CN1200730C - Pneumococcal and Meningococcal vaccines formulated with interleukin-12 - Google Patents

Abstract

This invention pertains to vaccine compositions comprising a mixture of antigen, such as pneumococcal or meningococcal antigen, and interleukin IL-12, which may be adsorbed onto a mineral in suspension. The pneumococcal or meningococcal antigen may be conjugated to a carrier molecule. These vaccine compositions modulate the protective immune response to the antigen.

Description

Streptococcus pneumoniae and meningococcus vaccine with the interleukin 12 preparation

Background of invention

Immune system adopts many mechanism to attack pathogen; Yet not all these mechanism all must activate after immunity inoculation.The inductive protective immunity of vaccination depends on the ability of vaccine-induced appropriate immunne response and resists or eliminating pathogen.According to pathogen, this may need cell-mediated immune responses and/or humoral immunoresponse(HI).

The example that helper T lymphocyte acts in immunne response is, they can be divided into cell subsets according to the cytokine that their produce, and observed unique cytokine distributes and determined their function in these cell subsets.This T cell model comprises two main subgroups: produce the TH-1 cell of IL-2 and interferon gamma (IFN-γ), it strengthens cell and humoral immunoresponse(HI) simultaneously; Produce the TH-2 cell of IL-4, IL-5 and IL-10, it strengthens humoral immunoresponse(HI).(Mosmann etc., Journal of Immunology.126：2348(1986))。Usually the immunogenicity that it is desirable to enhancement antigen is with the stronger immunne response of acquisition in the quilt organism of inoculating, and the enhancing host is to the resistance of antigenic substance.One class and the antigen of being inoculated are used together, and the immunogenic material of energy enhancement antigen is called as adjuvant.For example, known some lymphokine has adjuvanticity, can strengthen that (Nencioni is etc., Journal of Immunology to antigenic immunne response.139:800-804 (1987); EP285441 authorizes Howard etc.).

The invention summary

The present invention relates to vaccine combination, comprise one or more the streptococcus pneumoniae or the mixture of hitchens and Hansen antigen, interleukin IL-12 and mineral suspension.Interleukin IL-12 is adsorbable in mineral suspension or simple and its mixing.In the specific preference of the present invention one, IL-12 is adsorbed on the mineral suspension of Alumen (for example, aluminium hydroxide or aluminum phosphate) for example.These vaccine combinations are regulated antigenic protective immune response; That is, this vaccine combination can promote vaccination host's antibody response on amount and matter, strengthen cell-mediated immunity on amount, and the protective immune response to pathogen is provided.In a specific embodiment of the present invention, antigen is streptococcus pneumoniae or hitchens and Hansen antigen; Optional and the carrier molecule coupling of antigen is for example in streptococcus pneumoniae or meningococcal carbohydrate conjugates.

As herein described studies show that, IL-12 can improve with aluminum phosphate (AlPO ₄) humoral response of the streptococcus pneumoniae of preparation and the mice of meningococcus sugar coupling vaccination.The serotype of the concrete pneumococcal capsular polysaccharide that this paper exemplifies is serotype 1,4,5,6B, 9V, 14,18C, 19F, and 23F, (Pn1, Pn4, Pn5, Pn6B, Pn9V, Pn14, Pn18C, Pn19F, Pn23F), and meningococcal capsular polysaccharide is C type (Men C).Yet these serotypes are not construed to and limit scope of the present invention, use because other streptococcus pneumoniae and meningococcus serotype are also suitable in this article.And and the coupling of carrier molecule, for example CRM that exemplifies with this paper ₁₉₇Albumen coupling is optional, and this depends on the immunogenicity of selected streptococcus pneumoniae or hitchens and Hansen antigen and decides.This point is conspicuous to those skilled in the art.

Dosage 8ng to 1, the IL-12 of 000ng scope have strengthened the IgG1 to aluminum absorption Pn14 or Pn6B, IgG2a, and IgG2b and IgG3 reply.In addition, they have also strengthened replying the IgG2a of Pn4 and Pn9V.Dosage is about 5, and the IL-12 of 000ng has significantly reduced total IgG titre, the especially IgG1 of Pn14 and the titre of IgG2b.

The invention still further relates to preparation contains antigen mixture and has the immunogenic composition of IL-12 of mineral suspension or the method for vaccine combination.Particularly, IL-12 is adsorbed in the mineral suspension.The invention still further relates to and induce or strengthen by the method for the T cell of the generation IFN-γ of immune's protective immune response and complement associativity IgG antibody; comprise mammalian hosts; for example people or Primate host use the vaccine combination of effective dose, and said composition comprises antigen mixture, IL-12 and the mineral suspension of going up acceptable solution preparation with the physiology.Particularly, IL-12 is adsorbed in the mineral suspension.Described mineral suspension is the water slurry of aluminium hydroxide or aluminum phosphate preferably.

Detailed Description Of The Invention

Work described herein has disclosed IL-12 and has strengthened the Pnu-Imune 23 based on aluminum, (particularly serotype 14 and serotype 6B streptococcus pneumoniae sugar coupling vaccine), and meningococcus vaccine, the ability of the immunne response of (particularly C type) is to increase the ratio of complement associativity IgG2a and IgG2b antibody.As described herein, PnPs-14-CRM ₁₉₇Vaccine contains and avirulence diphtheria toxoid mutant (cross reaction material) (called after CRM ₁₉₇) link coupled serotype 14 pneumococal polysaccharides, and PnPs6B-CRM ₁₉₇Vaccine contains and CRM ₁₉₇Link coupled serotype 6B pneumococal polysaccharide.IL-12 is comparable to MPL  (3-O-deacetylation one phosphoric acid fat A; RIBI ImmunoChem Reaserch, Inc., Hamitton, Montana), the latter is a kind of strong adjuvant of Pnu-Imune 23 in mice.In the independent experiment that carries out in BALb/c mice body, it is vaccine-induced to exemplary aluminum coupling to have measured IL-12, the effect of the cytokine secretion of CRM specific T-cells.

IL-12 is produced by various antigen presenting cells, mainly is to be produced by macrophage and mononuclear cell.It is to induce T cell originally to become the key element of TH-1 cell.The generation of IL-12 or the ability that it is replied proved crucial in the development that protectiveness TH-1 class is replied for example, in parasitic infection, it should be noted that in the Li Shiman disease be exactly (Scott, etc., U.S. Patent number No.5,571,515) like this most.The effect of IL-12 is mediated by the IFN-γ that NK cell and helper T lymphocyte produce.Interleukin 12 (IL-12), the former natural kill cell stimulating factor that is called is a kind of heterodimer cytokine (Kobayashi etc., J.Exp.Med.170:827 (1989)).The expression and be separated in International Patent Application WO 90/05147 (nineteen ninety May 17 day open) of IL-12 albumen in recombinant host cell described to some extent.

Research described herein has disclosed IL-12 in streptococcus pneumoniae or meningococcus vaccine, particularly in streptococcus pneumoniae or the meningococcus sugar coupling vaccine as the application of adjuvant.Therefore, the present invention relates to contain the vaccine combination of the mixture of these antigens, IL-12 and mineral suspension.In specific embodiments of the invention, IL-12 is adsorbed in for example mineral suspension of Alumen (for example, aluminium hydroxide or aluminum phosphate).These vaccine combinations can be regulated antigenic protective immune response; That is, this vaccine combination can be induced by the inoculation host and be produced the complement fixation antibody required to the pathogen protective immune response.In specific embodiments of the invention, antigen is PNEUMOVAX-23, particularly pneumococal polysaccharide; This PNEUMOVAX-23 can randomly be coupled to a carrier molecule, for example streptococcus pneumoniae carbohydrate conjugates.The concrete pneumococal polysaccharide serotype that this paper exemplified is: serotype 1,4,5,6B, 9V, 14,18C, 19F, and 23F; Yet these serotypes should not be interpreted as limiting the scope of the invention, because other serotype also is fit to use in the present invention.

In another embodiment of the present invention, antigen is hitchens and Hansen antigen, particularly meningococcal polysacharide; The optional carrier molecule that is coupled to of this hitchens and Hansen antigen, for example meningococcus carbohydrate conjugates.What exemplify herein is Neisseria meningitidis C type; Yet the type should not be interpreted as limiting the scope of the invention, because other type also is fit to use in the present invention.

IL-12 can obtain from several suitable sources.It can be by recombinant DNA technology production; For example, the gene of coding human IL-12 is cloned in host system and expressed, enable the purified people IL-12 of mass production.Equally usefully biologic activity subunit or the fragment of IL-12 in the present invention.The merchandise resources of recombined human IL-12 and mice IL-12 comprises Genetics Institute, and Inc. (Cambridge, MA).

Antigen of the present invention, for example, streptococcus pneumoniae or hitchens and Hansen antigen or streptococcus pneumoniae or meningococcus carbohydrate conjugates are used in and induce in the mammalian hosts antigenic immunne response.For example, antigen can be that serotype 14 or 6B meningococcal polysacharide or its remain with the part that excites the immunne response ability.Other suitable antigen comprises polysaccharide and the conjugate thereof of other pod membrane bacterium, excretes poison and external membrane protein.

Method of the present invention comprises administration, the vaccine combination of (for example people or primates) immune effective dose, this vaccine combination comprises antigens mixed, for example PNEUMOVAX-23 or streptococcus pneumoniae conjugate, and the IL-12 that assists a ruler in governing a country dosage that is adsorbed in the mineral suspension.

Used in this article, " immunity effectively " dosage of vaccine combination is meant the dosage that is fit to induce immune response.IL-12 and antigenic concrete consumption be by this mammiferous age of being treated, body weight and medical condition decision, simultaneously also by the method decision of dispenser.The person skilled in the art is easy to determine proper dosage.This vaccine combination is optional to be formulated in materia medica or the physiology goes up acceptable carrier, for example normal saline or polyhydric alcohol, dispenser in (for example glycerol or propenyl).

This vaccine combination is optional to contain other adjuvant, for example vegetable oil or its Emulsion, surfactant, for example: hexadecylamine, the octadecane amino-acid ester, octadecylamine, LYSOLECITHIN SUNLECITHIN A, dimethyl-two (octadecyl bromination ammonium), N, two octadecyl-the N ' of N--N ' two (2-ethoxy-propanediamine), methylol cetyl glycerol, and Pluronic polyols; Polyamine, for example: pyrans, dextran sulfate, poly IC, carbopol; Peptide, for example: muramyldipeptide, dimethylglycine, tuftsin, immunostimulating complex, oil emulsion; Lipopolysaccharide, for example: MPL  and mineral coagulant.Antigen of the present invention also can be impregnated in liposome, be total to chelate (cochleates), biodegradable polymer is polylactide for example, poly-Acetic acid, hydroxy-, bimol. cyclic ester and polylactide-co-glycolide, or ISCOMS (immunostimulating complex), and the active component that adds also can use.Antigen of the present invention also can be co-administered with bacteriotoxin and attenuation derivant thereof.Antigen of the present invention also can with other lymphokine, include, but not limited to IL-2, IL-3, IL-15, IFN-γ and GM-CSF are co-administered.

Vaccine can be administered to the human or animal by number of ways, includes but not limited to parenteral, intradermal, percutaneous (for example by using the slow release polymer), intramuscular, intraperitoneal, intravenous, subcutaneous, oral cavity or intranasal drug delivery route.The antigen amount of using in these vaccines is looked antigenic characteristic and difference.Use together with conventional carriers antigen, the judgement of the antigenic dosage range of conventional carriers that suitable vaccine of the present invention uses and operation are just in time in those skilled in the art's ability.Vaccine of the present invention is intended for use in treating teenage and adult homoiothermic animal, particularly people.Usually, IL-12 and antigen are with coupling; Yet, in some cases, one skilled in the art will appreciate that IL-12 uses before or after can being close to antigen inoculation.

Streptococcus pneumoniae of the present invention and hitchens and Hansen antigen can regulate with the carrier molecule coupling or enhance immunity is replied.Suitable carrier protein comprises by chemistry or the genetic method bacteriotoxin of safe, with to administration and as effective carrier on the immunology.Example comprises pertussis, diphtheria and tetanus toxoid and avirulence mutein (cross reaction material (CRM)), the avirulence variant of diphtheria toxoid for example, CRM ₁₉₇Natural toxin or anatoxic contains the fragment of at least one t cell epitope, and is also the same with antigen vectors and outer membrane protein composite useful.The method for preparing PNEUMOVAX-23 and carrier molecule conjugate is known in the art, and can be at for example Dick and Burret, Contrib Microbiol Immunol.10:48-114 (Cruse JM, Lewis RE Jr, eds; Basel, Krager (1989)) and U.S. Patent number No.5, find among 360,897 (Anderson etc.).

The adjuvant effect of IL-12 has many important meanings.The adjuvant effect of IL-12 can increase by the concentration of immunity inoculation organism at the antibody that protecting function is arranged that antigen produced.IL-12 as adjuvant can enhance immunity the ability of the faint or antigen induction immunne response that immunogenicity is poor of originality.Concentration in the effectively common requirement of immunity is under the deleterious situation, and it can provide safer immunity inoculation.By reducing the antigen amount, the danger of toxic reaction has also reduced.

Usually, the vaccination scheme requires administration of antigens in the time in several weeks or several months, to excite protective immune response.Protective immune response be enough to protect by the organism of immunity opposing vaccine at the immunne response of productive infection of one or more special pathogens.

Shown in an embodiment, in the vaccine of aluminum prescription, comprise and be adsorbed on AlPO ₄On IL-12 and and CRM ₁₉₇Link coupled serotype 14 or serotype 6B pneumococal polysaccharide, this vaccine cause that usually by IgG1 be main replying, and the IL-12 of 0.2 μ g can increase IgG2 and the IgG3 subclass in BALB/c and the Swiss Webster mice greatly, but to IgG1 almost or invalid fully.The enhancing of the IgG2b of anti-Pn14 can be seen in Swiss Webster mice; 0.2 the IL-12 of μ g replys at the IgG of anti-Pn14 subclass with 25 μ g MPL  identical effectiveness is arranged, point out higher at least 100 times than MPL  biological activity at IL-12 aspect this.Distribution is estimated as the IgG subclass, especially having strengthened IgG2a replys, accepted the mice of 0.2 μ gIL-12, wanting of its sero-fast Pn14 streptococcus pneumoniae opsonophagocytosis specific activity contrast is high, has been equivalent to inoculate the mice of vaccine of the MPL  prescription of much bigger amount.

Say that simply IgG2a and IgG2b antibody are very effective to the activating complement system, IgG1 antibody is then invalid.Complement system is made of a series of accumulating in antigen (for example, antibacterial) link coupled IgG2a or IgG2b plasma protein on every side, forms a macromolecular complex.This complex deposits at bacterium surface, by membrane perforation (bactericidal activity) or promote the identification of phagocyte (for example polymorphonuclear cell (PMN) that uses) to antibacterial in this research, thereby absorbs antibacterial and kills their (conditioning phagocytosis).

The dosage that increases IL-12 has reduced replying of IgG1 and IgG2b greatly.The minimizing of these immunoglobulin subclasses singly is not owing to the dynamic (dynamical) change of antibody response, as (Buchanan, Van Cleave and Metzger make a summary 1945 observed in HEL (HEL) system; The 9th immunology international conference (1995)), because these subclass all reduce at the time point of all tests.The conversion of B cell generates these subclass needs IL-4, and (a kind of TH-2 cytokine, its generation are subjected to IL-12 and suppress) can predict the effect to IgG1.Yet the minimizing of IgG2b is not predicted, because the increase of IgG2b level is relevant with the existence of TH-1 class T cell in the research formerly.The non-IFN-γ of possibility, or the cytokine except that IFN-γ has played effect in IgG2b regulates.For example, Germann etc. (European Journal of Immunology 25:823-829 (1995)) find that having suppressed IL-12 enhancing IgG2a with anti--IFN-γ treatment mice replys, but do not strengthen the ability that IgG2b replys.Other research prompting TGF-β is the important factor (J.Stavnezer summary, Journal of Immunology .155:1647-1651 (1995)) of inducing IgG2b.Be not wishing to be bound by theory, IL-12 that might high dose has influenced that TGF-β produces or to its reactivity.

IFN-γ is to inducing at T cell dependent protein matter antigenic IgG2a antibody (Finkelman and Holmes, Annu.Rev.Immunol.8:303-33 (1990)) and do not rely on antigenic IgG3 at the T cell to reply (Snapper etc., J.Exp.Med.175:1367-1371 (1992)) be vital.Enhanced IFN-γ replys and is common in including IL-12 and AlPO ₄Vaccine (PnPs-14-CRM ₁₉₇) after the single needle inoculation, and after the booster immunization.IL-12 it seems the time of depending on inoculation back results lymphoid cell to the effect of TH-2 cytokine IL-5 and IL-10, and may depend on specific cytokine.The IL-12 of external source has eliminated the IL-5 of primary vaccination lymph-node cell (LNC) antigenic specificity of results after one week and the generation of IL-10 fully.For the second time after the inoculation, the visible difference of these two kinds of cytokines; The generation of the IL-5 of LNC or splenocyte is eliminated fully by 1 μ g IL-12 in the vaccine, but being created in behind the booster immunization of IL-10 is almost uninfluenced.Whether not clear these difference are owing to set up due to the cell culture at different time, or have reflected the amplification of TH-2 class cell mass to inoculating.Back one probability and Wolf and colleague's thereof data consistent (Bliss etc., Journal of Immunology, 156:887-894 (1996)), show that the T cell that produces IL-4 can formerly obtain with the vaccine immunity that contains IL-12 and with reclaiming in the BALB/c mouse of soluble antigen booster immunization.In their research,, also can detect IL-4 even in secondary vaccine, contain IL-12.Behind the booster immunization TH-2 cytokine exist solublely, why in BALB/c mouse, to be reduced to contrast (aluminum coupling vaccine) below horizontal even high-caliber IL-12 can not reply secondary IgG1.Do not resemble BALB/c mouse, high dose IL-12 severe inhibition the IgG1 of Swiss Webster mice reply.Whether this reduces relevant it be unclear that with inoculation back TH-2 cytokine generation for the second time.

In the present invention's research, IL-12 shows only to be had immunoregulatory activity or shows as " classics " adjuvant simultaneously and the immunomodulator activity, and this depends on vaccine.To PnPs14-CRM ₁₉₇Research in, to the IgG of vaccine reply (especially primary response) not the existence because of IL-12 significantly improve, and some subclass, for example, IgG2a and IgG3 can improve, other is then constant or reduce.Therefore, IL-12 is useful to the humoral response that adjusting has immunogenic vaccine.Possible, in these researchs, the adjuvanticity of IL-12 is covered by the existence of aluminum, and aluminum itself is the enough strong adjuvant of high immunogenicity PnPs-14 conjugate.The adjuvant character of IL-12 is not having under the situation of aluminum, better shows by the dosage of minimizing conjugate or the conjugate of use immunogenicity poorness.Therefore, carried out further evaluation, promptly used IL-12 by having under aluminum or the aluminum-free situation with PnPs6B coupling vaccine, this vaccine is more weak than PnPs-14 coupling vaccine immunogenicity in the SwissWebster mice.

Designed the adjuvanticity of other research explanation IL-12 to the streptococcus pneumoniae conjugate of immunogenicity poorness.Selected the Pn18C conjugate, because it and AlPo ₄Immunogenicity poorness during prescription, that is it is induced low titre IgG and not all mice all it to be produced and replys.When with MPL or QS-21 prescription, can obtain the higher IgG titre and the respondent of bigger ratio.

One hectogamma MPL  adds AlPO ₄Or 20 μ g QS-21 ^TMBe the best adjuvant of in this research Pn18C being replied, because they have induced the respondent of maximum rate to this serotype.But, IL-12 in the mice that has inoculated this conjugate to carrier protein CRM ₁₉₇IgG replied remarkable effect.And this effect of cytokines is because of existing AlPO in the vaccine ₄And change.IL-12 is not obviously having AlPO ₄Played adjuvant effect in the vaccine of prescription, cause first or for the second time the dose dependent of inoculation back IgG titre increase.IL-12 has strengthened CRM ₁₉₇IgG2a reply, this helps to induce the ability of TH-1 class accessory cell (IFN-γ Producer) to conform to its.Yet IL-12 is also first or strengthened CRM after the inoculation for the second time ₁₉₇IgG1 reply.IgG1 antibody is associated with the TH-2 class accessory cell that produces IL-4 usually.

The IL-12 of 0.1 μ g is added based on AlPO ₄Pn18C coupling vaccine (itself can induce high 10 times CRM ₁₉₇Reply) in invalid but significantly increased the titre of IgG2a to IgG1.The IgG2a titre that obtains with 0.1 μ gIL-12 is at least with 5 μ gIL-12, and do not have AlPO ₄Equally high.Yet, it should be noted that AlPO ₄The existence enhancing that do not hinder IL-12 that IgG1 is replied.With being coupled to AlPO ₄The Pn14 immune mouse, the IL-12 of 0.2 μ g dosage has increased anti-CRM ₁₉₇IgG1, IgG2a and IgG2b titre.Difference to the effect of IgG1 may reflect CRM ₁₉₇IgG replys this two kinds of immunogenic differences of conjugate; Be coupled to AlPO ₄Pn14 induced low 10 times CRM ₁₉₇Therefore the IgG titre, still has IL-12 to strengthen the space that IgG1 replys, but in immunity inoculation be coupled to AlPO ₄The mice of Pn18C in do not have this space.MPL  and QS-21 ^TMIn immunity inoculation be coupled to AlPO ₄The mice of Pn18C in the remarkable fact that increases the IgG1 titre show that IgG1 replys and farthest do not excited.In addition, the saccharide character on this conjugate may be a factor.In two experiments, the IL-12 of high dose causes anti-CRM ₁₉₇IgG1, the remarkable minimizing of IgG2a and IgG2b titre, this is a kind ofly to lack AlPO ₄The effect of Shi Weijian.

IL-12 and MPL  or QS-21 ^TMRendeing a service at its adjuvant of performance may be different.IL-12 has increased CRM significantly in Pn18C conjugate mice immunized ₁₉₇The IgG2a titre, but very little to the effect of IgG2b.On the contrary, MPL  and QS-21 ^TMIncreased the titre of these two kinds of IgG subclass.The separation prompting of these two kinds of subclass, IgG2b is the cytokine by non-IFN-γ, or (IFN-γ promotes immunne response and is converted to IgG2a) of the cytokine induction except IFN-γ, this type cytokines may mediate the immunoregulation effect to IL-12.A kind of material standed for that can drive the IgG2b generation is TGFb.Yet this antigenic character can not be excluded, because in Pn14 conjugate mice immunized, 0.2 μ gIL-12 causes IgG2a and IgG2b to bring up to and similar level with the titre equivalence of the MPL  generation of 25 μ g.Utilization is by PnPs14-CRM ₁₉₇Conjugate is covalently attached to CRM with serotype 6B pneumococcal capsular polysaccharide ₁₉₇Conjugate (PnPs6B-CRM ₁₉₇) research carried out of bivalent vaccine that mix to form, confirm and extended above-mentioned discovery.The IgG that IL-12 has not only changed the Pn6B conjugate replys, and has also increased all the IgG titres to this conjugate.In addition, this work shows that further the adjuvanticity of the IL-12 of low dosage passes through and AlPO relatively ₄Fill a prescription and be enhanced.With the above-mentioned PnPs14-CRM that uses ₁₉₇The research difference of carbohydrate conjugates, IL-12/AlPO ₄Strengthen IgG1 and IgG2a subclass simultaneously, show that it may not be a vague generalization phenomenon that the not obvious enhancing of IL-12 Pn14 IgG1 replys at Pn6B.This work has supported that further the mechanism of the adjuvanticity of IL-12 and MPL  is viewpoint inequality.Two kinds of adjuvants all are strengthened to similar level with the IgG1 of Pn6B with the IgG2a titre, but MPL  is to promoting IgG2b and IgG3 antibody more effective.

IL-12/AlPO ₄Pn14 IgG can not replied adjuvant effect.Reason to this is unclear; Yet, not wish bound by theoryly, this has reflected probably in the former research that mice is the PnPs14-CRM with 1 μ g dosage ₁₉₇The carbohydrate conjugates immunity, that is amount ratio is the high 10 times fact in Pn6B research.IL-12 contains serotype 1,4,5 to the application employing of more complicated Pnu-Imune 23,6B, and 9V, 18C, the avirulence vaccine of 19F and the pneumococcal carbohydrate conjugates of 23F is proved.IL-12 and AlPO ₄Coupling except that PnPs6B and PnPs14, has increased the IgG2a antibody of anti-PnPs4 and PnPs9V, and has strengthened mice to using serotype 18C pneumococal polysaccharide (PnOs-18C-CRM ₁₉₇) responsibility of carbohydrate conjugates (it is the immunogenicity poorness in mice) of preparation.

In also having an example, tested IL-12 to the scorching sugared coupling vaccine of coccus (MenC) of C type neisseria meningitis with to (HbOC) effect of sugared coupling vaccine of B hemophilus influenza (Hemophilus influenzae).With 50ng IL-12 and AlPO ₄The vaccine of preparation has strengthened the titre to the IgG2a of MenC capsular polysaccharide, but does not increase the titre to HbOC.

Data provided herein show AlPO ₄Can strengthen the ability of IL-12 greatly, therefore can adopt this cytokine of enough low doses.A possible mechanism is IL-12 and AlPO ₄Coupling, thus its retention time in animal body increased; The IL-12 that studies show that in addition can be rapidly and aluminum coupling (data not shown).In addition, AlPO ₄The local inflammation effect can induce and make IL-12 have more bioactive cytokine.

Except understanding IL-12 and AlPO ₄The physiology interact outside, several other issues result from the current research work with the Pnu-Imune 23 of IL-12 preparation.Suppose AlPO ₄Strengthened the activity of IL-12, understood so and assist a ruler in governing a country streptococcus pneumoniae carbohydrate conjugates IgG and reply the minimum dose that needs this cytokine, and whether the T cell that produces IL-5 be that the sugared coupling vaccine that contains IL-12 activates should be useful.These two problems embodiment 4 describe to the BALb/c mice study in focus on.

Providing following embodiment purpose is to illustrate the present invention, limits the scope of the invention but can not be construed to.The content of all lists of references that this paper quoted is included in order to reference at this.

Embodiment

Embodiment 1:IL-12 Swiss Webster mice to being coupled to CRM in the aluminum phosphate ₁₉₇Serotype 14 pneumococcal capsular polysaccharide (PnPs-14-CRM/AlPO ₄) the IgG effect of replying

Research design

With with 100 μ g AlPO ₄1 μ gPnPs-14-CRM of preparation ₁₉₇, do not contain or contain 0.2 microgram, 1 microgram or 5 microgram IL-12, twice immunity (when the 0th and 3 weeks) Swiss Webster mice.All vaccines contain 0.25% normal mouse serum, to stablize used low concentration IL-12.PnPs-14-CRM ₁₉₇Be a kind of by reduction amination effect and the toxicide diphtheria toxin, diphtherotoxin (CRM of genetic method ₁₉₇) be covalently attached to the conjugate of serotype 14 pneumococcal capsular polysaccharides.Another winding is subjected to the MPL  (3-O-deacylated tRNA base one phosphoric acid fat A, RIBI ImmunoChem Reserch, Inc., Hamilton Montana) of 25 micrograms to replace IL-12.Vaccine is three all subcutaneous being applied at interval.At the 3rd week (primary response), the 5th and the 7th week (second set responses in 2 behind booster immunization and 4 weeks) is collected serum.Analyze the IgG antibody of the anti-PnPs-14 of this serum.

Also having analyzed this serum promotes human polymorphonuclear leukocyte (PMN) opsonophagocytosis to kill the pneumococcal ability of 14 types.14 type streptococcus pneumoniae are with the antiserum diluent with as complement source, the serum conditioning of removing C8.They and human polymorphonuclear leukocyte (PMN) cultivate together then, measure the bacteria living percentage rate by colony counting.

The result

Table 1 shows that the IL-12 of 1 microgram and 5 micrograms has significantly reduced and uses AlPO ₄The anti-PnPs-14 IgG of the conjugate immune mouse of preparation replys.It is invalid that cytokine lowest dose level (0.2 microgram) is replied total IgG, but cause the bigger variation of indivedual immunoglobulin subclass levels.When the 5th and 7 weeks (after being 2 and 4 weeks of booster immunization respectively), 0.2 microgram IL-12 has induced obviously higher IgG2a, IgG2b and IgG3 titre, but keep the IgG1 level constant substantially.0.2 microgram IL-12 inductive IgG subclass scattergram and do not have a difference with what 25 microgram MPL  obtained, and the serum of mice of accepting these adjuvants than those with only containing AlPO ₄The serum of mice of vaccine immunity higher opsonophagocytosis activity (table 2) is arranged.

More the IL-12 of high dose significantly reduces IgG1 antibody; When 5 microgram IL-12, the IgG1 titre is than hanging down 10 times at least without the IL-12 mice immunized.This effect during the primary response and after booster immunization all clearly.Increase IL-12 dosage and do not cause IgG2a, the further increase of IgG2b and IgG3, and also as IgG1, they also reduce, although the degree difference.IgG2b shows maximum the minimizing, thereby contains the vaccine-induced IgG2a of same titre that the vaccine of 1 microgram or 5 microgram IL-12 and those do not contain adjuvant.IgG2a and IgG3 are more insensitive to the effect of high dose IL-12; Promptly use 5 microgram IL-12, behind secondary inoculation, these subclass also compare according in higher.

These studies show that IL-12 can regulate prescription AlPO is arranged ₄PnPs-14-CRM ₁₉₇The IgG subclass of coupling vaccine is replied.0.2 the IL-12 of micrograms dose increases the IgG2a to Pn14, IgG2b and IgG3 reply, and do not reply and do not influence IgG1.The more high dose of IL-12 causes the remarkable minimizing of IgG1 and IgG2b titre.These dosage IgG2a and IgG3 titre are also shown reduction, but they are still than higher without the IL-12 mice immunized.Embodiment 2 has shown that the IgG subclass changes and produce the CRM of IFN-γ ₁₉₇The remarkable minimizing that strengthens with antigenic specificity IL-5 generation of inducing of specific T-cells is associated, and prompting helper T lymphocyte phenotype is changed into the TH-1 class from the TH-2 class.

Table 1:IL-12 is to PnPs-14-CRM ₁₉₇The effect of/aluminum vaccine immunogenicity

			PnPs14 IgG replys
								Time	Adjuvant	Dosage (μ g)	IgG	IgG1	IgG2a	IgG2b	IgG3
3 weeks	Do not have		56,035	8,394	481	298	1,312
									IL-12	5	13,137	480	2,417	388	2,398
	IL-12	1	26,131	1,521	3,249	736	3,858
									IL-12	0.2	90,220	13,779	4,731	1,454	7,944
	MPL	25	46,451	14,303	1,506	8,506	18,203
5 weeks	Do not have		531,270	189,571	5,507	14,463	18,158
									IL-12	5	231,015	16,900	28,719	6,002	56,982
	IL-12	1	198,044	36,327	27,420	11,841	30,740
									IL-12	0.2	722,360	305,623	60,701	89,397	99,794
	MPL	25	751,066	221,324	44,957	91,265	77,989
7 weeks	Do not have		694,741	244,212	1,801	6,849	9,245
									IL-12	5	177,438	17,232	20,276	3,494	26,859
	IL-12	1	183,571	44,213	21,246	5,063	13,447
									IL-12	0.2	852,292	251,157	37,104	37,717	88,933
	MPL	25	783,622	187,055	30,694	89,153	59,297

Table 2: the PnPs-14-CRM that IL-12 or MPL  are arranged with prescription ₁₉₇/ AlPO ₄Inoculation

The opsonophagocytosis activity of mice serum

	Bacteria living rate %
											The 5th all serum					The 7th all serum
	The initial serum dilution that detects	No IL-12	0.2μg IL-12	1μg IL-12	5μg IL-12	25μg MPL	No IL-12	0.2μg IL-12	1μg IL-12	5μg IL-12											25μg MPL
2											6	10	6	6	9	6	6	7	7	5
	8	12	7	9	9	7	21	4	10	9											6
16											32	4	24	25	3	47	8	17	26	8
	32	71	12	61	94	23	68	6	85	90											21
64											64	46	90	89	51	116	34	79	99	76

Embodiment 2: prescription has the streptococcus pneumoniae conjugate vaccines (PnPs14-CRM of IL-12 ₁₉₇/ AlPO ₄) the character of the complementary cell of inductive T

Research design

With prescription 100 microgram AlPO are arranged ₄1 microgram PnPs14-CRM with various dose IL-12 ₁₉₇Conjugate tail base portion subcutaneous vaccination eight (8) only be one group respectively organize the Balb/c mice.Contain normal mouse serum (0.25%) as carrier protein.After one week, preparation draining lymph node cell suspending liquid from every group half mice is with CRM ₁₉₇, lysozyme, ConA or in culture medium, cultivated 6 days separately.The 3rd day with the culture supernatant of collecting parallel culture on the 6th day, with elisa assay IFN-γ, IL-5 and IL-10.

When the 3rd week, to remaining mice blood drawing and immune again with the same vaccine formulation that uses in the 1st immunity.After 14 days (the 5th week), mice is drawn blood again the 2nd immunity.After 4 days, collect their draining lymph node cell and splenocyte, and and CRM ₁₉₇, lysozyme, ConA or in culture medium, cultivated 6 days separately.The 3rd day with the culture supernatant of collecting parallel culture on the 6th day, with elisa assay IFN-γ, IL-5 and IL-10.

The result

With PnPs14-CRM ₁₉₇/ AlPO ₄IgG2a and IgG3 to Pn14 when vaccine formulation strengthened for the 5th week greatly with low dosage IL-12 (0.12 microgram and 1.0 micrograms) reply, and do not reply (seeing Table 3) but do not increase IgG1.Visible several places difference between the result who in last experiment, obtains with Balb/c mice and Swiss Webster mice; IL-12 does not significantly increase IgG2b antibody at Pn14 in this experiment, and the IL-12 of 5 micrograms dose, with respect to the matched group of the acellular factor, also not significantly (＞10 times) reduce the titre of IgG1.

After one week of immunity, do not work as and use CRM with the lymph-node cell of IL-12 immune mouse ₁₉₇In stimulated in vitro, produce IFN-γ, IL-5 and IL-10 (seeing Table 4).Adding IL-12 in the vaccine significantly increases the antigenic specificity generation of IFN-γ, and has eliminated the ability of lymph-node cell generation IL-5 and IL-10.IL-12 (0.2 microgram) with lowest dose level has obtained maximum IFN-γ generation; Higher dosage, particularly 5 micrograms show the level that has reduced this cytokine.This is at the CRM with 1 mcg/ml ₁₉₇Seem the most obvious in the culture that stimulates.IFN-γ that higher dosage IL-12 causes reduces and may not reflect and generally suppress phenomenon because in to Con A reaction IFN-γ produce be identical and vaccine in the IL-12 dosage indifference.

For the second time after immune two weeks, with the lymph-node cell that contains the IL-12 vaccine immune mouse and splenocyte to CRM ₁₉₇Irritant reaction in and without the IL-12 mice immunized relatively, continue to have produced high-caliber IFN-γ (table 5).As back 7 days of first immunity was observed, 0.2 microgram was the dose,optimums that promote that IFN-γ replys to the IL-12 of 1.0 micrograms.Yet opposite, the generation of IL-5 and IL-10 is subjected to Different Effects.1.0 and the IL-12 of 5.0 micrograms dose eliminated IL-5 basically and replied, but compares with it, IL-10 produced have only very little effect.IL-12 (5.0 microgram) has eliminated the ability (table 5 and 6) of splenocyte rather than lymph-node cell generation IL-10.

IL-12 is to the PnPs14 based on aluminum in the table 3:BALb/c mice

The immunne response effect of sugar coupling vaccine

Week	IL-12 dosage	IgG	IgG1	IgG2a	IgG2b	IgG3
3	Do not have	41,480	7,347	1,387	895	2,333
								0.2	26,253	1,521	1,118	171	5,911
	1	26,124	966	2,155	248	5,991
								5	10,753	541	671	183	3,242
							5	Do not have	234,220	33,284	2,896	3,105	2,487
	0.2	674,996	71,808	18,245	6,789	107,470
								1	632,714	32,022	22,749	7,853	44,350
	5	224,832	19,495	10,083	1,287	25,212

Table 4: AlPO is arranged with prescription ₄Behind the PnPs-14 conjugate single immunization of IL-12

The cytokine that lymph-node cell produced that obtained in 7th

	Culture on the 6th
		Cytokine exo-antigen μ g/ml	There are not 0.2 μ g, 1.0 μ g, 5.0 μ g IL-12 IL-12 IL-12 IL-12
IFN-γ CRM 30	23.2 102.7 60.5 32.2
		(U/mL) CRM 1	＜0.75 65.2 28.6 8.7
Lysozyme 30	＜0.75 2.9 6.6 4.5
		Con A 1	43.8 97.1 107.1 105.4
Culture medium-	＜0.75 3.6 10.6 5.2
IL-5 CRM 30	7.2 ＜0.22 ＜0.22 ＜0.22
		(ng/mL) CRM 1	2.2 ＜0.22 ＜0.22 ＜0.22
Lysozyme 30	＜0.22 ＜0.22 ＜0.22 ＜0.22
		Con A 1	＜0.22 ＜0.22 ＜0.22 ＜0.22
Culture medium-	＜0.22 ＜0.22 ＜0.22 ＜0.22
IL-10 CRM 30	10.4 0.8 0.21 0.21
		(ng/mL) CRM 1	2.6 0.21 0.21 0.21
Lysozyme 30	＜0.14 0.21 0.21 0.21
		Con A 1	＜0.14 0.21 0.21 0.21
Culture medium-	＜0.14 0.21 0.21 0.21

Table 5: AlPO is arranged with prescription ₄PnPs-14 conjugate secondary with IL-12

The cytokine that immunity back two all splenocytes are produced

	Culture on the 6th
		Cytokine exo-antigen μ g/ml	There are not 0.2 μ g, 1.0 μ g, 5.0 μ g IL-12 IL-12 IL-12 IL-12
		IFN-γ CRM 30	7.0 98.4 83.2 50.9
(U/mL)
		CRM 1	1.0 89.2 76.8 16.4
Lysozyme 30	＜0.4 ＜0.4 ＜0.3 ＜0.3
		Con A 1	42.7 48.7 50.6 49.5
		IL-5 CRM 30	13.2 3.1 0.6 ＜0.2
(ng/mL) CRM 1	4.5 4.4 0.8 ＜0.2
		Lysozyme 30	＜0.3 ＜0.3 ＜0.2 ＜0.2
Con A 1	＜0.3 ＜0.3 ＜0.2 ＜0.2
IL-10 CRM 30	8.6 4 7.1 0.6
		(ng/mL) CRM 1	1.1 2.5 1.7 ＜0.2
Lysozyme 30	＜0.2 ＜0.2 ＜0.3 ＜0.2
		Con A 1	0.5 0.4 ＜0.3 ＜0.2

Table 6: AlPO is arranged with prescription ₄PnPs-14 conjugate secondary with IL-12

The cytokine that immunity back two all lymph-node cell are produced

	Culture on the 6th
		Cytokine exo-antigen μ g/ml	There are not 0.2 μ g, 1.0 μ g, 5.0 μ g IL-12 IL-12 IL-12 IL-12
		IFN-γ CRM 30	9.8 86.9 58.7 62.0
(U/mL) CRM 1	0.6 78.6 62.9 36.8
		Lysozyme 30	＜0.4 ＜0.4 ＜0.3 ＜0.3
Con A 1	17.7 57.6 45.7 69.0
IL-5 CRM 30	12.5 1.4 ＜0.2 0.5
		(ng/mL) CRM 1	4.8 2.2 ＜0.2 ＜0.2
Lysozyme 30	＜0.3 ＜0.3 ＜0.2 ＜0.2
		Con A 1	1.1 ＜0.3 ＜0.2 ＜0.2
		IL-10 CRM 30	11.3 9.9 7.2 3.6
(ng/mL) CRM 1	4.4 5.5 3.3 1
		Lysozyme 30	＜0.2 ＜0.2 ＜0.2 ＜0.2
Con A 1	＜0.2 ＜0.2 ＜0.2 ＜0.2

Embodiment 3:IL-12 is to the adjuvanticity of weak immunogenicity streptococcus pneumoniae conjugate

Research design

Swiss-Webster mice (10 every group) has 100 microgram AlPO with filling a prescription or filling a prescription ₄1 microgram Pn18C conjugate immunity.Vaccine is supplemented with IL-12 (0.2,1 or 5 microgram), 100 microgram MPL  or 20 microgram QS-21 respectively ^TMCome the IL-12 of stable dilution with normal mouse serum (0.5% ultimate density), and add in all vaccines, no matter which kind of compositions is.After three weeks, mice blood drawing and with the used identical vaccine formulation booster immunization of initial immunity.Blood drawing when the 5th and 7 weeks of this research (being respectively booster immunization 2 and 4 all backs).Combining anteserum (pooled sera) Pn18C and CRM when measuring for the 5th week ₁₉₇Total IgG and IgG subclass.For measuring Pn18C respondent's ratio,, measure the IgG antibody of anti-Pn18C with ELISA with each mice serum dilution 1/500.Optical density is represented as a result.

The result

The IgG of Pn18C replys and is presented in the table 7.Have at prescription and to add IL-12 in the coupling vaccine of aluminum the IgG of Pn18C is replied no lasting effect.The IgG titre is 3 times of increases in the 5th all serum that 5 microgram IL-12 dosage cause merging, and it seems with the vaccine of 1 microgram IL-12 preparation and not induce Pn18C to reply.Lowest dose level IL-12 (0.1 microgram) and the prescription that does not contain IL-12 have AlPO ₄Vaccine-induced identical replying.With MPL /AlPO ₄Vaccine-induced the replying of ceiling rate of preparation; 7/10 mice obtains OD＞0.2, QS-21 ^TM/ AlPO ₄Singly use AlPO ₄Then opposite, each only induces 4/10 respondent.With containing IL-12 and AlPO ₄The mice of vaccine immunity (being respectively 0.1 microgram at IL-12 dosage, when 1.0 micrograms and 5 micrograms) induce 2/10,0/10 and 1/10 respondent.

In this experiment, MPL  and QS-21 ^TMCause the IgG of Pn18C to reply maximum 3 to 4 times increase.There is not AlPO ₄The time, IL-12 replys no adjuvant effect to the IgG of Pn18C.The vaccine-induced Pn18C identical with not containing the IL-12 vaccine that contains the IL-12 of 1 micrograms dose replys.It seems that contain vaccine lower or more high dose IL-12 has induced comparison according to vaccine lower replying.MPL  and QS-21 ^TMIt seems that the IgG that does not strengthen Pn18C replys.At the AlPO that do not filled a prescription ₄Vaccine in, QS-21 ^TMThat has induced ceiling rate replys thing (7/10, OD＞0.2), has induced respondents of 4/10 and all other prescriptions are maximum.

In order to determine that the IL-12 in the vaccine has activity really, has estimated the CRM in these mices ₁₉₇IgG replys.After table 8 and 9 was presented at (the 3rd week) and secondary (the 5th week) inoculation for the first time, IL-12 caused with the AlPO that do not fill a prescription ₄The mice body of vaccine immunity in, CRM ₁₉₇IgG reply and be dose dependent and increase.In addition, when the 3rd week and the 5th week, IgG1 and IgG2a titre all are the IL-12 dose dependent to be increased, and when the 5th week, IgG2b also increases.During the 5th week, IgG1 is inductive similar with the vaccine institute with 100 microgram MPL  preparation with the IgG2a titre.Opposite, the IgG2b titre that IL-12 promotes is than inductive low 20 times with MPL .These Notes of Key Datas, IgG1 and IgG2a be by different mechanism control, and IgG2a depends on the IL-12 activate mechanism and mechanism control that IgG2b is relied on by non-IL-12.These data clearly show, IL-12 can be used as the adjuvant that the IgG of proteantigen replys and works.And IgG1 and IgG2a titre increase prompting simultaneously, are that IL-12 has strengthened no AlPO at least in this model ₄PnOs18C-CRM ₁₉₇Activated TH-1 class of coupling vaccine and TH-2 class accessory cell.

When joining prescription AlPO is arranged ₄Pn18C coupling vaccine the time, the IL-12 of 0.1 micrograms dose caused for the 3rd week to CRM hardly ₁₉₇The total IgG increase of replying, but the 5th week caused 3 times increase.Yet the IL-12 of this dosage has increased the IgG2a titre time in the 5th week, the effect that promotes titre altogether with containing the vaccine-induced similar of MPL or QS-21.IL-12 does not significantly increase the IgG2b titre.As previous experiment finding, more the IL-12 of high dose causes the rapid minimizing of IgG titre, and all subclass all are affected.

The effect that table 7:IL-12 replys the IgG of PnOs18C conjugate

	The IgG titre	IgG subclass during the 5th week
			Adjuvant (μ g/ agent)	The 5th week of the 3rd week	IgG1 IgG2a IgG2b IgG3
AlPO 40.1μg IL-12+AlPO 41.0μg IL-12+AlPO 45.0μg IL-12+AlPO 4100μg MPL/AlPO 4QS-21+AlPO 4	＜100 4,608 ＜100 3,681 ＜100 130 260 13,545 233 9,027 ＜100 7,989	4,591 116 ＜100 ＜100 1,472 265 259 450 ＜100 ＜100 ＜100 ＜100 7,820 1,426 ＜100 1,481 1,522 935 877 ＜100 879 1,395 1,062 1,004
			There are not 0.1 μ g IL-12,1.0 μ g IL-12,5.0 μ g IL-12,100 μ g MPL QS-21	107 10,768 ＜100 1,808 ＜100 22,257 ＜100 460 112 1,729 ＜100 3,573	5,238 345 ＜100 144 336 105 ＜100 ＜100 12,443 671 172 773 203 ＜100 ＜100 400 524 363 189 126 2,483 101 ＜100 113

Table 8:IL-12 is to the CRM of PnOs18C conjugate after 3 weeks of immunity ₁₉₇The effect that IgG replys

Adjuvant (μ g/ agent)	IgG	IgG1	IgG2a
				AlPO 40.1μg IL-12+AlPO 41.0μg IL-12+AlPO 45.0μg IL-12+AlPO 4100μg MPL/AlPO 4QS-21 TM/ALPO 4	70,964 103,589 26,927 19,579 651,315 572,255	8,706 4,754 506 241 92,245 116,583	3,516 13,025 2,926 2,665 79,508 38,419
There are not 0.1 μ g IL-12,1.0 μ g IL-12,5.0 μ g IL-12,100 μ g MPL /TEM QS-21 TM	7,630 32,403 60,987 128,697 462,289 556,440	452 3,475 4,615 10,498 40,010 111,533	1,023 3,713 5,951 10,686 24,979 53,799

Table 9:IL-12 is to the CRM of PnOs18C conjugate after 5 weeks of immunity ₁₉₇The effect that IgG replys (adds

After strong two weeks of immunity)

Adjuvant (μ g/ agent)	IgG	IgG1	IgG2a	IgG2b
					AlPO 40.1μg IL-12+AlPO 41.0μg IL-12+AlPO 45.0μg IL-12+AlPO 4100μg MPL/AlPO 4QS-21 TM/ALPO 4	634,631 2,225,000 105,765 71,618 4,384,000 ＞5,000,000	102,974 88,204 8,018 1,582 637,655 ＞1,000,000	45,955 317,083 12,598 13,806 371,652 873,674	8,812 16,869 1,096 744 111,646 144,132
There are not 0.1 μ g IL-12,1.0 μ g IL-12,5.0 μ g IL-12,100 μ g MPL /TEM QS-21 TM	62,341 296,791 1,026,060 1,367,771 4,173,765 ＞5,000,000	12,783 52,288 101,381 74,494 264,691 1,303,508	3,655 23,741 96,024 108,815 266,160 445,712	1,679 7,069 11,862 14,258 303,662 131,991

Embodiment 4:IL-12 to Swiss Webster mice at containing PnPs6B-CRM ₁₉₇And PnPs-14-CRM ₁₉₇The effect that the IgG of bivalent vaccine replys

Research design

Swiss Webster mice contains every dose 0.1 microgram PnPs6B-CRM at the 0th and the 3rd Zhou Shiyong ₁₉₇Carbohydrate conjugates (and CRM ₁₉₇The conjugate of the pneumococcal capsular polysaccharide of covalently bound serotype 6B) and every dose 0.1 microgram PnPs14-CRM ₁₉₇The subcutaneous immunity of the vaccine of carbohydrate conjugates.This vaccine and 0,8,40, or 200 nanogram IL-12 use together, or separately or and 100 microgram aluminum (AlPO ₄) together.Add normal mouse serum (0.25%) as the carrier protein of stablizing low concentration IL-12.The mice matched group has the vaccine immunity of 100 micrograms, one phosphoric acid fat A (MPL ) with prescription.Mice was drawn blood when the 3rd week (primary response) and the 5th week (second set response).IgG antibody with anti-Pn6B and Pn14 capsular polysaccharide in the ELISA mensuration serum.

The result

To replying of PnPs6B conjugate

Table 10 has illustrated that combining anteserum replys at the IgG of bivalent vaccine Pn6B component.If vaccine does not contain adjuvant or only uses AlPO ₄Preparation almost or fully detected less than replying at Pn6B when the 3rd week.Single the highest postvaccinal titre it seems be by contain MPL  or 8-40 nanogram IL-12 and aluminum altogether-prescription vaccine-induced.Yet these titres are low, that is, be lower than 3,000.After the replying of the 5th week is presented at booster immunization, with being added with AlPO ₄40 nanogram IL-12, or the vaccine-induced the highest IgG titre of MPL  preparation to Pn6B.When not having aluminum, the IL-12 of the dosage range of 8 to 200 nanograms does not strengthen the IgG titre to Pn6B.

In the 5th when week, replied the IgG subclass of Pn6B and be presented in the table 10.The titre of each IgG subclass and with the vaccine that does not contain adjuvant or use AlPO ₄Vaccine (no IL-12) mice immunized of preparation is similar.And, without AlPO ₄, do not change replying of IgG subclass with the vaccine of 8-200 nanogram IL-12 preparation.Opposite, the IL-12 of these dosage and AlPO ₄During associating, cause IgG1 and the IgG2a titre of Pn6B are significantly increased.These titres are similar with the vaccine acquisition of preparing with MPL .IL-12 has also increased prescription AlPO ₄Inductive IgG2b of vaccine institute and IgG3 titre; Yet these titres be it seems inductive more much lower than the vaccine institute with MPL  preparation.

In order to determine coupling IL-12 and AlPO ₄Whether the increase that obtains has statistical significance, has measured the IgG titre of the Pn6B of selected group of each mice.Its geometric mean titer (GMT) is presented in the table 11.Data show, the vaccine of no adjuvant prescription or singly use AlPO ₄The group of the vaccine immunity of preparation has the similar GMT at Pn6B.AlPO ₄Add that titre that the vaccine of 40 nanogram IL-12 preparation causes is than not containing 29 times of the inductive titre increases of Adjuvanted vaccines institute.When checking with ANOVA, all data (use JMP software analysis variance; SAS Institute, Cary, North Carolina), do not find statistically-significant difference.Compare each subgroup data, ANOVA shows and does not contain the vaccine of adjuvant, and uses AlPO ₄With the IL-12 of various dose preparation inductive the 5th week of vaccine reply comparison the time, statistically-significant difference is arranged.Wherein, use AlPO ₄Preparation, the vaccine that is added with 40 nanogram IL-12 is than the vaccine-induced obvious much higher Pn6B antibody titer of no adjuvant prescription.Improved immunogenic another index as this prescription, 7 only more than or equal to 50,000 Pn6B titre in 10 mices of this group, and without adjuvant or singly use AlPO ₄In every group of the mice of the conjugate inoculation of preparation 1 or 2 only in this way high titres are only arranged.

Table 10:IL-12 is to using bivalence PnPs6B/14 streptococcus pneumoniae sugar coupling vaccine immunity

The effect that mouse anti PnPs6B IgG replys

		Pn6B IgG titre *		Pn6B IgG subclass is replied
								The 5th week *
				Group	Adjuvant	The 3rd week	The 5th week					IgG1	IgG2a	IgG2b	IgG3
				P344	200ng IL-12+AlPO 4	630	161,867					19,474	22,664	2,954	7,333
P345	40ng IL-12+AlPO 4	2,609	429,006					61,364	24,172	4,117	9,830
				P346	8ng IL-12+AlPO 4	1,977	284,206					46,734	32,859	3,195	8,764
P347	AlPO 4(no IL-12)	279	120,999					8,767	2,199	688	301
P348	200ng IL-12	＜100	22,401					6,816	3,147	501	1,104
				P349	40ng IL-12	164	23,343					5,056	2,532	879	292
P350	8ng IL-12	642	81,748					17,703	3,573	5,151	1,786
				P351	Do not have	＜100	20,153					3,061	1,506	364	1,220
				P352	100μg MPL	2,872	840,513					84,660	30,813	43,505	25,749

* combining anteserum titre

Table 11: the Pn6B IgG titre of each mice

Group	P344	P345	P345	P347	P350	P351	P352
								Mus #	AlPO 4+ 200ng IL-12	AlPO 4+ 40ng IL-12	AlPO 4+ 8ng IL-12	AlPO 4(no IL-12)	8ng IL-12	No adjuvant	100μg MPL
1	1,957	596,886	13,457	306,012	833,148	3,544	7,556
								2	2,498	1,205	1,000,000	3,653	9,431	326	1,359,470
3	100	9,453	1,422	8,708	3,163	1,136	81
								4	11,830	70,278	168,481	41,395	109,399	24,140	583,097
5	1,823	157,427	16,454	677,407	252	50,785	86,656
								6	6,114	90,843	989	9,089	150,245	228	284
7	279	49,182	372,709	17,164	112	1,351	1,000,000
								8	756,503	408,348	425	7,329	393	36,805	473,652
9	1,000,000	1,000,000	667,988	100	13,622	22,817	927,213
								10	177	1,052,210	6,206	245	182,629	851	-
GMT	4,347	103,743	22,580	9,735	10,120	3,589	55,799
								The Mus number of titre 〉=50,000	2	7	4	2	4	1	6

Statistics is (ANOVA relatively; α=0.05)

AlPO ₄+ 40 nanogram IL-12 compare with no adjuvant: marked difference is arranged

To replying of PnPs14 conjugate

The IgG of vaccine PnPs14 component replied be shown in table 12.Data show IL-12 in 8-40 nanogram dosage range, independent or and AlPO ₄During prescription, behind first or secondary inoculation, do not strengthen replying to PnPs14.And subclass the analysis showed that IL-12 does not strengthen the titre of IgG2a when filling a prescription with IL-12.In this research, MPL  is not observed in the research of front, and PnPs14 has been replied adjuvant effect, and is like this when analyzing combining anteserum at least.In order to obtain every group of notion of replying degree of variation, analyzed the Pn14 IgG antibody of each serum with 1/300 dilution factor.The results are shown in table 13, prompting has an amount of replying on a large scale in every group, that is, the coefficient of variation (CV) scope from 0.229 to 0.587, exception be that its CV is 0.051 with the group of the vaccine immunity that contains MPL .Therefore, it seems MPL , rather than IL-12, may reply adjuvant effect to the IgG of Pn14, and reduce the variation between Mus and the Mus.

Table 12:IL-12 is to using bivalence PnPs6B/14 streptococcus pneumoniae sugar coupling vaccine immunity

The effect that mouse anti Pn14 IgG subclass is replied

	PnPs14 Ig *Titre *		PnPs14 IgG subclass *In the 5th week
							Adjuvant	The 3rd week	The 5th week	IgG1	IgG2a	IgG2b	IgG3
200ng IL-12+ AlPO 4	2,170	58,657	6,880	8,996	1,945	5,995
							40ng IL-12+ AlPO 4	1,641	53,557	8,646	3,003	3,684	2,745
8ng IL-l2+ AlPO 4	2,181	85,173	10,094	11,346	5,328	2,560
							AlPO 4(no IL-12)	2,102	201,082	54,989	4,030	6,402	3,745
							200ng IL-12	849	18,293	5,769	1,582	536	799
40ng IL-12	1,544	11,442	4,350	714	514	455
							8ng IL-12	113	12,169	5,286	354	245	330
Do not have	509	22,601	6,080	808	618	694
100μg MPL	18,616	77,106	15,745	4,275	10,205	3,916

* combining anteserum titre

Table 13: each mice is to Pn6B/Pn14 bivalence streptococcus pneumoniae

Replying of coupling vaccine Pn14 component ^*

Adjuvant	O.D. scope	O.D. meansigma methods	Standard deviation	Variable coefficient
					AlPO 4+200ng IL-12	0.034-0.990	0.788	0.318	0.404
AlPO 4+40ng IL-12	0.457-0.948	0.771	0.176	0.229
					AlPO 4+8ng IL-12	0.023-0.974	0.707	0.281	0.397
AlPO 4(no IL-12)	0.328-0.974	0.770	0.220	0.285
					8ng IL-12 (aluminum-free)	0.009-0.812	0.505	0.292	0.587
No adjuvant	0.030-0.876	0.614	0.343	0.558
					100μg MPL	0.791-0.918	0.863	0.044	0.051

^*Under 1/300 dilution factor, measure the anti-Pn14 IgG antibody of each serum with ELISA

Embodiment 5: when existing or not having aluminum IL-12 to mice at unit price PnPs14-CRM ₁₉₇The comparison of coupling vaccine immune response effect

Research design

BALB/c mouse (8 every group) contains or does not contain 100 microgram AlPO at the 0th Zhou Shiyong ₄1 microgram PnPs-14-CRM of preparation ₁₉₇Conjugate does not contain IL-12 or contains 8,40,200,1,000 or 5,000 nanogram IL-12 and does subcutaneous immunity.Add normal mouse serum (0.25%) as the carrier protein of stablizing low concentration IL-12.In the 1st week, preparation lymph-node cell suspension from the mice of every group of half, and the generation of the assessment exo-antigen specific cell factor.Collect their spleen and weigh.When the 3rd week, the blood-letting of residue mice, and immune again with the used same vaccine formulation of initial inoculation.When the 5th week, twice mice immunized blood-letting, its spleen of weighing is assessed the cytokine that their splenocytes produce.PnPs14 and CRM ₁₉₇IgG and IgG subclass titre detect with combining anteserum.When measuring with the serum of single mice, the result expresses with geometric mean titer (GMT).

The result

IL-12 is to the effect of one week of immunity back spleen weight

First immunisation is being accepted no AlPO after one week ₄5,000 nanogram IL-12, but be not the mice of lower IL-12 dosage, accept not contain the mouse spleen weight significantly higher (table 14) of aluminum and IL-12 vaccine than those.When preparing, contain AlPO with 40 to 5000 nanogram IL-12 ₄Vaccine-induced higher spleen weight.Pair-wise comparison shows that, with 200 or 1000 nanogram IL-12, adds AlPO ₄The vaccine of preparation is used the IL-12 of same dose than those, but does not contain AlPO ₄Vaccine-induced higher spleen weight.In a word, data show and use AlPO ₄Preparation has strengthened the biologic activity of this cytokine greatly with IL-12, that is, strengthen IL-12 and caused the ability that spleen weight increases after one week of inoculation.

The effect that IL-12 replys at the IgG at PnPs14

At first, measured the anti-PnPs14 IgG antibody (table 15) of combining anteserum.With containing AlPO ₄With observe adjuvant effect the most clearly behind the vaccine initial immunity of 8 to 40 nanogram IL-12.This combination is with respect to using no AlPO ₄Mice with the vaccine immunity of no IL-12 preparation causes the increase of 17 to 21 times of IgG titres.AlPO ₄Higher replying when causing using respectively than the two with the combination of IL-12; AlPO ₄The increase of 4 times of IgG titres and 5 times when the IL-12 of itself and 40 nanogram dosage caused for 3 weeks respectively.To containing AlPO ₄The analysis of each serum (table 16) of vaccine immune mouse shows 8 nanogram IL-12 than only using AlPO ₄The vaccine of assisting a ruler in governing a country is induced high 5 times PnPs14 IgG titre behind primary vaccination.This titre difference has statistical significance.IL-12 more high dose does not strengthen and replys.The IL-12 of 1,000 to 5,000 nanogram dosage causes the remarkable decline of PnPs14 IgG titre.After the immunity for the second time, have only the IL-12 of 40 nanogram dosage to cause based on AlPO ₄The remarkable rising (3 times) of vaccine-induced PnPs14 antibody titer.

This combining anteserum Notes of Key Data, AlPO ₄With 8-40 nanogram IL-12 be combined in initial immunity after increased the IgG1 titre.After twice inoculation, IL-12 does not strengthen with no AlPO ₄Anti-PnPs14 IgG1 titre in the conjugate mice immunized is as indicated in analyzing combining anteserum (table 15) and single serum (table 17).And, with containing AlPO ₄The mice of vaccine immunity in, add 8 to 200 nanogram IL-12 and do not cause higher IgG1 titre (table 17) after 2 inoculations.

The best effect of IL-12 is that the IgG2a of PnPs14 replys when significantly having increased by the 5th week.This is containing AlPO ₄Or without AlPO ₄The preparation vaccine immunity the time all can see (table 18).There is not AlPO ₄The time, using 8-1, the GMT that 000 nanogram IL-12 can obtain IgG2a has the increase (14 to 42 times) of statistical significance.Similar, 8-1,000 nanogram IL-12 have strengthened and have contained AlPO ₄The ability of vaccine-induced IgG2a antibody is though higher from statistics by the inductive titre of the IL-12 of 8 and 40 nanogram dosage in this research.In a word, the highest IgG2a titre is by AlPO ₄Inductive with the conjugate of 40 nanogram IL-12 preparation.This and by 40 nanogram IL-12 but there is not AlPO ₄The time inductive IgG2a titre significantly different, show that again aluminum has strengthened the adjuvanticity of IL-12.

The IgG2b and the IgG3 titre (table 15) of combining anteserum have only been measured.The IL-12 dosage of 8 to 1,000 nanogram scopes, when and AlPO ₄Fill a prescription altogether, but be not no AlPO ₄The time, behind first and secondary immunity, significantly promote the increase of IgG3 titre.Do not see the lasting effect of IL-12 to the IgG2b titre.

IL-12 is at CRM ₁₉₇The IgG effect of replying

Assessed CRM ₁₉₇IgG reply, to understand IL-12 to the protein carrier of conjugate and the effect between the polysaccharide part whether variant (table 19).At no AlPO ₄The time, 40 nanogram IL-12 it seems the anti-CRM of medium increase behind secondary inoculation ₁₉₇The IgG titre.Yet, use AlPO at the same time ₄During with 8-40 nanogram IL-12 preparation vaccine, obtained the highest anti-CRM ₁₉₇The IgG titre.Find 40 nanogram IL-12 and AlPO ₄The increase of 6 times of IgG titres and 17 times when causing for the 5th week respectively, but the two has increased by 147 times when making up, and shows IL-12 and AlPO ₄Prescription has the highest adjuvanticity altogether.And IL-12 strengthens CRM ₁₉₇IgG1 reply, no matter whether vaccine use AlPO ₄Preparation (table 19 and 20).With containing AlPO ₄Behind the vaccine immunity, anti-CRM when IL-12 has significantly increased by the 5th week ₁₉₇IgG2a titre (table 19).Show that again it is 40 nanograms that best IL-12 dosage be it seems.Obvious can the increasing of this cytokine contains AlPO ₄Vaccine-induced IgG2b titre.

IL-12 is to CRM ₁₉₇The effect of the cytokine scattergram that specific T-cells produces

The cytokine that the splenocyte that secondary inoculation two week backs (the 5th week) are obtained produces has disclosed IL-12 and has acted on the cell that causes generation IFN-γ and IL-5., when at external use CRM ₁₉₇When stimulating, no AlPO ₄But produced the IL-5 of detection level with the splenocyte of no IL-12 mice immunized, but do not produced IFN-γ (table 21).With the vaccine of IL-12 preparation obvious to producing inducing of IL-5 cell, when its peak activity appears at 40 nanogram IL-12.More the IL-12 of high dose causes the decline that IL-5 produces, and does not in fact produce cytokine with the mice of the coupling vaccine immunity that contains 1,000 to 5,000 nanogram IL-12.Compellent, the generation of IFN-γ only detects in the mouse boosting cell of the vaccine immunity of preparing with 5,000 nanogram IL-12.As vaccine AlPO ₄Preparation has caused exciting the cell that produces a large amount of IFN-γ when adding 8 nanogram IL-12, and when not having IL-12, the IL-5 that only detects antigenic specificity produces.It seems that the maximum generation that causes IFN-γ is the IL-12 of 40 to 100 nanograms.Adding 5,000 nanogram IL-12 have then eliminated vaccine and have caused the ability that produces the IL-5 cell.

Table 14: with or need not 100 microgram AlPO ₄With shown in the IL-12 of dosage

1 microgram PnPs-14-CRM of preparation ₁₉₇Conjugate is after the subcutaneous immune week

The spleen weight of BALb/c mice

Adjuvant prescription			Spleen weight (g)
					Group number	IL-12(ng)	AlPO 4	Meansigma methods	Standard deviation
P641	0	-	0.179	0.0225
					P642	8	-	0.148	0.0112
P643	40	-	0.162	0.0202
					P644	200	-	0.175	0.0431
P645	1,000	-	0.196	0.0068
					P646	5,000	-	0.357	0.0247
					P647	0	+	0.151	0.0158
P648	8	+	0.151	0.0332
					P649	40	+	0.217	0.0596
P650	200	+	0.290	0.0226
					P651	1,000	+	0.277	0.0919
P652	5,000	+	0.305	0.0545

Statistics is (ANOVA relatively; α=0.05)

P642, P643, P644, P645 are to P641: not remarkable

P646 is to P641: significantly

P648 is to P647: not remarkable

P649, P650, P651, P652 are to P647: significantly

P641 is to P647: not remarkable

P644 is to P650: significantly

P642 is to P648: not remarkable

P645 is to P651: significantly

P643 is to P649: not remarkable

P646 is to P652: not remarkable

Table 15: with IL-12 and AlPO ₄The PnPs-14-CRM of preparation ₁₉₇Conjugate

The anti-PnPs14 IgG of the BALb/c mice of immunity replys

		The PnPs14IgG titre of combining anteserum
												Adjuvant prescription		IgG		IgG1		IgG2a		IgG2b		IgG3
IL-12(ng)	AlPO 4	The 3rd week	The 5th week	The 3rd week	The 5th week	The 3rd week	The 5th week	The 3rd week	The 5th week	The 3rd week	The 5th week
												0	-	1,691	24,498	479	9,967	139	492	＜100	＜100	295	1,516
8	-	4,679	32,966	841	9,860	377	1,902	108	609	390	1,354
												40	-	6,484	50,096	1,235	17,631	207	1,209	58	724	1,633	4,017
200	-	5,330	51,240	385	7,568	715	3,748	290	1,397	1,091	4,515
												1,000	-	6,347	69,673	1,286	12,814	859	6,532	124	＜100	782	6,208
5,000	-	1,131	19,621	229	3,598	126	1,392	＜100	＜100	635	3,616
0	+	7,825	103,092	1,714	38,147	195	1,535	617	3,973	447	2,963
												8	+	29,506	195,069	7,444	58,046	1,207	6,697	693	4,843	5,669	25,407
40	+	35,567	295,361	4,945	46,030	2,883	17,267	1,371	9,911	5,797	22,602
												200	+	10,177	190,701	1,777	41,800	626	9,816	＜100	1,479	3,443	23,648
1,000	+	2,422	245,683	90	31,373	167	13,847	＜100	722	1,173	34,039
												5,000	+	1,304	35,333	91	5,228	＜100	1,429	＜100	＜100	772	8,065

Table 16: use AlPO ₄The PnPs-14-CRM of preparation ₁₉₇The conjugate immunity

The effect that IL-12 antagonism PnPs14 IgG replys in the mice

			PnPs14 IgG GMT (increase multiple)
							Group	IL-12(ng)	AlPO 4	The 3rd week		The 5th week
P647	0	+	3,037		27,027
							P648	8	+	16,68	(5.5)	55,855	(2.1)
P649	40	+	6,667	(2.2)	88,271	(3.4)
							P650	200	+	2,333	(0.8)	57,076	(2.1)
P651	1,000	+	611	(0.2)	30,886	(1.1)
							P652	5,000	+	617	(0.2)	10,989	(0.4)

Statistics is (ANOVA relatively; α=0.05)

The 3rd all titres

P648 is to P647: significantly

P651 is to P647: significantly

P649, P650, P651 are to P647: not remarkable

The 5th all titres

P649 is to P647: significantly

P648, P650, P651 are to P647: not remarkable

Table 17: with or without AlPO ₄With various dose IL-12 preparation

PnPs-14-CRM ₁₉₇Twice mice of coupling vaccine immunity

PnPs14 IgG1 titre

Adjuvant prescription
				Group number	IL-12(ng)	AlPO 4	IgG1 GMT (geometric mean titer)
P641	0	-	9,492
				P642	8	-	5,964
P643	40	-	14,028
				P644	200	-	4,628
P645	1,000	-	5,815
				P646	5,000	-	1,757
P647	0	+	15,283
				P648	8	+	35,730
P649	40	+	31,855
				P650	200	+	34,166
P651	1,000	+	15,347
				P652	5,000	+	4,022

Statistics is (ANOVA relatively; α=0.05)

P642, P643, P644, P645, P646, P647, P651 are to P641: not remarkable

P648, P649, P650 are to P641: significantly

P648, P649, P650, P651 are to P647: not remarkable

P652 is to P647: significantly

Table 18: with or without AlPO ₄With various dose IL-12 preparation

PnPs-14-CRM ₁₉₇Twice mice of coupling vaccine immunity

PnPs14 IgG2a titre

Group number	IL-12(ng)	AlPO 4	The IgG2a GMT in the 5th week (increases multiple *)
					P641	0	-	97
P642	8	-	1,418	(14.6)
					P643	40	-	1,509	(15.6)
P644	200	-	2,228	(23.0)
					P645	1,000	-	4,126	(42.5)
P646	5,000	-	289	(3.0)
P647	0	+	806
					P648	8	+	6,841	(8.5)
P649	40	+	13,252	(16.4)
					P650	200	+	4,740	(5.9)
P651	1,000	+	3,291	(4.1)
					P652	5,000	+	368	(0.5)

* with respect to the control vaccine that does not contain IL-12

Statistics is (ANOVA relatively; α=0.05)

P642, P643, P644, P645 are to P641: significantly

P646 is to P641: not remarkable

P648, P649 are to P647: significantly

P650, P651, P652 are to P647: not remarkable

P643 is to P649: significantly

Table 19: with IL-12 and AlPO ₄The PnPs-14-CRM of preparation ₁₉₇

Resisting of the BALb/c mice of conjugate immunity

CRM ₁₉₇IgG replys

Adjuvant	CRM 197IgG titre (combining anteserum)	IgG subclass (combining anteserum) during the 5th week
			IL-12(ng) AlPO 4	The 5th week of the 3rd week	IgG1 IgG2a IgG2b
0 - 8 - 40 - 200 - 1,000 - 5,000 - 0 + 8 + 40 + 200 + 1,000 + 5,000 +	3,843 8,965 2,456 14,389 3,200 53,758 1,666 13,419 4,999 3,663 2,841 3,641 4,870 153,075 89,558 1,515,87 19,566 1,319,10 6,884 315,071 7,292 545,827 7,213 7,029	703 1,269 792 4,674 ＜100 ＜100 14,073 3,403 ＜100 1,803 2,044 ＜100 ＜100 506 ＜100 ＜100 ＜100 ＜100 55,922 1,796 407 377,82 85,972 10,972 147,03 199,29 7,206 48,852 36,807 3,865 126,72 44,190 4,127 1,041 769 ＜100

Table 20: with IL-12 and AlPO ₄The PnPs-14-CRM of preparation ₁₉₇

The BALb/c mice of conjugate immunity

CRM ₁₉₇The IgG1 titre

Group number	IL-12(ng)	AlPO 4	IgG1 GMT	Increase multiple
					P641	0	-	317	-
P642	8	-	1,136	3.6
					P643	40	-	9,141	28.8
P644	200	-	1,627	5.1
					P645	1,000	-	100	0.3
P646	5,000	-	174	0.6
P647	0	+	22,061	-
					P648	8	+	119,130	5.4
P649	40	+	73,226	3.3
					P650	200	+	14,391	0.7
P651	1,000	+	33,468	1.5
					P652	5,000	+	317	0.01

Statistics is (ANOVA relatively; α=0.05)

P643, P644 are to P641: significantly

P642, P645, P646 are to P641: not remarkable

P648, P649, P650, P651 are to P647: not remarkable

P642 is to P648, and P643 is to P649, and P644 is to P650, and P645 is to P651: significantly

Table 21: have or do not have AlPO ₄The time, prepare with IL-12

PnPs14-CRM ₁₉₇Twice mice of immunity

The cytokine that splenocyte produces

	Irritation cell is used		Without AlPO 4The IL-12 dosage (ng) of the vaccine of prescription						Use AlPO 4IL-12 dosage (ng) in the vaccine of prescription
															Cytokine	Antigen	μg/mL	0	8	40	200	1.000	5,000	0	8	40	200	1.000	5,000
															IFN-γ	CRM 197	30	＜0.06	＜0.06	0.4	0.3	0.4	6.9	0.1	20.6	32.1	30.2	29.4	21.0
(U/mL)	CRM 197	10	＜0.06	＜0.06	0.3	0.2	0.3	3.4	0.1	16.5	31.7	30.2	27.6	21.0
																CRM 197	3	＜0.06	＜0.06	＜0.06	＜0.06	0.2	1.4	＜0.03	15.7	30.9	28.7	26.7	20.1
	CRM 197	1	＜0.06	＜0.06	＜0.06	＜0.06	0.2	0.4	＜0.03	12.8	30.9	28.2	27.9	18.0
																CRM 197	0.3	＜0.06	＜0.06	＜0.06	＜0.06	0.1	0.1	＜0.03	6.3	27.5	26.2	26.5	6.5
	Lysozyme	30	＜0.06	＜0.06	＜0.06	＜0.06	＜0.03	＜0.03	＜0.03	＜0.0	＜0.0	＜0.0	＜0.02	＜0.02
																Con A	1	11.9	15.4	15.8	15.9	20.6	21.0	21.1	17.0	20.8	13.7	23.4	17.7
	Culture medium	-	＜0.06	＜0.06	＜0.06	＜0.06	＜0.03	＜0.03	＜0.03	＜0.0	＜0.0	＜0.0	＜0.02	＜0.02
															IL-5	CRM 197	30	370	480	2560	960	60	70	2010	1440	5280	1640	880	＜10
(pg/mL)	CRM 197	10	150	300	1110	260	＜24	＜24	1220	920	4590	410	430	＜10
																CRM 197	3	30	90	910	200	＜24	＜24	1190	690	2830	1030	200	＜10
	CRM 197	1	＜4	50	200	40	＜24	＜24	880	400	2150	520	140	＜10
																CRM 197	0.3	＜4	30	70	＜4	＜24	＜24	670	180	440	270	90	＜10
	Lysozyme	30	＜4	＜4	＜4	＜4	＜24	＜24	＜24	＜24	＜10	＜10	＜10	＜10
																Con A	1	＜4	＜4	＜4	＜4	＜24	＜24	60	80	40	＜10	＜10	＜10
	Culture medium	-	＜4	＜4	＜4	＜4	＜24	＜24	＜24	＜24	＜10	＜10	＜10	＜10

Embodiment 6:IL-12/AlPO ₄Effect to the humoral response of the streptococcus pneumoniae of nine valencys sugar coupling vaccine

Research design

IL-12 is extended to by serotype 1,4,5 6B, 9V, 14,18C, the nine valency vaccines of 19F and 23F to the effect assessment that streptococcus pneumoniae sugar coupling vaccine IgG replys.Swiss Webster mice 0 and 3 whens week with 0.1,1, or 5 microgram vaccines (carbohydrate weight) immunity.Separately, and AlPO ₄(100 microgram) or and is mixed with the AlPO of 50,200 or 1,000 nanogram IL-12 together ₄Use vaccine together.In vaccine, do not comprise normal mouse serum.To serotype 4,6B, 9V, 14,18C and to carrier protein CRM ₁₉₇IgG reply in the 5th when week (that is, two weeks behind the booster immunization) and assess with ELISA.

The result

In the 5th when week, is to CRM ₁₉₇Reply

Containing AlPO ₄Vaccine in add IL-12 and cause CRM ₁₉₇IgG2a and IgG2b antibody be dose dependent and increase.These are all visible (table 22) in all dosage of tested conjugate.Accept 50 nanograms, be increasing of obviously visible IgG2a titre in the mice of 1,000 this cytokine of nanogram to the maximum.This and other research (maximum IgG2a titre is to obtain when adding this cytokine of 40-100 nanogram in based on the vaccine of aluminum, and more the IL-12 of high dose causes the immunne response that weakens) antithesis.The reason of this difference of dose response is unclear between two researchs.This may be relevant with the difference of vaccine, that is multivalence is to unit price, or the normal mouse serum that vaccine comprises in the research in front is used for stablizing the low concentration cytokine has been left in the basket.

To replying of pneumococal polysaccharide

Use AlPO ₄Prepare the IgG that nine valency vaccines can strengthen several serotypes (comprising PnPs4, PnPs6B, PnPs9V and PnPs14) and reply (table 24-27), when particularly using the coupling vaccine (0.1 μ g) of lowest dose level.Add IL-12 and it seems that the IgG that does not further strengthen these serotypes replys.Yet, PnPs18C is replied, containing AlPO ₄5 microgram vaccines in add 50 nanograms or 1000 nanogram IL-12 cause the geometric average IgG titre of this serotype higher, and have surpass 10000 PnPs18CIgG titre mice than higher (table 23).Do not estimate PnPs1,5,19F and 23F reply.

Containing AlPO ₄Nine valency vaccines in add IL-12 and cause PnPs4, PnPs6B, the IgG2a titre of PnPs9V and PnPs14 is dose dependent increases (table 24-27).Usually, the increase of IgG2a and CRM with the highest titre of 1,000 nanogram IL-12 acquisition ₁₉₇Reply quite.Opposite with the experiment of adopting unit price PnPs14 conjugate or bivalence PnPs6B/PnPs14 vaccine, 50 nanogram IL-12 dosage are replied seldom the IgG2a of these serotypes or to no effect.Exception be that IgG2a to PnPs14 replys because the IL-12 of this dosage shows reply (table 27) that can strengthen this serotype.

In a word, this studies show that IL-12 can strengthen replying at the complement associativity IgG2a antibody subclass of the multiple streptococcus pneumoniae serotype of polyvalent vaccine.

Table 22: use AlPO ₄The nine valency streptococcus pneumoniae sugar coupling of preparation

IL-12 is to CRM in the mice of vaccine immunity ₁₉₇The effect of replying

Vaccine formulation	CRM during the 5th week 197Reply
		Conjugate dosage IL-12 AlPO4 (μ g) is (μ g) (ng)	IgG IgG1 IgG2a IgG2b IgG3
5.0 do not have 5.0 0 100 5.0 50 100 5.0 200 100 5.0 1,000 100	391,021 138,784 1,687 3,277 102 1,419,910 609,704 4,328 11,349 181 3,226,410 896,621 27,736 18,086 728 2,991,990 584,991 87,732 28,855 2,937 16,224,900 906,192 303,656 87,726 3,023
		1.0 do not have 1.0 0 100 1.0 50 100 1.0 200 100 1.0 1,000 100	545,046 162,757 1,178 9,213 358 956,584 338,751 1,284 4,118 306 1,936,170 370,961 6,677 31,982 931 4,788,500 660,082 187,034 36,785 1,065 12,404,500 644,151 533,065 69,185 1,176
0.1 do not have 0.1 0 100 0.1 50 100 0.1 200 100 0.1 1,000 100	15,215 3,800 ＜100 ＜100 ＜100 561,952 157,362 1,437 7,744 ＜100 807,363 141,670 16,064 26,978 2,092 1,560,380 313,263 38,686 51,737 306 2,296,310 202,111 112,158 36,958 1,054

The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of dosage shown in mice was used when 0 and 3 weeks.Conjugate is independent, or uses AlPO ₄(100 microgram), or use AlPO ₄Add the IL-12 preparation.Analyzed the CRM of the serum of the 5th all blood-letting ₁₉₇IgG antibody.

Table 23: use AlPO ₄5 micrograms, the nine valency streptococcus pneumoniae sugar of preparation

IL-12 is right in the mice of coupling vaccine immunity

The effect that PnPs18C replys

Vaccine formulation			PnPs18C replys
					Conjugate dosage (microgram)	IL-12 (nanogram)	AlPO 4(microgram)	IgG titre (GMT)	The Mus number of titre＞10,000
5	0	0	2,933	1
					5	50	100	23,725	4
5	1,000	100	48,375	5

The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of dosage shown in mice was used when 0 and 3 weeks.Conjugate is independent, or uses AlPO ₄(100 microgram) or use AlPO ₄Add the IL-12 preparation.Analyzed the PnPs18C IgG antibody of each Mus serum of the 5th all blood-letting.

Table 24: use AlPO ₄The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of preparation

The effect that IL-12 replys PnPs4 in the mice

Vaccine formulation	PnPs4 during the 5th week replys
		Conjugate dosage IL-12 AlPO 4(μg) (ng) (μg)	IgG IgG1 IgG2a IgG2b IgG3
5.0 do not have 5.0 0 100 5.0 50 100 5.0 200 100 5.0 1,000 100	55,068 13,731 ＜500 ＜500 ＜500 233,008 55,620 ＜500 1,157 990 285,806 64,493 1,050 1,329 2,634 203,236 56,654 1,789 692 2,693 371,329 35,778 4,048 1,080 3,820
		1.0 do not have 1.0 0 100 1.0 50 100 1.0 200 100 1.0 1,000 100	77,714 9,070 ＜500 608 ＜500 141,371 14,829 ＜500 ＜500 542 97,999 14,336 449 814 1,034 137,674 17,380 752 569 816 214,739 25,056 4,685 1,260 4,055
0.1 do not have 0.1 0 100 0.1 50 100 0.1 200 100 0.1 1,000 100	4,726 706 ＜500 ＜500 ＜500 79,686 12,071 ＜500 869 ＜500 70,917 9,649 1,032 1,389 ＜500 46,503 7,799 885 1,056 572 87,762 6,788 1,725 ＜500 1,682

The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of dosage shown in mice was used when 0 and 3 weeks.Conjugate is independent, or uses AlPO ₄(100 microgram) or use AlPO ₄Add the IL-12 preparation.Analyzed the PnPs4 IgG antibody of the serum of the 5th all blood-letting.

Table 25: use AlPO ₄The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of preparation

The effect that IL-12 replys PnPs6B in the mice

The PnPs6B of vaccine formulation during the 5th week replys
	Conjugate dosage IL-12 AlPO 4Total IgG 1 IgG2a IgG2b IgG3 (μ g) is (μ g) IgG (ng)
5.0 do not have 64,734 20,221＜100 195 325 5.0 0 100 103,686 39,061 138 2,498 1,801 5.0 50 100 487,798 127,753 916 3,200 13,758 5.0 200 100 214,743 59,979 924 959 6,459 5.0 1,000 100 427,514 94,478 4,426 2,552 13,142
	1.0 do not have 165,588 37,646＜100 2,047 2,337 1.0 0 100 730,920 133,441 990 2,770 7,468 1.0 50 100 428,549 77,124 838 3,755 12,931 1.0 200 100 164,820 29,685 316 662 4,703 1.0 1,000 100 401,513 51,132 11,442 2,735 31,613
0.1 do not have 4,787 1,034＜100＜100＜100 0.1 0 100 370,177 71,287 603 11,372 5,712 0.1 50 100 137,091 25,447 1,029 3,346 3,411 0.1 200 100 128,428 31,634 434 2,698 1,891 0.1 1,000 100 524,385 67,301 9,611 11,587 8,711

The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of dosage shown in mice was used when 0 and 3 weeks.Conjugate is independent, or uses AlPO ₄(100 microgram) or with adding AlPO ₄Add the IL-12 preparation.Analyzed the PnPs6B IgG antibody of the serum of the 5th all blood-letting.

Table 26: use AlPO ₄The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of preparation

The effect that IL-12 replys PnPs9V in the mice

The PnPs9V of vaccine formulation during the 5th week replys
	Conjugate dosage IL-12 AlPO 4Total IgG 1 IgG2a IgG2b IgG3 (μ g) is (μ g) IgG (ng)
5.0 do not have 36,831 15,568 306 250 317 5.0 0 100 78,614 37,544 359 667 286 5.0 50 100 117,345 61,031 1,073 834 2,089 5.0 200 100 134,333 35,031 2,973 748 2,594 5.0 1,000 100 197,407 40,368 15,353 2,147 1,945
	1.0 do not have 81,932 34,845 546 2,232 735 1.0 0 100 100,448 55,608 660 1,274 699 1.0 50 100 157,316 47,285 1,084 2,036 4,730 1.0 200 100 154,672 48,318 1,765 860 2,044 1.0 1,000 100 168,614 54,223 10,037 1,469 3,006
0.1 do not have＜500 181＜100＜100＜100 0.1 0 100 86,952 26,425 206 485 1,285 0.1 50 100 20,746 6,381 579 726 353 0.1 200 100 19,966 5,501 778 325 235 0.1 1,000 100 50,219 3,511 1,290 1,036 714

The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of dosage shown in mice was used when 0 and 3 weeks.Conjugate is independent, or uses AlPO ₄(100 microgram) or use AlPO ₄Add the IL-12 preparation.Analyzed the PnPs9V IgG antibody of the serum of the 5th all blood-letting.

Table 27: use AlPO ₄The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of preparation

The effect that IL-12 replys PnPs14 in the mice

The PnPs14 of vaccine formulation during the 5th week replys
	Coupling agent IL-12 AlPO 4Total IgG 1 IgG2a IgG2b IgG3 (μ g) is (μ g) IgG (ng)
5.0 do not have 2,676 1,750＜100＜100＜100 5.0 0 100 11,792 15,704 124 580 1,723 5.0 50 100 56,712 31,056 6,144 2,854 11,840 5.0 200 100 5,049 3,050 1,588＜100 2,106 5.0 1,000 100 11,848 3,760 1,853 366 2,035
	1.0 do not have 4,846 3,116＜100 409 699 1.0 0 100 20,605 31,022 291 2,383 9,286 1.0 50 100 8,338 4,722 1,354 715 10,079 1.0 200 100 5,618 3,252 1,014＜100 583 1.0 1,000 100 13,026 3,551 2,879 671 2,070
0.1 do not have＜100 105＜100＜100＜100 0.1 0 100 114 392＜100＜100 710 0.1 50 100 2,140 2,838＜100 245 3,592 0.1 200 100 2,200 426＜100 622 759 0.1 1,000 100 394 378 219 100 658

The nine valency streptococcus pneumoniae sugar coupling vaccine immunity of dosage shown in mice was used when 0 and 3 weeks.Conjugate is independent, or uses AlPO ₄(100 microgram) or use AlPO ₄Add the IL-12 preparation.Analyzed the PnPs14 IgG antibody of the serum of the 5th all blood-letting.

Embodiment 7:IL-12 and AlPO ₄Immunne response effect to the sugared coupling vaccine of the scorching coccus C type (menC) of neisseria meningitis

Research design

IL-12 and scorching coccus C type (menC) vaccine of anti-neisseria meningitis have been assessed in this research.The SwissWebster mice is the independent preparation with 0.1 microgram or 1 microgram in 0 and 3 whens week, or adds AlPO ₄(100 microgram), or add IL-12 (50 nanogram) and AlPO ₄The menC carbohydrate conjugates immunity of co-formulated.In vaccine, do not add normal mouse serum.Mice blood-letting when 3 and 5 weeks is with the anti-menC polysaccharide of elisa assay serum IgG antibody.

The result

When with the conjugate immunity of high dose more, produced the IgG titre of the menC that equates, and no matter which kind of adjuvant preparation.Yet, in vaccine, add IL-12/AlPO ₄Cause than using AlPO ₄Preparation (but not having IL-12) or the vaccine of preparing without adjuvant have higher anti-polysaccharide IgG2a titre.

In the conjugate mice immunized with low dosage more, at vaccine AlPO ₄Obtain higher menC titre (table 28) during preparation.In this adjuvant, add IL-12 and do not strengthen the total IgG titre, but cause IgG2a antibody to increase more than 10 times really.These data show IL-12 and AlPO ₄Associating can promote inducing the complement associativity IgG subclass of menC sugar coupling vaccine.

Table 28:IL-12/AlPO ₄The effect that the IgG of menC sugar coupling vaccine is replied

	MenC replys
			Vaccine formulation	IgG	IgG subclass during the 5th week
MenC IL-12 AlPO 4Conjugate (ng) (μ g) (μ g)	The 5th week of the 3rd week	IgG1 IgG2a IgG2b IgG3
			1.0 50 100	33,176 598,027	83,662 7,218 4,351 1,436
0 100	34,553 404,111	71,017 1,383 3,085 1,006
			0 0	16,254 288,493	63,043 1,965 ＜100 502
0.1 50 100	2,584 68,678	9,604 3,440 1,967 512
			0 100	8,174 30,450	6,532 288 429 ＜100
0 0	1,724 7,894	1,767 ＜100 ＜100 ＜100

Embodiment 8:IL-12 and AlPO ₄Effect to the immunne response of haemophilus influenzae type b sugar coupling vaccine (HbOC)

Research design

IL-12 and influenza haemophilus b type (HbOC) vaccine have been assessed in this research.Swiss Webster mice (10 every group) contains haemophilus influenzae type b capsular polysaccharide (HibPs) and CRM in 0 and 3 whens week with 0.1 microgram or 1 microgram ₁₉₇Link coupled sugared coupling vaccine immunity.Independent or and the AlPO of this vaccine (HbOC) ₄(100 microgram) associating, or and IL-12 (50 nanogram) add AlPO ₄Mixture use together.In vaccine, do not add normal mouse serum.Mice blood-letting when 3 and 5 weeks.To the Farr test determination of the antibody response of HibPs, this test determination all and sugared link coupled antibody, and no matter which kind of isotype they are, that is, IgM, IgG and IgA.The IgG subclass is replied with ELISA and is measured.In addition, to CRM ₁₉₇IgG and IgG subclass reply also and measure with ELISA.

The result

The anti-HibPs antibody titer of the combining anteserum that (primary response) blood-letting of the 3rd week obtains is separately or use AlPO ₄, or IL-12 adds AlPO ₄There is not difference between the mice of vaccine immunity of preparation, no matter and be used for the conjugate dosage size (table 29) of immunity.The analysis of the combining anteserum of the 5th all blood-letting prompting adds the aluminum immune mouse with 1 microgram HbOC and IL-12 and causes than singly adding aluminum or high at least 10 times anti-HibPs (table 30) during without adjuvant.Yet, the analysis of each mice serum is shown that this is because mice has the titre of about 10,000 mcg/ml and causes.When the result expresses with geometric mean titer, there is not IL-12 to strengthen the evidence that HibPs replys.Being combined serum with ELISA has assessed the IgG subclass of HibPs has been replied.IL-12 and AlPO ₄Coupling show with having strengthened 3 times of IgG2a titres in the 1 microgram conjugate mice immunized.Yet, this and only use AlPO ₄The titre zero difference that the vaccine of assisting a ruler in governing a country obtains.In 0.1 microgram HbOC mice immunized, IL-12 adds AlPO ₄Do not strengthen IgG2a titre to HibPs.By antagonism-CRM ₁₉₇The analysis (table 31) of replying discloses IL-12/AlPO ₄Adjuvant combination has activity, and wherein the increase to the IgG2a titre of this carrier protein sees with arbitrary dosage conjugate mice immunized.

Table 29: with IL-12 and AlPO ₄In the HbOC mice immunized of preparation

Anti--the HibPs antibody response

	Anti--HibPs antibody response (μ g/mL)
			Vaccine formulation	The 3rd week	The 5th week
HbOC IL-12 AlPO 4(μg) (ng) (μg)	Combining anteserum	Combining anteserum GMT *
			1.0 50 100 0 100 0 0	9.73 10.04 5.12	469.16 26.92 42.55 21.30 33.19 2.25
0.1 50 100 0 100 0 0	3.18 4.06 3.03	30.95 ND 15.11 ND 14.05 ND

Table 30:IL-12 and AlPO ₄The effect that the IgG subclass of HbOC is replied

Vaccine formulation			Anti--HibPs IgG subclass during the 5th week is replied (ELISA terminal point titre)
					HbOC (μg)	IL-12 (ng)	AlPO 4 (μg)	IgG1	IgG2a
1.0	50	100	754,745	26,899
					0	100	122,637	12,880
	0	0	73,114	8,570
					0.1	50	100	46,673	17,290
0	100	68,176	14,971
				0		0	35,237	11,418

Table 31: with IL-12 and AlPO ₄In the HbOC mice immunized of preparation

Anti--CRM ₁₉₇IgG replys

Vaccine formulation	Anti-CRM during the 5th week 197Reply
			HbOC IL-12 AlPO 4(μg) (ng) (μg)	IgG	IgG1 IgG2a IgG2b
1.0 50 100 0 100 0 0	1,775,700 2,221,780 3,979,530	681,944 39,672 40,527 818,557 19,010 32,672 1,466,010 8,059 15,961
			0.1 50 100 0 100 0 0	761,027 891,251 874,805	292,448 38,258 21,008 346,728 6,546 14,832 151,397 1,899 3,517

The equivalence example

Those of skill in the art use the normal experiment method to be familiar with in this area, maybe can determine many equivalence examples of the specific embodiment of the invention as herein described.These equivalent examples should be included in the application's the claim.

Claims (24)

1. immunogenic composition is characterized in that: comprise and the link coupled pneumococcal capsular polysaccharide antigen of carrier molecule, assist a ruler in governing a country the mixture of the water slurry of the interleukin IL-12 of dosage and aluminium hydroxide or aluminum phosphate.

2. immunogenic composition as claimed in claim 1 is characterized in that: interleukin IL-12 is adsorbed in the water slurry of aluminium hydroxide or aluminum phosphate.

3. immunogenic composition as claimed in claim 1 is characterized in that: interleukin IL-12 is human interleukin IL-12.

4. immunogenic composition as claimed in claim 1 is characterized in that: PNEUMOVAX-23 is selected from pneumococcal capsular polysaccharide serotype 1,4,5,6B, 9V, 14,18C, 19F and 23F, and their combination.

5. immunogenic composition as claimed in claim 1 is characterized in that: described carrier molecule is selected from tetanus toxin, diphtheria toxin, diphtherotoxin, pertussis toxin, PT and their avirulence variant.

6. immunogenic composition as claimed in claim 5 is characterized in that: carrier molecule is CRM ₁₉₇

7. the purposes of the compositions of claim 1 is characterized in that, said composition is used to prepare the medicine of inducing the PNEUMOVAX-23 immunne response.

8. purposes as claimed in claim 7 is characterized in that: interleukin IL-12 is adsorbed in the water slurry of aluminium hydroxide or aluminum phosphate.

9. purposes as claimed in claim 7 is characterized in that: interleukin IL-12 is human interleukin IL-12.

10. purposes as claimed in claim 7 is characterized in that: PNEUMOVAX-23 is selected from pneumococcal capsular polysaccharide serotype 1,4,5,6B, 9V, 14,18C, 19F and 23F, and their combination.

11. purposes as claimed in claim 7 is characterized in that: described carrier molecule is selected from tetanus toxin, diphtheria toxin, diphtherotoxin, pertussis toxin, PT and their avirulence variant.

12. purposes as claimed in claim 11 is characterized in that: carrier molecule is CRM ₁₉₇

13. an immunogenic composition is characterized in that: comprise and the link coupled meningococcal capsular antigen of carrier molecule, assist a ruler in governing a country the mixture of the water slurry of the interleukin IL-12 of dosage and aluminium hydroxide or aluminum phosphate.

14. immunogenic composition as claimed in claim 13 is characterized in that: interleukin IL-12 is adsorbed in the water slurry of aluminium hydroxide or aluminum phosphate.

15. immunogenic composition as claimed in claim 13 is characterized in that: interleukin IL-12 is human interleukin IL-12.

16. immunogenic composition as claimed in claim 13 is characterized in that: the meningococcal capsular polysaccharide antigen is the capsular polysaccharide of Neisseria meningitidis C type.

17. immunogenic composition as claimed in claim 13 is characterized in that: described carrier molecule is selected from tetanus toxin, diphtheria toxin, diphtherotoxin, pertussis toxin, PT and their avirulence variant.

18. immunogenic composition as claimed in claim 17 is characterized in that: carrier molecule is CRM ₁₉₇

19. the purposes of the described compositions of claim 13 is characterized in that, said composition is used to prepare the medicine of inducing the meningococcus immunne response.

20. purposes as claimed in claim 19 is characterized in that: interleukin IL-12 is adsorbed in aluminium hydroxide or aluminum phosphate water slurry.

21. purposes as claimed in claim 19 is characterized in that: interleukin IL-12 is human interleukin IL-12.

22. purposes as claimed in claim 19 is characterized in that: hitchens and Hansen antigen is the capsular polysaccharide of Neisseria meningitidis C type.

23. purposes as claimed in claim 19 is characterized in that: described carrier molecule is selected from tetanus toxin, diphtheria toxin, diphtherotoxin, pertussis toxin, PT and their avirulence variant.

24. purposes as claimed in claim 23 is characterized in that: carrier molecule is CRM ₁₉₇