CN101325967A - Influenza vaccines with reduced amount of emulsion adjuvant - Google Patents
Influenza vaccines with reduced amount of emulsion adjuvant Download PDFInfo
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- CN101325967A CN101325967A CNA2006800463581A CN200680046358A CN101325967A CN 101325967 A CN101325967 A CN 101325967A CN A2006800463581 A CNA2006800463581 A CN A2006800463581A CN 200680046358 A CN200680046358 A CN 200680046358A CN 101325967 A CN101325967 A CN 101325967A
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Abstract
An influenza virus vaccine comprising oil-in-water emulsion adjuvant is known, which can reduce dosage of the emulsion adjuvant required by the influenza virus vaccine so as to prepare more vaccines with a given amount of emulsion and/or miniaturize the amount of produced emulsion that is necessary for obtaining the given amount of vaccine dosage. These vaccines can be conveniently prepared by blending (i) the oil-in-water emulsion and (ii) aqueous products of influenza virus antigen. In one hand, ingredients (i) and (ii) having substantially identical volume are employed; in the other hand, the ingredients (ii) having excess volume can be utilized. When the substantially identical volume is applied, concentration of hemagglutinin of the ingredients (ii) is more than 60 mug/influenza virus strain/ml. The ingredients (i) and (ii) can be provided in a form of kit.
Description
The All Files that this paper quotes is included this paper in as a reference in full.
Technical field
The invention belongs to adjuvant preparation vaccine provides protection to resist the field of influenza infection.
Background technology
Conventional at present influenza vaccines of using generally do not contain adjuvant.The 17th and 18 chapters of list of references 1 are described these vaccines in detail.They are based on the virus of live virus or deactivation, and inactivated vaccine can be based on the surface antigen (comprising hemagglutinin and neuraminidase) of complete virus, " cracking " virus or purification.Hemagglutinin (HA) is a main immunogens in the influenza vaccines of deactivation, and with reference to HA level standard vaccine dose, vaccine contains the 15 μ g HA/ strains of having an appointment usually.
The flu outbreak phase needs a large amount of influenza vaccines, thereby but is difficult to increase the satisfied so huge demand of vaccine supply.Therefore, except producing more multi-vaccine antigen, the someone proposes to use the antigen/strain of lower content, uses adjuvant to remedy the antigen dose of minimizing simultaneously.The somebody proposes to adopt identical scheme in popular interval, thereby for example can cover more crowds and need not to improve the level of production.
Can buy a kind of vaccine of adjuvant preparation already, i.e. FLUAD
TMProduct.Adjuvant in this vaccine is an O/w emulsion.FLUAD
TMProduct is pre-mixed antigen and emulsion adjuvant in pre-filled syringe and provides.Product data sheet shows that each dose volume is 0.5ml, contains 15 μ g HA/ strains, 9.75mg Squalene, 1.175mg polysorbate80,1.175mg anhydrosorbitol trioleate, 0.66mg sodium citrate, 0.44mg citric acid and water for injection.Disclosed as list of references 2, mix 2 * emulsion and 2 * antigenic solution preparing vaccine with 1: 1 volume ratio, thereby obtain containing the emulsion and the antigenic final solution of 1 * concentration.The 10th chapter of list of references 8 has further been explained this 1: 1 mixed proportion.
Other improvement influenza vaccines (for popular phase and the use of popular interval) and their preparation method that the purpose of this invention is to provide moisture bag oil emulsion.
Summary of the invention
Except FLUAD
TMProduct, the influenza vaccines of having checked experimental MF-59 adjuvant to prepare in the people comprise the vaccine that antigen dose reduces.For example, in list of references 3 and 4, checked the vaccine based on the MF-59 adjuvant preparation of H5N3 strain, wherein contained HA is normal dose (15 μ g), doubling dosage (30 μ g) or half-value dose (7.5 μ g).Yet in all situations, the patient all accepts to contain the 0.5ml volume premixing vaccine of full dose (1 *) MF59 adjuvant.
Now, the inventor recognizes the oil in water emulsion adjuvant that does not always need full dose.Therefore, the present invention has reduced the amount of the required emulsion adjuvant of influenza vaccines, thus can with the emulsion preparation of specified rate more multi-vaccine and/or make must production into the vaccine dose institute that obtains to give determined number emulsion amount minimum.If need fast by a large amount of (vaccine), the aggregate demand that reduces emulsion adjuvant is favourable.
This area is known already, by mix (i) O/w emulsion and (ii) the water-based product of influenza antigen can prepare these vaccines easily.Yet mixed method of the present invention all is different from prior art in every respect, with FLUAD
TMProduct is compared, and the selection of method has various influences to dosage and volume.For example:
The same with prior art, use the component (i) of substantially the same volume and (ii).Yet, be to use the emulsion that is lower than 0.25ml can reduce the required volume of the final vaccine of preparation with the different of prior art.Reduce to remedy volume if increase the antigen concentration of component in (ii), then can keep the antigen amount (15 μ g/ strain) in the final vaccine; If (15 μ g/ strains/0.25ml) reduce to remedy volume, then the HA dosage of final vaccine reduces to keep the antigen concentration of component in (ii).
Unlike the prior art be can utilize excess volume component (ii).If the amount of the reduction of antigen concentration and increase is complementary, then can keep the antigen amount (15 μ g/ strain) in the final vaccine; If antigen concentration reduces the amount that increases that surpassed, then final antigen dose reduces (＜15 μ g/ strain); If (15 μ g/ strains/0.25ml), then the HA dosage of final vaccine increases (＞15 μ g/ strain) to keep antigen concentration.
Therefore, the invention provides the method for the influenza vaccines of preparation adjuvant preparation, comprise (i) O/w emulsion that mixes equal volume basically and the (ii) step of influenza antigen water-based product, wherein the concentration of the hemagglutinin of component in (ii) is greater than 60 μ g/ influenza virus strain/ml.Mix by equal-volume, HA concentration after mixing＞30 μ g/ influenza virus strain/ml.Therefore, this method obtains the compositions of final HA concentration＞15 μ g/ strains in the 0.5ml volume, so give＜this vaccine of 0.5ml can realize the dosage of 15 μ g/ strains.
The present invention also provides the method for the influenza vaccines of preparation adjuvant preparation, comprises (i) O/w emulsion that mixes equal volume basically and the (ii) step of influenza antigen water-based product, and wherein the volume of vaccine is less than 0.5ml.Described volume can be between 0.05ml-0.45ml, between the 0.1ml-0.4ml, and between the 0.2ml-0.3ml, or the like.Therefore, described volume can be about 0.1ml, about 0.15ml, about 0.2ml, about 0.25ml, about 0.3ml, about 0.35ml, about 0.4ml or about 0.45ml, or the like.Concentration as the hemagglutinin of fruit component in (ii) is about 60 μ g/ influenza virus strain/ml, and then the final HA concentration of vaccine is about 30 μ g/ influenza virus strain/ml, so during volume＜0.5ml, HA dosage is lower than 15 μ g/ agent/strains.Thereby can reduce adjuvant and antigen demand.
The present invention also provides the method for the influenza vaccines of preparation adjuvant preparation, may further comprise the steps: (a) mix (i) O/w emulsion of equal volume basically and (ii) influenza antigen water-based product, thereby obtain bulk vaccine (bulk vaccine); (b) take out at least a unit dose from described bulk vaccine, the volume of wherein said unit dose as mentioned above＜0.5ml.
Therefore, the present invention also provides the method for the influenza vaccines of preparation adjuvant preparation, comprise (i) O/w emulsion that mixes equal volume basically and the (ii) step of influenza antigen water-based product, wherein in the vaccine concentration of hemagglutinin less than 30 μ g/ influenza virus strain/ml.Therefore, final HA concentration＜15 μ g/ strains in the compositions of the standard 0.5ml volume of this method acquisition.Can about 0.5ml or as mentioned above＜the described vaccine of 0.5ml dosage.
The present invention also provides the method for the influenza vaccines of preparation adjuvant preparation, comprises the step of the influenza antigen water-based product of the O/w emulsion that mixes first volume and second volume, and wherein said second volume is greater than described first volume.In some embodiments, the hemagglutinin concentration in described second volume is less than 60 μ g/ influenza virus strain/ml.
These methods can be implemented can distribute these vaccines and give the patient to obtain premixed vaccine during a large amount of the manufacturing.Perhaps, thus can in using, implement the temporary preparation that these methods obtain give the patient.In one situation of back, the invention provides a kind of test kit, it is equipped with: the adjuvant component that (i) comprises O/w emulsion; The antigen component that (ii) comprises the water-based product of influenza antigen.Component (i) does not comprise influenza antigen, and component (ii) is not an O/w emulsion.The volume of these reagent constituents is substantially the same, and in this case, the component (ii) concentration of middle hemagglutinin may be no more than 60 μ g/ influenza virus strain/ml.Perhaps, what reagent constituents volume (ii) can be greater than component (i), preferred hemagglutinin concentration is lower than 60 μ g/ influenza virus strain/ml.In some embodiments, described two kinds of reagent constituents volume separately is less than 0.25ml, thereby acquisition is less than the final volume (as mentioned above) of 0.5ml after mixing.
The present invention also provides and adopts the obtainable compositions of the inventive method.These compositionss comprise and the blended influenza antigen of O/w emulsion.In these compositionss, HA concentration can for example the invention provides the compositions that comprises O/w emulsion and influenza antigen greater than 30 μ g/ strain/ml, and wherein said compositions provides 15 μ g HA/ influenza virus strain/agent, and dose volume is less than 0.5ml.
The present invention also provides the vaccine combination that comprises O/w emulsion and influenza antigen, and the volume of wherein said vaccine (as mentioned above) is less than 0.5ml.The present invention also provides the vaccine combination that comprises influenza virus hemagglutinin and Squalene, the weight ratio of Squalene and hemagglutinin (for example, between the 100-1000) between 50-2000 wherein, and the volume of wherein said vaccine (as mentioned above) is lower than 0.5ml.In the vaccine HA concentration can＞30 μ g/ strain/ml.
The present invention also provides the compositions that comprises influenza virus hemagglutinin and Squalene, the mass ratio of Squalene and hemagglutinin (for example, between the 100-1000) between 50-2000 wherein, and wherein HA concentration is less than 30 μ g/ strain/ml.The immunity inoculation unit dose of said composition can be about 0.5ml, perhaps can be lower than 0.5ml as mentioned above.
The present invention also provides the compositions of the hemagglutinin that comprises O/w emulsion and influenza virus strain, and the weight ratio of its medium oil and hemagglutinin is lower than 640/n.Described ratio can be, for example≤600/n ,≤550/n ,≤500/n ,≤450/n ,≤400/n ,≤350/n ,≤300/n ,≤250/n ,≤200/n ,≤150/n ,≤100/n ,≤50/n, or the like.N value preferably 1,2,3 or 4.Described oil preferably comprises Squalene.The immunity inoculation unit dose of said composition can be about 0.5ml, perhaps can be lower than 0.5ml as mentioned above.
The present invention also provides the method for the unit price influenza vaccines of preparation adjuvant preparation, comprise (i) O/w emulsion that mixes equal volume basically and (ii) a kind of step of influenza antigen water-based product of strain, wherein said equal volume (for example is lower than 0.22ml, ≤ 0.21ml ,≤0.20ml ,≤0.19ml ,≤0.18ml ,≤0.16ml ,≤0.15ml ,≤0.14ml ,≤0.13ml ,≤0.12ml ,≤0.11ml ,≤0.10ml ,≤0.09ml ,≤0.08ml ,≤0.07ml ,≤0.06ml ,≤0.05ml, or the like).The reagent corresponding box also is provided.For popular influenza strain, these unit price compositionss and test kit particularly useful (vide infra).
The present invention also provides the method for the unit price influenza vaccines of preparation adjuvant preparation, comprises the step of the influenza antigen water-based product of the O/w emulsion that mixes first volume and second volume, and wherein said second volume is greater than described first volume.In some embodiments, the hemagglutinin concentration in described second volume is less than 60 μ g/ml.
The present invention also provides the method for the unit price influenza vaccines of preparation adjuvant preparation, comprise (i) O/w emulsion that mixes equal volume basically and (ii) a kind of step of influenza antigen water-based product of strain, the Squalene concentration of wherein said emulsion is lower than 38mg/ml.Therefore, the concentration of mixing back Squalene is lower than 19mg/ml.Also provide the reagent corresponding box, the preferably about 0.25ml of described in this case equal volume.For popular influenza strain, these unit price compositionss and test kit particularly useful (vide infra).Squalene concentration in the component (i) can be, for example≤37mg/ml, ≤ 36mg/ml, ≤ 35mg/ml, ≤ 34mg/ml, ≤ 33mg/ml, ≤ 32mg/ml, ≤ 31mg/ml, ≤ 30mg/ml, ≤ 29mg/ml, ≤ 28mg/ml, ≤ 27mg/ml, ≤ 26mg/ml, ≤ 25mg/ml, ≤ 24mg/ml, ≤ 23mg/ml, ≤ 22mg/ml, ≤ 21mg/ml, ≤ 20mg/ml, ≤ 19mg/ml, ≤ 18mg/ml, ≤ 17mg/ml, ≤ 16mg/ml, ≤ 15mg/ml, ≤ 14mg/ml, ≤ 13mg/ml, ≤ 12mg/ml, ≤ 11mg/ml, ≤ 10mg/ml, ≤ 9mg/ml, ≤ 8mg/ml, ≤ 7mg/ml, ≤ 6mg/ml, ≤ 5mg/ml, or the like.
Oil in water emulsion adjuvant
Found that O/w emulsion is specially adapted in the influenza virus vaccine of adjuvant preparation.Known various such emulsion, they comprise at least a oil and at least a surfactant usually, and wherein one or more oil and one or more surfactants are biodegradables (but metabolism) and biocompatible.Oil droplet general diameter in the emulsion is lower than 5 μ m, even can have sub-micron diameter, can realize these small sizes with the Micro Fluid instrument, thereby stable emulsion can be provided.Preferred size is less than the oil droplet of 220nm, because can carry out filtration sterilization to them.
The present invention can use the oil such as animal origin (for example fish) or plant origin.The vegetable oil source comprises nut, seed and corn.The example of macadamia nut oil is the most normal Oleum Arachidis hypogaeae semen of buying, soybean oil, cocos nucifera oil and olive oil.For example, can utilize the Jojoba oil that obtains from flash Fructus Crotonis (jojoba bean).Seed oil comprises safflower oil, Oleum Gossypii semen, Oleum Helianthi, Semen Sesami wet goods.In corn oil, Semen Maydis oil is the easiest to be buied, but the oil of other corn such as Semen Tritici aestivi, Herba bromi japonici, rye (Secale cereale L.), rice, Herba Eragrostidis pilosae (teff), black Semen Tritici aestivi etc. also can utilize.Nut and the seed oil initial substance suitable by hydrolysis, separation and esterification can prepare natural non-existent glycerol and 1 in the seed oil, the 6-10 carbocyclic aliphatic acid esters of 2-propylene glycol.The fat of mammal milk and oils are metabolizable, therefore can be used for implementing the present invention.Well knownly obtain the required separation of pure oil, purification, saponification and other method from animal origin.But most of fishes contain the metabolism oil of being not difficult to reclaim.For example, the example of the available several fish oil of the present invention is cod liver oil, shark liver oil and whale oil, as spermaceti.Can 5-carbon isoprene unit synthesize many side chain oil by biochemical method, these side chain oil are commonly referred to terpenoid.Shark liver oil contains the unsaturated terpenoid of side chain that is called Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-24 hexenes (tetracosahexaene)), and this is that the present invention is particularly preferred.Squalane is the saturated analogues of Squalene, and it also is preferred oil.The fish oil that comprises Squalene and squalane is not difficult to buy from commercial source, maybe can obtain by methods known in the art.Other preferred oil is tocopherol (vide infra).Can utilize the mixture of oil.
Can be according to " HLB " (hydrophilic) of surfactant with its classification.The HLB of preferred surfactant of the present invention is at least 10, and preferably at least 15, more preferably at least 16.The available surfactant of the present invention comprises but is not limited to: polyoxyethylene sorbitol acid anhydride ester surfactant (being commonly referred to tween), particularly polysorbate20 and polysorbate80; The copolymer of oxirane (EO), expoxy propane (PO) and/or epoxy butane (BO) is with DOWFAX
TMFor trade name is sold for example linear EO/PO block copolymer; Multiple ethyoxyl (oxygen-1,2-second two bases)) Octoxinol that the group number may be different, wherein interested especially is octoxynol 9 (triton x-100 or uncle's octylphenoxy polyethoxy ethanol); (Octylphenoxy) polyethoxy ethanol (IGEPAL CA-630/NP-40); Phospholipid, for example poly-choline (lecithin) of phosphatidyl; Nonyl phenol ethoxylate, for example Tergitol
TMNP series; Derived from the polyoxyethylene aliphatic ether (being called the Brij surfactant) of lauryl alcohol, spermol, stearyl alcohol and oleyl alcohol, for example trietbhlene glycol list lauryl ether (Brij 30); Polyoxyethylene-9-lauryl ether; And sorbitan esters (being commonly referred to SPAN), for example anhydrosorbitol trioleate (sorbester p37) and sorbitan monolaurate.Contained preferred surfactant is Tween 80 (Tween-81), sorbester p37 (anhydrosorbitol trioleate), lecithin and triton x-100 in the emulsion.
Can utilize surfactant mixtures, for example Tween 80/sorbester p37 mixture, or Tween 80/triton-X100 mixture.Polyoxyethylene sorbitol acid anhydride ester, for example Tween-81 (Tween 80) and Octoxinol, for example the mixture of uncle's octylphenoxy polyethoxy ethanol (triton x-100) also is suitable for.Another useful mixture comprises laureth 9 and adds polyoxyethylene sorbitol acid anhydride ester and/or Octoxinol.
The preferable amount of surfactant (weight %) is: polyoxyethylene sorbitol acid anhydride ester (for example, Tween 80) 0.01-1%, particularly about 0.1%; Octyl group-or Nonylphenoxy polyoxy ethanol (for example other detergent of triton x-100 or triton series) 0.001-0.1%, particularly 0.005-0.02%; Polyoxyethylene ether (for example laureth 9) 0.1-20%, preferred 0.1-10%, particularly 0.1-1% or about 0.5%.
The used concrete oil in water emulsion adjuvant of the present invention includes but not limited to:
The submicron emulsion of Squalene, Tween 80 and sorbester p37.By volume, the composition of described emulsion can be about 5% Squalenes, about 0.5% polysorbate80 and about 0.5% sorbester p37.By weight, these ratios can be 4.3% Squalenes, about 0.5% polysorbate80 and 0.48% sorbester p37.This adjuvant is called " MF59 " [5-7], is described in detail as the 12nd chapter of the 10th Zhanghe list of references 9 of list of references 8.The MF59 emulsion preferably comprises citrate ions, for example the 10mM sodium citrate buffer solution.
The emulsion of Squalene, tocopherol and Tween 80.This emulsion can comprise phosphate-buffered saline.It also can comprise sorbester p37 (for example, 1%) and/or lecithin.These emulsions can have 2-10% Squalene, 2-10% tocopherol and 0.3-3% Tween 80, and the weight ratio of Squalene and tocopherol is preferred≤and 1, because this can provide more stable emulsion.The volume ratio of Squalene and Tween 80 can be about 5: 2.Tween 80 can be dissolved in and obtain 2% solution among the PBS, then the mixture of this solution of 90ml with (5g DL-alpha-tocopherol and 5ml Squalene) be mixed, this mixture of Micro Fluid prepares a kind of like this emulsion subsequently.The emulsion that obtains can have the submicron oil droplet, for example average diameter between 100-250nm, preferably about 180nm.
The emulsion of Squalene, tocopherol and triton detergent (for example, triton x-100).This emulsion also can comprise 3d-MPL (seeing below).This emulsion can comprise phosphate buffer.
The emulsion that contains polysorbate (for example, polysorbate80), triton detergent (for example, triton x-100) and tocopherol (for example, alpha-tocofecol succinic acid ester).The mass ratio of these three kinds of components that this emulsion comprised (for example is about 75: 11: 10,750 μ g/ml polysorbate80s, 110 μ g/ml triton x-100s and 100 μ g/ml alpha-tocofecol succinic acid esters), these concentration should comprise these components and antigenic any ratio.This emulsion also can comprise Squalene.This emulsion also can comprise 3d-MPL (seeing below).Water can contain phosphate buffer.
Squalane, polysorbate80 and poloxamer 401 (" Pluronic
TMThe emulsion of L121 ").Can use phosphate-buffered saline, pH 7.4 these emulsions of preparation.This emulsion is a kind of useful muramyldipeptide delivery vector, with the threonyl-MDP coupling [10] of preparing with " SAF-I " adjuvant (0.05-1%Thr-MDP, 5% Squalene alkane, 2.5%Pluronic L121 and 0.2% polysorbate80).Can be not and the Thr-MDP coupling yet, for example use " AF " adjuvant (5% squalane, 1.25%Pluronic L121 and 0.2% polysorbate80) preparation [11].Preferred microfluidization.
Emulsion can have oil, 0.1-10% phospholipid and the 0.05-5% non-ionic surface active agent of 0.5-50%.As described in list of references 12, preferred phospholipid fraction is phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidylinositols, phosphatidyl glycerol, phosphatidic acid, sphingomyelins and cuorin.Preferred submicron droplets size.
The submicron O/w emulsion of nonmetabolizable oil (for example, light mineral oil) and at least a surfactant (for example, lecithin, Tween 80 or sorbester p17).Can comprise additive, for example QuilA saponin, cholesterol, saponin-lipophilic conjugate (for example, list of references 13 described GPI-0100), dimethyl two (octadecyl) ammonium bromide and/or N, N-two (octadecyl)-N, N-two (2-ethoxy) propane diamine.
Wherein saponin (for example, QuilA or QS21) and sterin (for example, cholesterol) are combined into the micellar emulsion of helical form [14].
The emulsion [15] that comprises mineral oil, nonionic lipotropy ethoxylized fatty alcohol and nonionic hydrophilic surfactant active (for example, ethoxylized fatty alcohol and/or polyox-yethylene-polyoxypropylene block copolymer).
The emulsion [15] that comprises mineral oil, nonionic lipotropy ethoxylized fatty alcohol and nonionic lipophilic surfactant (for example, ethoxylized fatty alcohol and/or polyox-yethylene-polyoxypropylene block copolymer).
These emulsions are preferably mixed with antigen in sending temporarily.Therefore, in the vaccine of packing or distribution, adjuvant and antigen are separately deposited usually, at last preparation immediately in use.Generally there is aqueous form in antigen, thereby finally can prepare vaccine by mixing two kinds of liquid.Given all concentration of component of concrete emulsion in above description, these concentration generally are undiluted, therefore mix the described concentration in back with antigenic solution and can reduce.
After antigen and adjuvant mixed, hemagglutinin antigen maintained in the aqueous solution usually, but himself may be distributed near oil/water termination.Enter the hemagglutinin usually few (if any) of the oil phase of emulsion.
If compositions comprises tocopherol, α, β, γ, δ, ε or ξ tocopherol are all available, but preferred alpha-tocopherol.Tocopherol can take several forms, for example different salt and/or isomer.Salt comprises organic salt, for example succinate, acetate, nicotinate or the like.D-alpha-tocopherol and DL-alpha-tocopherol are all available.Preferably comprise tocopherol in the used vaccine of gerontal patient's (for example, 60 years old or bigger), because it is reported that vitamin E has positive influences [16] to immunne response in this patient's group.They also have anti-oxidation characteristics, thereby help stable emulsion [17].Preferred alpha-tocopherol is the DL-alpha-tocopherol, and the preferred salt of this tocopherol is a succinate.Have now found that the part cooperation that succinate in vivo can be relevant with TNF-.In addition, known alpha-tocofecol succinic acid salt is compatible with influenza vaccines, is the useful antiseptic [23] of replacement for mercury chemical compound.The vaccine that does not especially preferably contain antiseptic.
Influenza antigen
The present composition comprises influenza antigen.Usually can prepare antigen from influenza virus particles, perhaps can in recombinant host (for example, utilizing the insect cell of baculovirus vector), express such as antigen such as hemagglutinin and with purified form and use [18,19].Yet antigen is generally from virion.
Antigen can adopt live virus, perhaps the more preferably form of inactivation of viruses.The chemical method of inactivation of viruses comprises one or more the following agent treated with effective dose: detergent, formaldehyde, formalin, beta-propiolactone or ultraviolet light.Other chemical method of deactivation comprises with methylene blue, psoralen, carboxyl fullerene (C60) or their any combined treatment.Other method of inactivation of viruses is known in this area, for example binary ethamine (binary ethylamine), acetyl group aziridine or gamma-rays.INFLEXAL
TMProduct is complete virion inactivated vaccine.
If use inactivation of viruses, vaccine can comprise the surface antigen (comprise hemagglutinin, also comprise neuraminidase usually) of complete virion, lytic virus granule or purification.
Can be by the whole bag of tricks from containing the liquid collecting virion of virus.For example, purification process can comprise the band centrifugation that utilizes linear Sucrose gradient solutions, and described sucrose solution contains detergent so that virion breaks.After the optional dilution, can be by the diafiltration purifying antigen.
With detergent (for example, ether, polysorbate80, dexycholate, tricresyl phosphate-N-butyl ester, triton x-100, triton N101, cetab, Tergitol NP9, or the like) the processing virion, comprise that " tween-ether " cleavage method produces the subviral particle goods, thereby obtained the lytic virus granule.The method of well known cracking influenza virus is for example referring to list of references 20-25 etc.Usually utilize the decomposition agent (splitting agent) of concentration (the disrupting concentration) that break to make infectious or noninfective intact virus is broken or the broken cracking of carrying out virus.Break and cause virus protein to dissolve wholly or in part, changed viral integrity.Preferred decomposition agent be nonionic and ionic (for example; cation) surfactant; alkyl polyglucoside for example; the alkyl sulfide glycosides; acyl group sugar; sulfobetaines; betanin; polyoxyethylene alkyl ether; N; N-dialkyl group-glucamide (Glucamides); Hai Kemaige (Hecameg); alkyl phenoxy-polyethoxy ethanol; quaternary ammonium compound; sarcosyl; CTAB (cetab); tricresyl phosphate-N-butyl ester; cetavlon (Cetavlon); the myristyl leptodactyline; lipofectin reagent; fat transfection amine; DOT-MA; octyl group-or Nonylphenoxy polyoxy ethanol is (for example; the triton surfactant is as triton x-100 or triton N101); polyoxyethylene sorbitol acid anhydride ester (tween surfactants); polyoxyethylene ether; polyoxyethylene ester etc.A kind of useful cleavage method utilizes the continuous action of NaTDC and formaldehyde, and cracking occurs in (for example, in sucrose density gradient solution) during the initial virion purification.Cracked virion usefully can be resuspended in the isotonic sodium chlorrde solution of sodium phosphate buffer.BEGRIVAC
TM, FLUARIX
TM, FLUZONE
TMAnd FLUSHIELD
TMProduct is split vaccine (split vaccine).
The SAV of purification comprises influenza surface antigen hemagglutinin, also comprises neuraminidase usually.These method of protein of well known preparation purified form.FLUVIRIN
TM, AGRIPPAL
TMAnd INFLUVAC
TMProduct is a subunit vaccine.
Influenza antigens can also the virion form exist [26] (the viral sample liposome particles that does not contain nucleic acid), for example INFLEXAL
TMAnd INVAVAC
TMProduct.
Influenza virus can be an attenuation.Influenza virus can be a temperature sensitive.Influenza virus can be cold adaptation.These three kinds of probabilities are specially adapted to live virus.
The difference of per season of influenza virus strain that is used for vaccine.In present popular interval, vaccine comprises two kinds of influenza A strains (H1N1 and H3N2) and a kind of influenza B strain usually, generally is trivalent vaccine.The present invention also can utilize epidemic isolates (promptly, for vaccine receptor and general population's immunogen strain just) virus, for example H2, H5, H7 or H9 hypotype strain (particularly influenza A virus), the influenza vaccines of epidemic isolates can be univalent vaccines, and the common trivalent vaccine that perhaps can add epidemic isolates is the basis.Yet; depend on antigenic character in season and the vaccine, the present invention can shield to resist following one or more influenza A viruss HA hypotype: H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.The present invention can shield to resist one or more influenza A viruss NA hypotype: N1, N2, N3, N4, N5, N6, N7, N8 or N9.
Other strain that can usefully be included in these compositionss is the strain of the strain (for example, tolerating oseltamivir [27] and/or zanamivir) of tolerance antiviral therapy, comprises drug resistance epidemic isolates [28].
Adjuvant composition prepared of the present invention is specially adapted to immunity inoculation to resist epidemic isolates.Can cause the influenza strain of epidemic diseases outburst to be characterised in that: (a) to compare with the hemagglutinin in present popular people's strain, it contains new hemagglutinin, promptly, in the crowd, occur to surpass 10 years strain (for example, H2), the strain of at all in the crowd, not seeing perhaps (for example, common H5, H6 or the H9 that only in flock of birds, finds), thus the crowd that makes is originally on immunology for the hemagglutinin of this strain; (b) it can horizontal transfer in the crowd; (c) it has pathogenic to the people.Resist epidemic influenza for immunity inoculation, preferably have the strain of H5 hemagglutinin type, for example the H5N1 strain.Other possible strain comprises H5N3, H9N2, H2N2, H7N1 and H7N7, and any epidemic isolates that other may occur.In the H5 hypotype, virus may belong to HA clade 1, HA clade 1 ', HA clade 2 or HA clade 3[29], wherein clade 1 is relevant especially with 3.
The present composition can comprise one or more (for example, 1,2,3,4 or more kinds of) influenza virus strains, comprises the antigen of influenza A virus and/or Influenza B virus.If vaccine comprises multiple influenza strain, cultivate different strains usually respectively, collect the mixing of virus back and prepare antigen.Therefore, the inventive method can comprise the antigenic step of mixing multiple influenza strain.Preferably comprise antigenic trivalent vaccine from two kinds of influenza A virus strains and a kind of Influenza B virus strain.In some embodiments of the present invention, compositions can comprise a kind of antigen of influenza A strain.In some embodiments, compositions can comprise the antigen of two kinds of influenza A strains, as long as these two kinds of strains are not H1N1 or H3N2.In some embodiments, these compositionss can comprise the antigen of two or more influenza A strains.
Influenza virus can be a recombined strain, can be by the reverse genetic acquisition that learns a skill.Reverse genetic learn a skill [for example, 30-34] can utilize plasmid to contain the influenza virus of required genome section in external preparation.This technology is usually directed to express (a) for example can be from the encode dna molecular of required viral RNA molecule of polI promoter, (b) for example can be from the dna molecular of polII promoter coding virus protein, thus make the DNA that in cell, expresses two types can assemble complete infectious viral particle.DNA preferably can provide all viral RNAs and protein, but also may utilize helper virus that in described RNA and the protein some are provided.The preferred plasmid method, thus can produce each viral RNA [35-37] with different plasmids, these methods also comprise utilizes plasmid to express all virus proteins or some of them (for example, PB1, PB2, PA and NP protein), and certain methods has been utilized 12 kinds of plasmids.
For reducing required plasmid number, nearest method [38] has made up a plurality of rna plymerase is and (has for example transcribed box (synthetic for viral RNA) on same plasmid, the sequence of coding 1,2,3,4,5,6,7 or all 8 influenza A vRNA sections), the a plurality of protein coding regions (for example, the sequence of coding 1,2,3,4,5,6,7 or all 8 influenza A mRNA transcripies) that contain the rna plymerase ii promoter on another plasmid, have been made up.The preferred aspect of list of references 38 described methods comprises: (a) PB1, PB2 and the PA mRNA coding region on the independent plasmid; (b) 8 vRNA coding sections of all on the independent plasmid.Comprise one on the plasmid NA and 6 other sections on HA section and another plasmid also be favourable.
Except with polI promoter coding viral RNA section, also may use bacteriophage polymerase promoter [39].For example, can use the promoter of SP6, T3 or T7 polymerase easily.Because the kind specificity of polI promoter, the bacteriophage polymerase promoter for many cell types (for example, MDCK) more convenient, though also must be with the plasmid transfection cell of encoding exogenous polymerase.
Other technology may adopt dual polI and polII promoter encode the simultaneously viral RNA and the effable mRNA[40,41 of a template].
Therefore, influenza A virus can comprise one or more RNA sections (6 of A/PR/8/34 sections normally, wherein HA and N section be from vaccine strain, promptly 6: 2 reassortants) of reassortant virus, when particularly cultivating virus with ovum.Also can comprise and be used for production vaccine production the A/WSN/33 virus of reprovision virus or one or more RNA sections of any other virus stain.Usually, the present invention's protection is resisted the able person to anthrochorous strain, so the genome of strain generally comprises at least one the RNA section that is derived from mammal (for example people) influenza virus.It can comprise the NS section that is derived from bird flu virus.
Available ovum (normally SPF ovum) or cell culture are cultivated the virus as the antigen source.The standard method utilization of cultivating influenza virus at present contains the egg of embryo and purified virus from egg inclusions (allantoic fluid).Yet, cultivate virus with animal cell culture in recent years, for speed and the allergic reason of patient, preferably this cultural method.If adopt egg to cultivate virus, then virus can be introduced in the allantoic fluid of egg [24] with one or more aminoacid.
Cellular material is the cell line in mammal source normally.Suitable mammalian cell source includes but not limited to: hamster, cattle, primates (comprising people and monkey) and canine cells.Can utilize various cell types, for example nephrocyte, fibroblast, retina cell, pneumonocyte etc.The example of suitable hamster cell is the cell line of BHK21 by name or HKCC.Suitable MC is, for example cercopithecus aethiops cell, for example nephrocyte in the Vero cell line.Suitable canine cells is, for example the nephrocyte in the mdck cell system.Therefore, suitable cell line includes but not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6; WI-38; Or the like.The preferred mammal cell line that is used to cultivate influenza virus comprises: the mdck cell [42-45] that is derived from Madin-Darby canine kidney; Be derived from the Vero cell [46-48] of cercopithecus aethiops (Cercopithecusaethiops) kidney; Or be derived from the PER.C6 cell [49] of people embryo retinoblast.These cell lines can be extensively available from, for example American Type Culture Collection (ATCC) [50], cell preservation center, bandit Lille (Coriell Cell Repositories) [51] or European cell culture preservation institute (ECACC).For example, it is the various different Vero cells of CCL-81, CCL-81.2, CRL-1586 and CRL-1587 that ATCC provides catalog number (Cat.No.), and catalog number (Cat.No.) is the mdck cell of CCL-34.PER.C6 can preserving number 96022940 available from ECACC.Time choosing as mammal cell line substitutes, and available avian cell lines is cultivated virus [for example, list of references 52-54], comprises fowl embryo stem cell [52,55] and is derived from the cell line of duck (for example, duck retina) or hen.Suitable fowl embryo stem cell comprises EBx cell line, EB45, EB 14 and the EB 14-074[56 that is derived from chicken embryonic stem cells].Also can utilize chick embryo fibroblast (CEF), or the like.
The most preferably cell line of cultivating influenza virus is mdck cell system.Original mdck cell system can CCL-34 available from ATCC, but also can use the derivant of this cell line.For example, list of references 42 has disclosed the mdck cell system (" MDCK 33016 " are with DSMACC 2219 preservations) that is adapted to grow in the suspension culture base.Similarly, list of references 57 has disclosed the MDCK derived cell system (" B-702 " is with FERM BP-7449 preservation) that is grown in the serum-free culture suspension.List of references 58 has disclosed the non-tumorigenic mdck cell, comprises " MDCK-S " (ATCC PTA-6500), " MDCK-SF101 " (ATCC PTA-6501), " MDCK-SF 102 " (ATCC PTA-6502) and " MDCK-SF103 " (PTA-6503).It is to comprise " MDCK.5F1 " cell (ATCC CRL-12042) that list of references 59 has disclosed infecting extremely sensitive mdck cell.Can use any of these mdck cell system.
If utilize mammal cell line to cultivate virus, then compositions does not preferably contain egg albumin (for example, ovalbumin and ovomucoid) and chicken DNA, thereby can reduce allergenicity.
If cultivate virus with cell line, then growth medium and the being used to virus inoculation thing that starts cultivation does not preferably contain (that is, obtain after tested pollute negative findings) herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, sars coronavirus, adenovirus, rhinovirus, reovirus, polyoma virus, birnavirus, circovirus virus and/or parvovirus [60].Especially preferably do not contain herpes simplex virus.
If cultivate virus with cell line, then every dose of compositions preferably contains and is less than the 10ng residual host cell DNA of (preferably being less than 1ng, more preferably less than 100pg), though can there be the host cell DNA of trace.In a word, the host cell DNA in the present composition can not be longer than 100bp.
Now, detecting residual host cell DNA is the conventional requirement of biological product, belongs to technical staff's general ability.Be used to detect the normally validation test of test [61,62] of DNA.Can utilize mathematics and performance characteristic that can quantitative term description validation test, identify the source of error that it is possible.Checked generally this test such as features such as accuracy, precision, specificitys.In case good certain test of verification (for example, with the host cell DNA of known standard amount) and check can routine be carried out quantitative DNA detection.Can adopt three kinds of main DNA quantitative techniques: hybridizing method, for example Sourthern trace or slot blot [63]; Method of immunity, for example Threshold
TMSystem [64]; And quantitative PCR [65].The technical staff knows these methods, though the accurate feature of each method depends on the host cell of being studied, and for example selection of hybridization probe, the selection of amplification primers and/or probe, or the like.The Threshold of molecular device company (Molecular Devices)
TMSystem is the quantitative test that is used for the total DNA of pik level, and it has been used for the contaminating dna level [64] of monitoring bio medicine.Typical test is included in that biotinylated ssDNA is conjugated protein, urase link coupled anti--ssDNA antibody and DNA between non-sequence-specific ground form and react complex.Complete total DNA that all test components are all packed into available from the manufacturer measures in the test kit (Total DNA Assay Kit).The extensive stock manufacturer provides the quantitative PCR that detects residual host cell DNA test, for example AppTec
TMLaboratory service company (Laboratory Services), BioReliance
TM, Arthur technology company (Althea Technologies), or the like.Detect host cell DNA pollutes in Human virus's vaccine chemiluminescence cross experiment and total DNAThreshold
TMThe document 66 that relatively sees reference of system.
Can adopt the standard purification method, for example chromatography etc. is removed contaminative DNA during vaccine production.Handle by nuclease, for example can promote to remove residual host cell DNA with the DNA enzyme.List of references 67 and 68 has disclosed and has reduced the facilitated method that host cell DNA pollutes, it relates to the processing of two steps, at first can use the DNA enzyme (for example, Benzonase) during Virus culture, can during breaking, use virion cationic detegent (for example, CTAB) then.Alkylating agent, for example beta-propiolactone is handled and also be can be used for removing host cell DNA, and this method also can be preferred for inactivated virus particle [69].
Preferably in per 15 μ g hemagglutinins, contain＜10ng (for example,＜1ng,＜100pg) vaccine of host cell DNA, for example every 0.25ml volume contains＜10ng (for example,＜1ng,＜100pg) vaccine of host cell DNA.More preferably in per 50 μ g hemagglutinins, contain＜10ng (for example,＜1ng,＜100pg) vaccine of host cell DNA, for example every 0.5ml volume contains＜10ng (for example,＜1ng,＜100pg) vaccine of host cell DNA.
The average length of preferred any residual host cell DNA is shorter than 500bp, and for example be shorter than 400bp, be shorter than 300bp, be shorter than 200bp, be shorter than 100bp, or the like.
For using cell line, for example mdck cell is cultivated, and available suspension is cultivated [42,70,71] or the cell of adhere-wall culture is cultivated virus.A kind of suitable mdck cell system that is used for the suspension cultivation is MDCK 33016 (preserving number is DSM ACC 2219).Perhaps, can adopt microcarrier to cultivate.
Preferably support the cell line that influenza virus is duplicated with the culture medium and/or the protein-free culture medium culturing of serum-free.The culture medium that the present invention will wherein not contain the serum additive in human or animal source is called serum-free medium.No protein is interpreted as representing taking place not containing protein, somatomedin, other proteins additive and non-serum proteins in the culture medium of cell proliferation, but can comprise the required protein of viral growth, for example trypsin or other protease.The cell of growing in this culture medium self can naturally contain protein.
Support cell line that influenza virus duplicates preferably growth [72] below 37 ℃ (for example, 30-36 ℃, or about 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃), for example during virus replication.
The method of propagative viruses generally includes following steps in cultured cells: to cultured cells inoculation strain to be cultivated, with the infected required time of cell culture and virus breeding, for example by virus titer or antigen presentation measure (as, inoculation back 24-168 hour), collect the virus of breeding.With the virus and the ratio of cell is 1: 500-1: 1, preferred 1: 100-1: 5, more preferably 1: 50-1: 10 (by PFU or TCID
50Detection) cell of inoculated and cultured.Virus can be added in the cell suspension, or put on cell monolayer, virus is in 25-40 ℃, and preferred 28-37 ℃, absorption is at least 60 minutes on cell, but is less than 300 minutes usually, preferably between 90-240 minute.Can remove the cell culture (for example, monolayer) of infection by freeze thawing or enzymatic catalysis, thereby increase the viral level in the culture supernatant of collecting.Make the liquid deactivation or the freezing preservation of collection then.With about 0.0001-10, preferred 0.002-5, more preferably the infection multiplicity of 0.001-2 (" m.o.i. ") infects cultured cells.More preferably with about 0.01 m.o.i. infection cell.Infect the back and collected the cell that infects in 30-60 hour.Preferred 34-48 hour collecting cell after infection.More preferably 38-40 hour collecting cell after infection.Usually during cell culture, add protease (normally trypsin) with releasing virus, can add protease by any suitable stage in the training period.
Hemagglutinin (HA) is the main immunogens in the inactivated influenza vaccine, makes the vaccine dose standardization with reference to the HA level, generally detects by one-way radiation shape immunodiffusion (SRID) test.Vaccine contains the 15 μ gHA/ strains of having an appointment usually, though for example child, or can use lower dosage under the popularity.Yet, for live vaccine, by (the TCID of intermediate value TCID
50) rather than HA content detect administration, the TCID of every kind of strain
50Generally 10
6-10
8Between (preferred 10
6.5-10
7.5).With higher dosage (for example, 3 * or 9 * dosage [75,76]) the same, used part dosage, for example
1/
2(that is 7.5 μ gHA/ strains),
1/
4With
1/
8[73,74].Therefore, vaccine can comprise 0.1-150 μ g HA/ influenza strain, preferred 0.1-50 μ g, and for example 0.1-20 μ g, 0.1-15 μ g, 0.1-10 μ g, 0.1-7.5 μ g, 0.5-5 μ g, or the like.Concrete dosage comprises, for example about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 1.9, about 1.5/ strain, or the like.The same with the present invention, when having adjuvant in the vaccine, these lower dosage are the most useful.Can select all components (for example, their volume and concentration) of test kit of the present invention and method, thereby these HA dosage can be provided in final product mix.
The used HA of the present invention can be the natural HA that finds in the virus, perhaps can be modified.For example, the known HA of modification causes virus the morbific determinant of birds camber (for example, around the hyperalkaline zone (hyper-basic region) of the cleavage site between HA1 and the HA2) to remove, otherwise these determinants can stop virus to be grown in ovum.
Compositions of the present invention can comprise detergent, polyoxyethylene sorbitan acid anhydride ester surfactant (being called " tween ") for example, Octoxinol (for example, octoxynol 9 (triton x-100) or uncle's octylphenoxy polyethoxy ethanol), cetab (CTAB), or NaTDC, particularly for cracking or SAV.The detergent that can only have trace.Therefore, contained Octoxinol-10, alpha-tocopherol hemisuccinic acid ester (α-tocopheryl hydrogen succinate) and polysorbate80 is lower than 1mg/ml separately in the vaccine.The residual component of other trace can be antibiotic (for example, neomycin, kanamycin, a polymyxin B).
Deactivation but the vaccine (for example, the SAV of split-virus vaccine or purification) of non-full cell can comprise stromatin, thus benefit from other t cell epitope that is positioned at this antigen.Therefore, the non-whole-cell vaccines (particularly split vaccine) that comprise hemagglutinin and neuraminidase also can comprise M1 and/or M2 stromatin.If there is stromatin, but preferably comprise the M1 stromatin of detection level.Also can there be nucleoprotein.
Mix two kinds of components
Method of the present invention comprises mixes two kinds of components: (i) O/w emulsion and (ii) influenza antigen water-based product.In first aspect, use (i) of substantially the same volume and (ii), component HA concentration (ii) is greater than 60 μ g/ influenza virus strain/ml.In second aspect, use volume (ii) excessive.In described first aspect, utilize to be lower than FLUAD
TMThe dose volume of product is realized standard antigen dosage.In described second aspect, can reduce the required emulsion volume of specified rate antigen.In two kinds of situations, obtain the required emulsion volume of final vaccine and all be lower than existing FLUAD
TMProduct is used.
Can mix all components at production period, thereby obtain bulk vaccine, perhaps can mix all components in use, thereby obtain the vaccine of instant administration.If mix at production period, then mixed volume is usually greater than 1 liter, for example 〉=5 liter, 〉=10 liters, 〉=20 liters, 〉=50 liters, or the like.If mix in use, then mixed volume is usually less than 1 milliliter, for example≤0.6ml ,≤0.5ml ,≤0.4ml ,≤0.3ml ,≤0.2ml, or the like.If use the emulsion and the antigen emulsion of substantially the same volume, the ratio of mixed volume is 1: 1 basically, for example between 1.1: 1 and 1: 1.1, preferably between 1.05: 1 and 1: 1.05, more preferably between 1.025: 1 and 1: 1.025.Utilize＞60 μ g HA/ influenza virus strain/ml, the present invention can obtain the vaccine of the standard HA dosage of transmissibility 15 μ g/ strains, but the administration volume is lower.And FLUAD
TMThe per 15 μ g/ strains of product need the 0.25ml emulsion, and the present invention can reduce required emulsion amount.
If utilize the excessive antigenic solution of volume, described excessive normally at least 1.5: 1, for example 〉=2: 1, 〉=2.5: 1, 〉=3: 1, 〉=4: 1, 〉=5: 1, 〉=7.5: 1, 〉=10: 1, or the like.Describedly excessively be no more than 25: 1 usually, for example≤20: 1 ,≤15: 1 ,≤10: 1, or the like.By the HA concentration in the antigenic solution being reduced to＜60 μ g/ strain/ml, can in the finished product of 0.5ml dosage, realize 15 μ g/ strain HA dosage of standard, if for example excessive is 3: 1, HA concentration before then mixing should be 40 μ g/ strain/ml, is 15 μ g/ strain/0.5ml dosage thereby obtain ultimate density.
Make the minimizing of dosage surpass the excessive of volume, then can after the maintenance dose volume, obtain low antigen dose vaccine.For example, if 20 μ g/ strains/ml solution adopt 3: 1 excessive, then the HA dosage that provides of 0.5ml dose volume is 7.5 μ g/ strains.
Therefore, antigen concentration and excess volume ratio can be changed, thereby any required final antigen concentration and/or dose volume can be obtained.For example, when the adjuvant that adopts: the antigen volume ratio is 1: during α, for obtaining the final antigen dose of 15 μ g/ strain/0.5ml, HA concentration should be 30 (α+1)/α in the aqueous antigen product.The final antigen dose that lower HA concentration provides is＜15 μ g/ strain/0.5ml.
If HA concentration in the antigenic solution in the inventive method or the test kit＜60 μ g/ influenza virus strain/ml, then this level can be, for example≤55 μ g/ strain/ml ,≤50 μ g/ strain/ml ,≤45 μ g/ strain/ml ,≤40 μ g/ strain/ml ,≤35 μ g/ strain/ml ,≤30 μ g/ strain/ml ,≤25 μ g/ strain/ml ,≤20 μ g/ strain/ml ,≤15 μ g/ strain/ml ,≤10 μ g/ strain/ml, or the like.Can be chosen in and anyly give selected accurate concentration in the stable condition, reduce thereby meet used any volume.
Pharmaceutical composition
The present composition is pharmaceutically acceptable.They comprise the component except that antigen and adjuvant usually, and for example, they comprise one or more pharmaceutical carriers and/or excipient usually.To discussing fully of these components document 77 that sees reference.
Compositions can comprise one or more antiseptic, for example thimerosal or 2-phenyl phenol.Yet these vaccines preferably are substantially free of (that is, being less than 5 μ g/ml) hydrargyrum material, for example do not contain thimerosal [23,78].More preferably not mercurous vaccine.
Preferably comprise physiology salt, for example sodium salt is with control tension force.Sodium chloride (NaCl) between the preferred 1-20mg/ml.Other salt that can exist comprises potassium chloride, potassium dihydrogen phosphate, dehydration sodium hydrogen phosphate (disodiumphosphate dehydrate), magnesium chloride, calcium chloride etc.
The osmolality of compositions between 200mOsm/kg-400mOsm/kg, between the preferred 240-360mOsm/kg, more preferably is in the 290-310mOsm/kg scope usually.Had in the past and reported that osmolality did not influence [79] to the pain due to the vaccination, but preferably osmolality was maintained in this scope.
Compositions can comprise one or more buffer.Typical buffer comprises: phosphate buffer; The Tris buffer; Borate buffer; The succinic acid buffer; Histidine buffering liquid; Or citrate buffer solution.Contained buffer scope generally is 5-20mM.
The pH of compositions is usually between 5.0-8.1, and is more common between 6.0-8.0, for example between 6.5 and 7.5.Therefore, the inventive method can comprise the step that the first pH that regulates bulk vaccine packs again.
Compositions is preferably aseptic.The preferred apyrogeneity of compositions, for example every dosage contain＜1EU (endotoxin unit, gauge), preferred every dosage＜0.1EU.The preferred GF of compositions.
Compositions can comprise the once material of immunity, maybe can comprise the repeatedly material (that is " multiple dose " test kit) of immunity.In the multiple dose configuration, preferably comprise antiseptic.Except in multi-dose compositions, comprising antiseptic, also compositions can be placed the container that the sterile adapter that is used for transfer of material is housed.
The vaccine dose volume that generally gives is about 0.5ml, though can give the child with half-value dose (that is about 0.25ml).One of advantage of the present invention is to be not difficult the volume of preparation＜0.5ml.
Antigen and emulsion in the common blend compositions are though they can temporarily blended independent component be present in the kit form at first.
Compositions and test kit are preferably kept between 2 ℃-8 ℃.Should be freezing they.Should avoid light direct beam.
Test kit of the present invention
As mentioned above, can when sending, prepare the present composition temporarily.Therefore, the invention provides the test kit that instant blended each component is housed.Described test kit is preserved O/w emulsion and antigen stand-by respectively.Any other component can be included in one of these two kinds of reagent constituents, perhaps can be the part of the 3rd reagent constituents.
All components in the test kit are physical separation each other, can adopt the whole bag of tricks to realize this separation.For example, all components can be in independent container, for example in the bottle.Can take out the inclusions of a bottle then and it is added another bottle, perhaps take out the inclusions of two bottles respectively and in the 3rd container, mix and the inclusions of mixing two bottles.
In preferred configuration, one of all components of test kit are stored in the syringe, and other component is stored in container, for example in the bottle.Can utilize syringe (for example, syringe needle being housed) that its inclusions is injected second container and mix, then with in this syringe of mixture suction.Give the patient with the mixing inclusions in this syringe subsequently, utilize new aseptic syringe needle usually.A kind of component is packaged in need not to utilize another syringe to give the patient in the syringe.
In another preferred disposition, two kinds of reagent constituents are kept at same syringe respectively, for example in the double-chamber syringe, disclosed as list of references 80-87 etc.Use the inclusions of promptly having mixed in this syringe (for example, giving during the patient) in this two Room.This configuration need not independent blend step in use.
If the present invention utilizes the antigenic solution of excess volume, then the volume of bottle, syringe compartment etc. can become thereupon.
The inclusions of each assembly of test kit all can be in aqueous form usually.In some configurations, certain component (generally being antigen component rather than emulsion components) is in dried forms (for example, freeze dried form), and another component is in aqueous form.Can mix described two kinds of components and do component, thereby obtain to give patient's waterborne compositions with reactivate.Freeze-dried component is stored in bottle rather than the syringe usually.Exsiccant component can comprise stabilizing agent, for example lactose, sucrose or mannitol and their mixture, for example lactose/sucrose mixture, sucrose/mannitol mixture etc.A kind of possible configuration is stored in the water-based emulsion component in the pre-filled syringe, and the freeze-dried antigen component is stored in the bottle.
Packaging compositions or reagent constituents
The suitable vessel of the present composition (or reagent constituents) comprises bottle, syringe (for example, disposable syringe), nasal atomizer etc.These containers should be aseptic.
If compositions/component is stored in the bottle, described bottle is preferably made by glass or plastic material.Preferably make the bottle sterilization add compositions more earlier.For avoiding the patient to latex problem hypersensitive, preferably with the plug seal bottle of no latex, all packaging material preferably all do not contain latex.Bottle can be equipped with single dose vaccine, multiple dose (" multiple dose " bottle) maybe can be housed, for example 10 doses.Preferably make bottle with flint glass.
Bottle can be equipped with lid (for example, the Luer snap close), thereby the syringe of filling in advance can be inserted in the lid, the inclusions of syringe is pushed in the bottle (for example, rebuild freeze-dried material wherein), the inclusions of bottle is drawn back in the syringe again.After syringe taken out, load onto syringe needle from bottle, give the patient compositions.Lid is positioned at seals or covering, seal or covering contacts lid again thereby will remove earlier.Bottle can have lid, thus its inclusions of the aseptic taking-up of energy, particularly for multiple dose vials.
If certain component is packaged into syringe, syringe can be equipped with syringe needle.If be unkitted syringe needle, can provide independent syringe needle to be used for assembling and use with syringe.This syringe needle can have sheath.The preferred security syringe needle.Commonly No. 3,1 in2, No. 5,1 in2 and No. 5 syringe needles of 5/8 in2.Syringe can be equipped with peelable label, has printed the effect duration of lot number, influenza season and inclusions on it, thereby has helped scorekeeping.The plunger of syringe preferably has brake, thereby can prevent that plunger accident during aspirating from dropping out.Syringe can be equipped with latex rubber lid and/or plunger.Disposable syringe can contain single dose vaccine.Before loading onto syringe needle, syringe generally is equipped with the pin medicated cap with apical end, and described pin medicated cap is preferably made by butyl rubber.If syringe and syringe needle be packing separately, then preferably the butyl rubber cover is housed to syringe needle.Preferred syringe is with " Tip-Lok "
TMThose that put goods on the market for trade name.
Can do the labelling that shows the half-value dose volume to container, for example to help to be delivered to the child.For example, the syringe that contains 0.5ml dosage can have the labelling that shows the 0.25ml volume.
If use glass container (for example, syringe or bottle), the then container that preferably uses pyrex rather than soda-lime glass to make.
(for example, being contained in the same box) inset can be housed with test kit or compositions, and this inset comprises the details of vaccine, the operation instructions of administration for example, antigenic details etc. in the vaccine.Operation instructions also can comprise warning, for example prepare epinephrine solution in case the anaphylaxis after the vaccination, or the like.
The present composition is fit to the administration of human patient, the invention provides the method that produces immunne response in patient's body, comprises the step that the present composition is given the patient.
The present invention also provides test kit, perhaps is used as the present composition of medicine.
The present invention also provides the water-based product of substantially the same volume (i) influenza antigen, and wherein hemagglutinin concentration is greater than 60 μ g/ influenza virus strain/ml; (ii) oil in water emulsion adjuvant is used for producing the application of the medicine of immunne response in patient's body in preparation.
The present invention also provides the water-based product of substantially the same volume (i) influenza antigen, and wherein hemagglutinin concentration is less than 60 μ g/ influenza virus strain/ml; (ii) oil in water emulsion adjuvant is used for producing the application of the medicine of immunne response in patient's body in preparation.
The present invention also provides (i) to have the water-based product of the influenza antigen of first volume; The oil in water emulsion adjuvant that (ii) has second volume is used for producing the medicine of immunne response in preparation in patient's body application, described second volume is less than described first volume.
The immunne response that these methods and applications produce generally includes antibody response, and preferred protection antibody is replied.The method of the postvaccinal antibody response of well known assessment influenza virus vaccine, neutralising capacity and protective effect.For example, human research proof is at the antibody titer of human influenza virus's hemagglutinin relevant with protective effect (during the blood serum sample hemagglutination-about 30-40 of inhibition titre, the protective effect of homology viral infection being about 50%) [88].Generally detect antibody response by hemagglutination inhibition, microneutralization, single radiation immunity diffusion (SRID) and/or single radial hemolysis (SRH).Well known these experimental techniques.
Can give the present composition by the whole bag of tricks.Most preferred immunization route is intramuscular injection (for example, being injected in arm or the lower limb), but other available approach comprises subcutaneous injection, intranasal [89-91], oral [92], intradermal [93,94], transdermal, percutaneous [95] etc.
The vaccine of the present invention's preparation can be used for treating child and adult.Influenza vaccines recommend to be used for department of pediatrics and adult's immunity inoculation at present, and the age was from 6 months.Therefore, the patient can be less than 1 years old, 1-5 year, 5-15 year, 15-55 year or at least 55 years old.The preferred old people of patient who accepts vaccine (for example, 〉=50 years old, 〉=60 years old, preferred 〉=65 years old), youngster (for example, ≤ 5 years old), inpatient, health care personnel, army and army personnel, the gravid woman, chronic disease, immunodeficiency patient take antiviral compound (for example, oseltamivir or zanamivir chemical compound in preceding 7 days accepting vaccine; For example oseltamivir phosphate vide infra) the patient and the people who goes abroad.Yet these vaccines are not only applicable to these colonies, also can be applicable to widely among the crowd.For epidemic isolates, preferably give all age colonies.
Treatment can be single dose schedule or multiple dose drug regimen.Multiple dose can be used for initial immunization flow sheet and/or booster immunization vaccination schedule.In the multiple dose drug regimen, can give various dosage by identical or different approach, for example parenteral sensitization and mucosa are strengthened, and mucosa sensitization and gastrointestinal add by force, or the like.Give multidose (normally two dosage) the first patient of immunogen, for example for the people who never accepts in the past influenza vaccines, or (for example at the epidemic diseases burst period) is particularly useful for the vaccine that new HA hypotype is resisted in inoculation.Multiple dose can give at interval (for example, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, or the like) usually at least one week.
1,2 or 3 of the CPMP standard that preferred composition of the present invention satisfy to be renderd a service.In adult's (18-60 year), these standards are: (1) 〉=70% serum protection; (2) 〉=40% seroconversion; And/or (3) GMT increase 〉=2.5-doubly.In old people (＞60 years old), these standards are: (1) 〉=60% serum protection; (2) 〉=30% seroconversion; And/or (3) GMT increase 〉=2-doubly.These standards are according at least 50 patients' open label research (open label study).
Can be basically simultaneously (for example with vaccine of the present invention and other vaccine, can be during the same medical consultation at health care expert or vaccination center or going to a doctor) give the patient, for example with following vaccine basically simultaneously: Measles Vaccine, mumps Vaccine, rubella vaccine, the MMR vaccine, chickenpox vaccine, the MMRV vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, the DTP vaccine, link coupled influenza B haemophilus vaccine, the poliovirus vaccine of deactivation, hepatitis B virus vaccine, the meningococcal conjugates vaccine (for example, tetravalence A-C-W135-Y vaccine), respiratory syncytial virus vaccine, the streptococcus pneumoniae conjugate vaccines, or the like.Basically give in the gerontal patient particularly useful simultaneously with Pnu-Imune 23 and/or meningococcus vaccine.
Similarly, can be with vaccine of the present invention and antiviral compound, the antiviral compound (for example, oseltamivir and/or zanamivir) of particularly effectively resisting epidemic disease influenza poison (for example, can during health care expert's a same medical consultation or going to a doctor) basically simultaneously gives the patient.These antiviral (chemical compound) comprise neuraminidase inhibitor; (3R for example; 4R; 5S)-4-acetyl-amino-5-amino-3 (1-ethyl propoxyl group)-1-cyclohexene-1-carboxylic acid or 5-(acetyl-amino)-4-[(amino imino methyl)-amino]-2; 6-dehydration-3; 4, the 5-three deoxidations-D-glyceryl-D-galactose ninth of the ten Heavenly Stems (galactonon)-2-olefin(e) acid (enonic acid) comprise their ester (for example ethyl ester) and their salt (for example phosphate).Preferred antiviral compound is that (3R, 4R 5S)-4-acetyl-amino-5-amino-3 (1-ethyl propoxyl group)-1-cyclohexene-1-carboxylic acid, ethyl ester, phosphate ester (1: 1), are also referred to as oseltamivir phosphate (TAMIFLU
TM).
The cytokine induction agent
The present composition can comprise the cytokine induction agent, has now found that this reagent of mixing and O/w emulsion obtain unexpectedly effectively immunogenic composition, have synergism to t cell response.Report that for the drift of tolerance influenza antigen, t cell response is better than antibody response.In addition, T cytological effect mechanism may be the vaccine-induced important determiner [96] of resisting the protective effect of serious disease among the gerontal patient, also may reply the relevant susceptibility [97] of age that reduces influenza by stronger interferon-.
When giving the patient, the cytokine induction agent that is comprised in the present composition can cause immune system and discharge cytokine, comprises interferon and interleukin.Preferred cytokine induction agent can cause the release of following one or more cytokines: interferon-; Il-1; Interleukin-2; Il-1 2; TNF-α; TNF-β; And GM-CSF.Preferred cytokine induction agent causes and the release of Th-1 type immunne response related cytokine, for example interferon-, TNF-α, interleukin-2.Preferred interferon-and the interleukin-2 of stimulating.
Therefore, accepting these compositionss makes patient's T cell can discharge required cytokine in the antigenic specificity mode when stimulating with influenza antigens.For example, the T cell of blood purification from them can discharge gamma interferon when the external contact influenza virus hemagglutinin.This method of replying comprises ELISA, ELISPOT, flow cytometry and PCR in real time in the cell of detection peripheral blood mononuclear known in the art (PBMC).For example, list of references 98 has been reported a research, the T cells with antigenic specificity mediation immunne response at tetanus toxoid has been monitored in the research, particularly gamma interferon is replied, finding that ELISPOT distinguishes antigen specific T T to induce and reply and spontaneous sensitive method of replying, is to detect the most effectual way of stimulation (re-stimulating effect) but adopt the Flow cytometry kytoplasm inner cell factor again.
Suitable cytokine induction agent includes but not limited to:
Immunostimulatory oligonucleotide, the oligonucleotide (the dinucleotide sequence that contains the non-cytosine that methylates that is connected with guanine by the phosphide key) that for example contains the CpG motif, or double-stranded RNA, or contain the oligonucleotide of palindrome or contain the oligonucleotide of poly-(dG) sequence.
(" 3dMPL " is also referred to as " MPL to 3-O-deacylated tRNA base monophosphoryl lipid A
TM") [99-102].
Imidazoquinolie compounds, for example imiquimod (" R837 ") [103,104], resiquimod (" R-848 ") [105] and their analog; With their salt (for example, hydrochlorate).Other details of immunostimulating imidazoquinolie document 106-110 that sees reference.
Thiosemicarbazones chemical compound, for example those of list of references 111 disclosures.List of references 111 has also been described the method for preparation, manufacturing and screening reactive compound.Thiosemicarbazones produces aspect cytokine such as the TNF-α effective especially at the cell of stimulation human peripheral blood monokaryon.
Couroupitine A chemical compound, for example those of list of references 112 disclosures.List of references 112 has also been described the method for preparation, manufacturing and screening reactive compound.Thiosemicarbazones produces aspect cytokine such as the TNF-α effective especially at the cell of stimulation human peripheral blood monokaryon.
Nucleoside analog, for example (a) Ai Shatuola shore (Isatorabine) (ANA-245; 7-thia-8-oxo guanosine) and prodrug:
(b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the described chemical compound of list of references 113-115; (f) chemical compound shown in the following formula:
In the formula:
R
1And R
2Independent separately be H, halogen ,-NR
aR
b,-OH, C
1-6The C of alkoxyl, replacement
1-6The heterocyclic radical of alkoxyl, heterocyclic radical, replacement, C
6-10The C of aryl, replacement
6-10Aryl, C
1-6The C of alkyl or replacement
1-6Alkyl;
R
3Do not exist, or H, C
1-6The C of alkyl, replacement
1-6Alkyl, C
6-10The C of aryl, replacement
6-10The heterocyclic radical of aryl, heterocyclic radical or replacement;
R
4And R
5Independent separately be H, halogen, heterocyclic radical, replacement heterocyclic radical ,-C (O)-R
d, C
1-6The C of alkyl, replacement
1-6Alkyl, or be combined together to form 5 yuan of rings, as R
4-5:
X
1And X
2Independent separately is N, C, O or S;
R
8Be H, halogen ,-OH, C
1-6Alkyl, C
2-6Thiazolinyl, C
2-6Alkynyl ,-OH ,-NR
aR
b,-(CH
2)
n-O-R
c,-O-(C
1-6Alkyl) ,-S (O)
pR
eOr-C (O)-R
d
R
9Be H, C
1-6The C of alkyl, replacement
1-6The heterocyclic radical of alkyl, heterocyclic radical, replacement or R
9a, R wherein
9aBe:
R
10And R
11Independent separately is H, halogen, C
1-6The C of alkoxyl, replacement
1-6Alkoxyl ,-NR
aR
bOr-OH;
R
aAnd R
bIndependent separately is H, C
1-6The C of alkyl, replacement
1-6Alkyl ,-C (O) R
d, C
6-10Aryl;
R
cIndependent separately is H, phosphate-based, diphosphonic acid ester group, triphosphoric acid ester group, C
1-6The C of alkyl or replacement
1-6Alkyl;
R
dIndependent separately is H, halogen, C
1-6The C of alkyl, replacement
1-6Alkyl, C
1-6The C of alkoxyl, replacement
1-6Alkoxyl ,-NH
2,-NH (C
1-6Alkyl) ,-NH (C of replacement
1-6Alkyl) ,-N (C
1-6Alkyl)
2,-N (the C of replacement
1-6Alkyl)
2, C
6-10Aryl or heterocyclic radical;
R
eIndependent separately is H, C
1-6The C of alkyl, replacement
1-6Alkyl, C
6-10The C of aryl, replacement
6-10The heterocyclic radical of aryl, heterocyclic radical or replacement;
R
fBe H, C independently of one another
1-6The C of alkyl, replacement
1-6Alkyl ,-C (O) R
d, phosphate-based, diphosphonic acid ester group or triphosphoric acid ester group;
N independently is 0,1,2 or 3 separately;
P independently is 0,1 or 2 separately; Perhaps
(g) (a) each pharmaceutically acceptable salt-(f), (a)-(f) in each tautomer, or the pharmaceutically acceptable salt of this tautomer.
Loxoribine (Loxoribine) (7-pi-allyl-8-oxo guanosine) [116].
The chemical compound that list of references 117 discloses comprises: the acyl piperazine chemical compound; indole dione (Indoledione) chemical compound; tetrahydroisoquinoline (THIQ) chemical compound; benzo ring diketone (Benzocyclodione) chemical compound; amino azepine vinyl (Aminoazavinyl) chemical compound; amino benzimidazole quinolinones (Aminobenzimidazole quinolinone) is chemical compound [118 (ABIQ); 119]; hydroxyl phthalic amide (Hydrapthalamide) chemical compound; benzophenone cpd isoxazole compound; sterol compounds; quinazolone (Quinazilinone) chemical compound; azole compounds [120]; anthraquinone compounds; quinoxaline compounds; triaizine compounds; pyrazoles pyrimidine (Pyrazalopyrimidine) chemical compound and benzazole (Benzazole) chemical compound [121].
The chemical compound that list of references 122 discloses.
Chemical compound shown in formula I, II or the III, or their salt:
As described in list of references 123, as ' ER 803058 ', ' ER 803732 ', ' ER 804053 ', ' ER804058 ', ' ER 804059 ', ' ER 804442 ', ' ER 804680 ', ' ER 804764 ', ' ER 804057 ' (structure is as follows):
Or ' ER 803022 ' (structure is as follows):
Aminoalkyl glucosaminide phosphate derivative is as RC-529[124,125].List of references 126 has reported that RC-529 is at CD
4+The ability of T cell moderate stimulation cytokine response.
Phosphonitrile, poly-[two (carboxyl phenoxy group) phosphonitrile] for example described in the list of references 127 and 128 (" PCPP ", poly[di (carboxylatophenoxy) phosphazene]).
The chemical compound that contains the lipid that is connected in the acyclic main chain of phosphoric acid, for example TLR4 antagonist E5564[129,130]:
Micromolecule immunostimulant (SMIP), for example:
N2-methyl isophthalic acid-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;
N2, N2-dimethyl-1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2,4-diamidogen;
N2-ethyl-N2-methyl isophthalic acid-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;
N2-methyl isophthalic acid-(2-methyl-propyl)-N2-propyl group-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;
1-(2-methyl-propyl)-N2-propyl group-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;
N2-butyl-1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;
N2-butyl-N2-methyl isophthalic acid-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;
N2-methyl isophthalic acid-(2-methyl-propyl)-N2-amyl group-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;
N2-methyl isophthalic acid-(2-methyl-propyl)-N2-third-2-thiazolinyl-1H-imidazo [4,5-c] quinoline-2, the 4-diamidogen;
1-(2-methyl-propyl)-2-[(phenyl methyl) sulfur]-1H-imidazo [4,5-c] quinoline-4-amine;
1-(2-methyl-propyl)-2-(rosickyite base)-1H-imidazo [4,5-c] quinoline-4-amine;
2-[[4-amino-1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2-yl] (methyl) amino] ethanol;
2-[[4-amino-1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-2-yl] (methyl) amino] ethylhexoate;
4-amino-1-(2-methyl-propyl)-1,3-dihydro-2H-imidazo [4,5-c] quinoline-2-one-;
N2-butyl-1-(2-methyl-propyl)-N4, two (phenyl methyl)-1H-imidazo [4, the 5-c] quinoline-2 of N4-, 4-diamidogen;
N2-butyl-N2-methyl isophthalic acid-(2-methyl-propyl)-N4, two (phenyl methyl)-1H-imidazo [4, the 5-c] quinoline-2 of N4-, 4-diamidogen;
N2-methyl isophthalic acid-(2-methyl-propyl)-N4, two (phenyl methyl)-1H-imidazo [4, the 5-c] quinoline-2 of N4-, 4-diamidogen;
N2, N2-dimethyl-1-(2-methyl-propyl)-N4, two (phenyl methyl)-1H-imidazo [4, the 5-c] quinoline-2 of N4-, 4-diamidogen;
1-{4-amino-2-[methyl (propyl group) amino]-1H-imidazo [4,5-c] quinoline-1-yl }-2-methyl propan-2-ol;
1-[4-amino-2-(propyl group amino)-1H-imidazo [4,5-c] quinoline-1-yl]-2-methyl propan-2-ol;
N4, N4-dibenzyl-1-(2-methoxyl group-2-methyl-propyl)-N2-propyl group-1H-imidazo [4,5-c] quinoline-2,4-diamidogen.
The used cytokine induction agent of the present invention can be the regulator and/or the agonist of Toll sample receptor (TLR).For example, they can be one or more agonist in people TLR1, TLR2, TLR3, TLR4, TLR7, TLR8 and/or the TLR9 albumen.Preferred derivant is the agonist (for example, imidazoquinolines) of TLR7 and/or the agonist (for example, CpG oligonucleotide) of TLR9.These derivants can be used for activating the innate immunity approach.
Can each stage during preparation of compositions add the cytokine induction agent.For example, it can be added in the antigen composition, then this mixture be added in the O/w emulsion.Perhaps, it can be added in the O/w emulsion, can earlier derivant be added reemulsification in the emulsion components in this case, or can first emulsifying add in the emulsion again.Similarly, derivant can condense in emulsion droplets.Its hydrophile/lipophile characteristic is depended in the location and the distribution of final composition inner cell factor derivant, and for example derivant can be arranged in aqueous phase, oil phase and/or oil-water interface place.
Can be with cytokine induction agent and different medicines, for example antigen (as CRM197) coupling.List of references 131 has been summarized the micromolecule coupling technology.Perhaps, adjuvant can combine with other medicines are non-covalent, for example by hydrophobicity or the interaction of centrifugal property.
Two kinds of preferred cytokine induction agent are (a) immunostimulatory oligonucleotide and (b) 3dMPL.
Immunostimulatory oligonucleotide
Immunostimulatory oligonucleotide can comprise nucleotide modification/analog, modifies as thiophosphate, can be double-stranded or (except dsRNA) strand.List of references 132,133 and 134 has disclosed possible similar replacement, for example uses 2 '-deoxidation-7-denitrogenation guanosine to replace guanosine.List of references 135-140 has further discussed the adjuvant effect of CpG oligonucleotide.The CpG sequence can relate to TLR9, for example motif GTCGTT or TTCGTT[141].But CpG sequence specificity is induced the Th1 immunne response, CpG-A ODN (oligodeoxynucleotide) for example, or induce B cell response, for example CpG-BODN more specifically.List of references 142-144 has discussed CpG-A and CpG-B ODN.The preferred CpG-AODN of CpG.Preferably the CpG oligonucleotide is configured to 5 ' end and is easy to be receptor identification.Optional that two CpG oligonucleotide sequences are continuous to form " immune aggressiveness " (immunomer) at their 3 ' end.Referring to, for example list of references 141 and 145-147.Useful CpG adjuvant is CpG7909, and it is also referred to as ProMune
TM(Gray Pharm Pur GmbH (Coley Pharmaceutical Group, Inc.)).
Except that using the CpG sequence, also can use TpG sequence [148].These oligonucleotide can not contain unmethylated CpG motif.
Immunostimulatory oligonucleotide can be rich in pyrimidine.For example, it can comprise a plurality of successive thymidine nucleotide (for example, list of references 148 described TTTT), and/or its nucleotide form can contain＞25% thymidine (for example,＞35%,＞40%,＞50%,＞60%,＞80%, or the like).For example, it can comprise a plurality of successive cytidylic acids (for example, list of references 148 described CCCC), and/or its nucleotide form can contain＞25% cytosine (for example,＞35%,＞40%,＞50%,＞60%,＞80%, or the like).These oligonucleotide can not contain unmethylated CpG motif.
Immunostimulatory oligonucleotide comprises at least 20 nucleotide usually.They can comprise and be less than 100 nucleotide.
3dMPL
3dMPL (be also referred to as 3 take off-O-acidylate monophosphoryl lipid A or 3-O-deacylated tRNA base-4 '-monophosphoryl lipid A) be in the monophosphoryl lipid A 3 of reducing end glycosamine by the adjuvant of deacylation.3dMPL is from the preparation of the no heptose mutant (heptoseless mutant) of salmonella minnesota (Salmonella minnesota), and it chemically is being similar to lipid A but lacks sour unsettled phosphoryl and alkali labile acyl group.The cell of its activated monocyte/macrophage pedigree stimulates several release of cytokines, comprises IL-1, IL-12, TNF-α and GM-GSF (being also shown in list of references 126).List of references 149 has been described the preparation of 3dMPL at first.
3dMPL can take the form (for example, having 3,4,5 or 6 acyl chains that length may be different) of the different correlation molecule mixture of acidylate degree.By the N-acyl groupization, 3 ' also by the O-acyl groupization for the 2-position carbon (that is, 2 and 2 ') of two glycosamines (being also referred to as 2-deoxidation-2-amino-glucose) monosaccharide.The group that links to each other with carbon 2 is suc as formula-NH-CO-CH
2-CR
1R
1' shown in.The group that links to each other with carbon 2 ' is suc as formula-NH-CO-CH
2-CR
2R
2' shown in.The group that links to each other with carbon 3 ' is suc as formula-O-CO-CH
2-CR
3R
3' shown in.Representational structure is:
Radicals R
1, R
2And R
3Independently be-(CH separately
2)
n-CH
3The n value preferably between 8-16, more preferably between 9-12, most preferably 10.
Radicals R
1', R
2' and R
3' independently be separately: (a)-H; (b)-OH; Or (c)-O-CO-R
4, R wherein
4Be-H or-(CH
2)
m-CH
3, wherein the m value preferably between 8-16, more preferably 10,12 or 14.At 2, m preferred 14.In 2 ' position, m preferred 10.In 3 ' position, m preferred 12.So radicals R
1', R
2' and R
3' preferred dodecylic acid, tetradecanoic acid and hexadecanoic acid-the O-acyl group.
Work as R
1', R
2' and R
3' all be-during H, 3d-MPL have only 3 acyl chains (2,2 ' and 3 ' position on respectively have one).Work as R
1', R
2' and R
3' in have only two and be-during H, 3d-MPL can have 4 acyl chains.Work as R
1', R
2' and R
3' in have only one and be-during H, 3d-MPL can have 5 acyl chains.Work as R
1', R
2' and R
3' in none be-during H, 3d-MPL can have 6 acyl chains.The used 3d-MPL adjuvant of the present invention can be the mixture with these forms of 3-6 bar acyl chain; but comprise 3d-MPL in the preferred mixture with 6 acyl chains; to guarantee that particularly 6 acyl chain forms account for 10% of total 3d-MPL at least by weight, for example 〉=20%, 〉=30%, 〉=40%, 〉=50% or higher.The 3d-MPL that discovery has 6 acyl chains is the most activated adjuvant form.
Therefore, the 3D-MPL most preferred form that comprises of the present composition is:
When adopting the 3D-MPL of form of mixtures, mention the consumption of 3d-MPL in the present composition or consumption or the concentration that concentration refers to all kinds of blended 3d-MPL in this mixture.
Under aqueous conditions, 3d-MPL can form micelles agglomerate or the granule that varies in size, for example diameter＜150nm or＞500nm.The present invention can use any or the two, can select preferable granule by routine test.Smaller particles is preferred for the present invention's (for example, thereby the enough little clear aqueous suspension that can obtain 3d-MPL) [150] because of its preferable activity.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 applications, average diameter can be less than 50nm.These granules are enough little, thereby are suitable for filtration sterilization.Can be by the routine techniques assessment particle diameter of dynamic light scattering, this technology can disclose average particulate diameter.When mentioning particulate diameter and be xnm, distribution of particles generally near this meansigma methods, but in quantitative terms at least 50% the particulate diameter of (for example, 〉=60%, 〉=70%, 〉=80%, 〉=90% or more) in x ± 25% scope.
Preferably all 3dMPL are positioned at the aqueous phase of emulsion basically.
The common consumption of 3dMPL is 10-100 μ g/ agent in the vaccine, for example about 25 μ g or about 50 μ g.
Can use 3dMPL separately, or with one or more other chemical compound couplings.For example, but known coupling 3dMPL and QS21 saponin [151] (being included in [152] in the emulsion), aluminum phosphate [153] or aluminium hydroxide [154].
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.
Word " basically " is not got rid of " fully ", and for example the compositions of " essentially no " Y can not have Y fully.This word " basically " can optionally save from the present invention's definition.
The term " about " relevant with numerical value x represent, for example x ± 10%.
Unless special statement is arranged, comprises that certain method of the step of mixing two or more components does not require any concrete order by merging.Therefore, can any order mix all components.If three kinds of components are arranged, then can earlier two kinds of components be mixed with each other, again this mixture is mixed with the third component, or the like.
If utilize animal (particularly cattle) material cultured cell, these materials should never contain can infect spongiform encephalopathy (TSE), and the source that does not particularly contain bovine spongiform encephalopathy (BSE) obtains.Generally, preferred cultured cell in the material that does not contain animal origin fully.
If give health with chemical compound as the part of compositions, or available suitable prodrug substitutes this chemical compound.
If cellular material is used for reprovision or reverse genetics method, preferred approval is used for the cellular material of people's production of vaccine, for example among the European Pharmacopoeia summary section 5.2.3.
The accompanying drawing summary
Fig. 1 has shown and adopts different emulsions: the compositions of antigen volume ratio preparation is resisted the serum ELISA antibody titer (Log of H1N1 strain
10).Fig. 2 has shown the identical data of H3N2, and Fig. 3 has shown the identical data of Influenza B virus strain.
Embodiment of the present invention
The virus that utilization is cultivated on the mdck cell culture prepares the influenza subunit vaccine.Described strain is: (i) A/Wyoming/H3N2; (ii) A/New-Caledonia/H1N1; (iii) B/Jiangsu.Utilize the vaccine of these vaccine production adjuvant preparations, adopt 0.2 μ gHA/ strain/vaccine dose by intramuscular approach immune mouse.Adjuvant in the vaccine is MF59.The adjuvant and the aqueous HA antigen that mix different proportion.In every kind of situation, mice is accepted the material of equal volume, and the aqueous HA antigen consumption in all experiments is constant.Yet, the volume of MF59 is reduced from peak at 1: 1.The volume ratio that is adopted is 0.75,0.50,0.25 and 0.10.Contrast is without adjuvant.
As shown in Figure 1, the consumption of MF59 emulsion being reduced maximum 10 times does not have or almost not influence whole immunogenicity.Therefore, emulsion adjuvant consumption that can influenza vaccines are required is from FLUAD
TMIn used 1: 1 ratio reduce, thereby emulsion preparation that can usefulness specified rate multi-vaccine more.
Will be appreciated that just and described the present invention by embodiment, can make any improvement and still belong to scope of the present invention and the design in.
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Claims (27)
1. method for preparing the influenza vaccines of adjuvant preparation, described method comprises (i) O/w emulsion that mixes equal volume basically and the (ii) step of influenza antigen water-based product, and wherein the concentration of the hemagglutinin of component in (ii) is greater than 60 μ g/ influenza virus strain/ml.
2. the method for claim 1 is characterized in that, the unit dose volume of described vaccine is less than 0.5ml.
3. method for preparing the influenza vaccines of adjuvant preparation, described method comprise (i) O/w emulsion that mixes equal volume basically and the (ii) step of influenza antigen water-based product, and the unit dose volume of wherein said vaccine is less than 0.5ml.
4. method for preparing the influenza vaccines of adjuvant preparation said method comprising the steps of: (a) mix (i) O/w emulsion of equal volume basically and (ii) influenza antigen water-based product, thereby obtain bulk vaccine; (b) take out at least a unit dose from described bulk vaccine, the volume of wherein said unit dose is less than 0.5ml.
5. as claim 3 or 4 described methods, it is characterized in that the component (ii) concentration of middle hemagglutinin is about 60 μ g/ influenza virus strain/ml.
6. method for preparing the influenza vaccines of adjuvant preparation, described method comprises (i) O/w emulsion that mixes equal volume basically and the (ii) step of influenza antigen water-based product, and the concentration of hemagglutinin is less than 30 μ g/ influenza virus strain/ml in the wherein said vaccine.
7. method for preparing the influenza vaccines of adjuvant preparation, described method comprise the step of the influenza antigen water-based product of the O/w emulsion that mixes first volume and second volume, and wherein said second volume is greater than described first volume.
8. method as claimed in claim 7 is characterized in that, the hemagglutinin concentration in described second volume is about 60 μ g/ influenza virus strain/ml.
9. method as claimed in claim 7 is characterized in that, the hemagglutinin concentration in described second volume is less than 60 μ g/ influenza virus strain/ml.
10. can be by the compositions of the described method acquisition of above each claim.
11. a vaccine combination that comprises O/w emulsion and influenza antigen, the volume of wherein said vaccine is less than 0.5ml.
12. a vaccine combination that comprises influenza virus hemagglutinin and Squalene, wherein the weight ratio of Squalene and hemagglutinin is between 50-2000, and the volume of wherein said vaccine is less than 0.5ml.
13. vaccine as claimed in claim 12 is characterized in that, contained HA concentration is about 30 μ g/ strain/ml.
14. vaccine as claimed in claim 12 is characterized in that, contained HA concentration＞30 μ g/ strain/ml.
15. vaccine as claimed in claim 12 is characterized in that, contained HA concentration＜30 μ g/ strain/ml.
16. a compositions that comprises the hemagglutinin of O/w emulsion and n kind influenza virus strain, wherein, the weight ratio of oil and hemagglutinin is less than 640/n.
17. compositions as claimed in claim 16 is characterized in that, the n value is 1,2,3 or 4.
18. described method of above each claim or compositions is characterized in that described O/w emulsion comprises Squalene.
19. described method of above each claim or compositions is characterized in that described O/w emulsion comprises polysorbate80.
20. described method of above each claim or compositions is characterized in that described influenza antigen is an inactivation of viruses.
21. method as claimed in claim 20 or compositions is characterized in that, described influenza antigen comprises the surface antigen of complete virus, lytic virus or purification.
22. described method of above each claim or compositions is characterized in that, described influenza antigen is from H1, H2, H3, H5, H7 or H9 subtypes of influenza A virus.
23. described method of above each claim or compositions is characterized in that, prepare described influenza antigen from the influenza virus of cultivating with ovum.
24. described method of above each claim or compositions is characterized in that, prepare described influenza antigen from the influenza virus of cultivating with cell culture.
25. method as claimed in claim 24 or compositions is characterized in that, described cell culture is the mdck cell culture.
26. method as claimed in claim 24 or compositions is characterized in that, described compositions does not contain ovalbumin, ovomucoid and chicken DNA.
27. test kit for preparing each described compositions among the claim 10-26.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
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| US73402605P | 2005-11-04 | 2005-11-04 | |
| US60/734,026 | 2005-11-04 | ||
| US60/757,058 | 2006-01-05 | ||
| US60/801,680 | 2006-05-19 |
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| CN101325967A true CN101325967A (en) | 2008-12-17 |
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| CNA2006800476045A Pending CN101365485A (en) | 2005-11-04 | 2006-11-06 | Emulsions with free aqueous-phase surfactant as adjuvants for split influenza vaccines |
| CNA2006800476098A Pending CN101370515A (en) | 2005-11-04 | 2006-11-06 | Influenza vaccines including combinations of particulate adjuvants and immunopotentiators |
| CNA2006800487317A Pending CN101346152A (en) | 2005-11-04 | 2006-11-06 | Adjuvanted vaccines with non-virion antigens prepared from influenza viruses grown in cell culture |
| CN2006800476007A Active CN101365483B (en) | 2005-11-04 | 2006-11-06 | Adjuvanted influenza vaccines including cytokine-inducing agents |
| CNA2006800463581A Pending CN101325967A (en) | 2005-11-04 | 2006-11-06 | Influenza vaccines with reduced amount of emulsion adjuvant |
Family Applications Before (4)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006800476045A Pending CN101365485A (en) | 2005-11-04 | 2006-11-06 | Emulsions with free aqueous-phase surfactant as adjuvants for split influenza vaccines |
| CNA2006800476098A Pending CN101370515A (en) | 2005-11-04 | 2006-11-06 | Influenza vaccines including combinations of particulate adjuvants and immunopotentiators |
| CNA2006800487317A Pending CN101346152A (en) | 2005-11-04 | 2006-11-06 | Adjuvanted vaccines with non-virion antigens prepared from influenza viruses grown in cell culture |
| CN2006800476007A Active CN101365483B (en) | 2005-11-04 | 2006-11-06 | Adjuvanted influenza vaccines including cytokine-inducing agents |
Country Status (4)
| Country | Link |
|---|---|
| CN (5) | CN101365485A (en) |
| CA (1) | CA2907149A1 (en) |
| ES (1) | ES2367194T3 (en) |
| SI (2) | SI1951300T1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102970976A (en) * | 2010-06-10 | 2013-03-13 | 葛兰素史密丝克莱恩生物有限公司 | Process for the production of an oil in water emulsion |
| CN105125488A (en) * | 2010-05-12 | 2015-12-09 | 诺华股份有限公司 | Improved methods for preparing squalene |
| CN105727281A (en) * | 2009-02-10 | 2016-07-06 | 诺华股份有限公司 | Influenza vaccines with reduced amounts of squalene |
| CN117582491A (en) * | 2024-01-18 | 2024-02-23 | 江苏瑞科生物技术股份有限公司 | Influenza vaccine composition, preparation method and application thereof |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108042799A (en) * | 2010-07-06 | 2018-05-18 | 诺华股份有限公司 | cationic oil-in-water emulsion |
| FR2977800B1 (en) * | 2011-07-13 | 2014-03-14 | Sanofi Pasteur | VACCINE COMPOSITION WITH ALUMINUM HYDROXIDE NANOPARTICLES |
| GB201218195D0 (en) * | 2012-10-10 | 2012-11-21 | Istituto Zooprofilattico Sperimentale Delle Venezie | Composition |
| CN105363029B (en) * | 2014-08-12 | 2021-06-11 | 张世艳 | Novel adjuvant vaccine composition for HPV (human papillomavirus) |
| RU2761453C2 (en) * | 2016-12-23 | 2021-12-08 | Интервет Интернэшнл Б.В. | Combined porcine vaccine |
| CN111892648B (en) * | 2020-06-08 | 2022-08-26 | 中国科学院上海药物研究所 | Novel coronavirus polypeptide vaccine coupled with TLR7 agonist and application thereof |
| CN114184797A (en) * | 2021-12-06 | 2022-03-15 | 辽宁成大生物股份有限公司 | Detection method of influenza virus split vaccine monovalent stock solution hemagglutinin |
-
2006
- 2006-11-06 CN CNA2006800476045A patent/CN101365485A/en active Pending
- 2006-11-06 SI SI200631106T patent/SI1951300T1/en unknown
- 2006-11-06 CN CNA2006800476098A patent/CN101370515A/en active Pending
- 2006-11-06 CN CNA2006800487317A patent/CN101346152A/en active Pending
- 2006-11-06 SI SI200631278T patent/SI1951299T1/en unknown
- 2006-11-06 ES ES06808431T patent/ES2367194T3/en active Active
- 2006-11-06 CN CN2006800476007A patent/CN101365483B/en active Active
- 2006-11-06 CA CA2907149A patent/CA2907149A1/en not_active Abandoned
- 2006-11-06 CN CNA2006800463581A patent/CN101325967A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105727281A (en) * | 2009-02-10 | 2016-07-06 | 诺华股份有限公司 | Influenza vaccines with reduced amounts of squalene |
| CN105125488A (en) * | 2010-05-12 | 2015-12-09 | 诺华股份有限公司 | Improved methods for preparing squalene |
| CN102970976A (en) * | 2010-06-10 | 2013-03-13 | 葛兰素史密丝克莱恩生物有限公司 | Process for the production of an oil in water emulsion |
| CN117582491A (en) * | 2024-01-18 | 2024-02-23 | 江苏瑞科生物技术股份有限公司 | Influenza vaccine composition, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2907149A1 (en) | 2007-05-10 |
| CN101346152A (en) | 2009-01-14 |
| CN101370515A (en) | 2009-02-18 |
| SI1951300T1 (en) | 2011-10-28 |
| ES2367194T3 (en) | 2011-10-31 |
| CN101365483A (en) | 2009-02-11 |
| CN101365485A (en) | 2009-02-11 |
| CN101365483B (en) | 2012-08-22 |
| SI1951299T1 (en) | 2012-04-30 |
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