CN101346152A - Adjuvanted vaccines with non-virion antigens prepared from influenza viruses grown in cell culture - Google Patents
Adjuvanted vaccines with non-virion antigens prepared from influenza viruses grown in cell culture Download PDFInfo
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Abstract
An immunogenic composition comprises: (i) a non-virion influenza virus antigen, prepared from a virus grown in cell culture; and (ii) an adjuvant. Preferred adjuvants comprise oil-in-water emulsions.
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
At present, the SPF egg that contains embryo, purified virus from egg inclusions (allantoic fluid) are used in the standard method of cultivating influenza virus in the production of vaccine.Yet, cultivate virus with cell culture recently, this method may produce a large amount of antigens in a short time.In addition, this method can produce because of the pathogenic virus that can not cultivate with egg of birds.
Irving Baxter (Baxter) scientist's list of references 1 has compared from the trivalent whole virus particles vaccine (WVV) of the virus preparation of egg or Vero cell culture.The ability that has compared these two kinds of vaccine-induced body fluid and cell mediated immunity power.These authors reported Vero derive the immunogenicity of vaccine and egg derive vaccine quite, but the Vero vaccine of deriving is better with regard to t cell response.It is reported that for the drift of seasonal influenza virus antigenicity, t cell response more tolerates than antibody response, thereby improved year border immunity (year-to-year immunity).
Under these results' inspiration, Irving Baxter continues to have developed commodity PREFLUCEL by name
TMThe Vero derivative products.Yet in December, 2004, Irving Baxter has suspended its II/III phase clinical research, and is being seen because the ratio of heating and relevant symptom is higher than existing vaccine.
Therefore, the safe and effective vaccine of the influenza virus that need cultivate based on cell culture rather than egg.
Summary of the invention
There are various forms of influenza virus vaccines can use (the 17th and 18 chapters of the document 2 that for example, sees reference) at present.Many vaccines are based on live virus or inactivation of viruses, and wherein inactivated vaccine is based on the virion of complete virion, " cracking " or the surface antigen of purification (comprising hemagglutinin and neuraminidase).The PREFLUCEL of failure
TMProduct uses complete influenza virus particles.
Use the complete virion may relevant with reactionogenicity [3].For avoiding PREFLUCEL
TMThe being seen reactionogenicity problem of product, the virion antigen that the unfavorable usefulness of the present invention is complete promptly uses non-virion antigen (for example, the surface antigen of lytic virus granule or purification).These antigens are derived from the virus of cultivating with cell culture.The complete virion that has report [1] to say to utilize cell culture to cultivate can strengthen t cell response, yet the data show of this paper has only the t cell response of appropriateness when utilizing non-virion antigen.Therefore, for strengthening t cell response, non-virion antigen of coupling of the present invention and adjuvant.
PREFLUCEL
TMProduct does not contain adjuvant, and is relevant with potential hypersensitivity to add adjuvant in the forward direction influenza vaccines.For example, list of references 4 has reported that the influenza vaccines that contain adsorbed onto alum adjuvant can make Cavia porcellus sensitization, and do not have Adjuvanted vaccines not can, adjuvant has obviously improved egg protein activity hypersensitive has taken place.Similarly, list of references 5 has been reported with no adjuvant antigen and has been compared, and influenza antigen is adsorbed onto the egg albumen sensitization that causes on the aluminum salt early.In addition, list of references 6 has reported that the animal that former acceptance contains the ovalbumin of adsorbed onto alum adjuvant shows that at the commitment of influenza infection anaphylaxis increases the weight of.To the hypersensitivity of the vaccine component problem of influenza vaccines particularly, because give their common every year.Owing to avoided being used to cultivate the egg system of virus, the present invention has also advantageously avoided the relevant any problem of ovalbumin, this is more extensive and obviously (for example become more along with influenza vaccinations, along with immunity inoculation expand in the past indication want vaccinated patient group and along with patient's ratio of immunity inoculation in the described target colony increases).
Therefore, the invention provides a kind of immunogenic composition, it comprises: (i) the non-virion influenza antigen of the virus preparation of cultivating from cell culture; (ii) adjuvant.
The present invention also provides the method for preparing immunogenic composition, and this method comprises the step of mixing following component: (i) the non-virion influenza antigen of the virus preparation of cultivating from cell culture; (ii) adjuvant.
The present invention also provides a kind of test kit, and it is equipped with: first reagent constituents that (i) comprises the non-virion influenza antigen of the virus preparation of cultivating from cell culture; Second reagent constituents that (ii) comprises adjuvant.
Influenza antigen generally comprises influenza virus hemagglutinin.The preferred oil in water emulsion adjuvant of adjuvant, for example MF59 does not more preferably contain any aluminum salt.Have now found that O/w emulsion can strengthen the influenza specific T-cells and reply, these emulsions can also hypermnesis B cell response.In addition, they can improve the cross reactivity at allos variant (heterovariant) influenza strain, thereby make vaccine can induce protective immunity, even vaccine strain and epidemic isolates do not match.
In influenza vaccines, use the existing description of adjuvant.In list of references 7 and 8, the Vero that utilizes aluminium hydroxide preparation the to contain adjuvant complete virion vaccine of deriving.In list of references 9, the egg that utilizes the mixture preparation of aluminium hydroxide and aluminum phosphate the to contain adjuvant vaccine of deriving, wherein preferred vaccine are to resist the univalent vaccine that the egg of popular strain produces.In list of references 57, the MDCK that utilizes aluminium hydroxide preparation the to contain adjuvant inactivated virus particle (vaccine) of deriving.List of references 10 (for example in embodiment 7) but the complete equine influenza virus of prompting coupling adjuvant and deactivation.List of references 11 (for example in embodiment 5) but prompting coupling aluminium hydroxide and inactivation of viruses with the Embryo Gallus domesticus cell culture.In the embodiment 2 of list of references 12, the deutero-trivalent split vaccine of egg uses various adjuvant.In list of references 13, utilize the preparation of aluminum salt to contain the deutero-unit price complete virion of the egg vaccine of adjuvant.Yet, in these prior art situations of great majority, adjuvant and the coupling of complete virion vaccine, and not with antigen coupling derived from the virus of cell culture cultivation.In addition, attempt to use adjuvant to reduce every dose of required antigen amount, thereby can improve the dosage number under the popularity, rather than strengthen the t cell response of this vaccine.
Influenza antigen
The antigen that the present composition comprises is from cultivating the influenza virus particles preparation that the virus back obtains with cell line.Described antigen is non-virion antigen, comprises hemagglutinin usually.Therefore, the present invention does not comprise the vaccine that utilizes live virus or complete virion inactivation of viruses.Antigen of the present invention is non-virion antigen on the contrary, the surface antigen of for example cracked virion or purification (comprise hemagglutinin, also comprise neuraminidase usually).
Can from the liquid that contains virus, collect virion by the whole bag of tricks.For example, purification process can relate to the band centrifugation that utilizes linear Sucrose gradient solutions, and described 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) handle the virion of purification, comprise that " tween-ether " cleavage method produces the subviral particle goods, thereby obtained cracked virion.The method of well known cracking influenza virus is for example referring to list of references 14-19 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 Nonylphenoxy polyoxy ethanol is (for example; the triton surfactant is 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, and cracking occurs in (for example, in sucrose density gradient solution) during the initial virion purification.Therefore cleavage method can comprise and makes the material clarification (to remove non-virion material) that contains virion, concentrates the virion of collecting and (for example, adopts absorption method, as CaHPO
4Absorption), separate complete virion and non-virion material, (for example in the density gradient centrifugation step, utilize decomposition agent lytic virus granule, utilization contains decomposition agent, saccharose gradient (solution) as NaTDC), filter (for example, ultrafiltration) then to remove unwanted material.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 virion form have [20] (the viral sample liposome particles that does not contain nucleic acid), for example INFLEXAL V
TMAnd INVAVAC
TMProduct, but the present invention does not preferably use virion.Therefore, in some embodiments, influenza antigens is not the form of virion.
Influenza virus can be an attenuation.Influenza virus can be to responsive to temperature.Influenza virus can be cold adaptation.If utilize live virus as antigen, these three kinds of features are particularly useful.
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 the contained antigenic character of season and vaccine, the present invention can shield to resist following one or more influenza A virus hemagglutinin hypotypes: 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.
Except being suitable for immunity inoculation to resist popular interval strain, the adjunvant composition that contains of the present invention also 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 virus 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[21], wherein clade 1 is relevant especially with 3.
Colony's strain that can usefully be included in these compositionss is tolerance antiviral therapy (for example, tolerance oseltamivir [22] and/or zanamivir), comprises the strain [23] of drug resistance epidemic isolates.
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.Preferred univalent vaccine if vaccine comprises multiple influenza strain, is cultivated different strains respectively usually, mixes behind collection virus and the preparation antigen.Therefore, the inventive method can comprise the antigenic step of mixing multiple influenza strain.Preferred trivalent vaccine comprises two kinds of influenza A virus strains and a kind of Influenza B virus strain.
In some embodiments of the present invention, these compositionss can comprise a kind of antigen of influenza A strain.In some embodiments, these compositionss can comprise the antigen of two kinds of influenza A strains, as long as these two kinds of strains are not H1N2 and 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, 24-28] 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 [29-31] with different plasmids, these methods also comprise utilizes plasmid to express all virus proteins or some of them (for example, being PB1, PB2, PA and NP albumen), and certain methods has been utilized 12 kinds of plasmids.
For reducing required plasmid number, nearest method [32] 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 32 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 [33].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[34,35 of a template].
Therefore, virus, particularly 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.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 cell culture is cultivated the virus as the antigen source.Described cellular material is mammal cell line 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.Utilize mammalian cell to represent that vaccine does not contain chicken DNA and do not contain egg protein (for example, ovalbumin and ovomucoid).The preferred mammal cell line that is used to cultivate influenza virus comprises: the mdck cell [36-39] that is derived from Madin-Darby canine kidney; Be derived from the Vero cell [40-42] of cercopithecus aethiops (Cercopithecus aethiops) kidney; Or be derived from the PER.C6 cell [43] of people embryo retinoblast.These cell lines can be extensively available from, for example American Type Culture Collection (ATCC) [44], cell preservation center, bandit Lille (Coriell Cell Repositories) [45] 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, available avian cell lines [is for example cultivated virus, list of references 46-48], comprise and (for example be derived from duck, the duck retina) or hen, for example the cell line of chick embryo fibroblast (CEF), fowl embryo stem cell [46,49] comprises the EBx cell line, EB45, EB14 and the EB14-074[50 that are derived from chicken embryonic stem cells], 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 36 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 51 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 52 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 53 has disclosed infecting extremely sensitive mdck cell.Can use any of these mdck cell system.
The virus inoculation thing that is used to cultivate the cell culture of virus and is used to start 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 [54].Especially preferably do not contain herpes simplex virus.
Can cultivate [31,55,56] or the cell of adhere-wall culture with suspension and cultivate 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 use the culture medium and/or the protein-free culture medium culturing cell line 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.
These cell lines are preferably growth [57] 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.Can after infection, collect the cell of infection 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.Existing vaccine contains the 15 μ g HA/ strains of having an appointment usually, represents to use lower dosage though comprise adjuvant.With higher dosage (for example, 3 * or 9 * dosage [58,59]) the same, used part dosage, for example 1/2 (that is 7.5 μ g HA/ strains), 1/4 and 1/8[9,13].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 15, about 10, about 7.5, about 5, about 3.8, about 1.9, about 1.5/ strain, or the like.
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).
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, centering on the hyperalkaline zone (hyper-basic region) of the cleavage site between HA1 and the HA2) to remove.
Compositions 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 uncle's octylphenoxy polyethoxy ethanol), cetab (' CTAB '), or NaTDC, particularly for split vaccine or SAV.The detergent that can only have trace.Therefore, contained Octoxinol-10 and polysorbate80 can be lower than 1mg/ml separately in the vaccine.The residual component of other trace can be antibiotic (for example, neomycin, kanamycin, a polymyxin B).
Vaccine of the present invention can comprise stromatin, thereby benefits from other t cell epitope that is positioned at this antigen.Therefore, the vaccine (particularly split vaccine) that comprises hemagglutinin and neuraminidase also can comprise M1 and/or M2 stromatin.If there is stromatin, but preferably comprise the M2 stromatin of detection level.Also can there be nucleoprotein.
Prepare antigenic time as the influenza virus particles of cultivating the acquisition of virus back with cell line and select scheme, in some embodiments of the present invention, can prepare non-virion antigen by in recombinant host, expressing.For example, can in recombinant host (for example, utilizing the insect cell line of baculovirus vector), express hemagglutinin and/or neuraminidase [60,61].
Host cell DNA
If cultivate virus with cell line, the residual quantity that then reduces the cell line dna in the final vaccine as far as possible is standard operation, thereby can reduce any carcinogenic activity of this DNA as far as possible.
Vaccine of the present invention contains and is less than the 10ng residual host cell DNA of (preferably being less than 1ng, more preferably less than 100pg) for preferred every dose, though can there be the host cell DNA of trace.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 62 and 63 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.Use 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 [64].
Contain in preferred per 15 μ g hemagglutinins＜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.Contain in more preferably per 50 μ g hemagglutinins＜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.
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 [65,66] 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 on the whole 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 [67]; Method of immunity, for example Threshold
TMSystem [68]; And quantitative PCR [69].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 [68] 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 (AltheaTechnologies), or the like.Detect host cell DNA pollutes in Human virus's vaccine chemiluminescence cross experiment and total DNA Threshold
TMThe document 70 that relatively sees reference of system.
Adjuvant
The present composition comprises adjuvant, and its function is to strengthen the t cell response among the patient who accepts said composition, for example the stimulation specificity of influenza antigens is reacted and increases the T cell quantity that discharges cytokine among the patient.
List of references 7-13 has disclosed coupling aluminum salt adjuvant and influenza antigen.Though the present invention can utilize aluminum salt, preferably avoid, not the adjuvant that constitutes by one or more aluminum salt preferably promptly.The sensitization of aluminum salt appear in the newspapers [71-77].The used most preferably adjuvant of the present invention comprises O/w emulsion, and is as will be detailed later.Operable other adjuvant includes but not limited to:
Cytokine induction agent (vide infra).
The compositions that contains mineral comprises calcium salt and aluminum salt (or their mixture).Calcium salt comprises calcium phosphate (for example, " CAP " granule of list of references 78 disclosures).Aluminum salt (adjuvant that the present invention does not preferably use) comprises that hydroxide (for example, oxyhydroxide), phosphate (for example, hydroxyl phosphate, orthophosphate), sulfate etc. [for example, the 8th and 9 chapters of list of references 79], all salt can be taked any suitable form (as gel, crystal, amorphous etc.).Preferred these salt of absorption.The compositions that contains mineral also can be mixed with slaine granule [80].
Saponin [the 22nd chapter of list of references 118] is the heterogeneous mixture (heterologousgroup) that the steroline found at many floristic barks, leaf, stem, root even in spending and triterpene are glucosides.Isolating saponin is the adjuvant of broad research in Quillaia saponaria (the Quillaia saponaria Molina) bark.But saponin also commercialization available from beautiful colored Rhizoma Smilacis Chinensis (Smilax ornata) (sarsaparilla (sarsaprilla)), awl filigree Dianthus chinensis (Gypsophilla paniculata) (wedding gauze kerchief flower (brides veil)) and Saponaria officinalis (Saponariaofficianalis) (Radix saponariae).The saponin adjuvant preparation comprises the preparation of purification, for example QS21, and lipid formulations, for example ISCOM.The commodity of QS21 are by name
HPLC and RP-HPLC purification astragalin composition have been utilized.Identified specific part, comprised QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C with these technology purification.The preferred QS21 of saponin.List of references 81 has disclosed the preparation method of QS21.The saponin preparation also can contain sterin, for example cholesterol [82].But coupling saponin and cholesterol form the unique granule [the 23rd chapter of list of references 118] that is called immunostimulating complex (ISCOM).ISCOM generally also contains phospholipid, for example PHOSPHATIDYL ETHANOLAMINE or phosphatidylcholine.Any known saponin can be used among the ISCOM.ISCOM preferably contains one or more among QuilA, QHA and the QHC.List of references 82-84 has further described ISCOM.Optional other detergent [85] that do not contain of ISCOM.The summary of saponin adjuvant exploitation see reference document 86 and 87.
The lipid A derivant of escherichia coli (Escherichia coli), for example OM-174 (described in the list of references 88 and 89).
Antibacterial ADP-ribosylation toxin (for example, heat-labile enterotoxin of E, coli " LT ", cholera toxin " CT " or pertussis " PT ") and detoxification derivant thereof for example are called the sudden change toxin [90] of LT-K63 and LT-R72.List of references 91 has been described the application of detoxification ADP-ribosylation toxin as mucosal adjuvants, and list of references 92 has been described its application as the parenteral adjuvant.
Biological adhesive (bioadhesive) and mucoadhesive (mucoadhesive), the hyaluronic acid microsphere [93] of for example esterification or chitosan and derivant [94] thereof.
By biodegradable and non-toxic material (for example, poly-(alpha-hydroxy acid), poly butyric, poe, polyanhydride, polycaprolactone etc.), the microgranule that preferred poly-(lactide-co-glycolide) forms (promptly, the about 100nm of diameter is to 150 μ m, and preferably about 200nm is to about 30 μ m, and most preferably from about 500nm is to the granule of about 10 μ m), optional these microgranules are processed into (for example has electronegative surface, use SDS) or the surface (for example, using cationic detegent, as CTAB) of positively charged.
Liposome (the 13rd and 14 chapters of list of references 118).The example that is suitable as the Liposomal formulation of adjuvant is described in list of references 95-97.
Polyoxyethylene ether or polyoxyethylene ester [98].This preparation also comprises polyoxyethylene sorbitol ester surfactant [99] and at least a other non-ionic surface active agent of coupling, for example polyoxyethylene alkyl ether of Octoxinol or the ester surfactant [100] of coupling Octoxinol.Preferred polyoxyethylene ether is selected from: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-stearyl (steoryl) ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene-23-lauryl ether.
Muramyl peptide; for example N-acetyl group-muramyl-L-Threonyl-D-isoglutamine (thr-MDP), N-acetyl group-nor-muramyl-L-alanyl-D-isoglutamine (nor-MDP), the different paddy-L-Ala-two palmityl propionic acid amide .s of N-acetyl-glucosamine-N-acetyl muramyl-L-Al-D-(N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide) (" DTP-DPP ", or " Theramide
TM"), N-acetyl muramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1 '-2 '-two palmityls-sn-glyceryl-3-hydroxyl phosphorus acyloxy)-ethamine (MTP-PE).
Coupling is from the outer membrane protein albuminous body goods of first gram negative bacteria preparation with derived from lipolysaccharide (liposaccharide) goods of second gram negative bacteria, and wherein said external membrane protein albuminous body and lipolysaccharide goods form stable non-covalent adjuvant complex.This complex comprises " IVX-908 ", a kind of complex that comprises Neisseria meningitidis (Neisseria meningitidis) adventitia and lipopolysaccharide.They have been used as the adjuvant [101] of influenza vaccines.
Methylinosine 5 '-phosplate (" MIMP ") [102].
Polyhydroxylated pyrrolizidine chemical compound [103] or its pharmaceutically acceptable salt or derivant, for example shown in the following formula:
Wherein R is selected from down group: hydrogen, straight or branched, do not replace or replace, saturated or undersaturated acyl group, alkyl (for example, cycloalkyl), thiazolinyl, alkynyl and aryl.Example includes but not limited to: casurin (casuarine), casurin-6-α-D-glucopyranose, 3-table-casurin, 7-table-casurin, 3, and 7-two table-casurins, or the like.
γ inulin [104] or derivatives thereof, for example algammulin.
The preparation of cation lipid and common lipid (co-lipid) (normally neutral), for example aminopropyl-dimethyl-macene oxygen base (myristoleyloxy)-bromination third ammonium (propanaminium bromide)-two phytane acyl (diphytanoyl) phosphatidyl-ethanolamine (" Vaxfectin
TM") or aminopropyl-dimethyl-two-dodecyl oxygen base-bromination, third ammonium-dioleoyl phosphatidyl-ethanolamine (" GAP-DLRIE:DOPE ").Preferably contain (±)-N-(3-aminopropyl)-N, N-dimethyl-2, the preparation [105] of 3-two (syn-9-tetradecene oxygen base)-1-third ammonium salt (propanaminium salt).
The CD1d part; for example alpha-glycosylceramides [106-113] (for example; the alpha-galactoside ceramide), the alpha-glycosylceramides, OCH, the KRN7000[(2S that contain phytosphingosine; 3S; 4R)-1-O-(α-D-galactopyranoside)-2-(N-hexacosane acyl group (hexacosanoyl) amino-1; 3,4-octadecane triol), CRONY-101,3 " O-sulfo group (sulfo)-galactosyl ceramide, or the like.
List of references 118 and 119 is described these and other adjuvanticity material in detail.
Compositions can comprise two or more described adjuvants.For example, they can preferably comprise O/w emulsion and cytokine induction agent simultaneously, because the cytokine response that this combination energy enhanced flow influenza vaccine is caused, for example interferon-is replied, and this potentiation is better than single being seen with emulsion or derivant.
Oil in water emulsion adjuvant
Have now 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 said one or more oil and one or more surfactants are biodegradables (but metabolism) and biocompatible.The diameter of oil droplet is usually less than 5 μ m in the emulsion, 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 MF59 O/w emulsion has been described and can be used as egg the derive adjuvants [114] of influenza vaccines, for example FLUAD
TMIn the product, but and FLUAD
TMProduct is compared, and vaccine of the present invention can more be widely used for the general population, because they have been avoided egg protein, for example ovalbumin and egg stick mucin risk hypersensitive.
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 sorbitan esters 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 (Brjj 30); Sorbitan esters (being commonly referred to SPAN), for example anhydrosorbitol trioleate (sorbester p37) and sorbitan monolaurate.Preferred nonionic surfactants.Contained preferred surfactant is Tween 80 (polyoxyethylene sorbitan monoleate), sorbester p37 (anhydrosorbitol trioleate), lecithin and triton x-100 in the emulsion.
Can utilize surfactant mixtures, for example Tween 80/sorbester p37 mixture.Polyoxyethylene sorbitan esters, for example polyoxyethylene sorbitan monoleate (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 sorbitan esters and/or Octoxinol.
The preferable amount of surfactant (weight %) is: polyoxyethylene sorbitan esters (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, 0.5% polysorbate80 and 0.48% sorbester p37.This adjuvant is called " MF59 " [115-117], as list of references 1] the 12nd chapter of the 10th Zhanghe list of references 119 of 8 described in detail.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.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.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 [120] 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 [121].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 122, 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-the lipophilic conjugate (for example; list of references 123 described GPI-0100; by fatty amine is added deacylated tRNA basis soap glycosides through the carboxyl of glucuronic acid), 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 [124].
The emulsion [125] 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 [125] 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.The volume ratio that is used for blended two kinds of liquid can different (for example, between 5: 1 and 1: 5), but normally about 1: 1.
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 effect [126] to immunne response in this patient group, and obviously influence relates to the equilibrated gene expression of Th1/Th2 [127].They also have anti-oxidation characteristics, thereby help stable emulsion [128].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 [17] of replacement for mercury chemical compound.
The cytokine induction agent
When the cytokine induction agent that the present composition is contained gave the patient, it can cause immune system and discharge cytokine, comprises interferon and interleukin.Known cytokine response relates to the early stage and decision stage [129] of the host defense of resisting influenza infection.Preferred derivant can cause following one or more release of cytokines: interferon-; Il-1; Interleukin-2; Il-1 2; TNF-α; TNF-β; And GM-CSF.Preferred derivant 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.It is reported that derive interferon-ALPHA that influenza vaccines cause and β of egg replys and be higher than MDCK-or the Vero-influenza vaccines [130] that derive.
Therefore, accepting the present composition 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 131 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") [132-135].
Imidazoquinolie compounds, for example imiquimod (" R837 ") [136,137], resiquimod (" R-848 ") [138] and their analog; With their salt (for example, hydrochlorate).Other details of immunostimulating imidazoquinolie document 139-143 that sees reference.
Thiosemicarbazones chemical compound, for example those of list of references 144 disclosures.List of references 144 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 145 disclosures.List of references 145 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 146-148; (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) [149].
The chemical compound that list of references 150 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 [151 (ABIQ); 152]; hydroxyl phthalic amide (Hydrapthalamide) chemical compound; benzophenone cpd isoxazole compound; sterol compounds; quinazolone (Quinazilinone) chemical compound; azole compounds [153]; anthraquinone compounds; quinoxaline compounds; triaizine compounds; pyrazoles pyrimidine (Pyrazalopyrimidine) chemical compound and benzazole (Benzazole) chemical compound [154].
Polyethylene-the bridged piperazine derivatives of polyoxidonium polymer [155,156] or other N-oxidation.
The chemical compound that list of references 157 discloses.
Chemical compound shown in formula I, II or the III, or their salt:
As described in list of references 158, as ' ER 803058 ', ' ER 803732 ', ' ER 804053 ', ' ER804058 ', ' ER 804059 ', ' ER 804442 ', ' ER 804680 ', ' ER 804764 ', ER 803022 or ' ER 804057 ', for example:
Aminoalkyl glucosaminide phosphate derivative is as RC-529[159,160].
Phosphonitrile, poly-[two (carboxyl phenoxy group) phosphonitrile] for example described in the list of references 161 and 162 (" 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[163,164]:
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 165 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 can comprise nucleotide modification/analog, modifies as thiophosphate, can be double-stranded or (except RNA) strand.List of references 166,167 and 168 has disclosed possible similar replacement, for example uses 2 '-deoxidation-7-denitrogenation guanosine to replace guanosine.List of references 169-174 has further discussed the adjuvant effect of CpG oligonucleotide.The CpG sequence can relate to TLR9, for example motif GTCGTT or TTCGTT[175].But CpG sequence specificity is induced the Th1 immunne response, CpG-AODN (oligodeoxynucleotide) for example, or induce the B cell response more specifically, for example CpG-B ODN.List of references 176-178 has discussed CpG-A and CpG-B ODN.The preferred CpG-A ODN 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 175 and 179-181.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 [182].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 182 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 182 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 (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 183).List of references 184 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, 3dMPL 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, 3dMPL can have 4 acyl chains.Work as R
1', R
2' and R
3' in have only one and be-during H, 3dMPL can have 5 acyl chains.Work as R
1', R
2' and R
3' in none be-during H, 3dMPL can have 6 acyl chains.The used 3dMPL adjuvant of the present invention can be the mixture with these forms of 3-6 bar acyl chain; but comprise 3dMPL in the preferred mixture with 6 acyl chains; to guarantee that particularly 6 acyl chain forms account for 10% of total 3dMPL at least by weight, for example 〉=20%, 〉=30%, 〉=40%, 〉=50% or higher.The 3dMPL that discovery has 6 acyl chains is the active adjuvant form of tool.
Therefore, the 3dMPL most preferred form that comprises of the present composition is:
When adopting the 3dMPL of form of mixtures, mention that the consumption of 3dMPL in the present composition or concentration refer to all kinds of blended 3dMPL in this mixture.
Under aqueous conditions, 3d-MPL can form micellar aggregates 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 3dMPL) [185] 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 x nm, 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.
3dMPL preferably with the O/w emulsion coupling.Basically all 3dMPL are positioned at the aqueous phase of emulsion.
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 [186] (being included in [187] in the O/w emulsion), with immunostimulatory oligonucleotide, with QS21 and immunostimulatory oligonucleotide, with aluminum phosphate [188], with aluminium hydroxide [189] or with aluminum phosphate and aluminium hydroxide.
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 190 that sees reference.
Compositions is taked aqueous form usually.Antigen and adjuvant be mixture normally.
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 [17,191].More preferably not mercurous vaccine.The vaccine that does not especially preferably contain antiseptic.
Preferably comprise physiology salt, for example sodium salt is with control tension force.Preferred 1-20mg/ml sodium chloride (NaCl).Other salt that can exist comprises potassium chloride, potassium dihydrogen phosphate, dehydration sodium hydrogen phosphate (disodium phosphatedehydrate), magnesium chloride, calcium chloride etc.
The osmolality of compositions between 200-400mOsm/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 [192] 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 (particularly aluminium hydroxide adjuvant); 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 or between 7.0 and 7.8.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).
Compositions and test kit are preferably kept between 2 ℃-8 ℃.Should be not freezing.Should avoid light direct beam.
Test kit of the present invention
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 adjuvant and antigen stand-by respectively.This is configured in when utilizing oil in water emulsion adjuvant particularly useful.
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 two independent containers, for example in the bottle.For example 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 193-200 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.
Each component of test kit is in aqueous form usually.In some configurations, certain component (generally being antigen component rather than adjuvant component) 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 aqueous adjuvant component in the pre-filled syringe, and the freeze-dried antigen component is stored in the bottle.
The packing of 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.
Using of Therapeutic Method and vaccine
The present composition is fit to give human patients, 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 of the present invention or compositions, and it is used as medicine.
The present invention also provides (i) the non-virion influenza antigen of the virus preparation from cultivating with cell culture; (ii) adjuvant is used for producing the application of the medicine of immunne response in patient's body in preparation.
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.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%) [201].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 [202-204], oral [205], intradermal [206,207], transdermal, percutaneous [208] 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; Vide infra) the patient, to egg people hypersensitive and the people that 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).
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 the CD4 that produces the antigenic specificity cytokine response when stimulating with HA
+T cell percentage ratio.
Fig. 2-4 has shown the Log10 serum antibody titer (ELISA) that utilizes the different components immune mouse.Arrow has shown with MF59 emulsion adjuvant composition prepared.Fig. 2 has shown the result of H1N1; Fig. 3 shows H3N2; Fig. 4 has shown influenza B.
Fig. 5 has shown the serum HI titre of different adjuvants.
Fig. 6 is similar to Fig. 1, has shown the effect that adds CpG in various adjuvants.Each right left post has shown the cell % with antigenic specificity cytokine response; Right post has shown and has shown the cell % that the antigenic specificity interferon-is replied.
Fig. 7 is similar to Fig. 5, has shown when using 0.1 μ g antigen, contains (blank, prospect) and does not contain the HI titre of adjuvant (1)-(4) of (hypographous, background) CpG.
Fig. 8 has shown and has adopted different adjuvants and the combination results GMT (AU/ml) at the IgG of H3N2 strain.The left post of each centering has shown IgG1; Right post has shown IgG2a.Scale is logarithmic.
Embodiment of the present invention
Cultivate influenza virus strain Wyoming H3N2 (A), New-Caledonia H1N1 (A) and Jiangsu (B) respectively with mdck cell, thereby avoid existing in the final vaccine the deutero-protein of any egg (particularly ovalbumin).Preparation trivalent surface glycoprotein vaccine, with its 0th day and the 28th day with the Balb/C mice at the beginning of the immune immunogen of two kinds of dosage (0.1 and 1 μ g HA/ strain).Gathered the blood of mice and carried out various tests at the 42nd day: the HI titre with this blood; Recording anti--HA by ELISA replys; With the CD4 that discharges cytokine in the antigenic specificity mode
+The T cellular level comprises that independent detection discharges those cellular levels of gamma interferon.For IgG1 or IgG2a, special detection IgG replys.
List of references 1 report when utilizing the antigen of the influenza virus purification of from mammalian cell cultures, cultivating, the t cell response enhancing, but in contrast be to have only the CD4 of moderate quatity
+The T cell discharges cytokine in the antigenic specificity mode.For improving these results, add one of following adjuvant to vaccine: (1) aluminium hydroxide, with the 1mg/ml use and contain the 5mM histidine buffering liquid; (2) contain the MF59 O/w emulsion of citrate buffer solution, mix with antigenic solution with 1: 1 volume ratio; (3) calcium phosphate uses and contains the 5mM histidine buffering liquid with 1mg/ml; (4) microgranule that forms by poly-(lactide-co-glycolide) 50: 50 copolymer compositions, inner viscosity 0.4 (" PLG ") contains the antigen of absorption; (5) has the CpG immunostimulatory oligonucleotide of thiophosphate main chain; (6) resiquimod; Or (7) do not contain the negative control of adjuvant.
Fig. 1 has shown the T cell quantity that discharges cytokine with one of described 7 kinds of compositionss immunity back in the antigenic specificity mode.Each has all strengthened t cell response in described 6 kinds of adjuvants, but emulsion compositions (arrow) obtains best enhancing result.
Fig. 2-4 has shown that anti--HA ELISA replys.Similar result can find out once more that the emulsion-based compositions obtains the best and replys from these accompanying drawings and Fig. 5.The data of Fig. 5 also show utilizes immunostimulatory oligonucleotide to have good anti--HA to reply.
Fig. 6 has shown that adding CpG oligonucleotide (5) is to the effect of T cell in adjuvant (1)-(4).Wherein emulsion (2) is little to the effect of overall t cell response, but the ratio of interferon-secretory cell is very high, has shown that more TH-1 sample replys.In calcium phosphate (3), add CpG and observe similar action, but total t cell response strengthens.In aluminum hydroxide adjuvant, add CpG t cell response is not had beneficial effect.
Observe equally to the TH-1 sample among Fig. 8 and reply skew.List all shows it mainly is that IgG1 replys (TH2) with adjuvant (1)-(4), and is single the same with CpG (5).In all situations, CpG is added the level that has all improved IgG2a (TH1) in adjuvant (1)-(4), comprise that aluminium hydroxide and PLG adjuvant have produced IgG2a and replied, and this when not existing, CpG does not observe.In addition, CpG added in O/w emulsion (2) and the calcium phosphate (3) make IgG reply skew, thereby cause IgG2a to preponderate.Generally, CpG is added each adjuvant and improved HI titre (Fig. 7).Therefore, except that aluminum salt, in all adjuvants, add CpG and all strengthened t cell response and B cell response.
Therefore, opposite with the result who utilizes the antigenic list of references 1 of complete virion, described antigen is derived from the virus of cultivating with mammalian cell cultures, when finding not have adjuvant, a little less than replying at the specific T-cells of the influenza antigens of purification.Yet, add adjuvant and can strengthen t cell response.Specifically, O/w emulsion all is good adjuvants for t cell response and anti--HA antibody.With regard to these two standards, the MF59 emulsion is better than aluminum salt adjuvant.
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 (19)
1. immunogenic composition, described compositions comprises: (i) from the non-virion influenza antigen of the virus preparation of cultivating with cell culture; (ii) adjuvant.
2. compositions as claimed in claim 1 is characterized in that described compositions does not contain chicken DNA, ovalbumin and ovomucoid.
3. the described compositions of above each claim is characterized in that described compositions comprises the antigen of multiple influenza virus strain.
4. the described compositions of above each claim is characterized in that described compositions comprises the antigen of influenza A virus and the antigen of Influenza B virus.
5. the described compositions of above each claim is characterized in that described influenza antigen is a lytic virus.
6. as each described compositions among the claim 1-4, it is characterized in that described influenza antigen comprises the surface antigen of purification.
7. the described compositions of above each claim is characterized in that described influenza antigen is from following subtypes of influenza A virus: H1, H2, H3, H5, H7 or H9.
8. the described compositions of above each claim is characterized in that, contains 0.1-20 μ g hemagglutinin/virus stain in the described compositions.
9. the described compositions of above each claim is characterized in that the cell culture host's that described compositions contains cell DNA is less than 10ng.
10. the described compositions of above each claim is characterized in that described adjuvant comprises O/w emulsion.
11. compositions as claimed in claim 10 is characterized in that, one or more oil and one or more surfactants in the described emulsion are biodegradables and biocompatible.
12., it is characterized in that described emulsion has submicron droplets as claim 10 or 11 described compositionss.
13., it is characterized in that described emulsion comprises terpenoid as each described compositions among the claim 10-12.
14., it is characterized in that described emulsion comprises Squalene as each described compositions among the claim 10-12.
15., it is characterized in that described emulsion comprises tocopherol as each described compositions among the claim 10-12.
16., it is characterized in that described emulsion comprises polyoxyethylene sorbitan esters surfactant, Octoxinol surfactant and/or sorbitan esters as each described compositions among the claim 10-12.
17. the described compositions of above each claim is characterized in that, described compositions comprises 3-O-deacylated tRNA base monophosphoryl lipid A.
18. a method for preparing immunogenic composition, described method comprises the step of mixing following component: (i) the non-virion influenza antigen for preparing from the virus of cultivating with cell culture; (ii) adjuvant.
19. a test kit, described test kit is equipped with: (i) comprise first reagent constituents of non-virion influenza antigen, the virus preparation of described antigen from cultivating with cell culture; Second reagent constituents that (ii) comprises oil in water emulsion adjuvant.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US73402605P | 2005-11-04 | 2005-11-04 | |
| US60/734,026 | 2005-11-04 | ||
| US60/735,658 | 2005-11-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101346152A true CN101346152A (en) | 2009-01-14 |
Family
ID=40189139
Family Applications (5)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006800487317A Pending CN101346152A (en) | 2005-11-04 | 2006-11-06 | Adjuvanted vaccines with non-virion antigens prepared from influenza viruses grown in cell culture |
| CNA2006800476098A Pending CN101370515A (en) | 2005-11-04 | 2006-11-06 | Influenza vaccines including combinations of particulate adjuvants and immunopotentiators |
| CN2006800476007A Active CN101365483B (en) | 2005-11-04 | 2006-11-06 | Adjuvanted influenza vaccines including cytokine-inducing agents |
| CNA2006800476045A Pending CN101365485A (en) | 2005-11-04 | 2006-11-06 | Emulsions with free aqueous-phase surfactant as adjuvants for split influenza vaccines |
| CNA2006800463581A Pending CN101325967A (en) | 2005-11-04 | 2006-11-06 | Influenza vaccines with reduced amount of emulsion adjuvant |
Family Applications After (4)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006800476098A Pending CN101370515A (en) | 2005-11-04 | 2006-11-06 | Influenza vaccines including combinations of particulate adjuvants and immunopotentiators |
| CN2006800476007A Active CN101365483B (en) | 2005-11-04 | 2006-11-06 | Adjuvanted influenza vaccines including cytokine-inducing agents |
| CNA2006800476045A Pending CN101365485A (en) | 2005-11-04 | 2006-11-06 | Emulsions with free aqueous-phase surfactant as adjuvants for split influenza vaccines |
| CNA2006800463581A Pending CN101325967A (en) | 2005-11-04 | 2006-11-06 | Influenza vaccines with reduced amount of emulsion adjuvant |
Country Status (4)
| Country | Link |
|---|---|
| CN (5) | CN101346152A (en) |
| CA (1) | CA2907149A1 (en) |
| ES (1) | ES2367194T3 (en) |
| SI (2) | SI1951300T1 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2396032B1 (en) * | 2009-02-10 | 2016-09-28 | Seqirus UK Limited | Influenza vaccines with reduced amounts of squalene |
| WO2011141819A1 (en) * | 2010-05-12 | 2011-11-17 | Novartis Ag | Improved methods for preparing squalene |
| GB201009671D0 (en) * | 2010-06-10 | 2010-07-21 | Glaxosmithkline Biolog Sa | Novel process |
| MX343410B (en) * | 2010-07-06 | 2016-11-04 | Novartis Ag * | Cationic oil-in-water emulsions. |
| 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) |
| KR102632419B1 (en) * | 2016-12-23 | 2024-01-31 | 인터벳 인터내셔널 비.브이. | Combination vaccine for pigs |
| 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 |
| 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 |
-
2006
- 2006-11-06 CN CNA2006800487317A patent/CN101346152A/en active Pending
- 2006-11-06 CA CA2907149A patent/CA2907149A1/en not_active Abandoned
- 2006-11-06 ES ES06808431T patent/ES2367194T3/en active Active
- 2006-11-06 CN CNA2006800476098A patent/CN101370515A/en active Pending
- 2006-11-06 CN CN2006800476007A patent/CN101365483B/en active Active
- 2006-11-06 SI SI200631106T patent/SI1951300T1/en unknown
- 2006-11-06 CN CNA2006800476045A patent/CN101365485A/en active Pending
- 2006-11-06 CN CNA2006800463581A patent/CN101325967A/en active Pending
- 2006-11-06 SI SI200631278T patent/SI1951299T1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CN101365483B (en) | 2012-08-22 |
| CN101365485A (en) | 2009-02-11 |
| ES2367194T3 (en) | 2011-10-31 |
| SI1951300T1 (en) | 2011-10-28 |
| CA2907149A1 (en) | 2007-05-10 |
| CN101365483A (en) | 2009-02-11 |
| SI1951299T1 (en) | 2012-04-30 |
| CN101370515A (en) | 2009-02-18 |
| CN101325967A (en) | 2008-12-17 |
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