CN1238696A

CN1238696A - Vaccines

Info

Publication number: CN1238696A
Application number: CN97180166A
Authority: CN
Inventors: N·加康; M·弗里德
Original assignee: SmithKline Beecham Biologicals SA
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 1996-10-05
Filing date: 1997-09-30
Publication date: 1999-12-15
Also published as: CO4910170A1; GB9620795D0; NZ334734A; NO991524L; AU714930B2; IL128985A0; CA2267191A1; AU4781297A; EP0939650A1; AR009958A1; ZA978868B; WO1998015287A1; CZ116799A3; JP2001501640A; KR20000048866A; TR199900729T2; PL332633A1; NO991524D0; HUP9904549A3; HU9904549A

Abstract

The invention relates to a vaccine composition comprising an antigen, an immunologically active saponin fraction and a sterol.

Description

Vaccine

The present invention relates to the novel vaccine preparation, relate to the method and the purposes of described vaccine in medical science of producing described vaccine.Particularly, the present invention relates to vaccine, described vaccine comprises vitriol, antigen, such as the immunologic competence component and the sterol that derive from Quillaia saponaria (Quillaja Saponaria Molina) bark of QS21.

The immunologic competence saponin component that derives from South America Quillaia saponaria bark known in the art has adjuvanticity.For example QS21 is also referred to as QA21, and a kind of Quillaia saponaria component through the Hplc purification at United States Patent (USP) the 5th, 057, discloses (as QA21) its production method in No. 540.At Int.Archs.Allergy.Appl.Immun., 1985,77,409. is open as adjuvant by Scott etc. for quillaja saponin.Yet QS21 uses as adjuvant and has certain shortcoming.For example, when QS21 is expelled in the mammal as free molecule, find necrosis, that is to say in described injection site local organization death to occur.

WO96/33739 discloses and has comprised a kind of antigen, a kind of by such as the immunologic competence component that derives from the Quillaia saponaria bark of QS21 and a kind of bacterin preparation of sterol.

Present wonderful discovery, the fusion vitriol has not only strengthened humoral response in the bacterin preparation that comprises MPL, QS21 and SUV, and strengthened cell response, and the bacterin preparation that comprises MPL, QS21, SUV and vitriol is nontoxic and has good reactionogenicity to distribute and enhanced adjuvanticity.In addition, combination adjuvant seems to help the TH1 reaction.

In first aspect, the invention provides the vaccine adjuvant that comprises vitriol, a kind of immunologic competence saponin component and a kind of sterol.Term " vitriol " is meant aluminium hydroxide or aluminum phosphate.

Adjunvant composition of the present invention preferably comprises the described immunologic competence saponin component of pure form substantially.The present composition preferably comprises the QS21 of pure form substantially, that is to say QS21 at least 90% purity, preferably at least 95% purity and most preferably at least 98% purity.

Other immunologic competence saponin component that can be used in the present composition comprises QA17/QS17.The present composition that comprises QS21 and cholesterol shows to compare with the compositions that does not have this cholesterol and reduced reactionogenicity, and kept adjuvant effect.In addition, know QS21 pH be approximately 7 or bigger primary condition under degrade.Another advantage of this compositions is that QS21 has increased the stability of alkali mediated hydrolysis in comprising the preparation of cholesterol.

Preferred sterol comprises cupreol, stigmasterol, ergosterol, ergocalciferol and cholesterol.These sterols are that this area is extensively known, for example at Merck lndex, the 11st edition, the 341st page disclosed, as the cholesterol of the sterol of the natural generation in Animal fat, found.Most preferred sterol is a cholesterol.

Preferred compositions of the present invention is the formation liposome structure, is those compositionss of small unilamellar vesicle (SUV).The compositions that described sterol/immunologic competence saponin component forms the ISCOM structure has also constituted one aspect of the present invention.

QS21: the ratio of sterol generally is about 1: 100 to 1: 1 (w/w).Preferably have excessive sterol, QS21: the sterol ratio is at least 1: 1 (w/w).The scope that generally gives human QS21 and sterol that exists in vaccine is approximately 1 μ g to about 100 μ g, and the about 10 μ g of preferred every dosage QS21 are to about 50 μ g.

Preferred described liposome comprises neutral lipid, for example preferably at room temperature is amorphous phosphatidylcholine, for example egg yolk lecithin phatidylcholine, dioleoyl phospholipid phatidylcholine or two Laurel phosphatidyl cholines.Described liposome also can comprise charged lipid, and described lipid has increased liposome-QS21 structure stability to the liposome be made up of saturated lipid.In these cases, the amount of charged lipids is preferably 1-20%w/w, most preferably is 5-10%.Sterol is 1-50% (mol/mol) to the ratio of phospholipid, most preferably is 20-25%.

Preferred and the favourable especially compositions of the present invention comprises MPL (3-deacylation monophosphoryl lipid A is also referred to as 3D-MPL).Known that the 3D-MPL from GB 2 220 211 (Ribi) is the mixture of three types the deoxidation acidylate monophosphoryl lipid A with 4,5 or 6 acidylate chains, and by Ribi Immunochem, Montana produces.Preferred form is disclosed in international patent application 94/21292.

The suitable compositions of the present invention is those compositionss, does not have MPL when wherein beginning to prepare liposome, adds MPL then, is preferably the 100nm microgranule.So in little vacuolar membrane, do not comprise described MPL (being called outside MPL).The compositions (being called inner MPL) that comprises described MPL in little vacuolar membrane also forms one aspect of the present invention.In little vacuolar membrane or in vesicle film outside, can comprise described antigen.Soluble antigen preferably externally, and hydrophobicity or fatization (lipidated) antigen or be included in the inside of described film perhaps is included in the outside of described film.

Of the present invention preferred aspect, at first in QS21, add liposome/SUV, mix with vitriol then, this causes that described QS21 is bonded to described vitriol (through the interaction by described liposome) with tangible ratio.When injecting such preparation, expect that it is because storage influence of described vitriol if be free or do not comparing in the fixed lipid plastid with described QS21, causes the release that QS21 is slower in vivo.The described preparation that comprises MPL, QS21, SUV and vitriol, because they are nontoxic and hyperimmunization originality, so advantageous particularly.

Described bacterin preparation will preferably comprise can cause antigen or the antigen composition of human or animal to the pathogen immunne response.So provide the vaccine combination that comprises vitriol, antigen, immunologic competence saponin component and sterol aspect first of the present invention.

Can in chemical compound of the present invention, use antigen known in the art or antigen composition, comprise polysaccharide antigen, derive from following antigen or antigen composition: HIV-1 (such as gp120 or gp160); Any feline immunodeficiency virus; Human or animal's herpesvirus such as the gD or derivatives thereof; Or such as early protein immediately from the ICP27 of HSV1 or HSV2; Cytomegalovirus (particularly human) (such as the gB or derivatives thereof); Varicella zoster virus (such as gp I, II or III); Or such as the hepatitis virus of hepatitis B virus (for example hbs antigen or derivatives thereof), hepatitis A virus, hepatitis C virus and hepatitis E virus; Or other viral pathogen, such as respiratory syncytial virus (for example at United States Patent (USP) 5,149,650 disclosed HSRVF and G albumen or its immunogenic fragments or at United States Patent (USP) 5,194,595 disclosed comprising from the chimeric polyeptides of HSRV F and the proteic immunogenic fragments of G FG glycoprotein for example; Such as first, second and the meningitic meningitis strain of third type, streptococcus pneumoniae, human papillomavirus, influenza virus, hemophilus influenza B (Hib), Epstein-Barr virus (EBV) or such as the bacterial pathogens of Salmonella, eisseria, Borrelia (for example OspA or OspB or derivatives thereof) or chlamydia or Bordetella (for example P.69, PT and FHA) or such as the parasite of plasmodium or toxoplasma.

HSV glycoprotein D (gD) or derivatives thereof is preferred vaccine antigen.It is positioned on the viromembrane, has also found its (Eisenberg R.J. etc. in the Cytoplasm of infected cell; J ofVirol 1,980 35 428-435).It comprises 393 aminoacid that comprise signal peptide, and molecular wt is approximately 60kD.This may be fullest characterized (Cohen etc., J.Virology 60 157-166) in all HSV envelope glycoproteins.Known it be attached in the cell membrane in virus in vivo and play the role of a nucleus.And, show that glycoprotein D can be drawn neutralizing antibody in vivo, and watch for animals and avoid lethal factor invasion and attack.The gD molecule of clipped form does not have the C-terminal anchorage zone, and can be used as the soluble protein that outputs in the cell culture supernatant and produce in mammalian cell.The gD of this soluble form preferably.The production of the gD of clipped form has been described in EP 0 139 417.Preferred described gD is derived by HSV-2.One embodiment of the invention are HSV-2 glycoprotein Ds of 308 amino acid whose truncates, and described glycoprotein D comprises amino acid/11-306, the glycoprotein of the natural generation of asparagine and glutamine is arranged in addition at the C-terminal of the truncated protein that does not have the film anchorage zone.Described proteic this form comprises described signal peptide, cuts described signal peptide so that 283 amino acid whose albumen of the sophisticated solubility of secretory host cell.

In another aspect of this invention, hbs antigen is preferred vaccine antigen.

Expression used herein " hbs antigen " or " HBsAg " comprise the antigenic HBsAg antigen of any demonstration HBV surface antigen or its fragment.Should be appreciated that, except antigenic 226 the amino acid whose sequences of described HBsAg (referring to Tiollais etc., Nature, 317,489 (1985) and list of references wherein), if desired, HBsAg as herein described can be included in above-mentioned list of references and in the preceding S sequence of all or part described in the EP-A-0 278 940.Particularly, described HBsAg can comprise such polypeptide, the aminoacid sequence that it comprises comprises the proteic residue 12-52 corresponding to the L-of the HBsAg of the open reading frame on the ad serotype hepatitis B virus, after meet residue 133-145, (this polypeptide is called L* to meet residue 175-400 after again; Referring to EP 0 414 374).HBsAg within the scope of the present invention also can be included in before the preceding S1-described in the EP 0 198 474 (Endotronics) the S2-S polypeptide or such as those at its close analog described in the EP 0 304 578 (Mc Cormick and Jones).HBsAg as herein described also can the phalangeal process variant, for example at ' escape mutant ' described in WO91/14703 or european patent application 0 511 855A1, is the HBsAg that is substituted glycine by arginine at the 145th aminoacid particularly.

Described HBsAg generally is a particulate form.Described microgranule can comprise for example independent S albumen or can be composite particles, and for example (L*, S), wherein L* is defined above, and S represents the S-albumen of HBsAg.The form that described microgranule is preferably expressed in yeast.

The preparation of hbs antigen has many file records.Referring to for example, Harford etc. (1983) are 54, the 125 pages of Develop.Biol.Standard; Gregg etc. (1987) are at Biotechnolgy, and 5, the 479 pages, EP 0 226 846, EP 0 299 108 and list of references wherein.

In another embodiment, described vaccine antigen is a RSV antigen.Particularly be F/G antigen.United States Patent (USP) 5194595 (Upjohn) has been described the chimeric glycoprotein albumen of the immunogenicity section of the F that comprises RSV and G glycoprotein, and suggestion can comprise that antibacterial, yeast, mammal (for example Chinese hamster ovary celI) and insect cell (with for example baculovirus) express this albumen by various systems.

Wathen etc. (J.Gen.Virol.1989,70,2625-2635) the specific RSV FG chimeric glycoprotein albumen of expressing with baculovirus vector is described, described carrier is made up of the described F proteic amino acid/11-489 that is connected with the proteic aminoacid 97-279 of described G.

Preparation in the scope of the invention also can comprise tumor-resistant antigen, and can be used for immunotherapy treatment cancer.

Generally prepare vaccine according to method described in editors' such as Voller the New Trends and Developments inVaccines (University of Park Press, Baltimore, Maryland, the U.S., 1978).For example the United States Patent (USP) 4,235,877 of Fullerton has been described the encapsulation in the liposome.For example the United States Patent (USP) 4,474,757 of the United States Patent (USP) 4,372,945 of Likhite and Armor etc. discloses albumen and has puted together with macromolecular.

Protein content in each vaccine dose is selected by induction of immunity protective reaction in general vaccine and the amount of not having a significant adverse side effect.This amount depends primarily on used specific immunogen and how it exists (present).In general, expect that each dosage will comprise the albumen of 1-1000meg, preferred 2-100meg, most preferably 4-40mcg.Can determine the optimised quantity of specific vaccine by research on standard, described research on standard is included in the suitable immunne response of observation among the curee.After vaccination first, the curee can accept the booster dose immunity inoculation once or several times of enough intervals.

Preparation of the present invention both can be used to prevent purpose, also can be used for the treatment of purpose.

So more on the one hand, therefore the present invention provides vaccine combination of the present invention to be used for the treatment of patient's purposes.Therapeutic Method provided by the invention comprises the vaccine of the present invention that gives patient's effective dose.Particularly, the invention provides treatment virus, antibacterial, parsitism or a method for cancer, described method comprises the vaccine of the present invention that gives patient's effective dose.

Following embodiment and data declaration the present invention.The preparation method of vaccine 1.1 SUV of embodiment 1 preparation aluminiferous, SUV MPL and QS21

Under vacuum, (perhaps flow down) at noble gas dry in organic solvent lipid (such as from or egg yolk or synthetic phosphatidylcholine) and the mixture of cholesterol.Add aqueous solution (such as phosphate buffered saline(PBS)) then, and stirred vessel is in the suspension until all lipids.With this suspension Micro Fluid, reduce to 100nm then, then by 0.2 μ m filter aseptic filtration until the size of described liposome.Can replace this step with extruding or supersound process.General described cholesterol: the phosphatidylcholine ratio is 1: 4 (w/w), and adding described aqueous solution, to make the cholesterol ultimate density be 5 to 50mg/ml.

1.2 the vitriol in water (for example aluminium hydroxide or aluminum phosphate) (100-500 μ g) adds antigen (1-500 μ g, preferred 10-100 μ g).Select the volume of water so that final volumes of formulation is 500 μ l.After incubation 15-30 minute, add 50 μ g MPL (WO94/21292) with the form of small particle MPL.At room temperature described MPL was adsorbed on vitriol 15-30 minute.Add with such volume then and concentrate 10 times phosphate buffered saline(PBS) (1.5M sodium chloride, the sodium phosphate of 0.5M pH7.5), so that final preparation becomes etc. and to ooze.At room temperature this preparation of incubation is 15-30 minute.

Add QS21 (50 μ g) to SUV (cholesterol that comprises 50-250 μ g) then.This mixture is joined in above-mentioned vitriol/antigen/MPL/ buffer solution mixture.If desired, the antibacterial (50 μ g) of adding such as thimerosal.Embodiment 2

Table 1 has shown that QS21 comprises the dioleoyl phospholipid phatidylcholine of 25% (w/w), and uses than QS21 and measure excessive five times cholesterol with the combination of vitriol under the situation of liposome in the described liposome being with or without.

Preparation	????SUV	Bonded QS21 μ g
Preparation	????SUV	Bonded QS21 μ g	500 μ g vitriol+50 μ gQS21	?0	????＜10
500 μ g vitriol+50 μ gQS21	250 μ g cholesterol+1mgDOPC	????＞40	500 μ g vitriol+50 μ gQS21	?0	????＜10

In order to increase the combination of QS21, can reduce the amount of liposome with vitriol.This has reduced the ratio of cholesterol: QS21, and still for 1: 1 or bigger cholesterol: the QS21 ratio, described QS21 still shows nontoxic.Table 2 shows that if reduce the amount (reducing to 100 μ g by 500 μ g) of vitriol, the amount of bonded QS21 significantly reduces.By adding less liposome, still kept 1: 1 or bigger cholesterol: the QS21 ratio, the QS21 of recruitment and MPL can with the vitriol combination.

Preparation	Cholesterol/QS21	Bonded QS21 μ g	Bonded MPL μ g
Preparation	Cholesterol/QS21	Bonded QS21 μ g	Bonded MPL μ g
500 μ g vitriol+50 μ gQS21+50 μ gMPL	?????5/1	????42	???＞48
500 μ g vitriol+50 μ gQS21+50 μ gMPL	?????5/1	????42	???＞48	100 μ g vitriol+50 μ gQS21+50 μ gMPL	?????5/1	????17	???＞40
100 μ g vitriol+50 μ gQS21+50 μ gMPL	?????2/1	????30	???＞45	100 μ g vitriol+50 μ gQS21+50 μ gMPL	?????5/1	????17	???＞40
100 μ g vitriol+50 μ gQS21+50 μ gMPL	?????2/1	????30	???＞45	100 μ g vitriol+50 μ gQS21+50 μ gMPL	?????1/1	????40	???＞45

Embodiment 3

Test antigen when being with or without vitriol (gD2t that is expressed by herpes simplex virus-2 in Chinese hamster ovary celI comprises 283 aminoacid from the ripe N-terminal of described ripe glycoprotein) and MPL and with the adjuvant effect of the compositions of the QS21 of liposome combination.Test described preparation with cercopithecus aethiops.

Add 50 μ g MPL with 20 μ g gD2t and add the QS21 that 50 μ g are with or without liposome (250 μ g cholesterol add 1 μ g DOPC) and are with or without 500 μ g vitriol, make twice immunity of cercopithecus aethiops (the 0th, 28 day).Analyzed described immunne response at the 42nd day.

Summarized the result among Fig. 1 to 4 hereinafter.

To measure humoral response with respect to the proteic IgG of described gD.Fig. 1 shows the inductive height of tiring when the compositions ratio of MPL+QS21+SUV+ vitriol does not have vitriol.Fig. 2 shows that preparation of the present invention provides more superior antigenic specificity propagation.

Data show, fusion aluminium hydroxide in the bacterin preparation that comprises MPL and QS21 and SUV, strengthened humoral response and cell response the two.This is unexpected a discovery, help the reaction of Th2 type because people it is generally acknowledged aluminum as the adjuvant trend, and result displayed proves herein, and described reaction comprises undiminished a large amount of Th1 component when adding vitriol.

The preparation that comprises MPL and QS21 and SUV and vitriol is nontoxic and high immunogenicity.Embodiment 4 produces RSV FG Chinese hamster ovary celI source protein

The pEE14-FG plasmid comprises the chimeric formation thing of being made up of the fusions between aminoacid sequence F (1-525) and the G (69-298), and same A.BOLLEN (ULB/CRI, Belgium) cooperation obtains.This FG fusion rotein comprises 755 aminoacid altogether.It begins at the N-terminal signal sequence of F, and does not have C-terminal membrane-spanning domain (525-574) grappling territory of F glycoprotein.Connect the extracellular region that does not have the G in amino terminal district glycoprotein then, described amino terminal district comprises signal/grappling territory of typical II class glycoprotein G.

By will be from pNIV2857 (A.Bollen, ULB/CRI, Belgium) FG coded sequence is inserted into the Smal site of pEE14 (Celltech) as aspartic acid 7181 (flush end) 5 '-Hind III (flush end) 3 ' restricted fragment (2188bp), produces the pEE14-FG expression plasmid.Constitute the Kozak sequence that produces the alternative initial ATG of FG in the thing as following at pNIV 2857: pEE14---ccc gtacc ATG GAG-----x-----CAG TAG aagct ggg---pEE14

(Smal) Metl Glu (298) stops

Asp718I (Ke Lienuo) Hind III (Ke Lienuo)

5’F(1-525)-------x--------G(69-298)3’

Described F sequence in pEE14-FG is from SS2 RSV bacterial strain, and please doctor PRINGLE be prepared as with open arms the cowpox carrier (Baybutt and Pringle, J.Gen.Virol., 1987,2789-2796) cDNA in constitutes thing.Described G sequence is from A2 RSV bacterial strain, and produced by the reorganization G vaccinia virus that derives from doctor G.WERTZ (Alabama, the U.S.).The protein stabilized expression of CHO K1 transfection and FG

With calcium phosphate-glycerol transfection method, with the pEE14-FG plasmid DNA of twice of 20 μ g CsCl purification, transfection is by the deutero-CHO K1 of MCB 024M (Celltech) cell.According to GS (glutamine synthetase) expression system (BioTechn. such as Crocett, 1990, the 8th volume, 662) method is selected cell clone, and under the situation that has 25 micro-molar concentration methionine Sulphoximine (MSX), in the GMEM culture medium of the FBS (hyclone) that does not contain glutamine, additional 10% dialysis, increase.After transfection, screening 39 MSX resistance clones in 24 orifice plates, and the secretion of test FG fusion rotein.Measuring with specific " interlayer " ELISA (is the anti-FG serum/antigen of rabbit polyclonal/mAB19), confirm that all transfection bodies are positive to the F antigen presentation.Monoclonal antibody 19 identifications one conformation F1-epi-position, and be neutralizing antibody.

In the LDA with 0.07 cell in every hole on 96 orifice plates, carry out the best FG-of 3 of unicellular sub-clones and produce clone (n ° 7,13 and 37).Obtain 59 positive sub-clones altogether, and by Western blotting and 16 best FG-Producers of the further characterized of ELISA.8 best FG-sub-clone further amplifications again, the FG that estimates them under the situation that is with or without sodium butyrate (2mM) or DMSO (1 or 2%) expresses.6 sub-clones that increase, and preparation cell bottle (cell vial) are stored in-80 ℃ and liquid nitrogen.At last, select 3 best FG-sub-clones.These are CHO K1 FG ° 7.18, ° 13.1 and ° 37.2.

Western blot analysis (non-reduced condition) with monoclonal mAB19 shows that at 135kDa a FG master tape is arranged.By the FG albumen of recombinant baculovirus FG infection cell (UPJOHN) purification under similar trace condition+/-main broadband appears in 100kDa, and+/-other band appears in 70kDa.

According to described sub-clone and growth of cell culture condition, adding sodium butyrate in the CHO-FG cell culture medium has increased by 3 to 12 times with the FG expression.Particularly, in the presence of butyrate (WB/ELISA), sub-clone CHO-FG 13.1 expresses many 8-10 FG albumen doubly.

The shaft-like albumen of FG (baculo protein) by using purification carries out expression mensuration as the ELISA (mAB19 or MoAb AK13) of standard and with the western blot analysis of serial dilution.

Measure and the cell culture condition according to ELISA, CHO-FG 13.1 expressions after butyrate is handled are 5-12 μ g FG/ml.Under accumulation conditions and replacing culture medium (3 to 5 days), the output of acquisition is 16 to 28 μ g secretion FG albumen/ml.

Use (proprietary) growth medium of patent, make CHOK1 FG13.1 cell line be suitable under suspension and serum-free (S/SF) condition, growing.The output that cell line CHO-FG 13.1 S/SF that grow in not having the culture medium of butyrate the express output that the parental generation attached cell system of growing in the culture medium of butyrate expresses that coexists is similar.The butyrate that adds to CHO-FG 13.1 S/SF culture medium is to the almost not influence (increasing by 1.5 to 2 times) of output of FG.

Long-term expression is estimated and the evaluation of preliminary inherited characteristic shows, 13.1 cell lines that CHO-FG 13.1 and S/SF adapt to are stable, comprises the complete FG expression cassette of the single mRNA band that produces about 3000 nucleotide long (DNA and RNA analysis).Further clone 13.1 S/SF and produce FG antigen with described CHO-FG.

Use vitriol/MPL/QS21/SUV to strengthen cercopithecus aethiops to the proteic immunne response of FG from RSV (respiratory syncytial virus).

In Chinese hamster ovary celI, express described FG albumen (comprising F and the proteic fusion rotein of G) and to its purification from RSV.Go up the albumen of absorption 20 μ g purification at the vitriol (500 μ g) that adds monophosphoryl lipid A (MPL:50 μ g).After 30 minutes, add phosphate buffered saline(PBS) in the room temperature incubation.Add SUV then or separately or add SUV and the mixture of QS21 (50 μ g QS21 comprise the SUV of 250 μ g cholesterol and 1mg DOPC).To cercopithecus aethiops injection three times or have only the FG on the vitriol or do not have vitriol and these preparations MPL, SUV and the blended FG of QS21.

Fig. 5 has hereinafter shown that RSV neutralization that each preparation obtains is tired and FG-ELISA tires.Clearly, vitriol/MPL/QS21/SUV group inductive tire the highest.Embodiment 5 contrasts comprise the preparation that contains QS21/SUV and the introduction of vitriol preparation of the antigenic hepatitis B vaccine of SL*

Step according to No. 414373 statement of european patent application is produced SL*.

The Balb/c Mus is carried out immunogenicity research, comprised the inductive humoral response of preparation of QS21-SUV at 3 o'clock being with or without Al (OH) with contrast.MPL dosage is 5 μ g, and QS21 dosage is 5 μ g, and SUV contains 25 μ g cholesterol and 100 μ gDOPC.

In material and method, testing program has been described.In simple terms, on lower limb,, mice is carried out twice intramuscular immunity with the interval in 4 weeks with the SL* vaccine that comprises carrier, immunostimulant or the two combination.Analyze anti--HBs humoral response (IgG and isotype IgG).

In described research, comprise following several groups:

1．SL*(2μg)????Al(OH) ₃(50μg)

2．SL*(2μg)????Al(OH) ₃(50μg)/MPL/QS21-SUV

3．SL*(2μg)????Al(OH) ₃(50μg)/QS21-SUV

(4.SL* 2 μ g) MPL/QS21-SUV result

According to measuring humoral response by Elisa described in material and the method.Analyze two time points: inject after back 28 days (after the I 28) and the booster injection 14 days (after the II 14) first.

In Fig. 6, listed after the I that pooled serum is analyzed and anti-HBs reaction after the II.

These data show that in primary response, all inductive antibody titers of preparation that comprise QS21-SUV are suitable, and use Al (OH) separately ₃The time observed reply a little less than.

In replying once more, by comprising Al (OH) ₃Vaccine-induced antibody response also minimum.Yet all preparations that comprise QS21-SUV do not show identical situation.

Comprise Al (OH) ₃QS21-SUV (+/MPL) two inductive antibody responses of preparation the strongest (higher 2 times) than the inductive antibody response of MPL/QS21-SUV.

Although do not carry out statistical analysis, the result of indivedual serum has confirmed this observation.

Al (OH) ₃And QS21-SUV (+/-MPL) combination also influences the distribute immunne response (Fig. 7) of expression of homotype by described humoral response qualitatively.

Al (OH) ₃Induce the immunne response (having only 3%IgG2a) of tangible TH2 type, and Al (OH) ₃/ QS21-SUV (+/-MPL) the inductive IgG2a of preparation is up to 46%.Conclusion

Vitriol and QS21-SUV (+/-MPL) the inductive antibody titer of combination is than the inductive antibody titer height of the preparation that comprises independent carrier or immunostimulant.Material and method immunity

The vaccine that contains 2 μ gSL* with 50 μ l with around the interval to 10 groups of 5 female Balb/c Mus (6-8 week) in twice intramuscular immunity of shank (gastrocnemius (gastrocnemien)), described vaccine is at Al (OH) ₃(the Al of 50 μ g a great deal oves ³⁺), Al (OH) ₃/ QS21-SUV, Al (OH) ₃Prepare among/MPL/QS21-SUV, the MPL/QS21-SUV.Use the immunostimulant of 5 μ g-agent.

(after the II 14) to the animal blood drawing, detected antibody by Elisa the 28th day (after the I 28) and the 42nd day.Preparation

Use the batch of material of a plurality of various ingredients.Compound method

At the water-reducible Al of 50 μ g (OH) ₃Last absorption or do not adsorb SL* (2 μ g) 15 minutes.

If desired, in 15 minutes, add the suspension of 5 μ g MPL as 100nm microgranule (outside MPL) to prepared product.If desired, adding before 5 μ gQS21 and liposome mix with 1/5 QS21/ cholesterol weight ratio, adding and concentrate ten times buffer.

After adding QS21/SUV 15 minutes, in described preparation, add thimerosal.

Comprise the PBS buffering of the preparation of QS21-SUV, and in the PBS of pH6.8, prepare other preparation with pH7.4.Serology

By using Elisa, carry out the quantitative analysis of anti-HBs antibody as the HBs (Hep286) of envelope antigen.50 μ l antigen and antibody-solutions are used in every hole.Be 1 μ g/ml with antigen diluent to ultimate density in PBS, antigen spends the night in 4 ℃ of hole internal adsorption at 96 hole microtitration plates (MaxisorbImmuno-plate, Nunc, Denmark).Then in 37 ℃ with described plate with the PBS incubation 1 hour that comprises 1% bovine serum albumin and 0.1% polysorbas20 (saturated buffer).Add the twice diluent (1/100 begin dilution) of serum in described saturated buffer to described HBs bag in by plate, and in 37 ℃ of incubations 1 hour 30 minutes.Wash described plate four times with the PBS0.1% polysorbas20, and in each hole, be added in anti-mice IgG1, IgG2a, IgG2b or the described three kinds of antibody (Amershan that are diluted to 1/1000 biotin-conjugated in the saturated buffer, Britain) mixture was in 37 ℃ of incubations 1 hour 30 minutes.After washing step, be added in and be diluted to streptavidin-biotinylation peroxidase (peroxydase) complex (Amershan, Britain) of 1/5000 in the saturated solution, in 37 ℃ of incubations 30 minutes again.As above-mentioned wash plate and with 0.04% o-phenylenediamine (Sigma), the solution incubation of 0.03% hydrogen peroxide in the structure rafter acid buffer of 0.1% polysorbas20,0.05M pH4.5 20 minutes.With 2N H ₂SO ₄Stop described reaction and at the 492/620nm reading.With SoftmaxPro (using four parametric equations), calculate ELISA by reference and tire and represent with EU/ml.

Claims

1. the adjunvant composition that comprises vitriol, immunocompetence saponin component and sterol.

2. according to the adjunvant composition of claim 1, wherein said immunocompetence saponin component is QS21.

3. according to the adjunvant composition of claim 1 or 2, wherein said saponin combines with the liposome that comprises phospholipid and sterol.

4. according to each adjunvant composition in the claim 1 to 3, wherein said sterol is a cholesterol.

5. according to claim 2,3 or 4 adjunvant composition, wherein QS21: the ratio of sterol is by 1: 100 to 1: 1.

6. according to each the adjunvant composition in the claim 1 to 5, comprise 3-deoxidation acidylate monophosphoryl lipid A in addition.

7. the adjuvant and the antigenic vaccine that comprise claim 1 to 6.

8. the claimed vaccine combination of this paper; comprise and derive from human immunodeficiency virus, feline immunodeficiency virus, varicella zoster virus, herpes simplex types 1 virus, herpes simplex types 2 virus, human cytomegalic inclusion disease virus, first, second, third or any antigen or antigen composition of hepatitis E, respiratory syncytial virus, human papillomavirus, influenza virus, Hib, meningitis virus, Salmonella, eisseria, Borrelia, chlamydia, Bordetella, plasmodium or toxoplasma, and with the compositions of claim 1 to 6 as adjuvant.

9. claimed vaccine in arbitrary right, wherein said antigen is tumor antigen.

10. claimed vaccine in claim 8, wherein said antigen is selected from hepatitis B, HSV gD2t or the deutero-SL* of RSV FG chimeric protein.

11. the purposes of the compositions of each definition in the claim 1 to 6 is used to produce the vaccine of preventative therapy of virally, antibacterial or parsitism.

12. the purposes of the compositions of each definition in the claim 1 to 6 is used for the vaccine that the production immunotherapy is treated virus, antibacterial, parsitism or cancer.

13. suffer from pathogenic infection or to the mammiferous Therapeutic Method of its sensitivity, comprise give safe and effective amount according to each compositions in the claim 1 to 5.

14. suffer from the mammiferous Therapeutic Method of cancer, comprise give safe and effective amount according to each compositions in the claim 1 to 5.

15. the production method according to the vaccine combination of claim 1 comprises vitriol, immunocompetence saponin component and cholesterol is mixed with antigen and antigen composition.