuvant system that induced a strong Th1 antibody response with high lgG2a titers (Fig. 14).
  • the other adjuvant systems only induced a Th2 antibody response with high IgGI titer (Fig. 13).
  • Posintro TM also induced a strong Th2 response.
  • the Posintro TM induces both IFN-gamma (a Th1 induced cytokine) (Fig. 15) and IL-5 and IL-13 (both Th2 induced cytokines) (Figs. 16 and 17, respectively).
  • Posintro TM induces higher titer of IFN-gamma and significantly higher titers of IL-5 and IL-13 than the commercial vaccine Twinrix ® .
  • Posintro TM and Fendrix ® induce the same amount of IFN-gamma but Posintro TM induced higher titers of both IL-5 and IL-13.
  • NMRI mice were immunized parenteral with Prevenar ® together with HBsAg combined with PosintroTM or the commercial Hepatitis vaccines Twinrix ® (with Al as adjuvant) or Fendrix ® (with Al and MPL as adjuvant).
  • Anti-Hepatitis B IgG titer was measured three weeks after second immunization in serum (Fig 18).

Abstract

La présente invention concerne des compositions vaccinales comprenant un complexe immunostimulant qui est associé à une protéine A support, des antigènes saccharidiques attachés à une protéine B support, et un adjuvant contenant de l'aluminium. Le complexe immunostimulant comprend des saponines et des stérols et est capable de lier un acide nucléique. Le complexe immunostimulant peut comprendre en outre un résidu lipophile. Les compositions vaccinales sont capables d'induire une réponse immunitaire à l'antigène saccharidique et/ou à la protéine support A. La présente invention concerne aussi des utilisations de ces vaccins dans le traitement d'infections, de préférence dans le traitement prophylactique d'infections.
PCT/DK2009/050047 2008-02-28 2009-02-27 Compositions vaccinales comprenant des antigènes saccharidiques WO2009106085A1 (fr)

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WO2011110241A1 (fr) * 2010-03-09 2011-09-15 Glaxosmithkline Biologicals S.A. Composition immunogène comprenant des polysaccharides de s. pneumoniae conjugués à des protéines porteuses
US9107906B1 (en) 2014-10-28 2015-08-18 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
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Cited By (12)

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WO2011110241A1 (fr) * 2010-03-09 2011-09-15 Glaxosmithkline Biologicals S.A. Composition immunogène comprenant des polysaccharides de s. pneumoniae conjugués à des protéines porteuses
US9107906B1 (en) 2014-10-28 2015-08-18 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US9714283B2 (en) 2014-10-28 2017-07-25 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US9815886B2 (en) 2014-10-28 2017-11-14 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US9969793B2 (en) 2014-10-28 2018-05-15 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US10683343B2 (en) 2014-10-28 2020-06-16 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US11339206B2 (en) 2014-10-28 2022-05-24 Adma Biomanufacturing, Llc Compositions and methods for the treatment of immunodeficiency
US11780906B2 (en) 2014-10-28 2023-10-10 Adma Biomanufacturing, Llc Compositions and methods for the treatment of immunodeficiency
US11951165B2 (en) 2016-12-30 2024-04-09 Vaxcyte, Inc. Conjugated vaccine carrier proteins
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
US11084870B2 (en) 2017-03-15 2021-08-10 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
US11897943B2 (en) 2017-03-15 2024-02-13 Adma Biomanufacturing, Llc Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection

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