CN101330926B - Influenza vaccines extemporaneously adsorbed to aluminium adjuvants - Google Patents

Abstract

Antigen and adjuvant components of an adjuvanted influenza vaccine are not mixed during manufacture, but are provided as separate components for extemporaneous mixing at the time of use, for example as a kit comprising: (i) an antigen component, comprising an influenza virus antigen; and (ii) an adjuvant component, comprising an aluminium salt.

Description

Be adsorbed to the influenza vaccines on aluminium adjuvant temporarily

The All Files that this paper quotes is included this paper in as a reference in full.

Government's rights and interests

The present invention makes under the support of and infectious disease academy's (National Institute of Allergy and Infectious Diseases) irritated in NIH (National Institutes of Health)/country the contract HHSN266200400032C of U.S. government in whole or in part.Therefore, U.S. government enjoys some right to the present invention.

Technical field

The invention belongs to adjuvant preparation vaccine provides protection to resist the field of influenza infection.

Background technology

FLUAD except Chiron vaccine company (Chiron Vaccines) ^TMProduct comprises outside oil in water emulsion adjuvant, and the influenza vaccines of using are not at present prepared with adjuvant usually.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.

In recent years, someone proposes Alum adjuvant to be included in influenza vaccines (document 2-5 for example, sees reference).Need extra blend step so that overall manufacturing (speed) slows down, include these salt in also relevant with variety of issue except production period.For example, make the antigen of absorption precipitate from suspension because they are soluble, therefore preparing each dosage from bulk vaccine wants significant care.In addition, the combination of antigen and salt makes the quality control of final vaccine become complicated.Particularly, the in vitroimmunoassay that the effect of some influenza vaccines is tested institute's foundation requires not combination of antigen, namely is adsorbed onto these tests of expression nothing employing on adjuvant.

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 the adjuvant preparation.

Summary of the invention

According to the present invention, not in antigen and the adjuvant component of the influenza vaccines of production period mixing adjuvant preparation, but provide as independent component, mix in use temporarily.The effective reason of the present invention just is to find the absorption moment generation and irreversible under the condition that experiences during vaccination basically of (referring to the embodiment of this paper) antigen and adjuvant.Therefore, the present invention can avoid implementing to mix at production period the variety of issue that produces.

Therefore, test kit provided by the invention is equipped with: the antigen component that (i) comprises influenza antigen; (ii) comprise the adjuvant component of aluminum salt.Component (i) does not comprise Alum adjuvant, and component (ii) does not comprise influenza antigen.The antigen component that the present invention also provides (i) to comprise influenza antigen, and (ii) comprise the adjuvant component of aluminum salt, both use respectively simultaneously or use in order.

The present invention also provides the immunogenic composition that comprises influenza antigen and Alum adjuvant, and interim hybrid antigen and adjuvant prepare said composition in use.

The present invention also provides the method for preparing influenza vaccines, and the method comprises the following steps: (i) preparation comprises the antigen component of influenza antigen; (ii) preparation comprises the adjuvant component of aluminum salt; (iii) this antigen and adjuvant combination of components are placed in test kit.The method also can provide step (iv) to mix this antigen and adjuvant component to give the patient, and nonproductive personnel implement but step (iv) is usually by health care professional.

The present invention also provides preparation and gives the method for influenza vaccines, and the method comprises the following steps: (i) all components of mix reagent box, this test kit are equipped with the antigen component that comprises influenza antigen and comprise the adjuvant component of aluminum salt; (ii) give the patient with the component of mixing.The method generally includes: vibration adjuvant component is to disperse the aluminum salt of any precipitation; With the aseptic adjuvant component that adds of antigen component; The component that upset or soft vibration mix; With the component suction syringe that mixes; Give the patient with the component of mixing.Give the patient usually rear 24 hours of mixing with interior (for example, ≤ 18 hours ,≤12 hours ,≤6 hours ,≤3 hours ,≤2 hours ,≤1 hour ,≤30 minutes ,≤20 minutes ,≤10 minutes ,≤5 minutes ,≤2 minutes ,≤1 minute, etc.) carry out.

Test kit

Test kit of the present invention is equipped with two kinds of components, a kind of antigen that contains, a kind of adjuvant that contains.These two kinds of components are deposited respectively in test kit until determine to prepare vaccine and are given the patient, obtain antigen and are adsorbed onto vaccine on adjuvant thereby mix all components this moment.

Therefore, these two kinds of components physical separation each other in test kit can adopt the whole bag of tricks to realize this separation.For example, these two kinds of components can be placed on different containers, for example in bottle.Then can be for example by taking out the inclusions of a bottle, it is added another bottle, or by taking out respectively the inclusions of two bottles, they are mixed this two bottle in the 3rd container inclusions.

In a preferred configuration, one of kit components is contained in syringe, and another component is contained in container, for example in bottle.Can utilize pre-filled syringe (for example, syringe needle being housed) that its inclusions is inserted in second container and mix, then with in this syringe of mixture suction.Give the patient with the mixing inclusions in this syringe subsequently, usually utilize new aseptic syringe needle.Therefore, a kind of component is packaged in need not to utilize another syringe to implement patient's administration in pre-filled syringe.

In another preferred disposition, two kinds of kit components are contained in respectively same syringe, for example in double-chamber syringe, as disclosed in list of references 6-13 etc.Use the inclusions of namely having mixed in this syringe (for example, give the patient during) in this two Room.This configuration need not independent blend step in use.Inclusions in two Room is all aqueous form usually.

In some configurations, one of component (being generally antigen component rather than solution component) is in dried forms (for example, the form of lyophilizing), and another component is in aqueous form.Can mix described two kinds of components and do component with reactivation, thereby obtain to give patient's waterborne compositions.In the configuration of other time choosing, two kinds of components all are in dry form.Freeze-dried component is stored in bottle rather than syringe usually.Dry component can comprise stabilizing agent, for example lactose, sucrose or mannitol and their mixture, such as lactose/sucrose mixture, sucrose/mannitol mixture etc.

A kind of preferred configuration using is contained in the aqueous adjuvant component in pre-filled syringe, and the freeze-dried antigen component is contained in bottle.

If two kinds of components are all aqueouss, can various volume ratios, for example 1: 5 (aqueous antigen volume is excessive) and (aqueous adjuvant volume excessive) between mix they at 5: 1.Ratio between preferred 1: 2 and 2: 1, for example approximately 1: 1.

The suitable vessel that is used for test kit comprises bottle and disposable syringe.These containers should be aseptic.

Be contained in bottle as fruit component, bottle is preferably made by glass or plastic material.Preferably first make the bottle sterilization add again compositions.For avoiding the patient to the problem of latex allergy, preferably use the plug seal bottle without latex, preferably all packaging material 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.

The dose volume of the influenza vaccines that generally give is about 0.5ml, although half-value dose (that is, approximately 0.25ml) can be given the child.Can do the labelling of upper demonstration half-value dose volume to container, 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.

Bottle can be equipped with lid (for example, the Luer snap close), thereby pre-filled syringe can be inserted in lid, the inclusions of syringe is pushed in bottle (for example, rebuild freeze-dried material wherein), then the inclusions of bottle is drawn back in syringe.After syringe is taken out, load onto syringe needle from bottle, give the patient with compositions.Preferably make lid be positioned at the sealing or covering, thereby to first remove the sealing or covering contact again lid.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 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 lot number and the effect duration of inclusions on it, thereby has helped scorekeeping.The plunger of syringe preferably has stopper, 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 cap with apical end, and described pin cap is preferably made by butyl rubber.If syringe and syringe needle be packing separately, preferably to syringe needle, the butyl rubber cover is housed.Preferred syringe is with " Tip-Lok " ^TMThose that put goods on the market for trade name.

If use glass container (for example, syringe or bottle), the container that preferably uses pyrex rather than soda-lime glass to make.

Test kit can be equipped with (for example, being contained in same box) inset, and this inset comprises the details of vaccine, the operation instructions of administration for example, details of antigen etc. in vaccine.Operation instructions also can comprise warning, for example prepare epinephrine solution in case the anaphylaxis after vaccination, etc.

Test kit is preferably kept between 2 ℃-8 ℃.Should be freezing they.

Influenza antigen

One of all components of test kit comprise influenza antigen.Usually can prepare these antigen from influenza virus particles, perhaps can express in the recombinant host (for example, utilizing the insect cell of baculovirus vector) such as the antigen such as hemagglutinin and use [14,15] with purified form.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, usually also comprise neuraminidase) of complete virus, lytic virus 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 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, etc.) the processing virion, comprise that " tween-ether " cleavage method produces the subviral particle goods, thereby obtained lytic virus.The method of well known cracking influenza virus is such as referring to list of references 16-21 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, thereby 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), tributyl phosphate (tri-N-butyl-phosphate), cetavlon (Cetavlon), the myristyl leptodactyline, lipofectin reagent, fat transfection amine, DOT-MA, octyl group-or the Nonylphenoxy poly(ethylene oxide)polymers is (for example, the triton surfactant, as triton x-100 or triton N101), polyoxyethylene sorbitan esters (tween surfactants), polyoxyethylene ether, polyoxyethylene ester etc.A kind of useful cleavage method utilizes the continuous action of NaTDC and formaldehyde, during cracking occurs in initial virion purification (for example, in sucrose density gradient solution).Therefore cleavage method can comprise and makes the material clarification (to remove non-viral particulate matter) that contains virion, and the concentrated virion of collecting (for example, adopts absorption method, as CaHPO ₄Absorption), separate complete virion and non-viral particulate matter, (for example utilize decomposition agent lytic virus granule in the density gradient centrifugation step, utilization contains decomposition agent, saccharose gradient (solution) as NaTDC), then filter (for example, ultrafiltration) to remove unwanted material.The virion of cracking 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, usually also comprises neuraminidase.These method of protein of well known preparation purified form.FLUVIRIN ^TM, AGRIPPAL ^TMAnd INFLUVAC ^TMProduct is subunit vaccine.

Influenza proteins except HA and NA also can be used as influenza antigens, comprises the fragment of native protein.Also can use their combination.

Can also the virion form there be [22] in influenza antigens.

Influenza virus can be attenuation.Influenza virus can be to responsive to temperature.Influenza virus can be cold adaptation.These three kinds of probabilities are specially adapted to live virus.

The per season of influenza virus strain that is used for vaccine is different.In present popular interval, vaccine comprises two kinds of influenza A strains (H1N1 and H3N2) and a kind of influenza B strain usually, is generally trivalent vaccine.The present invention also can utilize epidemic isolates (namely, 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; the character that depends on season and the contained antigen of 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 compositions of adjuvant preparation 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 at present popular people's strain, it contains new hemagglutinin, namely, (for example occur to surpass the strain of 10 years in the crowd, H2), the strain of at all not seeing in the crowd perhaps (for example, H5, the H6 or the H9 that usually only find in flock of birds) is originally thereby make the crowd 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[23], wherein clade 1 and 3 is relevant especially.

Other strain that can usefully be included in these compositionss is the strain of the strain (for example, tolerance oseltamivir [24] and/or zanamivir) of tolerance antiviral therapy, comprises drug resistance epidemic isolates [25].

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, usually cultivate respectively different strains, collect the rear mixing of virus and prepare antigen.Therefore, the inventive method can comprise the step of the antigen that mixes multiple influenza strain.Although the trivalent vaccine that preferably comprises the antigen of two kinds of influenza A virus strains and a kind of Influenza B virus strain is univalent vaccine also useful (for example, for epidemic isolates).

Influenza virus can be recombined strain, can obtain by reverse Genetics Technique.Reverse Genetics Technique [for example, 26-30] 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 DNA molecular of the required viral RNA molecule of polI promoter coding, (b) for example can be from the DNA molecular of polII promoter coding virus protein, thus the DNA that makes two types of cells can assemble complete infectious viral particle.DNA preferably can provide all viral RNAs and protein, but also may utilize helper virus that some in described RNA and protein are provided.The preferred plasmid method, thereby can produce each viral RNA [31-33] 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 [34] 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), 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 have been made up on another plasmid.The preferred aspect of list of references 34 described methods comprises: (a) separately PB1, the PB2 on plasmid and PA mRNA coding region; (b) 8 vRNA coding sections of all on independent plasmid.Comprise one on plasmid NA and 6 other sections on HA section and another plasmid be also favourable.

Except with polI promoter coding viral RNA section, also may use bacteriophage polymerase promoter [35].For example, can use easily the promoter of SP6, T3 or T7 polymerase.Due to the kind specificity of polI promoter, the bacteriophage polymerase promoter for many cell types (for example, MDCK) more convenient, although also must be with the plasmid transfection cell of encoding exogenous polymerase.

Other technology may adopt dual polI and polII promoter encode simultaneously viral RNA and the effable mRNA[36,37 of a template].

Therefore, virus (particularly influenza A virus) can comprise one or more RNA sections (6 of A/PR/8/34 sections normally of reassortant virus, wherein HA and N section be from vaccine strain, namely 6: 2 reassortants), when particularly cultivating virus with ovum.One or more RNA sections that also can comprise or any other virus stain viral with the A/WSN/33 of reprovision virus for the production of the vaccine preparation.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 SPF ovum or cell culture are cultivated the virus as the antigen source.The standard method utilization of cultivating at present influenza virus 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, virus can be introduced together with one or more aminoacid in the allantoic fluid of egg [18].

Cellular material is mammal cell line normally.Suitable mammalian cell is originated and is included but not limited to: hamster, cattle, primates (comprising people and monkey) and canine cells.Can utilize various cell types, such as 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 Vero cell line.Suitable canine cells is, for example the nephrocyte in mdck cell system.Therefore, suitable cell line includes but not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6; WI-38; Etc..Utilize mammalian cell to represent that vaccine can not contain egg albumin (for example, ovalbumin and ovomucoid) and chicken DNA, thereby can reduce allergenicity.

The preferred mammal cell line that is used for the cultivation influenza virus comprises: the mdck cell [38-41] that is derived from Madin-Darby canine kidney; Be derived from the Vero cell [42-44] of cercopithecus aethiops (Cercopithecus aethiops) kidney; Or be derived from the PER.C6 cell [45] of people embryo retinoblast.These cell lines can be extensively available from, for example American Type Culture Collection (ATCC) [46], cell preservation center, bandit Lille (Coriell Cell Repositories) [47] 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 48-50], comprises the cell line that is derived from duck (for example, the duck retina) or hen, chick embryo fibroblast (CEF) for example, etc.Example comprises fowl embryo stem cell [48,51], comprises the EBx cell line, EB45, EB14 and the EB14-074[52 that are derived from chicken embryonic stem cells].

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 38 has disclosed the mdck cell system (" MDCK 33016 " are with DSMACC 2219 preservations) that is adapted to grow in suspension medium.Similarly, list of references 53 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 54 has disclosed the non-tumorigenic mdck cell, comprises " MDCK-S " (ATCC PTA-6500), " MDCK-SF101 " (ATCC PTA-6501), " MDCK-SF102 " (ATCC PTA-6502) and " MDCK-SF103 " (PTA-6503).It is to comprise " MDCK.5F1 " cell (ATCC CRL-12042) that list of references 55 has disclosed infecting extremely sensitive mdck cell.Can use any these mdck cells to be.

If utilize mammal cell line to cultivate virus, the antigen component in test kit does not preferably contain egg albumin (for example, ovalbumin and ovomucoid) and chicken DNA, thereby can reduce allergenicity.

If cultivate virus with cell line, growth medium and the virus inoculation thing be used for to start cultivated preferably do not 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 [56].Particularly preferably do not contain herpes simplex virus.

If cultivate virus with cell line, antigen component preferably contains and is less than the 10ng residual host cell DNA of (preferably being less than 1ng, more preferably less than 100pg) in every dose, although can there be the host cell DNA of trace.Can adopt the standard purification method, remove contaminative DNA such as chromatography etc. during the vaccine preparation.Process by nuclease, for example can promote to remove residual host cell DNA with the DNA enzyme.List of references 57 and 58 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, then can during breaking, use virion cationic detegent (for example, CTAB).Alkylating agent, for example beta-propiolactone is processed and also be can be used for removing host cell DNA, and the method also can be preferred for inactivated virus particle [59].

Contain in preferred every 15 μ g hemagglutinins＜10ng (for example,＜1ng,＜100pg) vaccine of host cell DNA, for example every 0.25ml volume contain＜10ng (for example,＜1ng,＜100pg) vaccine of host cell DNA.More preferably contain in every 50 μ g hemagglutinins＜10ng (for example,＜1ng,＜100pg) vaccine of host cell DNA, for example every 0.5ml volume contain＜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, etc.

For using cell line, for example mdck cell is cultivated, and available suspension is cultivated [38,60,61] or the cell of adhere-wall culture is cultivated virus.A kind of suitable mdck cell that is used for the suspension cultivation is to be MDCK 33016 (preserving number is DSM ACC 2219).Perhaps, can adopt microcarrier to cultivate.

Preferably support with culture medium and/or the protein-free culture medium culturing of serum-free the cell line that influenza virus is copied.The culture medium that the present invention will be wherein contain the serum additive in human or animal source is called serum-free medium.Be interpreted as representing occuring without protein not containing protein, somatomedin, other oroteins 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 copies preferably growth [62] below 37 ℃ (for example, 30-36 ℃, or approximately 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃), for example during virus replication.

The method of propagative viruses generally includes following steps in cultured cells: inoculate strain to be cultivated to cultured cells, with the required time of infected cell culture and virus breeding, for example by virus titer or antigen presentation measure (as, rear 24-168 hour of inoculation), collect the virus of breeding.Take virus and the ratio of cell as 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 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 usually is less than 300 minutes, preferably between 90-240 minute.Can remove by freeze thawing or enzymatic catalysis the cell culture (for example, monolayer) of infection, thereby increase the viral level in the culture supernatant of collecting.Then make liquid deactivation or the freezing preservation of collection.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.Can collect the cell of infection in 30-60 hour after infection.Preferred 34-48 hour collecting cell after infection.More preferably 38-40 hour collecting cell after infection.Usually add protease (normally trypsin) with releasing virus during cell culture, can add protease by any suitable stage in the training period.

Hemagglutinin (HA) is the main immunogens in 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, although for example child, or can use lower dosage under popularity.With higher dosage (for example, 3 * or 9 * dosage [63,64]) the same, used part dosage, for example 1/2 (that is, 7.5 μ g HA/ strains), 1/4 and 1/8[4,5].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, etc.Concrete dosage comprises, about 45, approximately 30, approximately 15, approximately 10, approximately 7.5, approximately 5, approximately 3.8, approximately 1.9, approximately 1.5/ strain for example, etc.The same with the present invention, when having adjuvant in vaccine, these lower dosage are the most useful.

For live vaccine, by the intermediate value (TCID of 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).

The present invention HA used can be the natural HA that finds in virus, perhaps can be modified.For example, the known HA of modification (for example causes the viral determinant that causes a disease at the birds camber to remove, around the hyperalkaline of the cleavage site between HA1 and HA2 zone (hyper-basic region)), otherwise these determinants can stop virus to be grown in ovum.

The antigen component of test kit of the present invention can comprise detergent, polyoxyethylene dehydration sorbitan esters surfactant (being called " tween ") for example, Octoxinol (for example, octoxynol 9 (triton x-100) or TRITON-X-100), cetab (CTAB), or NaTDC, particularly for cracking or SAV.Can only there be the detergent of trace.Therefore, in vaccine contained Octoxinol-10, alpha-tocopherol hemisuccinic acid ester (α-tocopheryl hydrogensuccinate) and polysorbate80 separately lower than 1mg/ml.The residual component of other trace can be antibiotic (for example, neomycin, kanamycin, 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 fragment of M2 stromatin or the M1 albumen of detection level.Also can there be nucleoprotein.

Adjuvant

In influenza vaccines, adjuvant used comprises chitosan [65]; O/w emulsion, for example MF59[66], water-in-oil-in-water compositions [67]; aluminum salt [2,5]; CpG oligodeoxynucleotide, for example CpG 7909[68], E.coli LT [69,87] and detoxification mutant [70-71] thereof; monophosphoryl lipid A [72] and 3-o-deacylated tRNA radical derivative [73] thereof; pertussis toxin, PT mutant [74], muramyldipeptide [75], etc.

Yet adjuvant component of the present invention is based on aluminum salt.These salt comprise the adjuvant that is called aluminium hydroxide and aluminum phosphate.These titles are usual, but just for convenience of and use, its actual chemical component the 9th chapter of document 76 [for example, see reference] is not accurately described.The present invention can be with any " hydroxide " that is conventionally used as adjuvant or " phosphate " adjuvant.

The adjuvant that is called " aluminium hydroxide " is generally aluminum oxyhydroxide salt, its common at least part of crystallization.Aluminum oxyhydroxide is with molecular formula AlO (OH) expression, itself and other aluminium compound, for example aluminium hydroxide Al (OH) ₃Difference be infrared (IR) spectrum, particularly at 1070cm ^-1There is absorption band in the place and at 3090-3100cm ^-1There is strong acromion [the 9th chapter of list of references 76] in the place.The width (WHH) of half-peak eminence diffraction zone has reflected the crystallization degree of aluminum hydroxide adjuvant, and the not good granule of crystallization shows stronger spectral line broadening because crystalline size is less.Surface area increases with the increase of WHH, and the adjuvant that the WHH value is larger shows that the ability of adsorption antigen is stronger.Aluminum hydroxide adjuvant is typical fibre morphology (for example, transmission electron micrograph is being seen).The pI of aluminum hydroxide adjuvant usually approximately 11, and namely adjuvant itself has positive surface charge under physiological pH.It is reported, during pH 7.4, the absorbability of aluminum hydroxide adjuvant is at every mg Al ⁺⁺⁺1.8-2.6mg between protein.

The adjuvant that is called " aluminum phosphate " is generally Adju-Phos, and these adjuvants also often contain a small amount of sulfate radical (that is, hydroxyl phosphoric acid aluminum sulfate).Can obtain these adjuvants by precipitation, the reaction condition during precipitation and concentration affects phosphate radical replace the degree of hydroxyl in this salt.PO in hydroxyl phosphate ₄/ Al mol ratio is usually between 0.3-1.2.Hydroxyl phosphate is because existing hydroxyl to be different from strict AlPO ₄For example, 3164cm ^-1IR band (for example, when being heated to 200 ℃) show and have structural hydroxyl [the 9th chapter of list of references 76].

The PO of aluminum phosphate adjuvant ₄/ Al ³⁺Mol ratio usually between 0.3-1.2, preferably between 0.8-1.2, more preferably 0.95 ± 0.1.Aluminum phosphate is normally unbodied, particularly hydroxyl phosphate.Typical adjuvant is PO ₄/ Al ³⁺Mol ratio contains 0.6mg Al between 0.84-0.92 ³⁺The amorphous Adju-Phos of/ml.Aluminum phosphate normally granular (for example, the dull and stereotyped sample granule seen in transmission electron micrograph).These granules are 0.5-20 μ m (for example, approximately 5-10 μ m) in the representative diameter of absorption after any antigen.It is reported, during pH7.4, the absorbability of aluminum phosphate adjuvant is at every mg Al ⁺⁺⁺0.7-1.5mg between protein.

The degree of the point of zero electric charge of aluminum phosphate (PZC) and phosphate radical substituted hydroxy is inversely proportional to, and should the replacement degree according to this salt of precipitation reaction condition used and reactant concentration and difference.Concentration that also can be by changing solution Free Phosphorus acid ion (phosphate radical more=PZC acidity is higher) or change PZC by adding such as buffer such as histidine buffering liquids (making PZC have more alkalescence).The PZC of the present invention's aluminum phosphate used usually between 4.0-7.0, more preferably between 5.0-6.5, for example approximately 5.7.

Aluminum salt suspension for the preparation of the present composition can contain buffer (for example, phosphoric acid or histidine or Tris buffer), but not necessarily.These suspensions are aseptic, apyrogeneity preferably.Suspension can contain free aqueous phosphate anion, for example concentration between 1.0-20mM, preferably between 5-15mM, 10mM more preferably from about.These suspensions also can contain sodium chloride.

In an embodiment of the invention, the adjuvant component comprises the mixture [4] of aluminium hydroxide and aluminum phosphate.In this case, aluminum phosphate may be more than aluminium hydroxide, and for example weight ratio is at least 2: 1, and for example 〉=5: 1, 〉=6: 1, 〉=7: 1, 〉=8: 1, 〉=9: 1, etc.

Give Al in patient's compositions ⁺⁺⁺Concentration preferably lower than 10mg/ml, for example≤5mg/ml ,≤4mg/ml ,≤3mg/ml ,≤2mg/ml ,≤1mg/ml, etc.Preferred scope is between 0.3-1mg/ml.Preferred maximum＜0.85mg/ agent.

Except one or more Alum adjuvants, the adjuvant component also can comprise one or more other adjuvant or immunostimulant.This other component includes but not limited to: 3D-MPL adjuvant (" 3d-MPL "); And/or O/w emulsion.3d-MPL is also referred to as 3D-MPL or 3D-MPL.This title shows 3 deacylated tRNA bases of reducing end glycosamine in monophosphoryl lipid A.It is prepared without heptose (heptoseless) mutant by salmonella minnesota (Salmonella minnesota), and its chemical constitution is similar to lipid A but lacks sour unsettled phosphoryl and alkali labile acyl group.The cell of its energy activated mononuclear cell/macrophage pedigree stimulates to discharge several cytokines, comprises IL-1, IL-2, TNF-α and GM-CSF.Preparation 3d-MPL is described in list of references 77 at first, and this product is by Kao Likesa company (Corixa Corporation) production and with trade name MPL ^TMSell.Other details document 78-81 that sees reference.

At last, in other embodiment of the present invention, replace aluminum salt with calcium salt.In these embodiments, the adjuvant component comprises synthos usually.

Pharmaceutical composition

The antigen component of test kit and adjuvant component are pharmaceutically acceptable, and the product of their mixing gained too.Blended product can comprise the component except antigen and adjuvant, and these components can be derived from antigen component and/or adjuvant component and/or the 3rd optional component.

Therefore, final mixture comprises one or more pharmaceutically acceptable carrier and/or excipient usually.To discussing fully of these carriers and the excipient document 82 that sees reference.

Final mixture can comprise 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 [17,83].More preferably not mercurous vaccine.Particularly preferably do not contain the vaccine of antiseptic.

Preferably comprise physiology salt, for example sodium salt is to control tension force.Sodium chloride (NaCl) between 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.

Compositions can comprise citrate ions.

Administration composition has the osmolality between 200mOsm/kg-400mOsm/kg usually, preferred 240-360mOsm/kg, more preferably 290-310mOsm/kg.Once reported in the past osmolality to the pain caused not impact [84] of vaccination, but still preferably osmolality was maintained in this scope.

Administration composition 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 is generally 5-20mM.

The pH of administration composition is usually between 5.0-8.1, and is more common between 6.0-8.0, for example between 6.5 and 7.5, or between 7.0 and 7.8.Therefore, the inventive method can comprise the step that the pH that first regulates bulk vaccine packs again.

Each component of test kit comprises container, and is preferably aseptic.

The preferred apyrogeneity of kit components, for example every dosage contains＜1EU (endotoxin unit, gauge), preferred every dosage＜0.1EU.

The preferred GF of kit components.

Kit components can comprise the material of primary immune response, maybe can comprise the repeatedly material (that is, " multiple dose " test kit) of immunity.Therefore, for example can the antigen of 10 doses be housed in a container, the adjuvant of 10 doses is housed in second container.Intra-operative, use the day morning mix this two kinds of components, thereby give a series of patients with 10 doses in this sky.Syringe with each dosage suction administrable.In the multiple dose configuration, preferably comprise antiseptic.Except comprise antiseptic in multi-dose compositions, also compositions can be placed in the container that is equipped with for the sterile adapter of transfer of material.

Giving of Therapeutic Method and vaccine

After mixing, the present composition is fit to give human patients, the invention provides the method that produces immunne response in patient body, comprises the step that compositions of the present invention is given the patient.

The present invention also provides test kit, perhaps is used as the present composition of medicine.

The adjuvant component that the present invention also provides (i) influenza antigen and (ii) comprises aluminum salt is for the preparation of the application in the medicine that produces immunne response in patient body, and wherein said medicine comprises as the antigen of separation component and adjuvant.

The immunne response that these methods and applications produce generally includes antibody response, and preferred protection antibody is replied.The method of well known assessment antibody response, neutralising capacity and protective effect after the influenza virus vaccine inoculation.Human research proof is for the antibody titer of human influenza virus's hemagglutinin relevant with protective effect (blood serum sample hemagglutination-inhibition titre is approximately during 30-40, and the protective effect of homology viral infection is about 50%) [85].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 lower limb), but other available approach comprises subcutaneous injection, intranasal [86-88], oral [89], intradermal [90,91], transdermal, percutaneous [92] 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 patient who accepts vaccine be the old people (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, conceived women, chronic disease, immunodeficiency patient take antiviral compound (for example, oseltamivir or zanamivir compound in front 7 days accepting vaccine; For example oseltamivir phosphate, vide infra) the patient, to the people of egg allergy and the people who goes abroad.Yet these vaccines are not only applicable to these groups, also can be applicable to widely in the crowd.For epidemic isolates, preferably give all age group.

the vaccine that the present invention can be prepared and other vaccine are basically simultaneously (for example, can the same medical consultation at health care expert or vaccination center or medical during) 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, the influenza B haemophilus vaccine of coupling, the poliovirus vaccine of deactivation, hepatitis B virus vaccine, the meningococcal conjugates vaccine (for example, tetravalence A-C-W135-Y vaccine), respiratory syncytial virus vaccines, the streptococcus pneumoniae conjugate vaccines, etc..Basically give simultaneously in the gerontal patient particularly useful 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) of particularly effectively resisting epidemic disease influenza poison basically simultaneously (for example, can health care expert's a same medical consultation or medical during) give the patient.These antiviral (compound) comprise neuraminidase inhibitor; (3R for example; 4R, 5S)-amino-3 (1-ethyl the propoxyl group)-1-cyclohexene-1-carboxylic acids of 4-acetyl-amino-5-, comprise its ester (for example ethyl ester) and salt (for example phosphate).Preferred antiviral compound is amino-3 (1-ethyl the propoxyl group)-1-cyclohexene-1-carboxylic acids of (3R, 4R, 5S)-4-acetyl-amino-5-, ethyl ester, phosphate ester (1: 1), also referred to as oseltamivir phosphate (TAMIFLU ^TM).

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.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.Usually but at least one week of interval (for example, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 6 weeks, approximately 8 weeks, approximately 10 weeks, approximately 12 weeks, about 16 weeks, etc.) gives multiple dose.

Because the present composition and test kit comprise aluminium adjuvant, the component precipitation may occur between storage life.Therefore, should give again the patient by first set of oscillations compound.The compositions of vibration is muddy white suspension.

General introduction

Term " contain " comprise " comprising " and " by ... form ", the compositions that for example " contains " X can only be comprised 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 be fully without Y.This word " basically " can optionally save from the present invention's definition.

The term " about " relevant to 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, can first two kinds of components be mixed with each other, then this mixture is mixed with the third component, etc.

If certain antigen is described as " absorption " to certain adjuvant, preferably adsorbed this antigen of at least 50% (by volume), for example 50%, 60%, 70%, 80%, 90%, 95%, 98% or more.

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 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 in European Pharmacopoeia summary section 5.2.3.

Embodiments of the present invention

Due to the problems referred to above being arranged, determine that adjuvant and antigen component until keep in using that can research prepare wherein separate but the vaccine of antigen absorption wherein still can occur when using aluminum salt as influenza virus vaccine adjuvants.Be to determine the feasibility of the method, the purification surface antigen of certain influenza virus is mixed with the aluminium hydroxide suspension.After mixing, immediately by desk-top centrifugation aluminum salt, detect the amount (that is the amount of the protein that, does not adsorb) that remains in the protein in supernatant.

Present influenza A virus strain

From influenza virus A/New Caledonian (H1N1) or A/Wyoming (H3N2) purification hemagglutinin, it is diluted to 75 μ g HA/ml.Preparation 4.25mg/ml (about 1.5mg Al ⁺⁺⁺/ ml) aluminum hydroxide adjuvant.1ml adjuvant suspension is added in the invisible spectro 4ml antigenic solution of 15ml Falcon, and this mixture and cultivating in room temperature overturns.First at the 0th o'clock, then took a sample in the time of the 5th, 10,20,30,60,90 and 120 minute.Contrast is independent antigen (10mM PBS, pH 7.7) or independent adjuvant (10mM PBS, pH7.7).Analyze with the material of precipitation absorption with the centrifugal sample of 4000rpm immediately.Pass through BioRad ^TMProtein determination and non-degeneration SDS-PAGE detect protein content.

The absorption result of study of two kinds of strains is as follows, only has the protein content of the contrast of antigen to be standardized as 100%:

Sample	A/New Caledonia protein in suspension	A/Wyoming protein in suspension
			Antigen control	100	100
The adjuvant contrast	0.0	0.3
			0 minute	3.8	1.2
5 minutes	1.7	0.7
			10 minutes	2.2	0.6
20 minutes	2.2	0.6
			30 minutes	1.6	0.8
60 minutes	1.4	0.5
			90 minutes	1.8	0.5
120 minutes	1.9	0.5

Therefore highly absorption occur very fast.The difference of each time point is not remarkable.For confirming these results, the SDS-PAGE of supernatant separates employing SYPRO ruby dyeing.Adjuvant contrasts, had no protein band in sample in 0,5,10,20 or 30 minute.Therefore any protein that exists is lower than the detectability of the method, thus the method enough sensitivity can detect the protein of 1-2ng.

Result shows that at least 97% antigen quick adsorption is to adjuvant.Unexpectedly, absorption is to occur moment basically, thereby can distribute the influenza vaccines of adjuvant preparation and need not to be adsorbed onto on adjuvant in advance.Therefore, can prepare more quickly Adjuvanted vaccines, this is the most useful in popularity.Utilize two kinds of different influenza A virus strains to obtain these results, can expect to observe effect same with other strain with based on other adjuvant of soluble aluminum salt fully.

Popular influenza A strain

The surperficial antigen preparation of purification of 2: 6 reassortants of preparation A/Vietnam/1203/2004 x A/PR8/8/34 (H5N1) influenza virus.Measure hemagglutinin content through SRID and be estimated as 41 μ g HA/ml.Utilize the Ultrafree-15 centrifugal filter device that 30ml A/H5N1 is concentrated into approximately 15ml.By the Bio-Rad protein test, utilize 0-50 μ g/ml gamma Globulin standard curve to record the protein total content of original and concentrated A/H5N1.Utilize this result to calculate the ratio of HA and total protein.Then calculate the required final volume of A/H5N1 solution of 60 μ g HA/ml with this value.

The aluminum hydroxide adjuvant of 0.7ml 2mg/ml is added in the 0.7ml 60 μ gHA/ml MBP of 1.5ml microcentrifugal tube.Mix all solution by upset, under room temperature, (approximately 20 ℃) are cultivated.The 5th minute, 10 minutes, 30 minutes, 2 hours, 8 hours and duplicate sampling in 24 hours.Contrast is 0.7mL 10mM PBS, and pH 7.7 adds 0.7mL 60 μ g HA/mL MBP; With 0.7mL 10mM PBS, pH 7.7 adds 0.7mL 2mg/mL aluminium hydroxide.Under room temperature with the centrifugal sample of 13000rpm 1 minute to remove the aluminium hydroxide of suspension, supernatant is poured in the 7ml sterile cuvette (bijou) of having made labelling.As mentioned above, utilize the SYPRO dyestuff to pass through BioRad ^TMProtein test and non-degeneration SDS-PAGE analysis result, result is as follows:

Sample	Protein in supernatant
		Antigen control	100
The adjuvant contrast	0.5
		5 minutes	3.7
10 minutes	2.1
		30 minutes	0.2
2 hours	0.3
		8 hours	1.1
24 hours	2.4

The result of A/H5N1 preparation is suitable with the result of utilizing A/New Caledonia of equal value and A/Wyoming goods to be tested.The supernatant of all samples is carried out the protein level that Baeyer-Lei De (Bio-Rad) protein test detects very low, prove that nearly all protein keeps being combined with aluminum hydroxide precipitate.After SDS-PAGE separates, sample is carried out sensitive SYPRO ruby dyeing and show in adjuvant contrast or arbitrary sample of 6 times there is no protein band.Therefore any protein that exists is lower than the detectability of the method.The protein concentration of point does not have notable difference any time.The all samples adsorptivity of aluminium hydroxide contrast and all time samples and protein concentration estimated value subsequently are all lower than the lower limit of 5-50 μ g/ml standard curve.

Therefore, data show that A/H5N1 protein moment is adsorbed onto Alum adjuvant, and can keep stable at least 24 hours.

People's clinical data

Report as list of references 93, in testing in the disclosed unmatchful photograph I phase of random tags, unit price cracking influenza A/Vietnam/1194/2004 (H5N1) bacterin preparation of 6 kinds of deactivations of 300 volunteers received comprises containing or the HA of 3 kinds of various dose of aluminium hydroxide adjuvant (7.5 μ g, 15 μ g or 30 μ g) not.All individual twice vaccination of accepting suppresses and microneutralization analyzing blood sample by hemagglutination.

Adopt approval for the production of VAXIGRIP ^TMPopular interval vaccine method [94], prepare vaccine in containing the egg of embryo.Vaccine strain is influenza A/Vietnam/1194/2004/NIBRG14 (H5N1) the reference strain of NIBSC preparation.This strain contains the modification hemagglutinin of high pathogenic avian influenza strain A/Vietnam/1194/2004 and other virus protein of neuraminidase and influenza A/PR/8/34 (H1N1).Modify the polybase acidic amino acid sequence (multibasic amino acid sequence) of hemagglutinin to remove cleavage site.

Prepare for 0.5ml syringe (No. 23,1 inch syringe needle) filling with the phosphate buffered saline(PBS) that does not contain adjuvant, the hemagglutinin level is the split vaccine of 7.5 μ g, 15 μ g or 30 μ g.For the vaccination of not using adjuvant, directly use these syringes in the patient.Yet, the same with the syringe contents of aluminium hydroxide adjuvant for the vaccination of using adjuvant, the inclusions of syringe is injected sterile vials.This mixing can be implemented at bedside just before use, mixes after 10 seconds, and the syringe that all inclusions suction is new (No. 23,1 inch syringe needle) softly reverberates so that antigen/adjuvant suspension homogenizes, and then intramuscular (triangular muscle) is injected into the patient.Volume injected is 0.5ml, except 30 μ g preparations (1ml volume) of adjuvant preparation.The final adjuvant content of vaccine is 600 μ g.The basic research of hybrid antigen and adjuvant shows that all three kinds of antigen doses have similar adsorption coefficient.

Each participant accepts twice intramuscular injection, interval 21 days (the 0th day and the 21st day).Gathered blood sample at the 0th, 21 and 42 day.

All 6 kinds of preparation well-tolerated did not have serious side effect between the 0th day and the 42nd day, there is no serious injection site pain, did not have oral temperature to surpass the incident of generating heat report of 38 ℃.

All preparations have all caused immunne response, and some individualities only namely detect after single administration and respond.Suppress for hemagglutination, it is 32 or higher that the 21st day each group has the titre of 6%-34%, increases to 28-67% the 42nd day this ratio.Those of the pattern of Neutralizing antibody response and hemagglutination inhibition are similar.30 μ g preparations of adjuvant preparation have been induced the strongest replying (hemagglutination after twice vaccination-inhibition seroconversion rate is 67%).Specifically, implement two dosage method with 30 μ g H5N1 vaccines of adjuvant preparation and show that immunne response meets the European management expectancy of approval seasonal current influenza vaccine.

Should be understood that just and described the present invention by embodiment, can make improvements and still belong to the design of scope of the present invention.

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Claims (17)

1. test kit, it is equipped with: the antigen component that (i) comprises influenza antigen; (ii) comprise the adjuvant component of aluminum salt, described component provides as independent component, mixes in use temporarily.

2. test kit as claimed in claim 1, is characterized in that, one of described component or both are contained in bottle.

3. test kit as claimed in claim 1, is characterized in that, one of described component or both are contained in syringe.

4. test kit as claimed in claim 1, is characterized in that, one of described component is contained in syringe, and another kind of component is contained in bottle.

5. test kit as claimed in claim 4, is characterized in that, described antigen component is contained in syringe.

6. according to any one of the preceding claims test kit, is characterized in that, described influenza antigen is inactivation of viruses.

7. test kit as claimed in claim 6, is characterized in that, described influenza antigen comprises the surface antigen of complete virus, lytic virus or purification.

8. according to any one of the preceding claims test kit, is characterized in that, described influenza antigen is from H1, H2, H3, H5, H7 or H9 subtypes of influenza A virus.

9. according to any one of the preceding claims test kit, is characterized in that, the influenza virus of cultivating from ovum prepares described influenza antigen.

10. test kit as described in any one in claim 1-8, is characterized in that, the influenza virus of cultivating from cell culture prepares described influenza antigen.

11. test kit as described in any one in claim 1-8 is characterized in that described influenza antigen component does not contain ovalbumin, ovomucoid and chicken DNA.

12. test kit as claimed in claim 10 is characterized in that, described influenza antigen component contains the cell DNA that is less than 10ng cell culture host.

13. test kit according to any one of the preceding claims is characterized in that, described influenza antigen component contains 0.1-50 μ g hemagglutinin/virus stain in this component.

14. test kit according to any one of the preceding claims is characterized in that, described adjuvant component comprises aluminum hydroxide adjuvant.

15. test kit according to any one of the preceding claims is characterized in that, described adjuvant component comprises the aluminum phosphate adjuvant.

16. a method for preparing test kit claimed in claim 1 said method comprising the steps of: (i) preparation comprises the antigen component of influenza antigen: (ii) preparation comprises the adjuvant component of aluminum salt; (iii) described antigen and adjuvant combination of components are contained in test kit, described component provides as independent component, mixes in use temporarily.

17. test kit as described in any one in claim 1-15, described test kit is used to medical science.