CN101267835A - Reducing interference between oil-containing adjuvants and surfactant-containing antigens - Google Patents

Abstract

Inclusion of fatty adjuvants in vaccine compositions can cause difficulties with certain antigenic components, particularly with antigens that include a surfactant component. A method for preparing an immunogenic composition comprising an antigen and a fatty adjuvant involves purification of the antigen substantially in the absence of surfactant. Where surfactants cannot be avoided, the following are combined: (i) an antigen component that includes a surfactant and (ii) a fatty adjuvant component, to give a composition in which the weight ratio of said fatty adjuvant to said surfactant is less than 1000:1.

Description

Reduce the oil-containing adjuvant and contain interference between the surfactant antigen

Include in all reference as cite herein that this paper quotes for referencial use.

Technical field

The present invention relates to produce the field that contains Adjuvanted vaccines.Specifically, the present invention relates in producing, use the aliphatic adjuvant based on the process that contains the antigenic vaccine of surfactant.

Background technology

The immunne response that adopts adjuvant can improve vaccine antigen is well-known (referring to for example list of references 1 and 2).For many years, be aluminum salt Approved by unique adjuvant of usefulness, but many countries have ratified to contain the vaccine of MF59 adjuvant and RC-529 adjuvant, comprise that Italy is (at the FLUAD of Chiron Corp (Chiron) ^TMIn the product) and Argentina (receive the SUPERVAX of biotechnology company (Berna Biotech) uncle ^TMIn the product).Other adjuvant that is in the human experimental stage in later stage comprises cholesterol and saponin, ISCOM, MPL ^TM, AS04, virion, SBA4 etc. mixture.

The common characteristic of some aluminum salt substitutes is to have aliphatic component.For example, the MF59 adjuvant comprises zamene (oil), MPL ^TMComprise and have the monophosphoryl lipid A that a plurality of fatty acid chains are connected in the deacylation form of didextrose amine main chain.

Contingent interference between the antigen that the present invention relates to avoid these aliphatic adjuvants and contain surface active agent composition.

Summary of the invention

The present inventor finds that the aliphatic adjuvant in the vaccine combination may be incompatible with the antigen that contains surface active agent composition.Yet, in many cases, still needing to merge aliphatic adjuvant and antigen/surfactant mixture, one of purpose of the present invention provides the mode of issuable difficulty in the process of avoiding doing like this.Another purpose provides and merges adjuvant and antigenic method, and it is incompatible that this method can be avoided.

These inconsistent problems when using the general antigen that adopts the surfactant purification, first aspect of the present invention provides a kind of method for preparing immunogenic composition, and wherein: (a) said composition contains antigen and aliphatic adjuvant; (b) basic this antigen of purification under the situation that does not have surfactant.Employing avoids using the alternative method of surfactant to come purifying antigen, can solve any incompatibility problem to the aliphatic adjuvant.

Yet, for clinical, history or rules reason, may be under the situation that does not adopt surfactant purifying antigen, perhaps remove the surfactant in the vaccine fully.In this case, the present invention has avoided interference by the ratio that reduces compositions medium oil and surfactant.The contained oil mass of typical case's MF59/HBsAg vaccine is far above surfactant, and oil surpasses 4000: 1 (weight ratio) with the ratio of surfactant.Target of the present invention is at utmost to reduce the content of aliphatic adjuvant in the compositions, and in second aspect, the oil mass that the present invention adopts is excessive 1000 times of weight at the most.Therefore, the invention provides a kind of method for preparing immunogenic composition, described method comprises that merging (i) contains the antigen component of surfactant and the (ii) step of aliphatic adjuvant component, with the weight ratio that obtains described aliphatic adjuvant and the described surfactant compositions less than 1000: 1.Similarly, the invention provides immunogenic composition, wherein: (a) described compositions contains antigen component and aliphatic adjuvant component; (b) described antigen component contains surfactant; (c) weight ratio of described aliphatic adjuvant and described surfactant was less than 1000: 1.

The aliphatic adjuvant

The aliphatic adjuvant has all been used in first and second aspects of the present invention, promptly contains the adjuvant component of aliphatic molecules.But typical aliphatic adjuvant contains metabolism oil, fatty acid and/or contains the molecule of fatty acid part.

Two kinds of preferred aliphat adjuvants be called ' MF59 ' and ' adjuvant of MPL '.' MF59 ' is the aliphatic adjuvant, but because it contains the zamene as metabolism oil.' MPL ' is the aliphatic adjuvant, because it contains the disaccharide that useful a plurality of fatty acid chain replaces.Except that MF59, also can adopt other oil in water emulsion adjuvant.

Therefore, preferred composition of the present invention comprises: (a) oil in water emulsion, as contain the submicron oil in water emulsion of zamene and polyoxyethylene sorbitan monoleate (optional be sorbitan trioleate); And/or (b) monophosphoryl lipid A of 3-O-deacylation.Polyoxyethylene sorbitan monoleate is also referred to as polyoxyethylene sorbitan monoleate.

Except that ' MF59 ' and ' MPL ', can be used for other aliphatic adjuvant of the present invention and include but not limited to: the glucosaminide phosphate derivative; N-acyl group-false dipeptides; Derive from the solubility adjuvant ' OM-174 ' of escherichia coli (Escherichia coli) lipid A; The chemical compound that contains the lipid that is connected in the acyclic main chain of phosphoric acid; With acyclic synthetic lipid A analogue.

When adopting, generally add isopyknic aqueous antigen composition, so that this Emulsion accounts for 50% of compositions cumulative volume as oil in water emulsion adjuvants such as MF59.When adopting the 3D-MPL adjuvant, dosage is generally 25 μ g/ml-200 μ g/ml, as 50-150 μ g/ml, 75-125 μ g/ml, 90-110 μ g/ml or about 100 μ g/ml.Usually each dosage is used the 3D-MPL of 25-75 μ g, as 45-55 μ g, or the about 50 μ g 3D-MPL of every dosage.As Emulsion adjuvants such as MF59, separate, independently providing in the container, mix in use temporarily with antigen; Perhaps can provide the Emulsion adjuvant with the blended state of antigen.The 3D-MPL adjuvant generally just mixed with antigen before being sold to the end user.

MF59

' Mp59 ' adjuvant is the oil in water emulsion that is formed by zamene, Tween 80 (polyoxyethylene sorbitan monoleate) and Span 85 (sorbitan trioleate).Make this Emulsion Micro Fluid, to obtain having the Emulsion of sub-micron droplet size.The preparation of MF59 is reported in list of references 3 at first, and Chiron Corp produces and sell this product.Other details can be referring to the 10th chapter of list of references 2, the 12nd Zhanghe list of references 4-6 of list of references 1.The volume of MF59 consists of: 5% zamene, 0.5% polyoxyethylene sorbitan monoleate and 0.5%Span 85.By weight, these ratios become 4.3% zamene, 0.5% polyoxyethylene sorbitan monoleate and 0.48%Span 85.Aspect second of the present invention, calculate oil: be not counted in the surface-active contents of MF59 self during the ratio of surfactant, because this ratio is based on antigenic surface-active contents.

MP59 Emulsion should contain citrate ion, as the 10mM sodium citrate buffer solution.

Other oil in water emulsion

As the substitute of MF59 adjuvant, can adopt other oil in water emulsion.Adjuvant Emulsion generally contains at least a oil and at least a surfactant, and described oil and surfactant are biodegradable (but metabolism) and biocompatible.The diameter of oil droplet less than 5 μ m, is generally sub-micron diameter usually in the Emulsion.Can adopt the Micro Fluid bed to realize small drop sizes, so that stable Emulsion to be provided.Preferably less than the drop of 220nm, because can carry out filtration sterilization to them.

Emulsion can comprise oil, and Tathagata is from the oil of animal (as fish) or plant, and non-mineral oil.The source of vegetable oil comprises nut, seed and grain.In the macadamia nut oil, modal example is Oleum Arachidis hypogaeae semen, soybean oil, cocos nucifera oil and olive oil.For example, also can adopt Jojoba oil available from flash Fructus Crotonis.Seed oil comprises safflower oil, cotton seed oil, Oleum Helianthi, Semen Sesami wet goods.In corn oil, Semen Maydis oil is the most common, but also can adopt other grain such as Semen Tritici aestivi, the oil of Herba bromi japonici, Fructus Hordei Vulgaris, Oryza sativa L., Herba Eragrostidis pilosae, black Semen Tritici aestivi etc.Though do not have glycerol and 1 under the natural situation in the seed oil, the 6-10 carbocyclic aliphatic acid esters of 2-propylene glycol can be a raw material with macadamia nut oil and seed oil, by wherein suitable material is hydrolyzed, separation and esterification prepare.Fat and oil from mammal milk are metabolizable, therefore can be used for implementing the present invention.It is well-known in the art obtaining the necessary separation of purified oil, purification, saponification and other step by animal origin.But most of fishes contain the metabolism oil that is easy to reclaim.For example, cod liver oil, shark liver oil and be to can be used for several fish oil of the present invention as whale oil such as spermaceti.With the biochemical synthetic many side chain oil of 5-carbon isoprene unit, they are generically and collectively referred to as terpenoid.Shark liver oil contains the unsaturated terpenoid of side chain, is called zamene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18, and 22-24 hexenes, it is the especially preferred terpenoid of this paper.The saturated analogues shark alkane of zamene also is preferred oil.Fish oil comprises zamene and shark alkane, can perhaps obtain by means known in the art available from commercial source.Other preferred oil is tocopherol (as follows).Can adopt the mixture of oil.

Can classify to surfactant by ' HLB ' (hydrophile/lipophile balance).The HLB of preferred surfactant of the present invention is at least 10, preferably at least 15, more preferably at least 16.Can be used for surfactant of the present invention comprises but is not limited to: polyoxyethylene sorbitan esters surfactant (so-called tween), particularly polysorbate 20 and polyoxyethylene sorbitan monoleate; The copolymer of oxirane (EO), expoxy propane (PO) and/or epoxy butane (BO), commodity are called DOWFAX ^TM, as straight chain EO/PO block copolymer; Hot benzene polysaccharide, wherein multiple ethyoxyl (oxygen-1,2-ethane two bases) quantity can change, and interested especially is hot benzene polysaccharide-9 (Triton X-100, or uncle's octylphenoxy polyethoxy ethanol); (Octylphenoxy) polyethoxy ethanol (IGEPAL CA-630/NP-40); As phosphatidylcholine phospholipid such as (lecithin); Derived from the polyoxyethylene aliphatic ether (being called the Brij surfactant) of lauryl alcohol, spermol, stearyl alcohol and oleyl alcohol, as 2,2'-ethylenedioxybis(ethanol). list lauryl ether (Brij 30); And sorbitan ester (so-called SPAN), as sorbitan trioleate (Span 85) and sorbitan monolaurate.The surfactant that comprises in the Emulsion is preferably Tween 80 (polyoxyethylene sorbitan monoleate), Span 85 (sorbitan trioleate), lecithin and TritonX-100.Surfactant mixture can adopt (for example) Tween 80/Span 85 mixture, or Tween 80/Triton-X100 mixture.

Being used for oil in water emulsion adjuvant of the present invention includes but not limited to:

The Emulsion of zamene, tocopherol and Tween 80.This Emulsion can comprise phosphate buffered saline (PBS).It also can comprise Span 85 (as 1%) and/or lecithin.These Emulsions can contain 2-10% zamene, 2-10% tocopherol and 0.3-3% Tween 80, and the weight ratio of zamene and tocopherol is preferred≤and 1, because this can provide more stable Emulsion.Tween 80 can be dissolved in PBS and obtain 2% solution, then the mixture of this solution of 90ml with (5g DL-alpha-tocopherol and 5ml zamene) be mixed, this mixture of Micro Fluid then, thus prepare a kind of this class Emulsion.The Emulsion that obtains can contain the submicron oil droplet, and for example average diameter is 100-250nm, preferably the oil droplet of about 180nm.

The Emulsion of zamene, tocopherol and Triton detergent (as Triton X-100).

Shark alkane, polyoxyethylene sorbitan monoleate and poloxamer 401 (" pluronics ^TMThe Emulsion of L121 ").Phosphoric acid salt buffer saline, pH 7.4 these Emulsions of preparation.This Emulsion is the delivery vector that can be used for muramyldipeptide, in " SAF-I " adjuvant [7] (0.05-1%Thr-MDP, 5% shark alkane, 2.5% pluronic L121 and 0.2% polyoxyethylene sorbitan monoleate) with Threonyl-MDP coupling.Also can adopt the adjuvant that does not contain Thr-MDP, as " AF " adjuvant [8] (5% shark alkane, 1.25% pluronic L121 and 0.2% polyoxyethylene sorbitan monoleate).Preferred Micro Fluid.

The Emulsion that contains 0.5-50% oil, 0.1-10% phospholipid and 0.05-5% nonionic surfactant.As described in list of references 9, preferred phospholipid fraction is phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidylinositols, phosphatidyl glycerol, phosphatidic acid, sphingomyelins and cuorin.Preferred submicron droplets size.

Can not metabolism oil (as light mineral oil) and the submicron oil in water emulsion of at least a surfactant (as lecithin, Tween 80 or Span80).Can comprise additive; (aliphatic amine is added to the GPI-0100 that produces on the deacylated tRNA basis soap glycosides as QuilA saponin, cholesterol, saponin-lipophilic conjugate as carboxyl by glucuronic acid; referring to list of references 10), dimethyl two (octadecyl) ammonium bromide and/or N; N-two (octadecyl)-N, two (2-ethoxy) propane diamine of N-.

Saponin (as QuilA or QS21) and sterin (as cholesterol) are coupled to the micellar Emulsion of spiral [11].

Preferably facing with preceding, mixed emulsion and antigen when sending.Therefore, in the vaccine of packing or sale, generally adjuvant and antigen are separately preserved, be mixed with final preparation in use.This antigen is generally aqueous form, so that finally prepare this vaccine by mixing two kinds of liquid.The volume ratio that is used for blended two kinds of liquid is variable (as 5: 1-1: 5), but be about 1: 1 usually.

When compositions contains tocopherol, can adopt in α, β, γ, δ, ε or the ξ tocopherol any, but preferred alpha-tocopherol.Tocopherol kind of the form of can peeking is as different salt and/or isomer.Salt comprises organic salt, as succinate, acetate, nicotinate etc.Can adopt D-alpha-tocopherol and DL-alpha-tocopherol.Preferred alpha-tocopherol is the DL-alpha-tocopherol, and the preferred salt of this tocopherol is a succinate.

3D-MPL

Monophosphoryl lipid A (the 3D-MPL of 3-O-deacylation; Be also referred to as 3 take off-monophosphoryl lipid A of O-acyl groupization or 3-O-deacylated tRNA base-4 '-monophosphoryl lipid A), it is known adjuvant.This title shows that 3 of the reducing end glycosamine in the monophosphoryl lipid A by deacylation.3D-MPL can chemically be similar to lipid A, but lacked the phosphoryl and the alkali-sensitive acyl group of acid labile by the no heptose mutant preparation of salmonella minnesota (Salmonella minnesota).The cell of its energy activated mononuclear macrophage cell lines, and stimulate several cytokines of release, comprise IL-1, IL-12, TNF-α and GM-CSF.The initial preparation of having described 3D-MPL in list of references 12, this product is produced and is sold with trade name MPL by Lasersoft Vision Inc. of section (Corixa Corporation).Other details can be referring to list of references 13-16.

3D-MPL can get the form of the mixture of the different correlation molecule of acyl groupization (as have 3,4,5 or 6 acyl chains, their length can be different).Two glycosamines (being also referred to as 2-deoxidation-2-amino-glucose) monosaccharide is the N-acyl groupization on the carbon of its 2-position (i.e. 2 and 2 ' position), and the O-acyl groupization is also arranged on 3 ' position.The group chemical equation that is connected in carbon 2 is :-NH-CO-CH ₂-CR ¹R ^{1 '}Be connected in carbon 2 ' group chemical equation be :-NH-CO-CH ₂-CR ²R ^{2 '}Be connected in carbon 3 ' group chemical equation be :-O-CO-CH ₂-CR ³R ^{3 '}Exemplary configuration is:

Radicals R ¹, R ²And R ³Be independently of one another-(CH ₂) _n-CH ₃The n value is preferably 8-16, and more preferably 9-12 most preferably is 10.

Radicals R ^{1 '}, R ^{2 '}And R ³Can respectively do for oneself: (a)-H; (b)-OH; Or (c)-O-CO-R ⁴, R wherein ⁴Be-H or-(CH ₂) _m-CH ₃, wherein the m value is preferably 8-16, and more preferably 10,12 or 14.On 2, m is preferably 14.On 2 ' position, m is preferably 10.On 3 ' position, m is preferably 12.So radicals R ^{1 '}, R ^{2 '}And R ^{3 '}Be preferably from dodecylic acid, tetradecanoic acid or hexadecanoic acid-the O-acyl group.

R ^{1 '}, R ^{2 '}And R ^{3 '}Be-during H, 3D-MPL only contain 3 acyl chains (2,2 ' and 3 ' position on respectively have one).R ^{1 '}, R ^{2 '}And R ^{3 '}In only have two and be-during H, 3D-MPL can contain 4 acyl chains.R ^{1 '}, R ^{2 '}And R ^{3 '}In only have one and be-during H, 3D-MPL can contain 5 acyl chains.R ^{1 '}, R ^{2 '}And R ^{3 '}In neither one be-during H, 3D-MPL can contain 6 acyl chains.The used 3D-MPL adjuvant of the present invention can be the mixture that contains these forms of 3-6 bar acyl chain; but preferably comprise 3D-MPL in the mixture with 6 acyl chains; specifically; the form that guarantees 6 acyl chains accounts at least 10% of 3D-MPL gross weight, for example 〉=20%, 〉=30%, 〉=40%, 〉=50% or more.The 3D-MPL that discovery has 6 acyl chains is the highest form of adjuvanticity.

Therefore, the most preferred form of the 3D-MPL that comprises of the present composition is:

When using 3D-MPL with form of mixtures, the content of 3D-MPL or concentration refer to the total amount or the total concentration of the various 3D-MPL materials that merge in the mixture in the present composition.

Under aqueous conditions, 3D-MPL can form micellar aggregates or the granule that varies in size, as diameter＜150nm or＞500nm.The present invention can adopt wherein one or both to adopt together, can select granule preferably by routine test.The present invention preferably adopts than granule (enough producing clarifying 3D-MPL aqueous suspensions as little arriving), because its active high [17].The average diameter of preferred particulates is less than 220nm, is more preferably less than 200nm or less than 150nm or less than 120nm, average diameter even can be less than 100nm.Yet in most of the cases, average diameter is not less than 50nm.These granules are little of enough adapting to filtration sterilization.By the routine techniques assessment particle diameter of dynamic light scattering, this technology can disclose average particulate diameter.When claiming that certain particulate diameter is x nm, this granule distributes near this meansigma methods usually, but the particle diameter of at least 50% quantity (for example＞60%,＞70%,＞80%,＞90% or more) is in the scope of x ± 25%.

3D-MPL can be used alone as adjuvant, perhaps with one or more other adjuvant compound couplings.For example, known to 3D-MPL and QS21 saponin [18]; With QS21 and oil in water emulsion [19]; With aluminum phosphate [20]; With aluminium hydroxide [21]; Or with saponin and il-1 2[22] coupling.

Preferred adjuvants mixture (when particularly using with HBsAg) comprises the mixture of 3D-MPL and aluminum salt, and aluminum salt is preferably aluminum phosphate.3D-MPL should be adsorbed on the aluminum salt.Preferably, the 3D-MPL of at least 50% (by weight) absorption, for example＞60%,＞70%,＞80%,＞90%,＞95%,＞98% or more.

The aluminium adjuvant that uses is commonly referred to as " aluminium hydroxide " or " aluminum phosphate " adjuvant at present.Yet they are just in order to call conveniently, are not the accurate description the 9th chapter of list of references 2 (for example referring to) in esse chemical compound.For with as aliphatic adjuvant couplings such as 3D-MPL, the present invention can use usually any as in " hydroxide " or " phosphate " of adjuvant.

The adjuvant that is called " aluminium hydroxide " generally is an aluminum oxyhydroxide salt, usually it to small part be crystallization.Aluminum oxyhydroxide (chemical formula is AlO (OH)), by infrared (IR) spectrum, can be with it and as aluminium hydroxide Al (OH) ₃Wait other aluminium compound difference to come, 1070cm especially occurs ^-1The absorption band and the 3090-3100cm at place ^-1The strong shoulder (the 9th chapter of list of references 2) at place.

The adjuvant that is called " aluminum phosphate " generally is an Adju-Phos, also usually contains a small amount of sulfate.Can obtain them by precipitation, reaction condition in the precipitation process and concentration can influence the degree that phosphoric acid replaces the hydroxyl of this salt.The PO of hydroxyl phosphate ₄/ Al mol ratio is generally 0.3-0.99.Since there is hydroxyl, can be with the AlPO of hydroxyl phosphate and standard ₄Distinguish.For example, 3164cm ^-1The IR bands of a spectrum (for example being heated to 200 ℃) at place show and have structural hydroxyl (the 9th chapter of list of references 2).

The PO of aluminum phosphate adjuvant ₄/ Al ³⁺Mol ratio is generally 0.3-1.2, preferred 0.8-1.2, more preferably 0.95 ± 0.1.It is amorphous that aluminum phosphate is generally, particularly hydroxyl phosphate.Typical case's adjuvant is PO ₄/ Al mol ratio is the unbodied Adju-Phos of 0.84-0.92, comprises 0.6mg Al ³⁺/ ml.Aluminum phosphate is generally granule.The particulate general diameter in antigen absorption back is 0.5-20 μ m (5-10 μ m according to appointment).

The degree inverse relationship of the PZC of aluminum phosphate and phosphate radical substituted hydroxy, this replacement degree may depend on reaction condition and the reactant concentration by precipitation preparation salt.Also can be by changing the concentration (the stronger PZC of multi-phosphate=acidity) of free phosphate anion in the solution, or adding changes PZC as buffer such as histidine buffering liquid (make PZC alkalescence stronger).The PZC that is used for aluminum phosphate of the present invention is generally 4.0-7.0,5.0-6.5 more preferably, according to appointment 5.7.

The aluminum phosphate solution that is used to prepare the present composition can contain buffer (as phosphate or histidine or Tris buffer), but also not necessarily.Aluminum phosphate solution is preferably aseptic and pyrogen-free.Aluminum phosphate solution can comprise free aqueous phosphate anion, and for example concentration is 1.0-20mM, preferred 5-15mM, the more preferably from about phosphate anion of 10mM.Aluminum phosphate solution also can contain sodium chloride.Sodium chloride concentration is preferably 0.1-100mg/ml (as 0.5-50mg/ml, 1-20mg/ml, 2-10mg/ml), more preferably about 3 ± 1mg/ml.Having of NaCl helps the correct pH of mensuration before antigen absorption.

Preferably use aluminum phosphate with the aqueous solution form, in solution, add 3D-MPL (randomly, and antigen) (NB: usually the aqueous aluminum phosphate is called " solution ", but from the physical chemistry viewpoint of strictness, this salt is soluble, formation be suspension).Preferably aluminum phosphate is diluted to desired concn, and guarantees before adding 3D-MPL and/or antigen, to be uniform solution.

N-acyl group-false dipeptides

N-acyl group-false dipeptides adjuvant preferably comprises by the hydroxyl of the acid groups esterification of neutral or charged form.Acyl group is given false dipeptides aliphatic feature.The preferred chemical formula of this adjuvant is (I):

Wherein:

-R ₁Be the acyl group derived from saturated or undersaturated, the straight or branched carboxylic acid that contains 2-24 carbon atom, described carboxylic acid does not replace or replaces with one or more substituent groups that are selected from down group: hydroxyl, alkyl, alkoxyl, acyloxy, amino, acyl amino, acyl sulfenyl and ((C _1-24) alkyl) sulfenyl;

-R ₂Be the acyl group derived from saturated or undersaturated, the straight or branched carboxylic acid that contains 2-24 carbon atom, described carboxylic acid does not replace or replaces with one or more substituent groups that are selected from down group: hydroxyl, alkyl, alkoxyl, acyloxy, amino, acyl amino, acyl sulfenyl and ((C _1-24) alkyl) sulfenyl;

-X is the acid groups of hydrogen atom or neutral or charged form;

-Y is the acid groups of hydrogen atom or neutral or charged form;

-A is oxygen atom, sulphur atom or imino group-NH-;

-B is oxygen atom, sulphur atom or imino group-NH-;

-m is 1,2,3,4,5,6,7,8,9 or 10;

-n is 0,1,2,3,4,5,6,7,8,9 or 10;

-p is 1,2,3,4,5,6,7,8,9 or 10; With

-q is 1,2,3,4,5,6,7,8,9 or 10,

Restrictive condition is an at least a acid groups for neutrality or ionic species among substituent X or the Y.

Adjuvant can acid or salt form, with the coupling of organic or inorganic alkali.

Can be used for treating the saline and alkaline alkali metal base that mainly comprises of one-tenth of application, as sodium hydroxide, potassium hydroxide or Lithium hydrate, ammonium salt; Alkaline earth metal alkali is as calcium hydroxide or Strontium hydrate. and magnesium salt; Ferrous metal salt etc.; Organic base is as derived from the organic base as methylamine, diethylamine, monoethanolamine, diethanolamine, benzylamine, N-methylbenzylamine, veratrylamine, trimethoxy benzylamine etc. primary, the second month in a season, tertiary amine; Basic amino acid is as lysine and ornithine or amino sugar.The example that is not useable for treating the alkali of application is that brucine, strychnine, agmatine, dragon are neosine, glycosamine, N-methylglucosamine or N-methylmorpholine.

Preferred substituents X and Y are selected from:

-carboxyl [(C _1-5Alkyl]

-CH-[(CH ₂) _rCOOH] [(CH ₂) _SCOOH], wherein: r is 0,1,2,3,4 or 5; S is 0,1,2,3,4 or 5

-phosphono [(C _1-5) alkyl]

-dihydroxy phosphorus acyloxy [(C _1-5) alkyl]

-dimethoxy phosphoryl

-phosphono

-hydroxyl sulfonyl

-hydroxyl sulfonyl [(C _1-5) alkyl]

-hydroxyl sulfonyloxy [(C _1-5) alkyl]

When substituent X and/or Y are the acid groups of neutral form, refer to free carboxylic acid, sulfonic acid or phosphoric acid.When acid groups is charged form, refer to carboxylic acid, sulfonic acid or phos-phate forms, promptly by adding the salt that organic or inorganic alkali forms, described alkali is preferably the alkali that is used for the treatment of application.When X and/or Y were the two carboxyls of carboxyalkyl, thiazolinyl, hydroxyl sulfonyl, hydroxyl sulfonyl alkyl, hydroxyl sulfonyl-alkoxyl, phosphine acyl-alkyl or phosphoryl-alkoxyl, situation was similar.

When m=1 and n=0, described adjuvant is derived from serine.When m=2 and n=0, described adjuvant is derived from homoserine.If m=3 and n=0 refer to five-homoserine chemical compound.If m=4 and n=0 refer to six-homoserine chemical compound.

When p=3 and q=1, interested product can be citrulline, ornithine or arginine compounds.When p=4 and q=1, refer to homoarginine or lysine chemical compound.

R ₁And R ₂Comprise chain length difference, similar and different, saturated or undersaturated, the straight or branched acyl derivative of characteristic, they can contain the one or more substituent groups that are selected from down group: alkyl, amino, acyl amino, hydroxyl, alkoxyl, acyloxy, acyl sulfenyl and alkylthio group.

The example of this class acyl group substitutive derivative has Oleum Ricini acyl group, 1,2-hydroxyl stearyl, 2-hydroxy-3-methyl bytyry, the amino valeryl of 3-hydroxyl-2-, palmityl, anti-oleoyl, oleostearin acyl group (eleostearyl), Semen arachidis hypogaeae acyl group, arachidonic acyl group, suitable 9-eicosylene acyl group, Shan Yu acyl group, erucyl, 8-methyl capryl, 9-methyl capryl, two dodecahexaene acyl groups or eicosapentaenoic acyl group.

The preferred adjuvant chemical formula is (Ia), and wherein A and B are oxygen atoms:

In chemical formula (Ia), X and Y are preferably hydrogen atom or phosphono.

Suitable adjuvant chemical formula (Ia) comprising:

-3-(3-lauroyl oxygen base myristoyl amino) 9-(3-hydroxyl myristoyl amino) 4-oxo-5-azepine decane-1,10-glycol 1 and/or 10-dihydrogen phosphoric acid ester, with and the addition salts that forms with organic or inorganic alkali,

-3-(3-lauroyl oxygen base-myristoyl amino) 9-(3-hydroxyl myristoyl amino) 4-oxo-5-azepine decane-1,10-glycol 1, two (dihydrogen phosphoric acid) esters of 10-, with and the addition salts that forms with organic or inorganic alkali,

-3-(3-hydroxyl myristoyl amino) 9-(3-lauroyl oxygen base myristoyl amino) 4-oxo-5-azepine decane-1,10-glycol 1, two (dihydrogen phosphoric acid) esters of 10-, with and the addition salts that forms with organic or inorganic alkali,

-3-(3-lauroyl oxygen base myristoyl amino) 9-(3-hydroxyl myristoyl amino) 4-oxo-5-azepine decane-1,10-glycol 1-dihydrogen phosphoric acid ester, with and the addition salts that forms with organic or inorganic alkali,

-3-(3-hydroxyl myristoyl amino) 9-(3-lauroyl oxygen base myristoyl amino) 4-oxo-5-azepine decane-1,10-glycol 1-dihydrogen phosphoric acid ester, with and the addition salts that forms with organic or inorganic alkali,

-3-(3-hydroxyl myristoyl amino) 9-(3-lauroyl oxygen base myristoyl amino) 4-oxo-5-azepine decane-1,10-glycol 10-dihydrogen phosphoric acid ester, with and the addition salts that forms with organic or inorganic alkali.

List of references 23 discloses the method for the adjuvant of synthetic chemistry formula (I) in detail, and this method may further comprise the steps: respectively by being easy to carry out capping reagent protection diamino acid (q+1) position of acidolysis and hydrogenolysis (hydrogolysis) and the amine functional group of ω position; Make the reaction of free carboxylic acid functional and Reducing agent, produce corresponding alcohol; Make (q+1) position amine functional group go the protection, then by with chemical formula R ₂The reaction acyl groupization of the carboxylic-acid functional derivant of OH then discharges free terminal amine functional group by hydrogenolysis, obtains the diamino alcohol of general formula I I:

Under the situation that the peptide condensing agent exists in atent solvent, with of ω-hydroxyl, omega-amino-or the ω-sulfo-amino acid compound condensation of this amino alcohol with general formula III:

So that the dipeptide compound of chemical formula (IV) to be provided:

If necessary, the alcohol functional group of its free end can be protected (for example by alkyl or acyl group, or any other suitable protecting group), or be substituted (if desired) in the presence of coupling agent.Can carry out catalytic hydrogenation or other deprotection to the alcohol of protection and handle, to obtain the derivant of general formula I.

List of references 23 also discloses the method for synthetic chemistry formula (II) adjuvant in detail, and this method may further comprise the steps: respectively by being easy to carry out the protection reagent protection chemical formula H of acidolysis and hydrogenolysis ₂N (CH ₂) _pCHNH ₂(CH ₂) _Q+1The amine functional group of the diamino acid of COOH (q+1) and ω position; Make the reaction of free carboxylic acid functional and Reducing agent, produce corresponding alcohol; Make (q+1) position amine functional group go the protection, then by with chemical formula R ₂The reaction acyl groupization of the carboxylic-acid functional derivant of OH then makes terminal amine functional group go protection by hydrogenolysis, to obtain the amino alcohol of general formula I I; Under the situation that the peptide condensing agent exists in atent solvent, with of the ω-hydroxy-amino-acid functional derivatives condensation of this amino alcohol with general formula III a:

Wherein X is chemical formula (RO) ₂The dialkoxy of P-O-or two aryloxy group-phosphoryl, to obtain the peptide sample chemical compound of Formula I Va:

When R is when being easy to carry out the group of hydrogenolysis, if desired, wherein another alcohol functional group can be used the phosphorylation agent phosphorylation in the presence of coupling agent.Can carry out catalytic hydrogenation to the alcohol of protection on the one hand, so that acyl group R ₂Go up the optional alcohol functional group that exists and go protection, on the other hand, discharge phosphoric acid functional group, make second optional phosphoric acid functional group that exists go protection by hydrogenolysis then, to obtain the derivant of general formula V:

Randomly carry out next step, form salt with the organic or inorganic alkali reaction.

The initial aminoacid configuration that uses has determined the spatial chemistry of the chiral centre of acyl amino, and the spatial chemistry of acyl amino depends on the fatty acid configuration of initial use.One can be from having the diamino acid of L or D configuration or raceme characteristic.One can be from L, and the hydroxylation aminoacid or the racemic mixture of D configuration begin.All these class stereoisomers or diastereomer are included in the scope of the present invention.

Particularly preferred adjuvant, the especially chemical compound of list and bis phosphoric acidization are the chemical compounds [23] that is called " OM-294-MP " (chemical formula VI) and " OM-294-DP " (chemical formula VII):

OM-174

Can obtain OM-174 by chemosynthesis, it has kept the trigalloyl based structures unit from lipid A.List of references 24-26 is described in more detail.List of references 26 has provided the chemical formula of OM-1745:

Therefore, the same with natural lipid A, OM-174 has 1,4 '-the didextrose amine main chain of β (1 → the 6)-connection of diphosphate.OM-174 can form aggregate form with micelle H1 structure, and main chain was positioned on the periphery when promptly wherein lipid molecular was packed, and acyl chain is inside, and itself is packaged into hexagon cylinder.May there be cubic phase.Also may there be H ₁₁Phase.

The glucosaminide phosphate derivative

Since there is acyl chain, and aminoalkyl glucosaminide phosphate ester (AGP) derivant (as RC-529[27,28]) be the aliphatic adjuvant.Usually, these derivants can be following chemical formula [29]:

Wherein:

-X represents the oxygen or the sulphur atom of axial location or radial position;

-Y represention oxygen atom or NH group;

-" n ", " m ", " p " and " q " are identical or different, are the integer of 0-6;

-R ₁, R ₂And R ₃, it can be identical or different, is the fatty acyl residue that contains about 20 carbon atoms of 1-, wherein R ₁, R ₂Or R ₃One of optional be hydrogen;

-R ₄And R ₅Identical or different, be hydrogen or methyl;

-R ₆And R ₇Identical or different, be hydrogen, hydroxyl, alkoxyl, phosphono, phosphonato, sulfo group, sulphur oxygen base, amino, sulfydryl, cyano group, nitro, formoxyl or carboxyl and their ester and amide;

-R ₈And R ₉Identical or different, be phosphono or hydrogen, preferred R ₈And/or R ₉In at least one is a phosphono.

The configuration that is connected in 3 of normal fatty acyl residue ' three-dimensional generative center is R or S, but preferred R.R ₄Or R ₅The spatial chemistry of the carbon atom that connects can be R or S.All stereoisomers, enantiomer and diastereomer, and their mixture all fall into the scope of the invention.Suitable adjuvant comprises the salt of these chemical compounds.

Preferred AGP chemical compound is ' RC-529 ' to be prepared as described in list of references 30:

Acyclic synthetic lipid A analogue

The lipid A analogue that can be used as the adjuvant of the present composition is ER-112022[31], it is the phospholipid dimer that connects by acyclic main chain:

Each monomeric unit of ER-112022 contains 3 different aliphatic groups that are incorporated into di-phosphate ester indirectly.Aliphatic group comprises 10 carbon ether chains, is incorporated into the unsaturated 12 carbon acyloxy chains of ether chain, with the 14-carbon β-oxo-amide chain that is connected nearerly with di-phosphate ester.Therefore, compare with the lipid A from the natural generations of escherichia coli (E.coli), this chemical compound has 3 specific characteristics: (i) ER-112022 does not contain the ring-type sugar backbone; (ii) secondly, phosphate ester is the di-phosphate ester that mixes in the structure main chain boundary, and is different with the phosphate ester on the escherichia coli lipid A; (iii) symmetrical configuration (fatty acid of six symmetrical distributions on the non-sugar backbone).

Similar useful chemical compound comprises the chemical compound with chemical formula XIV, XV or XVI, or its salt:

Definition as list of references 32, as ' ER 803058 ', ' ER 803732 ', ' ER 804053 ', ER 804058 ', ER 804059 ', ' ER 804442 ', ' ER 804680 ', ' ER 804764 ', ' ER 803022 ' or ' ER 804057 ', for example:

The chemical compound that contains the lipid that is connected in the acyclic main chain of phosphoric acid also can be used as adjuvant, as TLR4 antagonist E5564[33,34]:

Surfactant

First aspect of the present invention has been avoided adopting surfactant substantially in the antigen purification process, makes in the antigen component that the present invention adopts not table of discovery surface-active agent.Yet, aspect second, adopt surfactant, but be controlled with respect to its level of aliphatic adjuvant.

Surfactant generally is a nonionic surfactant, particularly has been used for the surfactant of bacterin preparation.Preferred organic surface active agent.They generally are alkylene oxide (as oxirane) and aliphatic alcohol, fatty acid, alkyl phenol, alkylamine or the product that contains other suitable combination thing of at least one active hydrogen atom.In most of surfactants, the carbon chain lengths of modal alcohol, amine and acid is C ₈-C ₁₈Modal alkyl phenol is nonyl phenol and octyl phenol.The surfactant that especially preferably contains poly-(oxygen ethylene) residue.

For example, can be used for surfactant of the present invention comprises but is not limited to: polyoxyethylene sorbitan esters surfactant (being commonly referred to tween), especially polysorbate 20 and polyoxyethylene sorbitan monoleate; The copolymer of oxirane (EO), expoxy propane (PO) and/or epoxy butane (BO), commodity are called DOWFAX ^TM, as straight chain EO/PO block copolymer; Hot benzene polysaccharide, wherein multiple ethyoxyl (oxygen-1,2-ethane two bases) quantity is variable, and interested especially is hot benzene polysaccharide-9 (Triton X-100, or uncle's octylphenoxy polyethoxy ethanol); (Octylphenoxy) polyethoxy ethanol (IGEPAL CA-630/NP-40); Derived from the polyoxyethylene aliphatic ether (being called the Brij surfactant) of lauryl alcohol, spermol, stearyl alcohol and oleyl alcohol, as 2,2'-ethylenedioxybis(ethanol). list lauryl ether (Brij 30); And sorbitan ester (being generically and collectively referred to as SPAN), as sorbitan trioleate (Span 85) and sorbitan monolaurate.

Interested two kinds of specific surfactants are Triton X-100 and polysorbate 20.Therefore, specifically, the preferred implementation of first aspect present invention has been avoided adopting these two kinds of nonionic surfactant substantially, preferably avoids adopting any nonionic surfactant substantially.Similarly, the preferred implementation of second aspect present invention has adopted the antigen component that contains one of these two kinds of nonionic surfactant.

The present invention is particularly suitable for adopting polysorbate 20.Determine that this surfactant can be applied to the people safely, is included in the application in the vaccine.

Can classify to surfactant by ' HLB ' (hydrophile/lipophile balance).The HLB of surfactant of the present invention is preferably at least 10, preferably at least 15, more preferably at least 16.

In a second aspect of the present invention, be applied to the patient for fear of surfactant with heavy dose, surfactant concentrations preferably should be not more than 50 μ g/ml in the said composition, for example≤40 μ g/ml ,≤35 μ g/ml ,≤30 μ g/ml ,≤25 μ g/ml ,≤20 μ g/ml ,≤15 μ g/ml ,≤10 μ g/ml ,≤5 μ g/ml etc.Preferred≤20 concentration of μ g/ml.

Antigen

The present invention includes employing antigen.The present invention is particularly suitable for using the antigen of common employing surfactant purification.These antigens are generally lipotropy antigen.They comprise at least one usually and stride the film district, and this strides the film district in vivo has the function of Antigen Location in lipid bilayer, for example on the pathogen surface.The present invention is particularly suitable for adopting viral surface antigen.

Aspect second of the present invention, this antigen component comprises surfactant.Antigen and surfactant are preferably composite form, but not simple antigen and surfactant mixture.Antigen/surfactant complex comprises liposome and the non-ionic precursor (niosome) that contains antigenic surfactant-stabilisation, and it is the vesicle by synthetic nonionic compound formation.Preferred antigen/surfactant complex is the granular pattern hbs antigen of purification in the presence of surfactant.List of references 35 has been described the recombinant HBsAg granule and how to be retained in the polysorbas20 (per 100 μ g HBsAg are 25 μ g polysorbas20s at the most) that adopts in its purge process.

Therefore, the most preferred antigen that second aspect present invention adopts is hepatitis B virus (HBV) surface antigen, it is a particulate form spherical in shape substantially (average diameter is about 20nm), comprises containing phospholipid and as the lipidic matrix of nonionic surfactant such as polysorbate 20.In purge process, nonionic surfactant can be mixed in the granule.

Yet aspect first, preferred HBsAg form is the form without the nonionic surfactant Purification of HBsAg of the present invention, so that the HBsAg granule does not mix any surfactant, thereby has avoided the interference of aliphatic adjuvant.

Hepatitis B virus (HBV) is one of known substance that causes viral hepatitis.The HBV virion is made up of the internal core that outside protein coat or capsid surround, and virus core contains the viral DNA genome.The key component of capsid is the protein that is called the HBV surface antigen, and perhaps more normal being called be ' HBsAg ', and it generally is 226 amino acid whose polypeptide, and molecular weight is～24kDa.Existing all hepatitis B vaccines all contain HBsAg, and when this antigen was applied to normal vaccination person, it can stimulate and produces anti-HBsAg antibody, thereby protection is not infected by HBV.

In production of vaccine, available prepared in one of two ways HBsAg.First method comprises the antigen by chronic hepatitis B carrier blood plasma purification particle form, because synthesized a large amount of HBsAg in the liver and be released in the blood flow in the HBV course of infection.The second way comprises by the recombinant DNA method marking protein.The HBsAg that adopts in the inventive method is recombinant expressed in yeast cells.Suitable yeast comprises yeast (Saccharomyces) (as saccharomyces cerevisiae (S.cerevisiae)), Pichia sp. (Pichia) (as pichia pastoris phaff (P.pastoris)) or Hansenula yeast (Hanensula) (as multiple-shaped nuohan inferior yeast (H.polymorpha)) host.Perhaps, can express this antigen with recombinant mammalian cells (as Chinese hamster ovary (CHO) cell, COS cell, Bu3 cell etc.), insect cell (as adopting baculovirus vector) or plant cell.Yet, adopt yeast expression usually.

The HBsAg of yeast expression is preferably nonglycosylated.Different with natural HBsAg (being the HBsAg in the blood plasma purified product), the HBsAg of yeast expression is generally nonglycosylated, this is to be used for most preferred HBsAg form of the present invention, because it has high immunogenicity, can also prepare under the situation of the risk that does not have blood product pollution.The HBsAg granule of yeast expression can comprise phosphatidylinositols, and this does not have in natural HBV virion.This granule also can comprise the LPS of non-toxic, with stimulating immune system [36].

Many methods of Purification of HBsAg are (for example referring to list of references 37-62) known in the art.In these methods, first aspect of the present invention has adopted the method for not using nonionic surfactant.On the contrary, second aspect of the present invention adopted nonionic surfactant in purification, so that surfactant mixes in the final granule HBsAg product.When purification begins, destroy in the process of recombinant yeast cell, preferably adopt polysorbate 20, surfactant is introduced the HBsAg granule.

After destroying cell, preferred HBsAg purification process comprises: ultrafiltration; The size exclusion chromatograph; Anion-exchange chromatography; Ultracentrifugation; Desalination; And filtration sterilization.Destroy the precipitable lysate in cell (for example destroying) back, HBsAg is stayed in the solution, prepare to carry out ultrafiltration with Polyethylene Glycol.Before or after filtration sterilization, may stablize the HBsAg granule by formaldehyde treated.Can remove unnecessary formaldehyde by ultrafiltration or chromatography subsequently.Also can adopt filtration sterilization.

HBsAg is preferably from HBV hypotype adw2.

Except that ' S ' sequence, before surface antigen can comprise all or a part-the S sequence, as before all or the part-S1 and/or preceding-S2 sequence.

Can be used for other viral surface antigen of the present invention normally from the envelope glycoprotein of enveloped virus.Being used for viral surface antigen of the present invention can include but not limited to:

-Retroviridae albumen.Retroviridae comprises lentivirus and Spumavirus.Interested virus comprises HTLV-I, HTLV-II, feline immunodeficiency virus (FFV), HIV (human immunodeficiency virus) (HIV comprises HIV-I and HIV-2), simian immunodeficiency virus (SIV), chimpanzee foamy virus and Human foamy spumavirus.

-Paramyxoviridae albumen is as F albumen.Paramyxoviridae comprises (a) paramyxovirus subfamily, and it comprises paramyxovirus genus, Rubulavirus and Morbillivirus, and (b) pneumonitis virus subfamily, and it comprises that pneumonitis virus belongs to.Interested virus comprises parainfluenza virus (PIV), people paramyxovirus, rinderpest virus, PPR virus, Measles virus, mumps virus, respiratory syncytial virus (RSV), upright all kinds of diseases and ailments poison, Heng Dela virus, equine morbillivirus (EMV), rabies virus and Mei Nage virus (Menangle virus).

-filamentous form virus section albumen.Filamentous form virus section comprises that Marburg virus belongs to and Ebola virus belongs to.

The projection glycoprotein of-coronaviridae.Coronaviridae comprises coronavirus genus and Orbivirus.Interested virus comprises human corona virus's (comprising sars coronavirus), avian infectious bronchitis virus, feline infectious peritonitis virus, murine hepatitis virus, Porcine epidemic diarrhea virus, swine erythrocyte coagulation type encephalomyelitis virus, transmissible gastroenteritis of swine virus and berne virus.

-Rhabdoviridae albumen is as G albumen.Rhabdoviridae comprises baculovirus genus, Vesiculovirus genus, lyssavirus, fever virus genus, Cytorhabdovirus and Nucleorhabdovirus in short-term.Interested virus comprises vesicular stomatitis virus, rabies virus, mokola virus, cattle ephemeral virus.

-Togaviridae albumen.Togaviridae comprises α virus and rubella virus.Interested virus comprises sindbis alphavirus, east and west encephalitis, Semliki Forest virus, rubella virus, aura virus, crust companion's gram virus (Babanki virus), crust horse (Barmah) forest virus avis-A, BEB, Buckie Ke Like virus (Buggy Creek virus), Chikungunya virus, everglades virus, Fort Morgan virus, GET, Highland J virus, Ke Zila virus (Kyzylagach virus), Mayaro virus, MID, mucambo virus, ndumu virus, AudioCodes uncle's virus (Ockelbo virus), O nyong-nyong virus, pixuna virus, ross river virus, sagiyama virus, UNA, Venezuelan equine encephalitis virus and whataroa virus.

The peplos of-flaviviridae (' E ') glycoprotein.Flaviviridae comprises that Flavivirus, pestivirus belong to and Hepacivirus.Interested virus comprises dengue virus, hepatitis C virus, yellow fever virus, Japanese encephalitis virus, west Nile virus, Saint Louis' encephalitis virus, bovine diarrhea virus and tick borne encephalitis (TBE) virus.

-Bunyaviridae albumen.Bunyaviridae comprises that bunyavirus genus, Nairovirus, Phlebovirus, Hantaan belong to and tomato spotted wilf virus belongs to (Tospovirus).Interested virus comprises Bunyavirus, Bunyavirus, California encephalitis, clarke tin pest poison (La Cross virus), Hantaan virus, sin nombre virus, crimean-Congo hemorrhagic fever virus, sandfly fever Sicily virus and Rift Valley fever virus.

-Arenaviridae albumen.Arenaviridae comprises; Lymphatic chorion meningitis virus, ippy virus and Lassa virus.

-Hepadnaviridae albumen (comprising HBsAg).Hepadnaviridae comprises that positive Hepadnavirus belongs to and the birds Hepadnavirus belongs to.Except that the viruses of human hepatitis B, this Viraceae comprises that also ground squirrel hepatitis B virus belongs to, the marmot hepatitis B virus belongs to, hair monkey hepatitis B virus belongs to, arctic Sciurus vulgaris hepatitis virus belongs to, DHB belongs to, the heron hepatitis B virus belongs to and Luo Si goose hepatitis B virus belongs to.

-herpetoviridae albumen.Herpetoviridae comprises that Simplexvirus, Varicellavirus, Roseolovirus, cytomegalovirus genus, Muromegalovirus, Lymphocryptovirus belong to and Ladino Tobamovirus (Rhadino virus).Interested virus comprises as herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes virus hominis 6 (HHV6), herpes virus hominis 7 (HHV7) and human herpes virus 8 herpes virus hominises such as (HHV8).Suitable antigen can be selected from gB, gC, gD and gH glycoprotein (as the glycoprotein among the HSV).Preferred especially HSV gD2.

-orthomyxoviridae family albumen.Orthomyxoviridae family comprises Influenza Virus and Thogotovirus.Preferably, comprise surface antigen hemagglutinin (HA) and/or neuraminidase (NA) from the antigen of Influenza Virus (A, B or C type influenza virus).

General with these viral surface antigens of surfactant purification, so antigen component can comprise surfactant, if when particularly existing with the particle form that contains lipid.

The present invention also can adopt the particulate antigen based on hybridization that contains viral surface antigen and heterologous antigen or fusion rotein.For example, known with HBsAg sequence and heterologous antigen fusion, so that utilize the ability of HBsAg to be assembled into granule.

For example, list of references 63 has been reported the fusions of HIV-1 gp120 and HBsAg, obtaining the spontaneous granular protein that is assembled into, its granular size and density and natural HBsAg particulate species seemingly, wherein iipidomic into about account for 25% and each granule contain 100 gp120 that have an appointment.Gp120 can be folded into its native conformation in fusions, and keeps its biologic activity.Similarly, can improve HIV gp41 epi-position by forming inner fusions with HBsAg, fusion rotein self is assembled into 22nm hdl particle [64] in yeast.

Also the method can be used for malaria vaccine.List of references 65 reports will insert in the HBsAg sequence from the epi-position of the antigenic 61aa at the most of malaria gp190, and the hybrid particle of expression can cause the immunne response of anti--gp190 in animal.List of references 66 has been reported a kind of protein, contain in this albumen 16 proteic 4 unit sequences of multiple ring sporinite with HBsAg as expressing fusion protein.List of references 67 reports produce in yeast by Pfs16 and HBsAg and merge the virus-like particle of forming.List of references 68 discloses and has encircled the hybridization antigen that sporinite albumen is blended in HBsAg.List of references 69 discloses the fusions in the 1 proteic C-terminal zone, merozoite surface of Plasmodium vivax (P.vivax), and it has formed the immunogenicity granule of 20-45nm size.Therefore, with self assembling particle form HBsAg submission malaria antigen, be known in the art.

Therefore, the present invention can adopt the hybridization antigen that contains viral surface antigen and heterologous antigen.Heterologous antigen can insert in the viral surface antigen sequence, perhaps can be blended in viral surface antigen sequence of N end or C-terminal.If the natural viral surface antigen can be assembled into granule (as HBsAg), insert so or merge and can not hinder this assembling.

In these hybridization albumen, heterologous antigen can be from antibacterial, fungus, parasite, virus (is not HBsAg but limit heterologous antigen) etc.Hybridization can comprise complete heterologous antigen in the albumen, but more common be to comprise this antigenic antigenicity fragment.

Specifically, when viral surface antigen was HBsAg, heterologous antigen can be from HIV, Plasmodium falciparum (Plasmodium falciparum), Plasmodium vivax (Plasmodium vivax), malariae (Plasmodiummalariae) or Plasmodium ovale (Plasmodium ovale).The HIV antigen that is applicable to preparation HBsAg crossbred comprises envelope glycoprotein gp120 or its antigenicity fragment [63].The Plasmodium falciparum antigen that is applicable to preparation HBsAg crossbred can be based on the subunit of ring sporinite surface antigen (" CSP "), for example they can comprise 3-20 multiple NANP motif (SEQ ID NO:2), and/or they can comprise the C-stub area (but general last 12 aminoacid that do not contain C-terminal) of CSP.For example, the present invention can adopt the antigen that is called " RTS ", this antigen contains most of C-terminal (the aminoacid 210-398 from the CSP of NF54 of Plasmodium falciparum or 7G8 separator, it comprises the t cell epitope district at 19 NANP repetitive sequences and aminoacid 367-390 place), it is by the N-terminal fusion of four aminoacid with the HBsAg of S2 part before the HBsAg.When expressing in yeast, RTS forms the granule that isolating protein also contains lipid (mainly being phospholipid) outward.Therefore, the sequence of RTS can contain: (i) N-terminal methionine residues; (ii) Met-Ala-Pro; (iii) corresponding to 189 aminoacid of the proteic aminoacid 207-395 of CS of proteic aminoacid 210-398 of the CS of Plasmodium falciparum 7G8 or Plasmodium falciparum NF54; (iv) Arg or GIy; (v) from before the hepatitis B-the proteic Pro-Val-Thr-Asn of S2; (vi) HBsAg.From the sequence of the total length RTS of 7G8 separator shown in the SEQ ID NO:1 of list of references 70 (also referring to list of references 71 Fig. 5):

MMAPDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNA

NPNANPNKNNQGNGQGHNMPNDPNRNVDENNANNAVKNNNNEEPSDKHIEQYLKKIKNSISTEWSPCSVTCGNGIQ

VRIKPGSANKPKDELDYENDIEKKICKMEKCSSVFNVVNSRPVTNMENITSGFLGPLLVLQAGFFLLTRILTIPQS

LDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVC

PLIPGSTTTNTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWF

VGLSPTVWLSAIWMNWYWGPSLYSIVSPFIPLLPIFFCLWVYI(SEQ ID NO：1)

This hybridization albumen can adopt the sequence of coding SEQ ID NO:1 to express in yeast.Preferred hybridization albumen and normal HBsAg co expression in yeast.In the past, this method had been used for RTS in the past, and the product of co expression is called " RTS, S ".Can adopt RTS: the S ratio is about 1: 4.Preferably express in saccharomyces cerevisiae (S.cerevisiae), used plasmid contains the proteic sequence of the described hybridization of coding, and comprises: (1) is used to control the expression of coded sequence from the upstream promoter of glyceraldehyde-3-phosphate dehydrogenase gene; (2) the ARG3 transcription terminator in this coded sequence downstream.This plasmid generally also comprises: (3) LEU2 selected marker thing; (4) 2 μ plasmid sequences; (5) origin of replication of function is arranged in escherichia coli (Escherichia coli).

RTS, S can with wait other malaria antigen coupling as the unknown albumen of thrombospondin-dependency (TRAP).With RTS, the preferred aliphat adjuvant of S coupling comprises oil in water emulsion, 3d-MPL and QS-21 saponin.

Aliphatic adjuvant and surfactant ratio

In a second aspect of the present invention, the weight ratio of aliphatic adjuvant and antigenic surfactant was less than 1000: 1 in the compositions.

In the MF59 adjuvant, this weight ratio is based on zamene content.Contain in antigen component in the compositions of 0.1 μ g/ml surfactant, zamene concentration is less than 100 μ g/ml.Contain in antigen component in the compositions of 1 μ g/ml surfactant, zamene concentration is less than 1mg/ml.Contain in antigen component in the compositions of 10 μ g/ml surfactants, zamene concentration is less than 10mg/ml, and zamene concentration should be 43mg/ml (promptly 4300: 1) in the exemplary composition that with MF59 is adjuvant.

In the 3D-MPL adjuvant, weight ratio promptly comprises all different acyl group forms that may exist based on the total amount of 3D-MPL.Contain in antigen component in the compositions of 0.1 μ g/ml surfactant, the 3D-MPL total concentration is less than 100 μ g/ml.Contain in antigen component in the compositions of 1 μ g/ml surfactant, the 3D-MPL total concentration is less than 1mg/ml.

1000: 1 ratio is maximum.In order further to reduce in adjuvant medium oil and the antigen interferential probability between the surfactant, can reduce this ratio.Therefore, this ratio can≤500: 1 ,≤400: 1 ,≤300: 1 ,≤200: 1 ,≤100: 1 ,≤50: 1, perhaps even≤25: 1.Ratio≤100: 1 o'clock contains in the antigen component in the compositions of 10 μ g/ml surfactants, oil content (as zamene or 3D-MPL content) can≤1mg/ml.Ratio≤25: 1 o'clock contains in the antigen component in the compositions of 10 μ g/ml surfactants, oil content (as zamene or 3D-MPL content) can≤250 μ g/ml; On the contrary, contain in the compositions of 100 μ g/ml 3D-MPL, its antigen part contains the surfactant that is not less than 4 μ g/ml.

This ratio is preferably greater than 1.5: 1, for example 〉=2: 1, 〉=2.5: 1, 〉=3: 1, 〉=4: 1, 〉=5: 1 or higher.

In 3D-MPL, preferred proportion is 2.5: 1-25: 1, more preferably 2.5: 1-10: 1, further more preferably 2.5: 1-5: 1.Therefore, contain in the antigen component in the compositions of 10 μ g/ml surfactants, 3D-MPL content should be 25 μ g/ml-250 μ g/ml, is preferably 25 μ g/ml-100 μ g/ml, more preferably 25 μ g/ml-50 μ g/ml; On the contrary, contain in the compositions of 100 μ g/ml 3D-MPL, surface-active contents should be 4 μ g/ml-40 μ g/ml in the antigen, is preferably 10 μ g/ml-40 μ g/ml, more preferably 20 μ g/ml-40 μ g/ml.

Immunogenic composition

Except containing outside antigen and the aliphatic adjuvant (aspect second, low-level surfactant), the present composition can contain carrier, adjuvant, excipient, buffer etc., is described in further detail as following.These nonantigenic components can have various sources.For example, they can be present in one of the antigen that uses in the production process or Adjuvanting material, perhaps can separate adding with these components.

The preferred present composition comprises one or more pharmaceutical carriers and/or excipient.

In order to control tension force, preferably contain physiology salt, as sodium salt.Preferred sodium chloride (NaCl), its concentration can be 1-20mg/ml.

The osmotic pressure of compositions is generally 200mOsm/kg-400mOsm/kg, is preferably 240-360mOsm/kg, more preferably belongs to the scope of 280-320mOsm/kg.Reported in the past that osmotic pressure can not influence the pain [72] that vaccination causes, but still preferably osmotic pressure is remained in this scope.

The present composition can comprise one or more buffer.Typical buffer comprises: phosphate buffer; The Tris buffer; Borate buffer solution; The succinate buffer; Histidine buffering liquid; Or citrate buffer.Buffer is generally 5-50mM, preferred 5-20mM.

The pH of the present composition is generally 5.0-7.5, is more generally 5.0-6.0 to reach optimum stabilization, perhaps is 6.0-7.0.Therefore, the inventive method can be included in and be packaged into the step that the preparation container is adjusted the pH of bulk vaccine before.The pH of vaccine that contains diphtheria and tetanus toxoid is preferred 〉=and 6, to avoid the risk of toxoid (especially diphtheria toxoid) Poison Reverse, the pH that therefore contains toxoid and the antigenic vaccine of HBsAg is preferably 6.0-7.0.For comprising other vaccine of unit price HBsAg, can accept pH＜6.

The present composition is preferably aseptic composite.

The present composition is preferably the apyrogeneity compositions, and for example every dosage contains＜1EU (endotoxin unit, standard is weighed), preferred every dosage＜0.1EU.

The present composition is preferably the compositions of GF.

As when adopting the HBsAg of absorption, final vaccine product may be the suspension of outward appearance muddiness.This outward appearance means, is not easy to observe microbial contamination, and therefore, described vaccine preferably contains antimicrobial.When vaccine is packaged into this particular importance in the multi-dose container.The antiseptic that preferably comprises is 2-phenyl phenol and/or thimerosal.Yet, recommend not adopt in the methods of the invention to contain mercurial antiseptic, although in many existing HBV vaccines, thimerosal is arranged all.Yet for security consideration, preferred final composition contains less than about 25ng/ml hydrargyrum.More preferably, final vaccine product can not detect and contain thimerosal.In the inventive method, usually before antigen preparation is added, remove the mercurial antiseptic that contains in the antigen preparation, or avoid adopting thimerosal, thereby realize above-mentioned purpose in the process that preparation is used for the component of production said composition.Can dialyse to HBsAg (for example containing cysteine), with remove may in the HBsAg preparation process, adopt as any mercurial antiseptics [73,74] that contains such as thimerosal.

In process of production, use WFI (water for injection) that component is diluted to required ultimate density usually.

The concentration of any aluminum phosphate is (with Al in the present composition ³⁺Form represent) preferably less than 5mg/ml, for example≤4mg/ml ,≤3mg/ml ,≤2mg/ml ,≤1mg/ml etc.

The concentration of HBsAg is preferably less than 100 μ g/ml in the present composition, for example≤90 μ g/ml ,≤80 μ g/ml ,≤70 μ g/ml ,≤65 μ g/ml ,≤60 μ g/ml ,≤55 μ g/ml ,≤50 μ g/ml ,≤45 μ g/ml ,≤40 μ g/ml etc.The concentration of general about 40 μ g/ml or about 20 μ g/ml.

The present composition preferably is applied to the patient with 0.5ml dosage.Should be understood that 0.5ml dosage comprises normal difference, for example 0.5ml ± 0.05ml.

The present invention can provide and be fit to be packaged into individually dosed bulk material, the described individually dosed patient's use of being sold to.Above-mentioned concentration is the concentration in the common final packaging dosage, so the concentration in the bulk vaccine may higher (for example, can be reduced to final concentration by dilution).

The present composition is generally aqueous form.

The packing present composition

After mixing described antigen and adjuvant, the inventive method can comprise takes out the 0.5ml blend sample and is packaged into step in the container.Under the multiple dose situation, take out a plurality of dosage and be packaged in the single container.As mentioned above, under another kind was arranged, described antigen and adjuvant be packing separately, is facing with preceding interim mixing.

The inventive method can comprise vaccine is packaged into additional step stand-by in the container.Suitable vessel comprises medicine bottle and disposable syringe (preferred asepsis injector).

When the present composition was packaged in the medicine bottle, they were preferably made by glass or plastics.Preferably before adding described aseptic composite, described medicine bottle is carried out aseptic process.Problem for fear of latex-responsive type patient preferably seals described medicine bottle with the agalactia plug.Medicine bottle can comprise the vaccine of single dosage, perhaps can comprise an above dosage (' multiple dose ' medicine bottle), for example 10 dosage.When adopting the multiple dose medicine bottle, should under the aseptic condition of strictness, extract each dosage out, carefully avoid polluting vial content with aseptic syringe needle and syringe.Preferred medicine bottle is made by flint glass.

Medicine bottle can have the medicated cap (as syringe needle interlocking (Luer lock)) of cooperation, so that the syringe that will fill in advance inserts in the medicated cap, syringe contents can be injected medicine bottle,, then vial content is drawn back in the syringe.After taking off syringe from medicine bottle, connect syringe needle, said composition is applied to the patient.Medicated cap is preferably placed in certain sealer or the covering, makes before entering this medicated cap, must remove sealing thing or covering.

Preferably compositions is packaged in the syringe, syringe is not connected with syringe needle usually, but can provide independent syringe needle yet, so that with syringe assembling and use.The preferred security syringe needle.Generally be 1-in2 3-number, 1-in2 5-number and 5/8-in2 5-syringe needle.Syringe can be equipped with exfoliated label paper, is printing the lot number and the expiration date of content above, to help keeping records.The piston of syringe preferably has suppression device, comes off to prevent this stopper accident in aspiration procedure.Preferred butyl rubber piston suppression device.This syringe can have latex rubber medicated cap and/or piston.Disposable syringe contains the vaccine of single dose.This syringe has tip cap usually, so as before to connect syringe needle seal tips, tip cap is preferably made by butyl rubber.If separately pack syringe and syringe needle, this syringe needle butyl rubber guard shield of preferably packing into so.Preferred gray butyl rubber.Preferred syringe is commodity " Tip-Lok " by name ^TMSyringe.

When adopting glass container (as syringe or medicine bottle), preferably adopt Pyrex but not container that soda-lime glass is made.

After being packaged into compositions in the container, with this container encloses to as selling in the chests such as carton, this chest indicates the details of vaccine, as trade name, antigenic tabulation in the vaccine (for example ' hepatitis B reorganization thing ' etc.), Container Description (for example ' disposable pre-filling for general record gram (Tip-Lok) syringe ' or ' 10 * 0.5ml single dose medicine bottle '), its dosage (for example ' separately contain a 0.5ml dosage '), warning (for example ' only for the adult use ' or ' only for the child use '), expiration date, indication (for example ' hepatitis B virus (HBV) that the age is caused all known hypotypes greater than 15 years old renal insufficiency (comprise hemodialysis before and hemodialysis) patient infect produce active immunity ' etc.), the patent No. etc.Each chest can contain more than one packing vaccine, for example five or ten packing vaccines (specifically being medicine bottle).If vaccine is included in the syringe, this chest can show the picture of syringe so.

This vaccine can be sold (for example in same chest) with promotional pamphlet, and described promotional pamphlet comprises the vaccine details, the antigen details in for example administration description, the vaccine etc.Description also can contain warning, for example prepares epinephrine solution, with the anaphylaxis of protective inoculation behind the vaccine etc.

The packing vaccine preferably is stored in 2 ℃-8 ℃.Should be not freezing.

Treat and use the method for vaccine

The present composition is fit to be applied to human patients, the invention provides the method that causes patient's immunne response, and described method comprises the step that the present composition is applied to the patient.

The present invention also provides the present composition that is used for medicine.

The present invention also provides (i) antigen and the (ii) aliphatic adjuvant of basic purification under the situation that does not have surfactant, is applied to application in patient's the medicine in production.

The present invention also provide (i) comprise the antigen component of surfactant and (ii) the aliphatic adjuvant be applied to application in patient's the medicine in production, wherein (ii) described in the aliphatic adjuvant with (i) described in the weight ratio of surfactant less than 1000: 1.

Immunogenic composition of the present invention is preferably vaccine, for example is used to prevent and/or treat hepatitis b virus infected vaccine.6 week of immunity for the first time the back measure, patient's the serum of accepting the present composition is anti--HBsAg GM tires preferably 〉=500mIU/ml.More preferably, when measuring after 12 months, this is tired 〉=500mIU/ml.

Said composition is specially adapted to have now and contains Adjuvanted vaccines (as ENGERIX B ^TMProduct) invalid patient is to protect it to avoid hepatitis b virus infection and/or to treat that it is hepatitis b virus infected.The particularly suitable patient subgroups of said composition comprises: immunity weakens the patient; Hemodialysis patients; Patient before the hemodialysis; The renal insufficiency patient; Patients with renal failure; Need hemodialysis early stage patients with renal failure before; Wait for the patient of liver transplantation (for example in waiting list); Latter stage patients with renal failure; Accept the patient of organ transplantation (particularly liver transplantation), for example used for the first time the patient that vaccine of the present invention was accepted organ transplantation before in 6 months; Accept the patient of (or accepted, for example for the first time used vaccine of the present invention preceding 6 months) hepatitis B immune globulin (HBIg) treatment; HLADQ2 haplotype patient [75]; HLA DR3 haplotype patient [75]; HLA DR7 haplotype patient [75]; Patient [76] with HLA allele D QB 1*0202; The patient of infected by HIV; The Chronic HBV carrier; Accept the patient of blood transfusion recently; Accept the patient of immunosuppressive drug; AIDS patient; The patient that ascites is arranged; The sclerosis patient; Encephalopathy patient; Accept the particularly patient of ifn-α treatment of interferon; The smoking patient; The patient who smokes a cigar; Body Mass Index 〉=30kg/m ²The patient; With accept the HBsAg vaccine but patient's (for example, preliminary dosage regimen of standard, as use 3 doses of ENGERIX B of seroconversion do not take place ^TMTheir serum of back is anti--and HBsAg tires＜10mIU/ml).

These patient's creatinine clearances may be less than 30ml/min (the normal health scope be male～100-140ml/min, women 90-130ml/min).Patient preferably at least 15 years old, for example 15-40 year, 15-60 year, 40-60 year or even above 60 years old.Age, no matter the patient above 55 years old existed any disease, all can usefully treat.

The present composition can be used by intramuscular injection, for example triangle intramuscular injection.

When the present composition comprised aluminium base adjuvant, component may precipitate between the storage life.Therefore, before being applied to the patient, said composition should be shaken up.The compositions that shakes up is muddy white suspension.

The preferred process of the present invention and vaccine

The method for optimizing of preparation immunogenic composition may further comprise the steps: mix the adjuvant component that (i) comprises the HBsAg component of polysorbate 20 and (ii) contain the 3D-MPL that is adsorbed in aluminum phosphate, the weight ratio that obtains 3D-MPL and polysorbate 20 was less than 1000: 1 compositions.

Preferred immunogenic composition is the compositions that meets the following conditions: (a) said composition comprises HBsAg, polysorbate 20,3D-MPL and aluminum phosphate adjuvant; (b) weight ratio of 3D-MPL and polysorbate 20 was less than 1000: 1.3D-MPL and HBsAg preferably are adsorbed in aluminum phosphate.HBsAg concentration is about 40 μ g/ml.3D-MPL concentration is about 100 μ g/ml.Al ³⁺Concentration is about 1mg/ml.

Another kind of preferred composition of the present invention comprises (i) by the HBsAg of saccharomyces cerevisiae (S.cerevisiae) purification with (ii) contain the adjuvant of the mixture of aluminum phosphate and 3D-MPL.HBsAg concentration is about 40 μ g/ml.3D-MPL concentration is about 100 μ g/ml.Al ³⁺Concentration is about 1mg/ml.HBsAg comprises polysorbate 20, oil: the surfactant weight ratio (be 3D-MPL: the polysorbate 20 weight ratio) be 2.5: 1-100: 1, promptly the concentration of polysorbate 20 is 1 μ g/ml-40 μ g/ml (being that per 100 μ g HBsAg contain 2.5 μ g-100 μ g polysorbate 20s).This ratio is preferably 2.5: 1-25: 1, and promptly the concentration of polysorbate 20 is 4 μ g/ml-40 μ g/ml.3D-MPL and HBsAg all are adsorbed in aluminum phosphate.

General introduction

Term " contain " comprise " comprising " and " by ... form ", the compositions that for example " contains " X can only be made up of maybe X can comprise other material, for example X+Y.

Term " basically " is not got rid of " fully ", may not contain Y fully as the compositions of " being substantially free of " Y.In case of necessity, can from the present invention's definition, leave out term " basically ".

Term " about " is relevant with numerical value x meansigma methods, for example x ± 10%.

Except as otherwise noted, comprise that the method for the step of mixing two or more components is without any need for specific order by merging.Therefore, can any order mix these components.When three kinds of components were arranged, two kinds of components can be mixed earlier mutually, then blended component were mixed with the third component again etc.As mentioned above, each component can be mixed in process of production, or faces and use preceding mixing.

Antigen is described as " absorption " when adjuvant, preferably this antigen of at least 50% (weight ratio) is adsorbed, and for example 50%, 60%, 70%, 80%, 90%, 95%, 98% or more.Preferably, HBsAg adsorbs fully, promptly detect in the supernatant less than.

When using animal sources (particularly cattle) material in cell culture, they should be available from not suffering from Transmissible spongiform encephalopathy (TSE), particularly not suffering from the source of mad cow disease (BSE).

Butyl rubber comprises chlorobutyl rubber and brombutyl rubber.

Should be understood that the neutral form of ionogen shown in can this paper chemical formula exists, perhaps can charged form exist that this depends on pH.Therefore, phosphate group can be write-P-O-(OH) ₂, this chemical formula is only represented the neutral phosphor acid groups, and the present invention also comprises other charged form.Similarly, sugared ring can be opened or closed form exists, though shown closed form in this paper structural formula, the present invention also comprises opening mode.

Embodiments of the present invention

Studied three kinds of different HBsAg preparations:

In brewing yeast cell, express, use the HBsAg of nonionic surfactant purification.

In Hansenula yeast (Hanensula) cell, express, with the HBsAg of nonionic surfactant purification.

S2 sequence before in Chinese hamster ovary celI, expressing, containing, without the HBsAg of surfactant purification.

Studied two kinds of different adjuvants:

Aluminium hydroxide (1mg/ml)

MF59 (13mM) with the citrate buffer preparation

Prepare six kinds of preparations, contain 20 μ g/ml HBsAg separately, 0.15M NaCl and 0.01% thimerosal (merthiolate):

	A	B	C	D	E	F
							HBsAg	CHO	CHO	Hansenula yeast	Hansenula yeast	Saccharomyces cerevisiae	Saccharomyces cerevisiae
Adjuvant	Aluminium hydroxide	MF59	Aluminium hydroxide	MF59	Aluminium hydroxide	MF59
							NaCl	0.15M	0.15M	0.15M	0.15M	0.15M	0.15M
Thimerosal	0.01％	0.01％	0.01％	0.01％	0.01％	0.01％
							Surfactant	-	-	+	+	+	+

Report that MF59 can improve the antibody response [77] of Primate to recombinant HBsAg.With preparation A-D immunity cercopithecus aethiops.Every group of 6 monkeys were accepted the intramuscular immunity at the 0th day and the 28th day.In time 0 blood sampling, per then 2 week blood samplings once up to the 8th week, are measured anti--HBsAg tire (GMT) with ELISA.Each valence value of organizing the 14th day is normalized to 1.0, and the result after the standardization is as follows:

	A	B	C	D
					The host	CHO	CHO	Yeast	Yeast
Adjuvant	Aluminium hydroxide	MF59	Aluminium hydroxide	MF59
					Surfactant	-	-	+	+
The 14th day	1.0	1.0	1.0	1.0
					The 28th day	0.9	0.8	1.3	0.4
The 42nd day	19.2	159.8	108.8	85.2
					The 56th day	10.8	32.6	61.3	24.5

Observe and tired in the 42nd day and the 56th day, these data show, to the CHO-antigen expressed, the MF59 adjuvant can improve more significantly than aluminium adjuvant and tired (the 42nd natural gift you can well imagine high 160 times with 20 times), but the antigenic situation of expressed by Hansenula yeast just in time opposite (the 42nd natural gift you can well imagine high 85 times with 110 times).

Adopt preparation B, E and F to carry out similar experiment with baboon.After injecting 14 days for the first time, measure GMT value with respect to the F class value, as follows:

	A	B	E	F
					Surfactant	-	-	+	+
Adjuvant	Aluminium hydroxide	MF59	Aluminium hydroxide	MF59
					The 14th day	-	13.5	4.5	1.0

In this article, reply (B group and list of references 77), replace aluminium adjuvant to cause GMT to reply lower (relatively E group and F group) with MF59 though expectation can improve.

After adopting preparation E and F to inject 28 days for the first time, relatively anti--the HBsAg of macaque, cercopithecus aethiops and C3H mouse replys (NB: mice is accepted 60%HBsAg dosage).Except measuring the GMT value, also measure respondent's quantity, promptly tire＞number of animals of 10mIU/ml.The result that relative F group data are carried out after the standardization is as follows:

Therefore, opposite with mice, for the HBsAg of saccharomyces cerevisiae-expression, in Primate, cause GMT value and respondent's level significantly to descend as adjuvant with MF59.

Finally, detecting the GMT that produces with preparation E and the inoculation of F booster immunization in the cercopithecus aethiops of seropositivity reaction improves.Before booster immunization and booster immunization measures after 28 days and tires, the raising of tiring is as follows:

Group	Adjuvant	Improve (x)
			E	Aluminium hydroxide	91.5x
F	MF59	19.1x

Except the raising multiple that causes than aluminum hydroxide adjuvant hangs down, use booster immunization dosage after 28 days, the GMT value that MF59 produces is also lower.

Therefore, all in all, comparing with aluminium hydroxide, for the seemingly relatively poor adjuvant of the HBsAg MF59 of yeast expression, but is preferable adjuvant for the HBsAg that CHO-expresses in Primate.These results may be interpreted as (a) by residual surfactant among the HBsAg of yeast purification and (b) interaction between the excessive zamene oil in the MF59 adjuvant.As oil, zamene acts on mutually with surfactant under aqueous conditions, and vice versa, suggestion in the antigen surfactant and adjuvant in oily incompatible.Can be under without the situation of detergent Purification of HBsAg (for example, expressing in the material) at CHO-, or the excessive degree that guarantees oil is unlikely to disturb the surfactant in the antigen, thereby solves this incompatibility.

In the HBsAg compositions that with MF59 is adjuvant, the MF59 of a volume is mixed with the antigenic solution of a volume.Therefore, in the 100ml volume, contain 50ml MF59 and 50ml HBsAg solution.This 100ml contains the zamene of 2.15g from MF59, and HBsAg concentration is 40 μ g/ml, and it contains 2mg HBsAg.Polysorbate 20 concentration is 25 μ g[35 among per 100 μ gHBsAg] time, this 100ml contains the 0.5mg polysorbate 20.With MF59 is the vaccine medium oil of adjuvant: the surfactant ratio generally is 4300: 1.By excessive oil is reduced to less than 1000: 1, can reduce the interaction of surfactant in adjuvant medium oil and the antigen greatly, disturb thereby reduce.

Only should be understood that and described the present invention, can under the situation that does not exceed the scope of the invention and design, make amendment with way of example.

List of references (this paper is for referencial use with fitting in it)

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[17]WO 94/21292。

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[20]WO96/26741。

[21]WO93/19780。

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[23]WO00/00462。

[24] (2003) Vaccine 21:2485-2491 such as Meraldi.

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[26] (2000) Eur J Biochem 267:3370-7 such as Brandenburg.

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[29] United States Patent (USP) the 6th, 764, No. 840.

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[31] (200I) J Biol.Chem 276:1873-80 such as Lien.

[32]WO03/011223。

[33] (2003) J Clin Pharmacol 43 (7): 735-42 such as Wong.

[34]US2005/0215517。

[35] (1997) Rev.Inst.Med.trop.S.Paulo such as Costa the 39th volume, the 1st phase

Paulo Ene.

[36] (2005) J Gen Virol 86:323-31 such as Vanlandschoot.

[37] Hilleman (1993) Hepatitis B vaccines in clinical practice (Hepatitis B virus vaccine in the clinical practice), 17-40 page or leaf, ISBN 0-8247-8780-3.

[38] (1969) J Virol 4:163 such as Gerin.

[39] (1969) J Virol 7:569 such as Gerin.

[40] (1972) Acta Pathol Microbiol Scan sect B 80:935 such as Siebke.

[41] (1976) J Clin Microbiol 4:205 such as Pillot.

[42] Duimel and Krijnen (1972) Vox Sang 23:249.

[43] (1974) PNAS USA 71:2663 such as Neurath.

[44] Charm and Wong (1975) Biotechnol Bioeng 16:593.

[45] United States Patent (USP) the 4th, 857, No. 317.

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[47] Houwen etc. Y91S) J Immunol Methods 8:185.

[48] (1993) Hepatitis B vaccines in clinical practice (Hepatitis B virus vaccine in the clinical practice) such as Sitrin, 83-101 page or leaf, ISBN 0-8247-8780-3.

[49] (1985) PNAS USA 82:6830-4 such as Wampler.

[50] (1994) Ann N Y Acad Sci 721:365-73 such as Chi.

[51] (1994) J Chromatogr A 672:25-33 such as Agraz.

[52] (1992) PNAS USA 89:11745-9 such as Mason.

[53] (1999) J Virol Methods 79:205-17 such as Demi.

[54] (1999) J Chromatogr B Biomed Sci Appl 735:271-7 such as Ibarra.

[55]WO90/10058。

[56] Japanese patent application is JP63239234 number.

[57] United States Patent (USP) the 4th, 694, No. 074.

[58]EP-0341733。

[59] United States Patent (USP) the 5th, 242, No. 812.

[60] the Russ P application is RU-2128707 number.

[61] (2000) J Biotechnol 77:157-67 such as Hardy.

[62] (2000) Biotechnol Prog 16:435-41 such as Dogan.

[63] (2004) Virology 321 (1): 75-S6 such as Berkower.

[64] (1996) J Gen Virol 77:2001-8 such as Eckhart.

[65] (1991) Vaccine 9 (7): 477-84 such as von Brunn.

[66] (1991) Am J Trop MedHyg 45 (5): 533-8 such as Vreden.

[67] (1995) MolBiochem Parasitol 72 (1-2): 179-92 such as Moelans.

[68] (1997) N Engl J Med 336 (2): 86-91 such as Stoute.

[69] Wunderlich and del Portillo (2000) Mol Med 6 (3): 238-45.

[70] United States Patent (USP) the 5th, 928, No. 902

[71]WO93/10152。

[72] (2001) Vaccine 27:3645-51 such as Nony.

[73]WO02/12287。

[74]WO03/066094。

[75] (1998) Tissue Antigens 51:593-604 such as Desombere.

[76] (1997) Tissue Antigens 50:8-14 such as McDermott.

[77] (1996) J Infect Dis 174:1168-75 such as Traquina.

Sequence table

＜110〉NOVARTIS VACCINES ﹠ DIAGNOSTIC (NOVARTIS VACCINES AND DIAGNOSTICS SRL)

M. Ken Tuoni (CONTORNI Mario)

＜120〉reduce oil-containing adjuvant and contain interference between the surfactant antigen

<130>P041598WO

<140>PCT/IB2006/YYYYYY

<141>2006-08-02

<150>GB-0515906.6

<151>2005-08-02

<150>GB-0522599.0

<151>2005-11-04

<150>GB-0523923.1

<151>2005-11-24

<160>2

<170>SeqWin99，version 1.02

<210>1

<211>4

<212>PRT

＜213〉Plasmodium falciparum (Plasmodium falciparum)

<400>1

Asn Ala Asn Pro

1

<210>2

<211>423

<212>PRT

＜213〉Plasmodium falciparum (Plasmodium falciparum)

<400>2

Met Met Ala Pro Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala

1 5 10 15

Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala

20 25 30

Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala

35 40 45

Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala

50 55 60

Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala

65 70 75 80

Asn Pro Asn Lys Asn Asn Gln Gly Asn Gly Gln Gly His Asn Met Pro

85 90 95

Asn Asp Pro Asn Arg Asn Val Asp Glu Asn Asn Ala Asn Asn Ala Val

100 105 110

Lys Asn Asn Asn Asn Glu Glu Pro Ser Asp Lys His Ile Glu Gln Tyr

115 120 125

Leu Lys Lys Ile Lys Asn Ser Ile Ser Thr Glu Trp Ser Pro Cys Ser

130 135 140

Val Thr Cys Gly Asn Gly Ile Gln Val Arg Ile Lys Pro Gly Ser Ala

145 150 155 160

Asn Lys Pro Lys Asp Glu Leu Asp Tyr Glu Asn Asp Ile Glu Lys Lys

165 170 175

Ile Cys Lys Met Glu Lys Cys Ser Ser Val Phe Asn Val Val Asn Ser

180 185 190

Arg Pro Val Thr Asn Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro

195 200 205

Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr

210 215 220

Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly

225 230 235 240

Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn

245 250 255

His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met

260 265 270

Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu

275 280 285

Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys

290 295 300

Pro Leu Ile Pro Gly Ser Thr Thr Thr Asn Thr Gly Pro Cys Lys Thr

305 310 315 320

Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys

325 330 335

Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser

340 345 350

Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser

355 360 365

Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser

370 375 380

Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro

385 390 395 400

Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe

405 410 415

Phe Cys Leu Trp Val Tyr Ile

420

Claims (36)

1. method for preparing immunogenic composition, said method comprising the steps of, mix (i) and contain the antigenicity component of surfactant and (ii) aliphatic adjuvant component, the weight ratio that obtains described aliphatic adjuvant and described surfactant was less than 1000: 1 compositions.

2. immunogenic composition, wherein: (a) described compositions comprises antigen component and aliphatic adjuvant component; (b) described antigen component comprises surfactant; (c) weight ratio of described aliphatic adjuvant and described surfactant was less than 1000: 1.

3. method as claimed in claim 1 or 2 or compositions is characterized in that, but described aliphatic adjuvant comprises metabolism oil.

4. method as claimed in claim 3 or compositions is characterized in that, described aliphatic adjuvant comprises the submicron oil in water emulsion of zamene and polyoxyethylene sorbitan monoleate.

5. method as claimed in claim 1 or 2 or compositions is characterized in that, described aliphatic adjuvant comprises the molecule that contains fatty acid part.

6. method as claimed in claim 5 or compositions is characterized in that, the monophosphoryl lipid A (' 3D-MPL ') that described aliphatic adjuvant is a 3-deoxidation acyl groupization.

7. method as claimed in claim 6 or compositions is characterized in that, described 3D-MPL is the mixture of the different molecule of acyl group degree.

8. as claim 6 or 7 described method or compositionss, it is characterized in that described aliphatic adjuvant comprises:

9. as each described method or compositions among the claim 6-8, it is characterized in that described 3D-MPL is a particle form.

10. method as claimed in claim 9 or compositions is characterized in that, but described granule filtration sterilization.

11., it is characterized in that described 3D-MPL and the coupling of aluminum salt as each described method or compositions among the claim 6-10.

12. method as claimed in claim 11 or compositions is characterized in that, described aluminum salt is aluminum phosphate.

13. method as claimed in claim 12 or compositions is characterized in that, described 3D-MPL is adsorbed on the described aluminum salt.

14. each described method or compositions in the claim is characterized in that described surfactant is a polyoxyethylene sorbitan esters as described above.

15. method as claimed in claim 14 or compositions is characterized in that, described ester is a polysorbate 20.

16., it is characterized in that described surfactant concentrations is not higher than 50 μ g/ml as claim 14 or 15 described method or compositionss.

17. each described method or compositions in the claim is characterized in that described antigen is viral surface antigen as described above.

18. method as claimed in claim 17 or compositions is characterized in that, the granule hbs antigen (' HBsAg ') that described antigen is purification in the presence of surfactant.

19. method as claimed in claim 18 or compositions is characterized in that, described HBsAg expresses in yeast cells.

20. method as claimed in claim 19 or compositions is characterized in that, described yeast is a saccharomyces cerevisiae.

21., it is characterized in that described HBsAg is nonglycosylated, and/or comprise phosphatidylinositols as each described method or compositions among the claim 18-20.

22., it is characterized in that described HBsAg is from the adw2 hypotype of hepatitis B virus as each described method or compositions among the claim 18-21.

23. each described method or compositions in the claim is characterized in that as described above, described antigen is the hybridization albumen that comprises viral surface antigen and heterologous antigen.

24. method as claimed in claim 23 or compositions is characterized in that, described viral surface antigen is HBsAg, and described heterologous antigen is a malaria antigen.

25. method as claimed in claim 24 or compositions is characterized in that, described hybridization albumen comprises the proteic fragment of ring sporinite of HBsAg and Plasmodium falciparum.

26. method as claimed in claim 25 or compositions is characterized in that, described hybridization albumen comprises four or a plurality of tandem repetitive sequence and the HBsAg in the proteic C end parts of Plasmodium falciparum ring sporinite, ring sporinite albumen advantage immunity district.

27. each described method or compositions in the claim is characterized in that the weight ratio of described aliphatic adjuvant and described surfactant was less than 500: 1 as described above.

28. each described method or compositions in the claim is characterized in that the weight ratio of described aliphatic adjuvant and described surfactant was less than 50: 1 as described above.

29. each described method or compositions in the claim is characterized in that as described above, described aliphatic adjuvant is 3D-MPL, and the weight ratio of described aliphatic adjuvant and described surfactant is 2.5: 1-25: 1.

30. each described method or compositions in the claim is characterized in that as described above, the osmotic pressure of described compositions is 200mOsm/kg-400mOsm/kg.

31. each described method or compositions in the claim is characterized in that described compositions comprises phosphate buffer as described above.

32. each described method or compositions in the claim is characterized in that as described above, the pH of described compositions is 6.0-7.0.

33. (i) comprise the antigen component of surfactant and (ii) the aliphatic adjuvant be applied to application in patient's the medicine in production, wherein (ii) described in the aliphatic adjuvant with (i) described in the weight ratio of surfactant less than 1000: 1.

34. method for preparing immunogenic composition, said method comprising the steps of: mix the adjuvant component that (i) contains the HBsAg component of polysorbate 20 and (ii) contain the 3D-MPL that is adsorbed in aluminum phosphate, the weight ratio that obtains described 3D-MPL and polysorbate 20 was less than 1000: 1 compositions.

35. an immunogenic composition, wherein: (a) described compositions contains HBsAg, polysorbate 20,3D-MPL and aluminum phosphate adjuvant; (b) weight ratio of described 3D-MPL and polysorbate 20 was less than 1000: 1.

36. a method for preparing immunogenic composition, wherein: (a) described compositions comprises antigen and aliphatic adjuvant; (b) the basic described antigen of purification under the situation that does not have surfactant.