CN1549726A

CN1549726A - Innate immune system-directed vaccines

Info

Publication number: CN1549726A
Application number: CNA01814845XA
Authority: CN
Inventors: R・M・迈德齐托夫; R·M·迈德齐托夫
Original assignee: Yale University
Current assignee: Yale University
Priority date: 2000-07-31
Filing date: 2001-07-31
Publication date: 2004-11-24
Also published as: EP1322326A4; WO2002009748A1; CA2418036A1; AU2001286405B2; EP1322326A1; WO2002009748A9; AU8640501A

Abstract

The present invention provides novel vaccines, methods for the production of such vaccines and methods of using such vaccines. The novel vaccines of the present invention combine both of the signals necessary to activate native T-cells-a specific antigen and the co-stimulatory signal-leading to a robust and specific T-cell immune response.

Description

Innate immune system-directed vaccine

Invention field

The present invention relates to the production of new generation vaccine, this vaccine and use the method for this vaccine.More particularly, the invention provides comprised a kind of isolating pathogen association molecule pattern (PathogenAssociated Molecular Pattern) (PAMP) and a kind of antigenic unique vaccine molecule.More specific ground the invention provides and comprised a kind of isolating PAMP and a kind of antigenic new fusion protein matter, provides two kinds of required signals of natural T-cell-stimulating to such an extent as to inoculate with these fused proteins.New generation vaccine of the present invention provides a kind of effective way that single molecule is induced the T-cell immune response of reinforcement for preparing and utilize, this reacting activation other aspects of adaptive immunity reaction.Method and composition of the present invention provides design, production and has used with the strong approach of specific antigen as the vaccine of target, and antigen wherein comprises and pathogen, tumor, allergen and the related antigen of other diseases correlation molecule selected.

Background of invention

All articles of mentioning hereinafter, patent and other materials are all quoted as a reference clearly herein.

1. immunity

Multicellular organism has been developed the immune system of two routines to infectious medium.These two systems are geneogenous or natural immunity (being also referred to as " innate immunity ") and adaptability (acquired) or specific immunity.Main difference between two systems is that they discern the mechanism of infectious medium.

Innate immune system utilizes the receptor of an interplanting system coding to be identified in the conservative molecule pattern that exists in the microorganism.These molecule patterns appear in some constituent of microorganism, comprise: liopopolysaccharides, Peptidoglycan, lipoteichoic acid, phosphatidylcholine, antibacterial-specific protein comprise the composition of lipoprotein, DNA of bacteria, viral strand and double-stranded RNA, unmethylated CpG-DNA, mannan and many other antibacterials and fungal cell wall.Such molecule pattern also can appear in other molecules such as the plant alkaloid.The target of these innate immunitys identification is owing to be by microorganism rather than by the molecule pattern (PAMP) that is called as the pathogen association of the host living beings production of infection.(people (1989) Cold Spring Harb.Symp.Quant.Biol.54:1-13 such as Janeway; People such as Medzhitov (1997) Curr.Opin.Immunol.94:4-9).

The receptor of identification PAMPs in the innate immune system is called pattern identification receptor (PRRs).(people (1989) Cold Spring Harb.Symp.Quant.Biol.54:1-13 such as Janeway; People such as Medzhitov (1997) Curr.Opin.Immunol.94:4-9).These receptors structurally change and belong to several different protein familieses.Some Direct Recognition PAMPs in these receptors (for example CD14, DEC205, collectins), other (for example complement receptors) then discern the product that is obtained by PAMP identification.The member of these receptor families can be divided into three classes usually: 1) circulation body fluid receptor in blood plasma; 2) at the endocytosis receptor and 3 of immunocyte surface expression) can be at the frizzled receptor of cell surface or cell inner expression.(people (1997) Curr.Opin.Immunol.94:4-9 such as Medzhitov; People such as Fearon (1996) Science 272:50-3).

The PRRs of cell is expressed on the effector lymphocyte of innate immune system, is included in the cell of exercising antigen-presenting cell (APC) function of specialty in the adaptive immunity.Such effector lymphocyte is including, but not limited to macrophage, dendritic cell, bone-marrow-derived lymphocyte and epithelium.This expression way makes PRRs can directly induce congenital effector mechanism (innate effector mechanism), and also as mentioned belowly by the expression of inducing one group of endogenous signal such as inflammatory cytokine and chemotactic factor the existence of infectious medium is given the alarm to host living beings.The function in back makes the strength that can mobilize effector effectively to resist invader.

On the contrary, only the adaptive immune system of finding in vertebrates uses two types antigen receptor, and these receptors are to produce by somatic cell mechanism (somaticmechanism) in the growth of each bion.This two classes antigen receptor is T-cell receptors (TCR) and immunoglobulin receptor (IgR), and they are expressed in the cell type of two kinds of specializations respectively, i.e. T-lymphocyte and B-lymphocyte.The specificity of these antigen receptors be in lymphocytic maturation process by somatic cell gene reset, the random pair of receptor subunit and in rearrangement template dependent/non-dependent ground add nucleotide and generation randomly to the coding region.

Recently studies have shown that innate immune system is in the startup of control adaptive immune system with suitably play crucial effect in the inducing of cytological effect thing reaction.(people (1996) Science 272:50-3 such as Fearon; People such as Medzhitov (1997) Cell.91:295-8).In fact, confirmed that the lymphocytic activation of T-originally needs two kinds of different signals: a kind of is that another kind is so-called costimulatory signal B7 by the specific antigen peptide of TCR identification, and it is in the CD28 molecular recognition of expressing and being expressed on the T-cell on the APCs.(people (1996) Annu.Rev.Immunol.14:233-58 such as Lenschow).CD4 originally ⁺The lymphocytic activation of T-needs two kinds of signals (being specific antigen and B7 molecule) all to express on identical APC.If one originally CD4 T-cell when not having the B7 signal to exist, discerned antigen, this T-cell is will be by apoptosis dead.Therefore, the expression of B7 molecule on APCs controlled originally whether CD4 T-lymphocyte is activated.Because the activation of the CD8 T-cell of CD4 T-cell control tool cytotoxin function and the activation that tool produces the B-cell of antibody ability, the expression of B7 molecule determines then whether an adaptive immunity reaction is activated.

Recently research proves that also innate immune system plays crucial effect in the expression of control B7.(people (1996) Science 272:50-3 such as Fearon; People such as Medzhitov (1997) Cell.91:295-8).As above-mentioned, innate immunity identification is to be mediated by the PRRs that discerns PAMPs.PRRs causes controlling the activation of the signal path that multiple induction type Ir expresses to the identification of PAMPs, and wherein said gene comprises the gene of the coding necessary signal of activated lymphocyte such as B7, cytokine and chemotactic factor.(people (1997) Cell.91:295-8 such as Medzhitov; People such as Medzhitov (1997) Nature 388:394-397).Induce B7 to express by PRR by identification PAMPs, thus explained self/nonself distinguish and guarantee only to be specific to usually the deutero-antigenic T-cell of microorganism be activated.This mechanism has avoided being specific to the lymphocytic activation of id reaction of autoantigen usually.

Identified the receptor of the innate immune system of control B7 molecule and cytokine-expressing recently.(people (1997) Nature 388:394-397 such as Medzhitov; People such as Rock (1998) Proc.Natl.Acad.Sci.USA, 95:588-93).These receptors belong to class Toll receptor (TLRs) family, and name is because they and fruit bat Toll protein homology like this, the back of the body abdomen pattern development of this protein participation drosophila embryos and the immunoreation of adult fly.(people (1996) Cell86:973-83 such as Lemaitre).In mammalian organism, show the such PAMPs of TLRs identification such as bacterial product LPS, Peptidoglycan and lipoprotein.(people (1999) J.Biol.Chem.274:17406-9 such as Schwandner; People such as Yoshimura (1999) J.Immunol.163:1-5; People such as Aliprantis (1999) Science 285:736-9).

2. vaccine development

Use the mode of the disease that vaccine causes by infectious medium as a kind of antagonism traditionally.Yet along with the progress of vaccine technologies, vaccine has been used to other purposes, comprising but be not limited to control, the adjusting of hormonal action and the prevention or the treatment of tumor of mammal fertility.

The primary and foremost purpose that is used to resist the vaccine of disease is the immunological memory of inducing peculiar microorganism.More generally, need vaccine to induce immunoreation, no matter they belong to microorganism or expressed by tumor cell or other ill or unusual cells to specific antigen.Have B-and the lymphocytic division of T-that is specific to antigenic surface receptor and be differentiated to form specificity and memory.

In order to make a kind of vaccine can induce the immunoreation of protectiveness, it must satisfy following essential condition: 1) it must comprise specific antigen or its fragment that becomes the target of protective immunity after the vaccination; 2) it must can be presented such antigen by the form of immune system recognition with a kind of, for example, and a kind of form that before immunity identification, degraded is had resistance; And 3) it must activate APCs to CD4 ⁺T-presented by cells antigen, and CD4 ⁺The T cell is then induced B-cell differentiation and other immunoeffectors function.

Conventional vaccine comprises the suspension of microorganism attenuation or that kill such as virus or antibacterial, and they self can not induce serious infection, but can offset (or have virulence) species of unmodified after in being inoculated into the host.The use of this term now has been expanded and has consisted essentially of the preparation (preparation of (variant or mutant) microbial strains of the microbial strains that virulence is arranged that for example kills or the attenuation of living that any purpose is positive immunoprophylaxis; Microorganism, fungus, plant, protozoacide or metazoal derivant or product; Synthetic vaccine).Vaccine embodiment is including, but not limited to the recombinant protein to the vaccinia virus of smallpox vaccination, the antibacterial that prevents the whole inactivation of tetanic tetanus toxoid (tetanus toxoid), prevention pertussis (pertussis), the polysaccharide that prevents the pneumonia that streptococcus caused and prevention hepatitis B.

Although the vaccine of attenuation is normally immunogenic, their use is conditional, and this is because their effect usually need be to specific, the detailed knowledge of the molecule factor of determination of virulence.In addition, in vaccine the use of the pathogen of attenuation relevant with multiple risk factor, this has in most of the cases stoped their safe handlings in the mankind.

On the other hand, the problem of synthetic vaccine be they often be non-immunogenicity or unprotect.Utilize available adjuvant to increase the immunogenicity of synthetic vaccine often because unacceptable side-effects rather than a kind of selection of this adjuvant auto-induction.

Adjuvant is defined as the immunogenic material of any increase antigens mixed.Although chemical drugs such as Alumen often are considered to adjuvant, they exercise the effect similar to carrier, and they are likely by the interaction effect of stable antigen and/or promotion antigen and antigen-presenting cell.Best adjuvant is that those imitate the ability of microorganisms and activate innate immune system.Pure antigen can not the induction of immunity reaction because not inducing the necessary costimulatory signal of activated lymphocyte (for example B7.1 or B7.2).Therefore, adjuvanticity key mechanism is owing to inducing costimulatory signal by the composition that the carries PAMPs microorganism that belongs to the adjuvant conventional ingredient or quasi-microorganism.(people (1989) Cold Spring Harb.Symp.Quant.Biol.54:1-13 such as Janeway).As what above discussed, PRRs has induced lymphocyte activator (for example B7) and differentiation (effector cytokine) necessary signal to the identification of these PAMPs.

Thereby because often use adjuvant and with in the cell that target antigen contacts do not triggered geneogenous immunoreation many with the amount of the mole many than antigen, this non-specific innate immune system of inducing has produced over-drastic inflammatory reaction with the reaction that generation activation adaptive immunity reacts necessary signal, and this makes that many very effective adjuvants are inapplicable clinically.Ratified Alumen recently and can be used as clinical adjuvant (although it has relatively limited effect), thereby this is because it is not that a kind of innate immunity stimulus object can not cause over-drastic inflammation.Yet a kind of vaccine that comprises the innate immunity stimulus object and use in a kind of mode that does not cause excessive inflammation will be more effective than the vaccine that comprises antigen and adjuvant such as Alumen far away.Antigen and a kind of PAMP such as bacterial lipoprotein (BLP) merged to make the stoichiometry optimization of two kinds of signals and adjust their effects, reduce to minimum thereby make when antigen mixes the unwanted excessive inflammatory response that produces when increasing its immunogenicity with the adjuvant that includes the innate immunity stimulus object to identical APC.In addition, chimeric construct body of the present invention will stop the activation of not taking in antigenic APCs.In the absorption that does not have target antigen with when presenting, the activation of such APCs can cause inducing autoimmune response, this is again to stimulate one of problem that adjuvant interrelates, this problem to stop with innate immunity commonly used or limited the application of these adjuvants in the mankind.Especially, chimeric construct body display of the present invention conventional desired basic immune characteristic of the vaccine that has replenished adjuvant or character, but not often inductive over-drastic inflammatory reaction of inducing adjuvant of this chimeric construct body.Thereby, the vaccine method that the present invention describes makes undesired side effect (for example over-drastic inflammatory reaction, autoimmune) reduce to minimum or elimination, and still induce highly effective specific immunoreation, a kind of good replacement scheme to the vaccine that comprises antigen and adjuvant mixture is provided.

3. selectable vaccine strategy

Immunostimulating complex as vaccine.Immunostimulating complex (ISCOMS) is to comprise the systemic widely immunoreactive cage structure thing of inducing of Quil-A, cholesterol, activated saponin adjuvant and phospholipid.(people (1999) Immunol.Lett.65:133-140 such as Mowat; People such as Smith (1999) J.Immunol.162 (9): 5536-5546).ISCOMS is suitable for repeating giving body one by one with synantigen not, and this is because these complex allow antigens to enter into MHC I and II processing path.(people (1991) Immunol.72:317-322 such as Mowat).

ISCOMS is used with the soluble protein conjugate of modification.(people (1992) Vaccine 10 (9) such as Reid: 597-602).These complex also produce the reaction of a kind of Th1 type, as so a kind of vaccine desired like that.(people (1999) Methods19:94-102 such as Morein).

Yet opposite with molecule of the present invention, ISCOMS is more complicated structure, has caused the potential problems of repeatability and dosage; They do not contain the conjugate between antigen and the PAMP yet.Because ISCOMS does not point to APCs specifically, their use then can cause toxicity and lack specific problem.

The multiple antigenic recombiant vaccine.A plurality of U.S. Patent Publications by multiple antigenic peptide (MAPs) form as the chimeric protein of vaccine.For example, people such as Klein be awarded one group of patent (for example U.S. Patent number 6,033,668; 6,017,539; 5,998,169; With 5,968,776), the gene of coding polymer heterozygote (multimeric hybrids) has been described wherein, this heterozygote comprise be connected to from the immunogenicity zone of second pathogen from first antigenic proteinic immunogenicity zone.Though patent concentrates on human parainfluenza/respiratory syncytial virus protein chimera, first and second antigens can be selected from antibacterial and viral pathogen more widely.Although the vaccine of people such as Klein expectation is a fused protein, but all member's peptides all are to choose as antigenic effect (promptly by TCR or IgR identification) rather than according to the pairing of antigen and PAMPs according to them, so this vaccine is not that design stimulates innate immune system.

Sinugalia (U.S. Patent number 5,114,713) vaccine of forming by from the peptide of the circumsporozoite protein of Plasmodiumfalciparum (P.falciparum) is disclosed, this peptide can be used as can with link coupled general T-cellular antigens decision position, B-cellular antigens determination section position, for example derived from the surface protein of pathogenicity medium (for example antibacterial, virus, fungus or parasite).The peptide of these combinations can prepare by recombination form.These general T-cellular antigens decision positions are known not to be PAMPs, and they are by adaptive immune system rather than by the innate immune system effect.

People such as Russell-Jones (U.S. Patent number 5,928,644) disclose derived from the proteinic T-cellular antigens decision of the TraT of escherichia coli (E.coli) position, this protein is to be used to produce hybrid molecule to improve all types of target to be comprised the immunoreactive of parasite, soluble factor (for example LSH) and virus.Thereby these constructs provide increases the strategy of antigenic complexity of vaccine, thereby has promoted adaptive immunity reaction that strengthen or multiple.Yet known antigens decision position is not PAMPs, and they are by adaptive immune system rather than by the innate immune system effect.

Thereby aforesaid invention all has significant difference with the present invention on purpose, notion, strategy and binding mode.

4. to the overview of new generation vaccine of the present invention

New generation vaccine of the present invention comprises one or more the isolating PAMPs with one or more antigen combinations.Used antigen can be the antigen (for example including, but not limited to the relevant antigen of pathogen, the antigen that tumor is relevant, the antigen that allergy is correlated with, the antigen that neurological handicap is relevant, the antigen that cardiovascular disease is relevant, the antigen that rheumatoid arthritis is relevant, antigen, hormone, pregnant relevant antigen, embryonal antigen and/or the fetal antigen etc. that other diseases is relevant) of any kind in vaccine of the present invention.Can in Fig. 1, provide by the polytype antigenic example that the present invention produces.

In a preferred embodiment, vaccine is recombinant protein, reorganization lipoprotein or recombinant glycoprotein, and they contain PAMP (for example BLP or flagellin) and one or more antigens.The basic conception of preparation fused protein of the present invention provides in Fig. 1.

After giving the human or animal with vaccine of the present invention, this vaccine will interact with APCs such as dendritic cell and macrophage.This interaction will have two results: at first, the PAMP of vaccine part will interact with PRR such as TLR and stimulate a signal path, for example NF-κ B, JNK and/or p38 path.Secondly, because the interaction of PAMP and TLRs and/or other pattern recognition receptors, recombiant vaccine is taken in the size of dependent cells type, recombiant vaccine and the characteristic of PAMP in dendritic cell and the macrophage by phagocytosis, endocytosis or huge pinocytosis (macropinocytosis).

The activation of the inductive signal path of TLR-will cause dendritic cell and macrophage and inducing of expressing of the B-cell pair cell factor, chemotactic factor, adhesion molecule and costimulatory molecules in some cases.The absorption of vaccine will cause being fused to antigenic processing and their the presenting by MHC type i and MHC Type II molecule of PAMP.This will produce the originally necessary two kinds of signals of T-cell-stimulating---specific antigen signal and costimulatory signal.In addition, will be recruited by vaccine-induced chemotactic factor (because interaction of PAMP and TLR) that originally the T-cell is to APC and cytokine such as IL-12, they will induce the T-cell differentiation is Th-1 effector lymphocyte.As a result, will induce strong T-cell immune response, the latter activates other aspects of adaptive immunity reaction, for example activation of antigen-specific b-cell and macrophage more successively.

Thereby new generation vaccine of the present invention provides the effective way of the disadvantageous side effect that one or more specific antigen generation immunoreation are not associated with conventional vaccine usually.

Summary of the invention

The present invention relates generally to vaccine, prepare the method for vaccine and the method for use vaccine.

More clearly, the invention provides the vaccine that comprises a kind of isolating PAMP, its immunostimulation part or immunostimulation derivant and antigen or its immunogenicity part or immunogenic derivatives.The example of a preferred vaccine of the present invention is a kind of fused protein, and it comprises a kind of PAMP, its immunostimulation part or immunostimulation derivant and antigen or its immunogenicity part or immunogenic derivatives.

Vaccine of the present invention can comprise any PAMP peptide or protein, including, but not limited to following PAMPs: other protein components of Peptidoglycan, lipoprotein and lipopeptid, flagellin, outer membrane protein (OMPs), outer surface proteins (OSPs), bacteria cell wall and other PRR parts.

A kind of preferred PAMP of the present invention is BLP, comprises the BLP by the polypeptide SEQID NO:2 coding of illustrating in Figure 15.Outside the isolating protein PAMPs, the also peptide mimics of usefully any nonprotein PAMP in the present invention.

Useful antigen is including, but not limited to the relevant antigen relevant with other diseases of those microorganisms among the present invention, the latter including, but not limited to those allergens be correlated with relevant with cancer.The antigenic component of vaccine can derived from, but be not limited to antibacterial, virus, fungus, yeast, protozoacide, metazoa, tumor, malignant cell, plant, animal, people, allergen, hormone and amyloid-β peptide.Antigen, its immunogenicity part or immunogenic derivatives can be made up of peptide, polypeptide, lipoprotein, glycoprotein, mucin etc.

Various vaccine of the present invention including, but not limited to:

1) one or more PAMPs, its immunostimulation part or immunostimulation derivant, it is incorporated into one or more antigens, its immunogenicity part or immunogenic derivatives;

2) a kind of PAMP/ antigen coalescence protein matter comprises one or more PAMPs, its immunostimulation part or immunostimulation derivant, and one or more antigens, its immunogenicity part or immunogenic derivatives; With

3) a kind of antigen of modification, its immunogenicity part or immunogenic derivatives, it comprises one section targeting sequencing that is fused to fatization (lipidation) or glycosylated concensus sequence, and described concensus sequence further is fused to antigen or its immunogenicity part or immunogenic derivatives.

The present invention also comprises such vaccine, and this vaccine further comprises a kind of pharmaceutically acceptable carrier including, but not limited to Alumen.

More clearly, the invention provides and comprise one or more PAMPs, its immunostimulation part or immunostimulation derivant, and the fused protein of one or more antigens, its immunogenicity part or immunogenic derivatives.The PAMP domain of fused protein of the present invention can be made up of aminoacid, aminoacid polymer, Peptidoglycan, glycoprotein and lipoprotein or any other appropriate ingredients.The used a kind of preferred PAMP of fused protein of the present invention is BLP, comprises the BLP by polypeptide SEQ ID NO:2 coding.Flagellin is the used another kind of PAMP of fused protein of the present invention, and provides (but being not limited thereto) by access number P04949 (coli flagellum albumen) and A24262 (Salmonella flagellin).Antigenic structure territory useful in the fused protein of the present invention is including, but not limited to E α (a kind of peptide antigen derived from mice MHC Type II I-E), the molten born of the same parents' element in Listerella, PSMA, HIV gp120, Ra5G and TRP-2.In a preferred embodiment, fused protein of the present invention comprises a construct that comprises following composition: one section targeting sequencing that sends the signal of fatization or glycosylation concensus sequence, one section fatization and/or glycosylation concensus sequence and antigen.More clearly, fused protein of the present invention comprises that comprises the antigenic construct of targeting sequencing-CXXN-, and targeting sequencing wherein is the signal of concensus sequence fatization, and X wherein refers to any aminoacid, is preferably serine.The example of useful leader peptide is selected from the peptide of peptide SEQ ID NO:3 (as shown in figure 15), SEQ ID NO:4 (as shown in figure 16), SEQID NO:5 (as shown in figure 17), SEQ ID NO:6 (as shown in figure 18) and SEQ ID NO:7 (as shown in figure 19) including, but not limited to those among the present invention.

In another embodiment, the present invention also provides a kind of isolating PAMP and antigenic fused protein of comprising, and antigen wherein is autoantigen.

The present invention further provides the method for recombinant production fused protein of the present invention.Thereby, the invention provides the recombinant expression carrier of the nucleotide sequence that comprises the chimeric construct body of the present invention of encoding and with such recombinant expression carrier transformed host cells.Any cell that can express fused protein of the present invention all is suitable as host cell.Such host cell is including, but not limited to antibacterial, yeast, insecticide, plant and animal cell.For some PAMPs such as BLP host cell more preferably is bacterial cell.More preferably, host cell is the bacterial cell of the PAMP domain of a kind of can suitably modification the (for example fatization, glycosylation) fused protein, when must or want to carry out this type of modification.

The present invention also provides with vaccine of the present invention and has made animal obtain the method for immunity, wherein such method including, but not limited to parenteral, intravenous, oral, use suppository or give vaccine by mucomembranous surface.Be the people by premunitive animal in a preferred embodiment.

Immunoreation can utilize any suitable method to measure, including, but not limited to the tuerculoderma of the immunity of the immunoassay of direct measurement peripheral blood lymphocyte, natural killer cell cytotoxic assay, cell proliferating determining, immunocyte and hypotype and pair cell mediation.

Thereby the present invention also provides by giving a kind of vaccine of the present invention and stimulate the signal path of TLR-mediation to treat easily that allergen is produced allergic patient.

The present invention also provides by giving the patient that a kind of vaccine of the present invention is treated susceptible or suffered from Alzheimer disease, and wherein target antigen is a kind of and related peptide of Alzheimer disease or protein, including, but not limited to amyloid-peptide.

Thereby it is a kind of in animal moderate stimulation innate immunity reaction and strengthen method for the adaptive immunity reaction of external source or autoantigen that the present invention provides further, and this method comprises with external source or autoantigen and gives PAMP jointly.

The present invention also provides a kind of vaccine that comprises with external source or the bonded PAMP of autoantigen, thereby can and strengthen in the reaction of animal moderate stimulation innate immunity the adaptive immunity reaction of external source or autoantigen is not caused undesired level of inflammation.

In addition, the invention provides a kind of vaccine that comprises with external source or the bonded PAMP of autoantigen, when having given the treatment effective dose, thereby this vaccine can and be strengthened in animal moderate stimulation innate immunity reaction the adaptive immunity reaction of external source or autoantigen is not caused undesired level of inflammation.

The present invention also provides a kind of Therapeutic Method that comprises the step that gives individual a kind of vaccine, this vaccine comprises and external source or the bonded PAMP of autoantigen, thereby can and strengthen in animal moderate stimulation innate immunity reaction the adaptive immunity reaction of external source or autoantigen is not caused undesired level of inflammation.

The technical staff that the extra embodiment of the present invention is implemented vaccine production in this area and vaccine is well-known.Such obvious variation also is that the present inventor is desired among the present invention.

The accompanying drawing summary

Fig. 1 represents the example according to selectable fused protein of the present invention.But the change of these fused proteins and combination be the method according to this invention and preparing also.

Fig. 2 represents to produce the bare bones of different recombinant protein vaccines, and vaccine wherein contains different antigen and a signal (PAMP) that triggers the innate immunity reaction.Each antigen is represented by the difformity of vaccine middle body.

Fig. 3 represents BLP/E α construct.

Fig. 4 represents that BLP/E α is with dose dependent mode activation NF-κ B.

The production of IL-6 in the dendritic cell that Fig. 5 represents to stimulate with BLP/E α.

Fig. 6 represent dendritic cell activated induce with vaccine antigen by MHC Type II path processing and present.

Fig. 7 represents the immunostimulation of external chimeric construct body BLP/E α to specific T-cell.

Fig. 8 represents that chimeric construct BLP/E α is to the effect of specific T-cell proliferation in the body.

Fig. 9 represents that the activation of the inductive B-cell of CpG-depends on MyD88.MyD88 ^-/-, the MyD88 deficient cells; ICE ^-/-, the caspase-1-deficient cells; B10/ScCr is derived from the TLR4 deficient cells of C57BL/10ScCr mice; TLR2 ^-/-, the TLR2 deficient cells.

Figure 10 is illustrated in CpG stimulation and the inductive IkB α degraded of CpG-DNA-, produces IL-6 by macrophage and mediates by a signal path that depends on MyD88.

Figure 11 represents when stimulating with the CpG oligonucleotide, wild type with the B10/ScCr dendritic cell but be not from MyD88 ^-/-The dendritic cell of animal are produced IL-12.

Figure 12 represents the activation of flagellin fusant to NF-κ B.

Figure 13 represents flagellin fusant inducing NF-κ B in macrophage.

Figure 14 represents the NF-kB activity in the RAW κ B cell

Figure 15 represents SEQ ID NO:3.

Figure 16 represents SEQ ID NO:4.

Figure 17 represents SEQ ID NO:5.

Figure 18 represents SEQ ID NO:6.

Figure 19 represents SEQ ID NO:7.

Figure 20 represents SEQ ID NO:10.

Figure 21 represents SEQ ID NO:11.

Detailed Description Of The Invention

1. describe, in general terms

The invention discloses a kind of based on the vaccine design New Policy recently found of present inventor in the congenital immunity field. The method is not limited to any specific antigen or its immunogenicity part or derivatives thereof (such as microorganism relevant, that allergen is relevant or Tumor-assaciated etc.), also is not limited to any specific PAMP or its immunostimulation part or immunostimulation derivative. Therefore, term " vaccine " is used in reference to any treatment, immunogenic or immunostimulating composition that comprises feature of the present invention herein in general sense. Here one of relevant vaccine is defined in other places openly in more detail.

The activation of adaptive immunity reaction needs specific antigen or derivatives thereof and a kind of signal (such as PAMP) that can induce B7 to express at APCs. The present invention has made up the necessary two kinds of signals of inducing of adaptive immunity reaction in a single chimeric construct body---a kind of signal (PAMP) and a kind of signal (antigen) by antigen receptor identification by innate immune system identification.

According to the present invention, PAMP and antigen all needn't be comprised of polypeptide. Yet perhaps PAMP or antigen or both can be a kind of peptide or polypeptide. In one embodiment of the invention, when PAMP or its immunogenicity part or derivatives thereof and antigen or its immunostimulation part or derivatives thereof are polypeptide, can produce the chimeric construct body to be used for vaccine with recombinant DNA technology. Selectively, when peptide mimics is used for substituting nonprotein antigen, such as polysaccharide or nucleic acid etc., and/or when nonprotein PAMP such as lipopolysaccharides, CpG-DNA, DNA of bacteria, strand or double-stranded viruses RNA, phosphatid ylcholine, lipoteichoic acid etc., recombinant technique also can be used for producing a kind of protein chimeric construct body. The present invention considers to use the PAMPs of BLP, bacterial outer membrane albumen (OMP), bacterium outer surface proteins A (OspA), flagellin and other dna encodings in one embodiment in the recombinant production of chimeric construct body. Known these PAMPs can induce the activation of congenital immunity reaction, therefore are specially adapted to bacterin preparation. (people (1996) Microbiol.Rev.60:316-41 such as Henderson). In addition, shown that BLP can be identified by TLRs. (people (1999) the Science 285:736-9 such as Aliprantis). The details as the method for the PAMP domain of PAMP/ antigen coalescence protein matter with BLP has been described; Yet any inducer of innate immune system all is fit to purpose of the present invention equally.

In another embodiment, one or more PAMP analogies have substituted the PAMP in the fused protein.

The present invention further provides the method for producing the chimeric construct body, wherein PAMP or its immunostimulation part or derivatives thereof, perhaps antigen or its immunogenicity part or derivatives thereof, perhaps PAMP and antigen are nonproteins. Usually, these methods are attached to PAMP on the antigen with chemical method, thereby produce nonprotein chimeric construct body.

The present invention further provides the approach that in peptide vaccine is synthetic, utilizes recombinant DNA technology. Many surface antigens that exist in the purpose pathogen can not be subjected to the guidance of nucleic acid coding, and this is because they are not protein (for example lipopolysaccharides) or have low protein content (for example peptide glycan).

The present invention considers to use the peptide mimics of these surface antigens or its immunogenic protein or derivatives thereof, and the use of the peptide mimics in vaccine.

As discussed in detail herein, the present invention considers to comprise the vaccine of chimeric construct body, and chimeric construct body wherein comprises at least a antigen or its immunogenicity part or derivative and at least a PAMP or its immunogenicity part or derivative. Thereby the present invention comprises the vaccine that contains fused protein, and wherein one or more proteantigens are connected on the peptide mimics of one or more protein PAMP or PAMP. Preferably, fused protein has maximum immunogenicity and only induces appropriate inflammatory reaction.

Domain at target antigen or target antigen has relatively low molecular weight, and when not having enough immunogenicities owing to its little individuality, haptens can be served as in antigen or antigenic structure territory, and can be combined with larger carrier molecule, to such an extent as to the molecular weight of binding molecule will be enough high to cause the strong immune response to this antigen. In one embodiment of the invention, antigen self serves as carrier molecule. In another embodiment of the invention, PAMP serves as carrier molecule. In the another one embodiment, haptens by merge or in conjunction with and be combined to cause to haptenic antibody response with PAMP or the antigenic structure territory of vaccine. In the another one embodiment, (do not have restricted), when the molecular weight of antigen and PAMP all hangs down, preferably PAMP and antigen are combined with the 3rd molecule that serves as carrier. Such carrier molecule can be keyhole limpet hemocyanin or well known to a person skilled in the art in many haptenic carrier molecules any one. In the another one embodiment, fused protein contains a part or PAMP analogies and carrier protein or the carrier peptide of antigen or antigenic structure territory, PAMP or PAMP. Such carrier protein can be again keyhole limpet hemocyanin or well known to a person skilled in the art in many haptenic carrier proteins or the carrier peptide any one. Multiplet territory by utilizing multiple antigenic protein and/or identical or different antigen protein and/or the more aforesaid quantity that makes up to increase antigen or epitope are that the present invention considers. What also consider is wherein PAMPs or the fused protein that increases of the quantity of PAMP derivative or PAMP analogies. Determine that the optimal proportions of PAMP antagonism prodomain is so that immunogenicity maximization and inflammatory reaction is minimized is in technical staff's technical scope.

2. definition

" adaptive immunity " refers to adaptive immune system, wherein relates to the acceptor that two classes produce by body cell mechanism in each ontogeny. As employed herein, " adaptive immune system " phalangeal cell and humoral immunity. The immunity identification of adaptive immune system is mediated by antigen receptor.

" adaptive immunity reaction " refers to a kind of reaction that comprises above-mentioned " adaptive immune system " feature.

" adapter molecule " refers to a kind of molecule that can be combined with some TLRs momently, by the numerator mediated immunostimulation of innate immune system, and the signal granting of inducing of the mediated cell factor. " adapter molecule " is including, but not limited to marrow differentiation marker 88 (MyD88).

" allergen " but refer to induced metamorphosis reaction or a part or the derivative of anaphylactoid antigen or antigen.

" amino acid polymer " finger protein matter or peptide contain at least two amino acid whose polymers that connect with peptide bond with being connected, wherein such polymer or do not contain non-peptide bond or contain one or more non-peptide bonds. As used herein, " protein " comprises polypeptide and oligopeptides.

" antigen " refers to a kind of material of specifically being identified by the antigen receptor of adaptive immune system. Thereby as used herein, term " antigen " comprises antigen, the immunogenic antigenic derivant of tool or part and derived from the immunogenic molecules of antigen. Preferably, the used antigen of the present invention is the antigen that separates. Useful especially antigen is relevant, that allergen is relevant or disease association including, but not limited to those pathogen among the present invention.

" antigenic determinant " refers to the zone of given antigen receptor combination on the antigen.

" antigen presenting cell " or " APC " or " professional antigen presenting cell " or " professional APC " are that a kind of function is by taking in, process and trigger at its surface expression antigen the immune system cell of adaptive immunity reaction. Such effector cell is including, but not limited to macrophage, dendritic cells and B cell.

" antigen receptor " refers to two class antigen receptors of adaptive immune system: T-cell receptors (TCR) and immunoglobulin receptor (IgR), they are expressed in the cell type T-lymphocyte of two kinds of specializations and B-lymphocyte respectively. The secreted form of immunoglobulin receptor is called antibody. The specificity of antigen receptor be lymphocyte by somatic cell gene reset, the random pair of receptor subunit and in rearrangement template dependent/non-dependent ground add the process of nucleotides to the code area and produce at random during maturation.

" chimeric construct body " refers to comprise the construct of antigen and PAMP or PAMP analogies, antigen wherein and PAMP comprise such as amino acid, amino acid polymer, nucleotides, nucleotide multimer, carbohydrate, carbohydrate polymer, lipid, lipid polymer or other synthetic or naturally occurring chemicals or chemicals polymer molecule. As used herein, " chimeric construct body " refers to that wherein antigen comprises the construct of the molecule of a class and PAMP or the associating of PAMP analogies, and the molecule that this PAMP or PAMP analogies can comprise same type also can comprise dissimilar molecules.

" CpG " refers to a kind of dinucleotides, and one of them unmethylated cytimidine (C) residue abuts against 5 ' of guanine (G) residue and locates. As used herein, " CpG-DNA " refers to a kind of synthetic repetition CpG, the intact bacterial DNA that contains the CpG primitive or its derivative that contains CpG. CpG-DNA by the TLR mediation, and depends on " protein adapter molecule " to the immunostimulation of B-cell.

" derivant " refer to any structurally with original molecule or relevant deutero-molecule or the chemical compound of chemical compound.As used herein, " derivant " comprises peptide mimics (for example PAMP analogies).

" domain " refers to have the proteinic part of self function.The combination of domain has determined its general function in the single protein.One " antigenic structure territory " comprises a kind of antigen or immunogenicity of antigens part or derivant.One " PAMP domain " comprises the immunostimulation part or the derivant of PAMP or PAMP analogies or PAMP or PAMP analogies.

" fused protein " and " chimeric protein " all refers to any protein blend zoarium that comprises two or more domains, domain wherein is selected from: protein, peptide, lipoprotein, lipopeptid, glycoprotein, glycopeptide, mucin, mucopeptide, make at least two domains or encode like this from different species or by different genes, make that perhaps in two domains one is naturally occurring and second domain is not naturally occurring, perhaps also make a similar a kind of naturally occurring molecule in two domains and another not similar same a part.Term " fused protein " also refers to a kind of antigen or its immunogenicity part or derivant that contains one section aminoacid sequence through modifying, aminoacid sequence wherein can cause the post translational modification to this aminoacid sequence or its partial sequence, and wherein the sequence of post translational modification is a kind of part of PRR.As another definition to fused protein, in front in the sentence, cause post translational modification and the aminoacid sequence that forms a kind of part of PRR can comprise a concensus sequence, perhaps this aminoacid sequence can comprise a kind of targeting sequencing and a concensus sequence.

" hapten " but refer to a kind of self non-immunogenicity but conjugated antigen receptor and can combine with bigger carrier molecule and have an immunogenic micromolecule.

" with ... association " refers to a kind of reversible associating between two kinds of identical or different chemical individuals and forms a kind of more complicated material.

" with ... in conjunction with " refer to a kind of reversible or irreversible (for example covalency) associating between two kinds of identical or different chemical individuals and form a kind of more complicated material.

" immunostimulation " refers to that molecule activates the ability of adaptive immune system or innate immune system.As used herein, " antigen " is the molecule example that can stimulate adaptive immune system, and PAMPs or PAMP analogies are the molecule examples that can stimulate innate immune system.As used herein, each immune " activation " comprise in body fluid and/or the cell immune response production of the composition of antagonism molecules of immunization stimulus.

" innate immunity " refers to innate immune system, and it is different with " adaptive immune system ", uses the receptor of an interplanting system coding to be identified in the conservative molecule pattern that exists in the microorganism.

" innate immunity reaction " refers to comprise the reaction of above-mentioned " innate immune system " feature.

" isolating " refers to for mixture or naturally occurring material, material, cell, tissue or subcellular component isolating or that purify basically.

" connector " refers to any chemical individual that a kind of chemical part is connected with other chemical parts.Thereby those chemically or physically connect PAMP and the antigenic connector that is.The connector example is including, but not limited to complicated or simple hydrocarbon, nucleoside, nucleotide, phosphonate nucleotide (nucleotide phosphate), oligonucleotide, polynucleotide, nucleic acid, aminoacid, little peptide (small peptide), polypeptide, saccharide (for example monosaccharide, disaccharide, trisaccharide) and lipid.The detailed description that the additional examples of connector comprises herein partly provides.Not restrictedly, the present invention does not consider to use peptide bond or aminoacid or peptide connector to connect polypeptide PAMP and polypeptide antigen yet.The present invention further considers the molecule for preparing such connection by the recombinant DNA program.Connector also can be exercised the function of spacer.

" virulent " refers to invasive, diffusible tumor.

" microorganism " is to such an extent as to refer to the too little live organism that can not with the naked eye see.Microorganism is including, but not limited to antibacterial, fungus, protozoacide, trickle algae and virus.

" analogies " refer to very similarly molecule of a kind of and second kind of molecule, and have effect or function with second kind of molecular mimicry.

" part " refers to an ingredient in the molecule.Though two parts are arranged in individual molecule usually, the part more than two also can be arranged in individual molecule.

" molecule pattern " refers to a kind of chemical constitution or primitive, and it generally is the composition of microorganism or some other biological body, produces but generally can't help normal cell or other intact animal's cells.The molecule pattern is found in or by the molecular composition of following type: lipopolysaccharide, Peptidoglycan, lipoteichoic acid, phosphatidylcholine, lipoprotein, DNA of bacteria, viral strand or double-stranded RNA s, some viral glycoprotein, unmethylated CpG-DNA, mannan and many other antibacterials, fungus and zymic cell wall constituent etc.

" nonprotein chimeric construct body " or " nonprotein chimera " refer to that a kind of antigen or PAMP or PAMP analogies or two or more in them wherein are not aminoacid polymeric " chimeric construct bodies ".

" the molecule pattern of pathogen association " or " PAMP " refer to a kind of in microorganism and the molecule pattern of not finding in the mankind, and it can trigger the innate immunity reaction when combining PRR.Thereby as used herein, term " PAMP " comprises any such microorganism molecule pattern, and is not limited to that those are associated with pathogenic microbes or microorganism.As used herein, term " PAMP " comprises PAMP, immunostimulating PAMP derivant or part and derived from any molecules of immunization stimulus of any PAMP.These structure or derivatives thereofs are potential starting materials of innate immunity reaction, and are the parts that comprises the PRRs of Toll receptor and TLRs therefore." PAMPs " is found in or by some molecular immunostimulation structures, these molecules are including, but not limited to lipopolysaccharide; Phosphatidylcholine; Polysaccharide comprises Peptidoglycan; Teichoic acid comprises lipoteichoic acid; Protein comprises lipoprotein and lipopeptid; Outer membrane protein (OMPs), the protein component and the flagellin of outer surface proteins (OSPs) and other bacteria cell walls; DNA of bacteria s; Virus strand or double-stranded RNA s; Unmethylated CpG-DNA; Mannan; The mycobacteria cell membrane; Porin; And multiple other antibacterials and fungal cell wall composition, comprise that those are found in the yeast.The detailed description that extra PAMP example comprises herein partly provides." PAMP/ antigen conjugate " refers to the antigen and PAMP or the PAMP analogies that covalently or non-covalently connect.Conjugate can comprise protein PAMP or antigen and nonprotein PAMP or antigen.

" PAMP/ antigen fusions " or " PAMP/ antigen chimera " refers to any protein blend compound of forming between PAMP or PAMP analogies and antigen.

" passive immunity " refers to antibody or contacted antigenic lymphocyte gives individuality so that it obtains immunity.

" PAMP analogies " are not although referring to those is present in the microorganism, owing to can trigger the innate immunity reaction but the molecule of PAMP analog in conjunction with PRR and this combination.The PAMP analogies can be naturally occurring molecule or partially or completely synthetic molecule.Give an example, but be not limited to this, certain plants alkaloid such as paclitaxel are the PRR parts, have the immunostimulation to innate immune system, thereby show as the PAMP analogies.(people (2000) J.Biol.Chem.275 (4) such as Kawasaki: 2251-2254).

" pattern identification receptor " or " PRR " refer to a kind of member of innate immune system receptor family, and it can start the innate immunity reaction when combining PAMP, its immunostimulation part or derivant.Different and belong to several different protein familieses on member's structure of this receptor family.Some this receptor Direct Recognition PAMPs (for example CD14, DEC205, collectins), other (for example complement receptors) then discern the product that is produced by PAMP identification.The member of these receptor families can be divided three classes usually: 1) circulation body fluid receptor in blood plasma; 2) at the endocytosis receptor of immunocyte surface expression; With 3) can be at the frizzled receptor of cell surface or cell inner expression.The PRRs of cell can express on congenital immune effector lymphocyte, is the cell of the professional APCs in the adaptive immunity comprising function, also can express on the cell of the top layer epithelium that runs into pathogen when infecting at first.PRRs also can induce the expression of a cover endogenous signal, for example inflammatory cytokine and chemotactic factor.The example of the PRRs that the present invention is useful is including, but not limited to following: C-type clusterin (for example body fluid as collectins (MBL) and cell as macrophage C-type clusterin, macrophage mannose receptor, DEC205); Contain the multiple protein of rich leucine (for example Toll receptor and TLRs, CD14, RP105); Scavenger (scavenger) receptor (for example macrophage scavenger receptor, MARCO, WC1); And pentraxins (for example C-reactive protein, serum, amyloid P, LBP, BPIP, CD11b, C and CD18).

" peptide mimics " refers to the protein or the peptide that are very similar to the nonprotein molecule and have similar action or function with the nonprotein molecule.Selectable, peptide mimics can be nonprotein molecule or the non-peptide molecule that is very similar to peptide or protein and has similar action or function with peptide or protein.

" pharmaceutically acceptable carrier " refers to the carrier that receptor (generally being mammal) can tolerate.

" protein chimeric construct body " refers to that wherein antigen and PAMP or PAMP analogies are the polymeric chimeric construct body of aminoacid.

" recombinant " refers to the genetic stocks that makes by montage gene, gene derivative or other genetic stockss.As used herein, " recombinant " also refers to the product (for example recombinant protein) by the recombination generation.

" spacer " refers to any chemical individual that is used for physically separating these two parts between two chemical parts.Thereby the chemical individual between PAMP or PAMP analogies and antigen is spacer.The example of spacer is including, but not limited to nucleic acid (for example non-transcribed DNA between two sections DNA that transcribe), aminoacid, saccharide (for example monosaccharide, disaccharide, trisaccharide) and lipid.

" strong immunization reaction " refers to by the inductive immunoreation of chimeric construct body, its intensity with by approximately identical or higher with the inductive reaction of complete Freund's adjuvant (CFA) antigens mixed.

" treatment effective dose " refers to can produce the amount of the medium (for example vaccine) of measurable positive interaction in patient.

" class Toll receptor " (TLR) refers in the receptor protein family with the Toll protein homology of drosophila yellow gorilla (Drosophila melanogaster) any one.TLRs also refers to I type transmembrane signal receptor protein, it is characterized in that having the outer rich leucine repetitive structure territory of born of the same parents and the interior and interior homeologous domain of interleukin 1 receptor born of the same parents of born of the same parents.TLR family particularly comprises among the people at other species including, but not limited to mice TLR2 and TLR4 and congener thereof.The present invention has also defined Toll receptor protein and TLRs, other members' the nucleic acid of wherein encoding in this proteinic nucleic acid and coding Toll protein and TLR protein TLR2, TLR4 and the TLR family is compared the sequence identity that has at least about 70%, more preferably, have sequence identity, more preferably, have sequence identity at least about 85% at least about 80%, again more preferably, have sequence identity, most preferably, have sequence identity at least about 95% at least about 90%.In addition, the present invention also considers Toll receptor and TLRs, wherein such Toll receptor and the aminoacid sequence of TLRs are compared the sequence identity that has at least about 70% with Toll protein with the aminoacid sequence of TLRs, TLR2, TLR4 and congener thereof, more preferably, has sequence identity at least about 80%, more preferably, has sequence identity at least about 85%, again more preferably, has sequence identity at least about 90%, most preferably, has sequence identity at least about 95%.

" tumor " refers to a large amount of to lack the proliferative cell of normal growth control in various degree.As used herein, " tumor " comprises " pernicious " tumor (another kind of extreme case) that slowly outgrowth " optimum " tumor (a kind of extreme case) is invaded adjacent tissue to quick hypertrophy.

" vaccine " refers to comprise the compositions of a kind of antigen and selectable other accessory molecules, its purpose is to give an object to stimulate this antigenic immunoreation of antagonism specifically with such compositions, and preferably cause immunological memory, make this object can cause immunoreactive enhancing when certain time runs into this antigen in future.The example of other accessory molecules is adjuvant (they are nonspecific immunity stimulation molecules) and the pharmacokinetics of other enhancement antigens and/or the molecule of pharmacokinetic property.By convention, vaccine is usually by forming as the antigenic organism that causes disease (suitably attenuation or kill) or the some parts of pathogenic organism body.The virus of the organism of attenuation such as attenuation or the antibacterial of attenuation are operated some or all abilities that make that their forfeitures are grown in its natural host.There is a cover to be used to prepare the biological technique method of vaccine at present.(referring to for example W.Bains (1998) " biotechnology A is to Z " (Biotechnology From A to Z), SecondEdition, Oxford University Press).The whole bag of tricks is including, but not limited to following:

1) viral vaccine of forming by the virus that changes in the heredity.Can design virus makes that they are harmless but still can duplicate (though sometimes inefficiently) in the cultured animals cell.Another kind method is that the protein gene from a kind of pathogenic virus is cloned in another harmless virus, makes that the virus of gained has some immunological properties of pathogenic virus and do not cause any disease as a result.The example of a kind of method in back including, but not limited to, with the meat bait distribute as the cowpox and the adenovirus of the change of rabies vaccine and design the vaccinia virus of producing hemagglutinin and rinderpest virus fused protein;

2) the enhanced bacterial vaccine of forming by antibacterial, when wherein antibacterial is dead, strengthen with genetic engineering its value as vaccine (for example to the bacillus coli vaccine of pig, to salmon in the bacterial vaccine of furunculosis).Recombinant DNA technology can be used for deleting the Disease-causing gene in the antibacterial or the epitope of protectiveness in the pathogen is forwarded in a kind of safe antibacterial;

3) the bio-pharmaceutical vaccine is made up of protein or proteinic part, and the part of this protein or albumen egg is identical with shell or the protein in the cell wall of virus, fungus or antibacterial, can prepare by recombinant DNA method;

4) multiple antigenic peptide (MAPs) is (normally on the polylysine skeleton) peptide vaccine that chemically adheres to, and makes several vaccines to send simultaneously;

5) the polyprotein vaccine is made up of a kind of single protein by the genetic engineering preparation, to such an extent as to form the part of successive polypeptide chain from the different peptides of purpose organism; With

That 6) produces in transgenic plant can be used as food with vaccine that oral vaccine is provided the vaccine delivery of edible banana (for example by).

3. specific embodiment

A. fused protein

The present invention is based in part on unexpected discovery, and the vaccine (for example fused protein BLP/E α) that promptly comprises PAMP and antigenic chimeric construct body has shown that conventional vaccine is replenishing the basic amynologic characteristic or the character of expecting behind the adjuvant.

On the one hand, the present invention is based on BLP/E α induces the activation of NF-κ B and IL-6 respectively in macrophage and dendritic cell this discovery of production, proves that vaccine can activate innate immune system.The activity of BLP/E α and LPS quite, and its activity do not cause by endotoxic pollution, because verified its can not be suppressed by polymyxin B.

On the other hand, the present invention is based on this discovery of maturation that BLP/E alpha fusion protein matter can be induced dendritic cell, and this point is confirmed inducing of cell surface expression by costimulatory molecules B7.2.In addition, BLP/E α suitably targeting antigen processes and presents approach, and has produced a functional E α peptide/MHC Type II complex.This result is confirmed by the facs analysis of antibody that use is specific to the complex of E α peptide and MHC Type II.

In addition, the present invention be based on comprise BLP/E α chimeric construct body recombiant vaccine can external by stimulate dendritic cell activate and generate T-cell receptors sepcific ligands (E α/MHC-II) and this surprising discovery of activation antigen specificity T-cell effect.In addition, to the result of mouse immune and as a result of and the antigen specific T that takes place-cell effect proves recombiant vaccine activation antigen specificity T-cell effect in vivo with BLP/E α.For comparing, mice is carried out immunity with the E α peptide that has mixed complete Freund's adjuvant (CFA).Recombiant vaccine of the present invention has been induced the stronger immunoreation that is produced than by the E α peptide that has mixed CFA in mice.

The present invention is also based on this surprising discovery of minimum inflammatory reaction (with comparing by adjuvant is inductive) of having carried out immune induction with the recombiant vaccine that comprises chimeric construct body of the present invention.Yet As mentioned above, although lured the inflammatory reaction that weakens different, this vaccine has unexpectedly been induced intensive immunoreation.Thereby the vaccine method that the present invention describes makes undesired side effect (for example over-drastic inflammatory reaction) reduce to minimum and still induces highly effective specific immune response.The present invention also provides the fused protein that comprises at least a antigen molecule or antigenic structure territory and at least a PAMP or PAMP analogies to be used as vaccine.Preferably, this fused protein has maximum immunogenicity and only induces appropriate inflammatory reaction.Multiplet territory and/or the more aforesaid quantity that increases antigen or epitope that makes up by utilizing multiple antigen protein and/or identical or different antigen protein are that the present invention considers.Determine PAMP to the optimal proportion of antigen molecule so that the immunogenicity maximization and inflammatory reaction is minimized or controlled be in technical staff's technical scope.

Utilize emerging system to produce recombinant polypeptide and have several advantages.At first, heterologous protein or peptide are usually by host's proteasome degradation; This can be by utilizing a gene fusion expression system to be avoided, especially to little peptide.Secondly, set up general and effective purification scheme for several fusion partners.Utilize a kind of fusion partner can allow the sharp separation of recombinant peptide as affine handle.Once more, by using different fusion partners, recombinant products can be positioned different compartments or make its secretion; This strategy can cause the directed location of the purification of fusion partner compartment easier and/or fused protein.

In addition, there is the available method of various chemolysis or enzymolysis fused protein that the available strategy of the catabolite that acquisition wants in a large number can be provided.Often the emerging system that uses is: staphylococcal protein A emerging system and synthetic ZZ variant, and it has the IgG affinity and is used to produce antibody to small peptide; Glutathione S-transferase emerging system (people (1998) Gene 60 such as Smith); The beta galactosidase emerging system; With trpE emerging system (Yansura (1990) Methods Enzym.185:61).Some this systems are commercially available test kits, comprise carrier, purification composition and detailed description.

The present invention also considers to have the fused protein to the modification of the affinity of metal (metal ion) affinity substrate, so as to by in the fused protein sequence, add, disappearance or replace and introduce one or more specific melts combine or metalchelated amino acid residue serves as a mark.The suitableeest ground, with fusion partner such as antigen or PAMP sequence modification so that it contains metal-chelating aminoacid labelling; If yet change antigen and PAMP can not to ligand-binding site point, avtive spot or other functional sites have a negative impact and/or do not destroy tertiary structure relation important in the protein, also can provide a melts combine site by this change.These melts combine residues or metal-chelating residue can be identical or different, and can be selected from cysteine, histidine, aspartic acid, tyrosine, tryptophan, lysine and glutamic acid, and fused protein and melts combine or the chelating of location to allow to express.Histidine is preferred melts combine residue.Melts combine/chelating residue is located according to the overall tertiary structure of fused protein, and feasible combination to metal/chelating maximizes and expression or proteinic bioactive interference to fused protein are minimized.

The fusion sequence of antigen, PAMP and labelling optionally contains connection peptides.This connection peptides can be separated labelling and antigen sequence or PAMP sequence.If sequence with conventional chemical or alternative division of enzyme method or digestion of used like this connection peptides coding, this labelling can separate with the remainder of fused protein after purification so.For example, the division site of selecting in labelling can be enzyme division site.The example in suitable enzyme division site comprises the cleavage site of protease, as enterokinase, factor Xa, trypsin, collagenase and thrombin.Alternatively, the division site in the connector can be when can splitted site when (for example Bromine cyanide., azanol or low pH) in the chemical drugs that is exposed to a kind of selection.

Division in the division site of selecting makes labelling separate with antigen/PAMP fused protein.So can obtain the antigen/PAMP fused protein of the form of purifying, not contain any fragments of peptides of expressing or purifying that connected in the past to be easy to.Be inserted on the connector useful in the fusion sequence of the present invention if will divide the site, the present invention is not limited in this division site.Any division site of wanting all can be used for this purpose, and wherein many all are known in the field.

Selectable connection peptides can be served as the purposes except that the division site is provided in the fused protein of the present invention.As in one embodiment, but be not limited to this, connection peptides can be inserted between PAMP and the antigen to prevent or to alleviate sterically hindered between two domains.In addition, connection peptides can be supplied with the modification after the translation, includes, but are not limited to this, for example phosphorylation site, biotinylation site, sulfation site, carboxylation site, fat site, glycosylation site etc.

In one embodiment, fused protein of the present invention contains one and directly is fused to antigen sequence on the PAMP sequence at its aminoterminal or c-terminus.In another embodiment, the fused protein that comprises antigen and PAMP sequence of the present invention directly is fused on the labelled sequence at its aminoterminal or c-terminus.The fused protein of gained is a solubility kytoplasm fused protein as a result.In another embodiment, fused protein further comprises a connector sequence of inserting between antigen sequence and PAMP sequence or labelled sequence.This fused protein is also with solubility kytoplasm protein production.

B. antigen

As used herein, " antigen " is anyly can induce sensitivity and/or immunoreation sexual state after arbitrary incubation period (being generally a couple of days or a few weeks longer in the people), and in vivo or external may be obvious that with the antibody of the object of sensitization and/or the material of immune cell responses.Antigenic example including, but not limited to relevant antigen, the especially pathogen of: (1) microorganism as the antigen of virus, fungus or antibacterial or derived from their immunogenic molecules; (2) " self " antigen, comprise jointly cellular antigens (comprise and contain the relevant antigenic cell of normal transplantation antigen and/or tumor), RR-Rh antigen and peculiar in or be specific to the antigen of special cells or tissue or body fluid; (3) the relevant antigen of allergen, for example those are associated with environment allergen (for example grass, pollen, mould, dust, insecticide and squama), professional allergen (for example milk, squama, urethanes, epoxy resin), food (for example shellfish, Semen arachidis hypogaeae, egg, milk product), medicine (for example antibiotic, anesthetis); (4) vaccine (for example influenza vaccines).

The lymphocytic antigen of T-processing and the identification of the peptide showed very major part depended on antigenic aminoacid sequence rather than antigenic three dimensional structure.Thereby used antigen part can contain epitope or purpose ad hoc structure territory rather than full sequence in vaccine of the present invention.In fact, the antigen part of vaccine of the present invention can comprise one or more immunogenicity of antigens part or derivant rather than whole antigen.In addition, in order to make the B-lymphocyte antigenic space epitope is produced antibody response, vaccine of the present invention can contain the whole antigen with complete three dimensional structure or keep the antigenic part of antigenic determinant three dimensional structure.

1. the relevant antigen of pathogen.The antigenic specific examples that pathogen is relevant includes, but are not limited to cowpox, fowlpox virus, the turkey influenza virus, bovine leukemia virus, the cat leukemia virus, bird flu, chicken Pneumovirinae (chicken pneumovirosis virus), Canine Parvovirus, equine influenza, FHV, Avian pneumo-encephalitis virus (NDV), chicken/Pennsylvania/1/83 influenza virus, infectious bronchitis virus, dengue virus, Measles virus, rubella virus, pseudorabies, Epstein-Barr virus, HIV, SIV, EHV, BHV, HCMV, Hantaan virus, C.tetani, mumps virus, Measles virus (morbillivirus), herpes simplex types 1 virus, herpes simplex types 2 virus, human cytomegalic inclusion disease virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, respiratory syncytial virus, the human papillomavirus, influenza virus, Salmonella (Salmonella), neisseria (Neisseria), Borrelia (Borrelia), chlamydia (Chlamydia), Bordetella (Bordetella) and Plasmodium (Plasmodium) and toxoplasma (Toxoplasma), Cryptococcus (Cryptococcus), streptococcus (Streptococcus), staphylococcus (Staphylococcus), haemophilus (Haemophilus), Diptheria, tetanus (Tetanus), pertussis (Pertussis), Escherichia (Escherichia), Candida (Candida), aspergillus (Aspergillus), inner amoeba (Entamoeba), giardia lamblia (Giardia) and Trypanasoma.

2. the relevant antigen of cancer.Method and composition of the present invention also can be used for producing the vaccine to the proteantigen of antitumor association, and antigen wherein is the antigen of melanoma association, the antigen of breast carcinoma association, the antigen of colorectal carcinoma association, the antigen of carcinoma of prostate association etc.

Tumor example relevant or the tissue specificity proteantigen useful in such vaccine is including, but not limited to being selected from the antigen in the following table.

The cancer type	Antigen
The cancer type	Antigen	Prostate	Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), Her-2neu, SPAS-1
Melanoma	TRP-2, tryrosinase, Melan A/Mart-1, gp100, BAGE, GAGE, GM2 ganglioside	Prostate
Melanoma		Breast	Her2-neu, kinesin 2, the TATA element regulation factor 1, oncoprotein matter D52, MAGE D, ING2, HIP-55, the TGF-1 anti-apoptotic factor, HOM-Mel-40/SSX2
Testis	MAGE-1、HOM-Mel-40/SSX2、NY-ESO-1	Breast
Testis	MAGE-1、HOM-Mel-40/SSX2、NY-ESO-1	Colorectum	EGFR、CEA
Lung	MAGE?D、EGFR	Colorectum	EGFR、CEA
Lung	MAGE?D、EGFR	Ovary	Her-2neu
Several cancers	NY-ESO-1, glycoprotein MUC1 and MUC10 mucin, p53 (especially Tu Bian form), EGFR	Ovary	Her-2neu
Several cancers		Miscellaneous tumor antigen	CDC27 (comprising proteinic saltant), triose-phosphate isomerase

Tumor is in order to produce proliferative cell and malignant cell, and they must form blood vessel.Prevent strategy potentialization in treatment of tumor-blood-vessel growth.Method and composition of the present invention also can be used to produce the vaccine of antagonism tumor-blood-vessel growth.The example of the target antigen of such vaccine is VEGF, vascular endothelial growth factor receptor, fibroblast growth factor and fibroblast growth factor acceptor etc.

3. the relevant antigen of allergen.Method and composition of the present invention can be used to prevent or treat allergy and asthma.According to the present invention, one or more protein allergens can be connected on one or more PAMPs, make PAMP/ allergen chimeric construct body, and give that with it this antigen is had allergic object.Thereby method and composition of the present invention also can be used to make up and can suppress allergic vaccine.In this case, can start the related or combination of PAMP of Th1 reaction after allergen and a kind of TLR of being attached to go up, this PAMP is including, but not limited to BLP or flagellin.For example, the startup of innate immunity is trended towards having the feature of production justacrine cytokine such as IL-12 by TLR, this has caused a kind of so-called Th1 reaction rather than general Th2 reaction in the object, and the latter triggers the B-cell and produces IgE (general metamorphosis and/or anaphylactoid starting material).It is Th1 effector lymphocyte that the IL-12 that dendritic cell and macrophage produce when PAMP is attached on their TLRs will instruct the T-cell differentiation.To block the differentiation of the Th2 cell that produces IL-4 by cytokine such as interferon-that the Th1 cell produces, thereby stop as the IgE homotype production of antibodies that causes allergic reason.

The specific examples of the proteantigen that useful allergen is relevant in the method and composition of the present invention including, but not limited to: derived from the allergen of pollen, for example those are derived from such as Japanese cedar (Cryptomeria, Japanese cedar (Cryptomeria japonica)) and so on tree, those are derived from such as orchard grass (orchardgrass, orchardgrass (Dactylis glomerata)) and so on grass (grass family), those are derived from such as artemisiifolia (Ambrosia, artemisiifolia (Ambrosiaartemisiifolia)) and so on weeds; The specific examples that pollen allergy is former, comprise former Cry j 1 of Japanese cedar pollen allergy (J.Allergy Clin.Immunol. (1983) 71:77-86) and Cry j 2 (FEBS Letters (1988) 239:329-332) and artemisiifolia allergen Amb a I.1, Amb a I.2, Amb a I.3, Amb a I.4, Amb a II etc.; Allergen derived from fungus (aspergillosis, candidiasis, interlink spore genus etc.); Derived from demodicid mite (from the allergen of dermatophagoides pteronyssinus (Dermatophagoides pteronyssinus), dust demodicid mite (Dermatophagoides farinae) etc.; The specific examples of demodicid mite allergen comprises Der p I, Der p II, Der p III, Der p VII, Der f I, Derf II, Der f III, Der f VII etc.) allergen; The house dust; Allergen (for example former Fel d of cat allergy I) derived from animal skin fragment, Excreta and hair; Derived from the allergen of insecticide (for example the scale hair or the squama of moth, butterfly, Chironomidae etc., the poisonous substance of Vespidae, for example gold ring wasp (Vespa mandarinia)); Food allergy former (egg, milk, meat, seafood, beans, frumentum, fruit, nut and vegetable etc.); Allergen derived from parasite (for example ascarid and nematicide, as Anisakid nematode (Anisakis)); And based on the medicine (for example insulin) of protein or peptide.Many these allergens all are commercially available.

In another embodiment, can realize by giving a kind of protein PAMP chronic allergic prophylactic treatment.In a preferred embodiment, the PAMP of preventative vaccine is OMP, more preferably is OspA, most preferably is BLP.Alternatively, flagellin can be used as PAMP.

4. other diseases antigen.What also consider among the present invention is the antigenic vaccine of antagonism and the connection of the disease association except that cancer, allergy and asthma.One in many cases so, but be not limited thereto, the protein cleavage product that is called the beta amyloid precursor protein matter of " amyloid-β peptide " is associated extracellular gathering with the pathogeny of Alzheimer disease.(people such as Janus, Nature (2000) 408:979-982; People such as Morgan, Nature (2000) 408:982-985).Thereby used chimeric construct body can comprise antigenicity domain (as the antigenic portions of construct) and the PAMP or the PAMP analogies of amyloid-β peptide or amyloid-β peptide in the vaccine of the present invention.In the example of its side's disease, can produce vaccine, during described other disease is listed in the table below at oneself protein matter or self peptide.

Disease	Antigen
Disease	Antigen	Autoimmune disease	The HLA-allele that disease connects (for example HLA-DRB1, HLA-DR1, HLA-DR6 B1 protein or its fragment, chain gene (Chain gene)); TCR chain subgroup; CD11a (the antigen 1 of leukocyte function association; LFA-1); IFN γ; IL-10; The TCR analog; The IgR analog; 21-hydroxylase (for Addison's disease); Calcium sensory receptors (for acquired hypoparathyroidism); Tryrosinase (for vitiligo)
Cardiovascular disease	Ldl receptor	Autoimmune disease
Cardiovascular disease	Ldl receptor	Diabetes	Glutamate decarboxylase (GAD); Insulin B chain; PC-1; IA-2; IA-2b; GLIMA-38
Epilepsy	NMDA	Diabetes

C.PAMPs

PAMPs is by the total discrete molecular structure of a big group Institute of Micro-biology.They are the metabolic conservative products of microorganism, are difficult for producing antigenic variability and being different from autoantigen.(people (1997) Current Opinion in Immunology 9:4 such as Medzhitov).

PAMPs can be by the molecular composition of following type or is found therein, but is not limited to these molecules: flagellin, lipopolysaccharide (LPS), porin, the protein of lipid A-association (LAP), lipopolysaccharide, pilin matter, unmethylated CpG primitive, DNA of bacteria s, double-stranded viruses RNAs, mannan, the protein of cell wall association, heat shock protein, glycoprotein, lipid, the cell surface polysaccharide, polysaccharide (as Peptidoglycan), phosphatidylcholine, teichoic acid (for example lipoteichoic acid), mycobacteria cell wall constituent/film, bacterial lipoprotein (BLP), outer membrane protein (OMP) and outer surface proteins A (Osp A).(people (1996) Microbiol.Review60:316 such as Henderson; People such as Medzhitov (1997) Current Opinion in Immunology 9:4-9).

The preferred PAMPs of the present invention comprises that those contain DNA-encoded protein matter composition as BLP, eisseria porin, OMP, flagellin and OspA, because these can be used as fusion partner to prepare the preferred embodiment of the invention, promptly comprise the fused protein of PAMP and antigen (being preferably autoantigen).The used a kind of preferred PAMP of the present invention is BLP, because known BLP can induce activation people (1996) Microbiol.Review 60:316 such as () Henderson of innate immunity reaction and show and can be discerned by TLRs people (1999) Sciences 285:763 such as () Aliprantis.Similarly, confirmed that also flagellin can induce the character of innate immunity (people (2001) J.Immunol.166:1248 such as Eaves-Pyles; People such as Gewirtz (2001) J Clin Invest.107:99; Aderem, Presentation at KeystoneSymposium, Keystone, CO, 2001).

In addition, the present invention consider the derivant of the PAMPs that can be discerned by innate immune system, partly, part or peptide produce vaccine.As used herein, term " fragment of PAMP ", " part of PAMP ", " part of PAMP " and " peptide of PAMP " all refer to the immunostimulation part in the whole PAMP molecule.Thereby used PAMPs can comprise immunostimulation part or derivant rather than whole PAMP, for example the escherichia coli cell wall matter lipoprotein amino acid/11 to 24 of PAMP in vaccine of the present invention.

In another embodiment, the present invention considers the peptide mimics of nonprotein PAMPs.The peptide mimics of polysaccharide and Peptidoglycan is the example of the used peptide mimics of the present invention.The present invention considers to identify with the phage system of selection peptide mimics of these nonproteins PAMPs.For example, a kind of antibody that produces at nonprotein PAMP can be used to screen and contain the phage library of short peptide sequence at random.The sequence of selecting is separated and measure to determine its effectiveness as the protein derivatives of nonprotein PAMP in the chimeric construct body of the present invention.Such peptide mimics can be used for producing recombiant vaccine disclosed herein.

In the another one embodiment, the present invention considers the analog of the analogies of the analogue type (mimics) of PAMP or PAMP, and wherein the analog of aminoacid and/or peptide has substituted the PAMP that contains peptide or aminoacid and/or the peptide residue of protein PAMP respectively.

In another embodiment, the chimeric construct body is one and comprises CpG or CpG-DNA and a kind of antigenic construct.CpG or CpG-DNA can combine with protein or nonprotein antigen.In addition, can make the CpG of structure, function, antigenicity or the immunogenic properties of having imitated CpG or the peptide mimics of CpG-DNA, and use it for generation a kind of antigen-PAMP (wherein PAMP is a kind of CpG peptide mimics) protein chimeric construct body.This chimeric construct body can make by recombinant DNA technology, and the fused protein of expression can be used to the compositions and methods of the invention.

D. peptide mimics

The present invention also comprises the analogies of a kind of PAMP or antigenic three dimensional structure.Especially, the present invention also comprises the peptide of very similar non-peptide PAMPs and antigenic three dimensional structure.Such peptide provides alternative to non-polypeptide PAMPs or antigen respectively, this realizes by following advantage is provided, as: more economical production, better chemical stability, enhanced pharmaceutical properties (half-life, absorbability, usefulness, effectiveness) and/or specificity (for example broad-spectrum biological activity) and other advantages of changing.

On the contrary, the analog of PAMP and/or antigen protein can synthesize like this so that a kind of or both partially or entirely form by aminoacid and/or peptide analogues.Such analog can contain the aminoacid that non-natural exists, or natural existence but in protein uncommon aminoacid, including, but not limited to D-aminoacid or as the aminoacid of Beta-alanine, ornithine or canavanine etc., wherein many all is known in the field.Alternatively, PAMP and/or antigenic analog can synthesize like this so that a kind of or both partially or entirely form by the peptide analogues that contains non-peptide bond, wherein many examples all are known in the field.Such analog can provide specificity (for example broad-spectrum biological activity) and other advantages of better chemical stability, enhanced pharmaceutical properties (half-life, absorbability, usefulness and effectiveness etc.) and/or change when comparing with naturally occurring PAMP and/or antigen.

In one form, the molecular structure of consideration is the molecule that contains peptide of mimic protein secondary structure element.(referring to as people such as Johnson (1993) Peptide Turn Mimetics, inBiotechnology and Pharmacy, people such as Pezzuto, (editors) Chapman andHall).Expect that such molecule can allow the intermolecular interaction similar to natural molecule.

In another form, peptide analogues is used as the nonprotein medicine usually and is used for medicine and produces, and the character of its character and original peptide is similar.The non-peptide compound of these types is also referred to as " peptide mimics " or " analogies of peptide (peptidomimetics) " (Fauchere (1986) Adv.Drug Res.15,29-69; People such as Veber (1985) Trends Neurosci.8:392-396; People such as Evans (1987) J.Med.Chem.30:1229-1239) also develops with computerized molecular simulation usually.

Go up the structurally similar peptide mimics of useful peptide with treatment and can be used to produce equal treatment or preventive effect.Usually, peptide mimics structurally similar (for example a kind of polypeptide) with biochemical property or pharmaceutical active with the example polypeptide, but one or more peptide bond is arranged optionally by by being selected from-CH ₂NH-,-CH ₂S-,-CH ₂-CH ₂-,-CH=CH-(cis and trans) ,-COCH ₂-,-CH (OH) CH ₂-,-CH ₂The key of SO-etc. substitutes.(Morley (1980) Trends Pharmacol.Sci.1:463-468 (general remarks); People such as Hudson (1979) Int.J.Pept.ProteinRes.14:177-185 (CH ₂NH-, CH ₂-CH ₂-); People such as Spatola (1986) Life Sci.38:1243-1249 (CH ₂-S); Hann (1982) J.Chem.Soc.Perkin Trans.1:307-314 (CH=CH-, cis and trans); People such as Almquist (1980) J.Med.Chem.23:1392-1398 (COCH ₂-); People such as Jennings-White (1982) Tetrahedron Lett.23:2533 (COCH ₂-); People such as Holladay (1983) Tetrahedron Lett.24:4401-4404 (C (OH) CH ₂-); And Hruby (1982) Life Sci.31:189-199 (CH ₂S-); Wherein each is all quoted as a reference herein).

The labelling of peptide mimics is usually directed to directly or adds one or more labellings with covalent manner to the non-interference position of peptide mimics by a spacer (for example amide group), and non-interference position wherein is to be predicted by quantitative structure-activity data and molecular simulation.Non-interference position so generally is not form direct position contacting (for example, they are the noncontact sites in the PAMP-PRR complex in the present embodiment) with macromole, and wherein peptide mimics is incorporated on the described macromole to produce therapeutic effect.Derive (for example the adding labelling) of peptide mimics should not disturb the biology of wanting or the pharmaceutical active of peptide mimics in itself.

The PAMP peptide mimics can be built by substituting aminoacid with organic moiety by the drug design based on structure.(Hughes(1980)Philos.Trans.R.Soc.Lond.290：387-394；Hodgson(1991)Biotechnol.9：19-21；Suckling(1991)Sci.Prog.75：323-359)。

The design of peptide mimics can be by identifying that can strengthen or weaken the amino acid mutation that PAMP is attached to its PRR assists.Available method comprises yeast two-hybrid method (people (1991) Proc.Natl.Acad.Sci.USA 88:9578-9582 such as Chien) and phage display method.The double cross method is surveyed the protein protein interaction in the yeast.(people (1989) Nature340:245-246 such as Fields).The phage display method survey immobilization protein and the interaction between the protein of phage such as λ and M13 surface expression.(people (1993) Strategies 6:2-4 such as Amberg; People such as Hogrefe (1993) Gene 128:119-126).These methods allow the evaluation of just selecting and bearing selection and determine these interactional sequences to protein protein interaction.

The synthetic conventional method of peptide is known in the field.(synthetic (the Amino Acid and Peptide Synthesis) of Jones (1992) aminoacid and peptide, Oxford University Press; Jung (1997) combined peptide and non-peptide library handbook (Combinatorial Peptide andNonpeptide Libraries:A Handbook), John Wiley; The practical study course (Peptide Chemistry-A Practical Textbook) of people such as Bodanszky (1993) chemistry of peptides, SpringerVerlag).

E. flagellin PAMPs

Bacterial flagellum is made up of the structural protein flagellin, and this protein is induced the expression of chemotactic factor IL-8 and the activation of NF-κ B in people and mouse cell.In addition, flagellin activates mammalian cell by class Toll receptor TLR5.These find, and flagellin extremely conservative fact in antibacterial show flagellin be a kind of can be by the molecule pattern (PAMP) of the pathogen association of innate immune system identification.

Because flagellin is a kind of protein and PAMP, it also is suitable for producing the reorganization fusion bacterin.As the embodiment part is described below, checked a series of fusion constructs to activate the ability of mammal innate immune system.In experiment, the activation of NF-κ B is used as readout (read-out), because it is the important path that indication triggers class Toll receptor, and has held itself out to be a character of reorganization fusion bacterin.

The conservative modification of F.PAMPs

The present invention also considers to discern the conservative modification of naturally occurring protein PAMPs, PAMPs peptide and the PAMPs peptide mimics of corresponding PRRs.Such modification is the example of PAMP analogies.Conservative modification comprises with the another kind of amino acid whose sudden change in following one group of a kind of amino acid replacement:

1. little aliphatic, nonpolar or slight polar residue: Ala, Ser, Thr, Pro and Gly;

2. polar, electronegative residue and amide thereof: Asp, Asn, Glu and Gln;

3. polar, positively charged residue: His, Arg and Lys;

4. big aliphatic, nonpolar residue: Met, Leu, Ile, Val and Cys; With

5. aromatic residue: Phe, Tyr and Trp.

Selected alternative type can be based on the analysis (people such as Schulz of amino acid replacement frequency among the PAMPs of different plant species, protein structure principle (Principles of Protein Structure), Springer-Verlag, 1978, pp.14-16), also can form analysis (people (1974) the Biochemistry 13:211 such as Chou of potentiality based on structure by Chou and Fasman development; People such as Schulz, protein structure principle (Principles in Protein Structure), Springer-Verlag, 1978, pp.108-130) and based on by the protein hydrophobic pattern analysis of Kyte and Doolittle development people (1982) J.Mol.Biol.157:105-132 such as () Kyte.

The present invention also considers not influence the conservative modification that PAMP is attached to its PRR ability.The present invention includes and changed total electrical charge, structure, hydrophobicity/hydrophilic PAMPs, these changes be produce by amino acid replacement, insertion or disappearance and keep and/or improve and their ability of receptors bind.Preferably, the its corresponding wild type PAMP of PAMP of sudden change compares, have sequence identity, more preferably, have sequence identity at least about 80% at least about 70%, more preferably, have sequence identity, more more preferably, have sequence identity at least about 90% at least about 85%, most preferably, has sequence identity at least about 95%.

Numerous methods of determining percent homology are known in the field.6.0 the GAP computer program of version can be provided by University of Wisconsin's heredity computer set (University of WisconsinGenetics Computer Group), and has utilized the comparison method via Smith and improved Needleman of Waterman and Wunsch.(people (1970) J.Mol.Biol.48:443 such as Needleman; People such as Smith (1981) Adv.Appl.Math.2:482).Numerous methods of determining concordance percentage ratio also are known in the field, and it also is the FASTA computer program that is provided by University of Wisconsin's heredity computer set that a preferable methods is to use.

G. combined therapy

The invention provides the method for treatment target, comprise and give object antibody or activated immunocyte and make the individual immunity that obtains passively, and give a kind of vaccine that comprises fused protein of the present invention, preferably, wherein institute's administered antibodies or activated immunocyte are same antigenic at the fused protein of vaccine.Such treatment can be in sequence, carries out in order or simultaneously.This combined therapy considers to use antigenic monoclonal or the polyclonal antibody at PAMP/ antigen fusant.

The invention provides the method for treatment target, comprise and give object antibody or activated immunocyte and make the individual immunity that obtains passively, and give a kind of vaccine that comprises chimeric construct body of the present invention, preferably, wherein institute's administered antibodies or activated immunocyte are at identical with the antigen of chimeric construct body antigenic.Such treatment can be in sequence, carries out in order or simultaneously.This combined therapy considers to use antigenic monoclonal or the polyclonal antibody at PAMP/ antigen chimeric construct body.

It is combined that the present invention also considers to comprise the use and the treatment that second kind of non-immunity instructed of vaccine of chimeric construct body of the present invention.A non-restrictive example of such combined therapy is to comprise the vaccine of a kind of fused protein of the present invention and the combination of a kind of chemotherapeutics such as anti-cancer chemotherapeutic agents.A further non-restrictive example of such combined therapy is to comprise the vaccine of a kind of fused protein construct of the present invention and a kind of combination of anti-angiogenic agent.A further non-restrictive example of such combined therapy is vaccine and a kind of radiotherapy such as the anticancer radiocurable combination that comprises a kind of fused protein of the present invention.One of such combined therapy more further non-restrictive example be the vaccine that comprises a kind of fused protein of the present invention with surgical method as removing or the combination of the surgical method that the minimizing blood vessel is blockaded.

What the present invention also considered is more than a kind of other treatment means and the combination of vaccine of the present invention.A non-restrictive example is the combination of vaccine of the present invention and passive immunization therapy treatment and chemotherapeutic treatment.In such combined therapy, treatment can be order or simultaneously.

The PAMP domain can comprise the immunostimulation part of whole PAMP or PAMP.Preferably, fused protein has maximum immunogenicity and induces minimum inflammatory reaction.The character of wanting so for example can be by using two or more different antigens and/or different antigenic part, and/or by using more than the same antigen of a copy or the part of same antigen, and/or obtain by both combination.Alternatively, can use the part of two or more different PAMPs or different PAMPs, and/or the identical PAMP of two or more copies or the part of identical PAMP, and/or both combinations.A further embodiment consideration comprises the fused protein of the part of multiple antigenic and/or antigenic part and multiple PAMPs and/or PAMPs.The ratio of wanting of determining PAMP antagonism prodomain is so that immunogenicity maximization and inflammatory reaction is minimized is in technical staff's technical scope.

Utilize emerging system to produce recombinant polypeptide and have several advantages.At first, heterologous protein or peptide are usually by host's proteasome degradation; This can be by utilizing a gene fusion expression system to be avoided, especially for little peptide.Secondly, set up general and effective purification scheme for several fusion partners.Utilize a kind of fusion partner can allow the sharp separation and the purification of recombinant peptide as affine handle.Once more, by using different fusion partners, recombinant products can be positioned different compartments or make its secretion; This strategy can cause the directed location of the purification of fusion partner compartment easier and/or fused protein.

In addition, there is the available method of various chemolysis or enzymolysis fused protein that the available strategy of the peptide that acquisition wants in a large number can be provided.Often the emerging system that uses comprises: have the IgG affinity and be used to produce staphylococcal protein A emerging system and synthetic ZZ variant to the antibody of small peptide; Glutathione S-transferase emerging system (people (1998) Gene 60 such as Smith); The beta galactosidase emerging system; With trpE emerging system (Yansura (1990) Methods Enzym.185:61).Some this systems are commercially available test kits, comprise carrier, purification composition and detailed description.

The present invention also considers to have the fused protein to the modification of the affinity of metal ion affinity substrate, by in the fused protein sequence, add, disappearance or replace and introduce one or more specific melts combine or metalchelated amino acid residue with mark mode.The suitableeest ground, with or modify so that it contains an additional metal-chelating aminoacid labelling for antigen or for the fusion partner of PAMP domain.If yet the sequence of change antigen or PAMP domain can not to ligand-binding site point, avtive spot or other functional sites have a negative impact and/or do not destroy tertiary structure relation important in the protein, also can provide a melts combine site by this change.These melts combine sites or metal-chelating site can be identical or different, and can be selected from cysteine, histidine, aspartic acid, tyrosine, tryptophan, lysine and glutamic acid, and fused protein and melts combine or the chelating of location to allow to express.Histidine is preferred melts combine residue.Melts combine/chelating residue is according to the overall tertiary structure location of fused protein, and feasible combination to metal/chelating maximizes and make expression and its bioactive interference to fused protein to minimize.

The fusion sequence of antigen, PAMP and labelling can contain a connection peptides.This connection peptides can be separated labelling and antigen sequence or PAMP sequence.If sequence with conventional chemical or alternative division of enzyme method or digestion of used like this connection peptides coding, this labelling can separate with the remainder of fused protein after purification so.For example, selected division site can be enzyme division site in labelling.Suitable enzyme division site example comprises the division site of protease, as enterokinase, factor Xa, trypsin, collagenase, thrombin etc.Alternatively, the division site in the connector also can be can splitted site when the chemical drugs that is exposed to a kind of selection or condition are for example in Bromine cyanide., azanol or low pH or other chemical drugss as known in the art or the condition.

Division in the division site of selecting makes labelling separate with antigen/PAMP fused protein.So can obtain the antigen/PAMP fused protein of the form of purifying, not contain any peptide derivant of expressing or purifying that connected in the past to be easy to.Be inserted on the connector useful in the fusion sequence of the present invention if will divide the site, the present invention is not limited in this division site.Any division site of wanting all can be used for this purpose, and wherein many all are known in the field.

Another purposes of connection peptides is to add to instruct man-hour the antigen division to present peptide so that be easy on the APC surface in cell.Suitable division site can be inserted by connector, makes fused protein do not divided before by the APC internalization.In this case, such peptide division site can be incorporated between PAMP and the antigen to produce the intracellular antigen of no PAMP by connector.Such orientation division also can be used for promoting the generation of APC particular peptide uniquely, has identified these peptides and specific HLA haplotype interaction.Alternatively, isolate, so that these epitopes can be by suitable processing to present on the APC surface from single antigen or more than the connector that a kind of antigenic different structure territory can involved division site.

Selectable connection peptides can be served as the purposes except that the division site is provided in the fused protein of the present invention.As in one embodiment, but be not limited to this, connection peptides can be inserted between PAMP domain and the antigenic structure territory to prevent or to alleviate sterically hindered between two domains.In addition, connection peptides provides some things can for the modification after the translation, includes, but are not limited to, for example phosphorylation site, biotinylation site, sulfation site, carboxylation site, glycosylation site, fat site etc.

In one embodiment, fused protein of the present invention contains an antigenic structure territory or an immunogenicity of antigens part on the immunostimulation part that directly is fused to PAMP domain or PAMP at its aminoterminal or c-terminus.In another embodiment, fused protein of the present invention comprise the immunostimulation part of PAMP domain or PAMP be inserted into the antigenic structure territory or the immunogenicity of antigens part in and the sequence that can produce PAMP in post translational modification.In the another one embodiment, fused protein of the present invention comprises antigenic structure territory or the immunogenicity of antigens part in immunostimulation part that is inserted into PAMP domain or PAMP or the sequence that can produce PAMP in post translational modification.In another embodiment, the fused protein that comprises antigen and PAMP sequence of the present invention directly is fused on the labelled sequence at its aminoterminal or c-terminus.The fused protein of gained is a solubility kytoplasm fused protein as a result.In another embodiment, fused protein further comprises a connector sequence of inserting between antigen sequence and PAMP sequence or labelled sequence.This fused protein is also with solubility kytoplasm protein production.

H. recombinant technique

Protein PAMPs, proteantigen and derivant thereof can utilize the standard peptide synthetic technology to generate.Alternatively, recombinant technique can be used to generate the nucleic acid molecules of coded protein PAMPs, proteantigen and derivant thereof.

The nucleic acid of coding PAMP/ antigen fusant (as synthetic widow-or polynucleotide) can be synthetic by chemical technology at an easy rate, people's ((1981) J.Am.Chem.Soc.103:3185-3191) such as Matteucci phosphotriester method or use automatic synthesis method for example.In addition, bigger nucleic acid can as the synthetic one group segmental oligonucleotide of various modules that defines the nucleic acid of coding PAMP/ antigen fusant, connect these oligonucleotide to make up complete nucleic acid molecules subsequently easily by well-known method preparation.

The present invention further provides the recombinant nucleic acid molecules of coding PAMP/ antigen coalescence protein matter.As used herein, " recombinant nucleic acid molecules " refers at external nucleic acid molecules through molecule manipulation.The method that generates recombinant nucleic acid molecules is known in the field.(people (1989) molecular cloning laboratory manual (Molecular Cloning:A Laboratory Manual) such as Sambrook, ColdSpring Harbor Press).In preferred recombinant nucleic acid molecules, the nucleotide sequence of a coding PAMP/ antigen fusant is operably connected on one or more expression control sequencs and/or the carrier sequence.

As known in the art, the carrier sequence that the nucleotide sequence of coding PAMP/ antigen fusant is operably connected among the present invention and/or the selection of expression control sequenc directly depend on the functional character of wanting (as protein expression) and want transformed host cells.The carrier that the present invention considers can instruct the nucleotide sequence reproduce of coding PAMP/ antigen fusant at least or be inserted into host chromosome, preferably also expresses described nucleotide sequence.

The expression control element that is used to regulate the protein coding sequence expression that is operably connected is as known in the art, and including, but not limited to inducible promoter, constitutive promoter, secretion signal, enhancer, transcription terminator and other regulating elements.Preferably, used a kind of inducible promoter that is easy to control, for example can respond culture medium nutrition.

In one embodiment, the carrier that comprises the nucleic acid molecules of a coding PAMP/ antigen fusant will comprise the protokaryon replicon, for example have and instruct self-replicating and make recombinant nucleic acid molecules maintain the nucleotide sequence of intrachromosomal ability in prokaryotic host cell that is transformed such as bacterial host cell.Such replicon is as known in the art.In addition, the carrier that comprises the protokaryon replicon can comprise that also its expression can authorize the gene of a detectable labelling such as drug resistance.General antibacterial drug resistance gene is that those are authorized ampicillin (Amp) or tetracycline (Tet) resistance.

The carrier that comprises the protokaryon replicon can further comprise the protokaryon or a viral promotors that can instruct PAMP/ antigen fusant at bacterial host cell such as expression in escherichia coli (transcribe and translate).Promoter is that formed by nucleotide sequence and allow the RNA polymerase combination and transcribe an expression control element.With the promoter sequence of bacterial host compatibility generally be that restriction site provides with the plasmid vector that inserts nucleic acid fragment of the present invention by containing easily.General such vector plasmid is that (Richmond, the pUC8 that CA) provides, pUC9, pBR322 and pBR329 are by Amersham Pharmacia Biotech, pPL that Piscataway, NJ provide and pKK223 by Biorad Laboratories.

With the expression vector of eukaryotic cell compatibility, be preferably the carrier of those and vertebrate cells compatibility, also can be used to express the nucleic acid molecules of the nucleotide sequence that contains coding PAMP/ antigen fusant.Eukaryotic expression vector is as known in the art and several commercial source is arranged.Usually, such carrier provides the restriction site easily that inserts the dna fragmentation of wanting.General such carrier is pSVL and pKSV-10 (Amersham Pharmacia Biotech), pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC, #31255), carrier pCDM8 described herein and other similar carrier for expression of eukaryon.

The carrier for expression of eukaryon that is used for making up recombinant molecule of the present invention can further be included in eukaryotic cell effective as selective labelling, is preferably the drug resistance selected marker.A preferred drug resistance labelling is the gene that its expression causes neomycin resistance, for example neomycin phosphotransferase (neo) gene.(people (1982) J.Mol.Anal.Genet.1:327-341 such as Southern).Alternatively, selected marker can be positioned on the different plasmids, two carriers is introduced by cotransfection host cell, and selected by cultivating when the suitable drug at selected marker exists.

The present invention further provides nucleic acid molecules transformed host cells with coding PAMP/ antigen coalescence protein matter of the present invention.That this host cell can be protokaryon or eucaryon.The eukaryotic cell that can be used for expressing PAMP/ antigen coalescence protein matter is unrestricted, as long as cell line and cell culture processes compatibility, and with the propagation of expression vector and the expression compatibility of fused protein.Preferred eukaryotic host cell is including, but not limited to yeast, insecticide and mammalian cell, for example is preferably those from mice, rat, monkey or human fibroblast cell line's vertebrate cells.

Any prokaryotic hosts all can be used to the express recombinant nucleic acid molecules.Preferred prokaryotic hosts is escherichia coli.At PAMP is in the embodiment of lipoprotein, and the expression of PAMP/ antigen coalescence protein matter in bacterial cell is preferred.The expression of nucleic acid in bacterial cell system is that the suitable post translational modification of the protein portion in the assurance lipoprotein is desired.Preferably, the host cell of selecting to express PAMP/ antigen fusant (for example lipoprotein/antigen fusant) is the cell that produces the lipoprotein in lipoprotein/antigen fusant natively.

Nucleic acid molecules conversion proper host cell with coding PAMP/ antigen fusant of the present invention is to realize by the well-known method that generally depends on carrier and used host system type.As for the conversion of prokaryotic host cell, electroporation and salt processing method are general the employings.(referring to as people such as Cohen (1972) Proc.Natl.Acad Sci.USA 69:2110; People such as Maniatis, molecular cloning laboratory manual (Molecular Cloning:A Laboratory Manual), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); People such as Sambrook (1989)).As for transforming vertebrate cells with the carrier that contains rDNAs, electroporation, cationic lipid or salt processing method are general the employings.(referring to as people such as Graham, Virology (1973) 52:456; People such as Wigler (1979) Proc.Natl.Acad.Sci.USA 76:1373-76).

The success cell transformed, as the cell that contains the nucleic acid molecules of the PAMP/ antigen fusant of the present invention of encoding can be identified by well-known technology.For example, the cell that is got by the nucleic acid molecules of introducing coding PAMP/ antigen fusant of the present invention can be by the clone to produce single-population.Can be from the cell of this colony by results, cracking, their nucleic acid compositions can with as Southern (1975) (J.Mol.Biol.98:503) or people (1985) such as Berent (Biotech.3:208) described method check that the existence of recombinant molecule or the protein of these cells generation can measure by immunological method.

The present invention further provides and used a kind of nucleic acid molecules described herein to produce the method for PAMP/ antigen coalescence protein matter.Briefly, the production of recombinant protein generally comprises following steps.

At first, obtain the nucleic acid molecules of coding PAMP/ antigen coalescence protein matter.As indicated above then, this nucleic acid molecules is preferably placed and the effective chain position of suitable control sequence.Transform a suitable hosts with ceneme, host transformed is cultivated under the condition that allows production PAMP/ antigen coalescence protein matter.Selectable, with fused protein and culture medium or cell separation; The recovery of fused protein and purify and to tolerate under the situation of a little impurity optional at some.

Aforesaid each step can multiple mode be carried out.For example, the coded sequence of wanting can obtain from genomic fragment and be directly used among the suitable host.The structure of exercisable expression vector is that appropriate combination by replicon and control sequence realizes in multiple host.Control sequence, expression vector and method for transformation all depend on the host cell type that is used for expressing gene, and go through in front.Those skilled in the art can easily reequip any host/expression system as known in the art, and are used to produce PAMP/ antigen coalescence protein matter jointly with nucleotide sequence described herein.

Endonuclease is the nuclease that can cut the inside phosphodiester bond in the nucleic acid molecules.The example of nuclease is including, but not limited to S1 endonuclease, deoxyribonuclease (DNase I) and restriction endonuclease from fungus aspergillus oryzae (Aspergillus oryzae).Cutting as DNA operation basis is to finish with the enzyme of ligase (connection) with being called restriction endonuclease (cutting) with connection procedure.If there is not normally available suitable restriction endonuclease cleavage site, the end that then these cleavage sites can be added to coded sequence is inserted in these carriers so that resectable nucleotide sequence to be provided.

In addition, also the restriction endonuclease cleavage site can be inserted in the nucleotide sequence of coding PAMP/ antigen coalescence protein matter.Preferably, these cleavage sites are designed between the nucleotide sequence of the identical or different PAMPs of coding, between the identical or different antigen or between coding PAMP and the antigenic nucleotide sequence.Well known to a person skilled in the art that suitable cleavage site is including, but not limited to following site: EcoR I, BamH I, Bgl/II, Pvu I, Pvu II, Hind III, HinfI, Sau3A, Alu I, Taq I, Hae III and Not I.(T.A.Brown (1996) gene clone outline (Gene Cloning:An Introduction), Second Edition, Chapman﹠amp; Hall, Chapter 4:49-83).

I. conjugate

The present invention also comprises and comprises two or more covalently bound or non-covalent connections but " conjugate " of the molecule that is mutually related.Thereby, vaccine of the present invention comprises PAMP/ antigen conjugate, such as, but be not limited to following: protein/nucleic acid conjugate, nucleic acid/protein conjugate, nucleic acid/nucleic acid conjugate, peptide mimics/nucleic acid conjugate, nucleic acid/peptide mimics conjugate, peptide mimics/peptide mimics conjugate, lipopolysaccharide/protein conjugate, lipoprotein/protein conjugate, RNA/ protein conjugate, CpG-DNA/ protein conjugate, nucleic acid analog/protein conjugate and mannan/protein conjugate.Also contain under the situation of another kind of chemical drugs at the PAMPs that identifies in the future, also can consider to contain the conjugate in such chemical drugs and antigenic structure territory.

The conjugation methods of polypeptide, saccharide and lipid and DNA is that the technical staff is known.Referring to as U.S. Patent number 4,191,668,4,650,625,5,162,515,5,700,922,5,786,461,6,060,056; And J.Clin.Invest. (1988) 82:1901-1907.

Nonprotein PAMPs such as CpG or CpG-DNA and lipopolysaccharide can be puted together by routine techniques with protein or nonprotein antigen.For example, PAMP/ antigen conjugate can be connected with the copolymer of D-lysine with D-glutamic acid by polymer such as PEG, poly-D-lysine, polyvinyl alcohol, polyvinylpyrrolidone (polyvinylpyrollidone), immunoglobulin.Puting together of PAMP and antigen and polymer connector can realize in the mode of any number, generally comprises one or more cross-linking agent and PAMP and antigenic functional group.Polypeptide PAMPs and antigen will contain amino acid side chain as amino, carbonyl or sulfydryl, and these side chains are useed as PAMP and the interconnective site of antigen.Residue with such functional group can be added on PAMP or the antigen.Such residue can be integrated into by solid phase synthesis technique or recombinant technique, and these two kinds of technology all are that the synthetic field of peptide is known.

Under the situation of sugar or fat analog, functional amino and mercapto groups can be integrated into by the conventional chemical method.For example, the primary amino radical group can by when sodium cyanoborohydride exists and reacting ethylenediamine be integrated into, sulfydryl can reduce with the standard disulfide reducing agent subsequently by the reaction of cysteamine dihydrochloride (cysteaminedihydrochloride) and introduce.If can a kind of similar mode not derive when the polymer connector also has the appropriate functional group to contain functional group yet.The isodigeranyl functional cross-link agent also is useful as sulfosuccinic acylimino (4-iodoacetyl) Aminobenzoate when PAMPs or antigenic ratio in increasing conjugate, and it is connected to the ε amino group of D-lysine residue in D-lysine and the D-glutamic acid copolymer on the sulfydryl side chain from aminoterminal residue in will link coupled peptide.

J. bacterin preparation and sending

Vaccine of the present invention contains one or more PAMPs, its immunostimulation part or immunostimulation derivant (for example domain of being discerned by innate immune system) and one or more antigens, its immunogenicity part or immunogenic derivatives (for example domain of being discerned by adaptive immune system).Because have in conjunction with PRRs and start the ability of innate immunity reaction according to definition PAMP analogies, so the bacterin preparation that the present invention considers comprises the PAMP analogies that substitute PAMP.Thereby the present invention considers to comprise the vaccine of chimeric construct body, and this chimeric construct body comprises at least one antigenic structure territory and at least one PAMP domain.In a specific embodiment, vaccine of the present invention comprises BLP/E alpha fusion protein matter.

The vaccine that comprises chimeric construct body of the present invention can prepare according to the known method of compositions of preparation pharmaceutically useful, whereby with chimeric construct body and pharmaceutically acceptable carrier combinations in a mixture.If can be stood by the receptor after a kind of compositions gives, and if said composition when making active component to arrive to need the site of effect, promptly be called as " pharmaceutically acceptable carrier ".Aseptic phosphate buffered saline(PBS) is an example of pharmaceutically acceptable carrier.Other appropriate carriers are known in those skilled in the art.(people such as Ansel, pharmaceutical doses form and drug delivery system (Pharmaceutical Dosage Forms and Drug Delivery System), 5 ^ThEdition (Lea ﹠amp; Febiger 1990); Gennaro (ed.), Remington ' sPharmaceutical Sciences 18 ^ThEdition (Mack Publishing Company1990)).

The example of other several excipient of considering comprises water, dextrose, glycerol, ethanol and its combination.Vaccine of the present invention can further contain auxiliary substance, for example moistening or emulsifying agent, pH buffer agent, stabilizing agent or other carriers, including, but not limited to the reagent of for example aluminium hydroxide or aluminum phosphate (Alumen), the solution that is used as 0.05 to 0.1% in the phosphate buffered saline(PBS) usually is to strengthen its effectiveness.

Chimeric construct body of the present invention also can be by being used as vaccine on the immunogenic carrier molecule that is attached to solubility.The appropriate carriers molecule comprises protein, and comprising keyhole limpet hemocyanin, it is a kind of preferred carrier protein.Utilize standard method the chimeric construct body can be attached on the carrier molecule.(people such as Hancock, " peptide is synthetic to be used as immunogen (Synthesis of Peptidesfor Use as Immunogens); " " molecular biology method: immunochemistry rules " (Methodsin Molecular Biology:Immunochemical Protocols), Manson (ed.), pages 23-32 (Humana Press 1992)).

In addition, the present invention considers a kind of vaccine combination that comprises pharmaceutically acceptable injection-type carrier.Vaccine of the present invention can the form with conventional carrier gives under the situation of other standard vectors being with or without with the form of injection-type solution or suspension.Improve the reagent of overall immune reaction in the optional comfortable immunologic process of the carrier that is added.

Propose with liposome as appropriate carriers.Insoluble aluminum salt is that aluminum phosphate or aluminium hydroxide are used as the carrier in the application of people's routine clinical.Polynucleotide and polyelectrolyte and water-solubility carrier such as muramyldipeptide have also been used.

The preparation of injection-type vaccine of the present invention comprises the chimeric construct body is mixed with muramyldipeptide or other carriers.The mixture of gained can emulsifying in mannide monooleate/Squalene or squalane carrier as a result.By volume every part of mannide monooleate uses four parts of Squalenes and/or squalane.The method for preparing vaccine combination is that those of ordinary skills are known.(Rola, immunoreagent and diagnostic dermatogen (Immunizing Agents and Diagnostic SkinAntigens).Be recorded in Remington ' s Pharmaceutical Sciences, 18thEdition, Gennaro (ed.), (Mack Publishing Company 1990) pages1389-1404).

Can adopt other pharmaceutical carriers to control vaccine in the action period for the treatment of in using.The sustained release preparation can be compound or absorb the chimeric construct body and prepare by using polymer.For example, the polymer of bio-compatible comprises the polyanhydride copolymer matrix of poly-(ethylene-common vinyl acetate) (poly (ethylene-co-vinylacetate)) substrate and stearic acid dimer and decanedioic acid.(people (1992) Bio/Technology 10:1446 such as Sherwood).The rate dependent that the chimeric construct body discharges from such substrate is in the molecular weight of construct, the amount of construct in substrate and the size of discrete particles.(people (1989) Biophys.J.55:163 such as Saltzman; People such as Sherwood, the same.; People such as Ansel, pharmaceutical doses form and drug delivery system (PharmaceuticalDosage Forms and Drug Delivery Systems), 5 ^ThEdition (Lea ﹠amp; Febiger1990); And Gennaro (ed.), Remington ' s Pharmaceutical Sciences 18thEdition (Mack Publishing Company 1990)).The chimeric construct body also can combine with Polyethylene Glycol (PEG) to increase stability and to expand biological utilisation number of times (people such as Katre for example; U.S. Patent number 4,766,106).

Vaccine of the present invention can the parenteral mode give.The normal mode that gives vaccine is intramuscular, subcutaneous and peritoneal injection.In addition, giving can be to be undertaken by inculcating single or multiple bolus continuously.

Also used particle gun and successfully sent and pass plasmid DNA inducing the immunity of pathogen in the pair cell, mainly depended on 1 type CD8.sup.+T-cell for the protection of this pathogen.(people (1999) J.Immun.163 (8) such as Kaufmann: 4510-4518).

Gene transfer-mediated inoculation method has become fast-developing field, and can be used for treating non-infectious and infective disease with such inoculation method compositions of the present invention, and noninfectious disease is including, but not limited to genetic diseases.(, be recorded in Goodman ﹠amp referring to as the treatment (Gene-Based Therapy) of people such as Eck (1996) based on gene; Gilman ' s ThePharmacological Basis of Therapeutics, Ninth Edition, Chapter 5, McGraw Hill).

Alternatively, vaccine of the present invention particularly uses flagellin as PAMP, can be with for causing that mode that the mucomembranous surface immunoreation designs prepares and send and pass.Thereby vaccine combination can be by giving mucomembranous surface as a nose or mouthful (gastric) approach.Other give pattern and comprise suppository and oral formulations.For suppository, binding agent and carrier comprise poly alkylene glycol (polyalkalene glycols) or triglyceride.Oral formulations comprises glucide, cellulose and the magnesium carbonate of used usually initial thing (incipient) as pharmaceutical grade.These compositionss can be solution, suspension, tablet, pill, capsule, slow releasing preparation or form of powder, and contain 1 to 95% the chimeric construct body of having an appointment.This vaccine to be giving with the corresponding to mode of dosage particles, and giving with immunogenic drug dose with effective, protectiveness.

The quantity of used vaccine obviously depends on the situation of patient age, body weight, height, sex, overall medical condition, medical history, treatment and the order of severity thereof and individual immunity system synthetic antibody and produces cell-mediated immunoreactive ability and different.Usually, provide dosage at the chimeric construct body of about 1 μ g reagent/kg patient body weight in 100mg reagent/kg patient body weight scope, although also can give lower or higher dosage to the receptor.Yet the accurate amount of active component depends on practitioner's judgement.Suitable dosage range can be determined at an easy rate by those skilled in the art, and depend on specific construct and at nanogram chimeric construct body on gram chimeric construct body rank.Preferably, the dosage range of active component is that nanogram arrives microgram; More preferably, arrive milligram for nanogram; Be most preferably microgram to milligram.The suitable scheme that initially gives with booster dose also is variable, but can comprise that follow-up initially giving of giving arranged subsequently.Dosage can be dependent on the approach that gives and changes according to the size of object.

The present invention includes and contain from the single creature style as from the antigen of a special pathogen and the vaccine of PAMPs.The present invention also comprises and contains from the antigenicity material of several separate sources and/or separate vaccine from the PAMP of several separate sources material.Such combination-vaccine contain as from various microorganisms or from the various bacterial strains of same microorganism or from the antigen and the PAMPs of the combination of various microorganisms.

For the purpose for the treatment of, give mammal with the effective amount of medicine with antigen/PAMP fused protein.If the amount that gives can produce measurable positive interaction in the receptor, then claim this bacterin preparation to give with " medicine is effectively measured ".Especially, if bacterin preparation of the present invention in the receptor, cause measurable body fluid and/or cell immune response then can in the receptor, produce positive interaction.Especially, the present invention considers that a medicine of wanting effectively measures, promptly wherein vaccine can cause measurable body fluid and/or cell immune response to target antigen in the receptor, but neither causes over-drastic nonspecific inflammation also not cause autoimmune response to non-target antigen.

As used herein, term " treatment " refers to that treatment is handled and preventative or protectiveness is handled both.In one embodiment, the present invention considers to use the patient that disclosed vaccine is treated needs this type of treatment.Patient can suffer from disease, such as, but be not limited to cancer, allergy, infectious disease, autoimmune disease, sacred disease, cardiovascular disease or the disease related with allergy.In another embodiment, the present invention considers that giving disclosed vaccine makes patient produce passive immunity to some diseases, and these diseases are such as, but be not limited to cancer, allergy, infectious disease, autoimmune disease, sacred disease, cardiovascular disease or the disease related with allergy.In another embodiment, the present invention considers to give disclosed vaccine makes patient produce immunity to the disease the disease of being mentioned in previous sentence, and wherein purpose is to remove specific molecular or the specific cells in the health.A non-restrictive example is for removing or prevent the deposition of speckle in cardiovascular disease.

K. treatment/immunostimulant

Vaccine of the present invention can be used to strengthen the immunity of the animal that needs this vaccine, more specifically is mammal, more specifically is people (for example patient).Immunostimulant is that treatment is diagnosed as the ideal goal as the patient of infection, leprosy, tuberculosis, herpes zoster, wart, herpes, malaria, gingivitis and the arteriosclerosis of cancer, acquired immunodeficiency syndrome, some part or whole body.

The advantage of vaccine of the present invention has been induced for them has only minimum bad inflammatory reaction to the strong immunoreation of target antigen, and the situation of having only minimum autoimmune disease.Such characteristic that has alleviated side effect has the advantage that obviously is better than other vaccine methods, especially at this respect that autoantigen is reacted, because for many other vaccine strategies, in order to cause strong reaction to autoantigen, use strong adjuvant, they cause over-drastic inflammation and can increase the danger of autoimmune disease.

As used herein, " immunostimulant " refers to any increase of organism to exogenous antigen or other target antigens such as those antigenic responding abilities related with cancer, and this comprises the immunocyte that increased quantity, in such antigenic cell surveys and eliminate the antigenic activity and the ability that have increased of such through immunity to attack at those.

Immunoreactive intensity available standards is checked and is measured, including, but not limited to following method: utilize methods known in the art directly to measure immunoassay (people (1992) Cytom.13:169-174 such as Loeffler of peripheral blood lymphocyte, natural killer cell cytotoxic assay (people (1992) J.Immunol.Meth.155:19-24 such as Provinciali), cell proliferating determining (people (1992) J.Immunol.Meth.149:133-135 such as Vollenweider), immunocyte and hypotype; People such as Rivoltini (1992) Can.Immunol.Immunother.34:241-251) and the skin test of cell-mediated immunity (people (1993) Cancer Res.53:1043-1050 such as Chang).For the outstanding article of the method for measuring immune intensity and analysis referring to as the up-to-date rules of people such as Coligan (Ed.) (2000) immunology (Current Protocols inImmunology), Vol.1, Wiley ﹠amp; Sons.

As above-mentioned checking measurements, any statistically evident increase all is considered to " enhanced immunoreation " or " immunostimulant " in immunoreation intensity.Making S-phase T-cell increase by 5 percent increase has realized by method of the present invention.Enhanced immunoreation is also indicated as fever and inflammation by the phenomenon of health, although may not observe a kind of or these two kinds of phenomenons for recombiant vaccine of the present invention.Enhanced immunoreation feature also is to cure general and local infection, reduce disease symptoms, as the tumor size reduction, simultaneous phenomenon such as the sxs such as Kaposi sarcoma, bronchial infection of leprosy, tuberculosis, malaria, naphthousulcers, herpetic and papilloma wart, gingivitis, arteriosclerosis, AIDS.

L. production of vaccine

Program of the present invention can be used for generating the chimeric construct body that comprises one or more purpose antigens and one or more PAMPs.The epitope labelling (for example His labelling) that can add a little non-immunogenic is so that the purification of the fused protein of expressing in antibacterial is easier.The combination of antigen and PAMP such as BLP or flagellin provides activation antigen specificity adaptability and innate immunity to react necessary signal.

A large amount of different fused proteins of synantigen and PAMPs combination that comprise not can utilize recombinant DNA technology well known in the art or put together chemistry and generate at an easy rate.By utilizing the method for identical technology, almost any antigen can generate vaccine.Therefore, this new method is that non-normal open is used.

Utilize bacterial expression system can generate a large amount of recombiant vaccine products.Product available standards technology is purified from bacterial cultures.Thereby this method is extremely economical and has cost-efficient.As what select, the recombiant vaccine product can be produced from the culture of yeast or other eukaryotic cells (including, but not limited to insect cell or mammalian cell) and purify.The nonprotein vaccine product of puting together also can produce with big relatively quantification student, particularly when PAMP and antigen both can by relative directly purification procedures obtain also then with simple and efficient relatively put together chemically conjugated together the time.

Alternatively, comprise protein component and non-protein component the chimeric construct body can by with the recombinant methods protein component with prepare non-protein component with chemical method and be connected these two kinds of compositions and acquisition easily with connector as known in the art chemistry then, the some of them method is described herein.In addition, because antigen and PAMPs that the present invention considers can be naturally occurring, they can be purified from their natural origin and chemically be linked together then.T-cell and B-cellular antigens can the method and are used to generate vaccine.

The fusion of antigen and PAMP such as BLP or flagellin makes the stoichiometry of two kinds of signals reach the suitableeest state, thereby has minimized undesired excessive inflammatory response (for example when antigen mix the excessive inflammatory response that is taken place when increasing their immunogenicity with adjuvant).

The fusion of antigen and PAMP such as BLP has increased the probability that in response to vaccine activated APC triggers the adaptive immunity reaction of wanting productively.The activation of such APC can not cause inducing of autoimmune response when having absorption and antigen-presenting, and this is again to prevent or limit one of problem of its adjuvant commonly used that uses in the people.

In a preferred embodiment, fused protein of the present invention comprises antigen or its immunogenicity part, this antigen or its immunogenicity part are modified to contain the aminoacid sequence that comprises targeting sequencing and concensus sequence, this aminoacid sequence causes the post translational modification to the part of concensus sequence or this sequence, and wherein the sequence of post translational modification is the part of PRR.The antigen of modifying is including, but not limited to containing the antigen of antibacterial fat concensus sequence CXXN (SEQ ID NO:1), and wherein X is any aminoacid, but is preferably serine.Numerous targeting sequencings are known in the field, but preferred targeting sequencing is described by preceding 20 aminoacid among the SEQ ID NO:2, and wherein preceding 20 aminoacid among the SEQ IDNO:2 are illustrated in SEQ ID NO:3.Describe among the extra SEQ ID NO:4-7 of suitable targeting sequencing in sequence list.Preferred chimeric construct body comprises the targeting sequencing that is fused to a certain sequence, and described a certain sequence comprises the antibacterial fat concensus sequence SEQ ID NO:1 (for example targeting sequencing-CXXN-antigen) that further is fused on the antigen.Although antigenic this modification can be called as fusion, this modification can be not by fusion dna by in the antigenic DNA of any purpose of coding, introducing targeting sequencing through mutation and CXX sequence subsequently realizes.The expression of the nucleic acid molecules of this chimeric construct body of coding has produced a kind of substrate in bacterial host cell, before this by the bacterialprotease effect, cutting targeting sequencing from the antigen of modifying, is the effect of antibacterial lipid transferring enzyme then, and fatization comprises sequence or its part of fat concensus sequence.The product of gained is as the PRR part and can stimulates congenital and chimeric construct body or fused protein adaptive immune system as a result.In embodiment in addition, chimeric construct body or fused protein comprise extra polarity or charged aminoacid do not change construct with the hydrophilic that increases chimeric construct body or fused protein immunogenicity or immunostimulatory properties.

Need not further describe, believe that those of ordinary skill in the art just can be with the illustrative embodiment of the description of front and back and put into practice method of the present invention.Therefore, the following examples have been pointed out the preferred embodiment of the invention especially, and are just illustrative, and should not be construed as by any way the restriction to the remainder of disclosure.Other common and specific structures are apparent to one skilled in the art.

Embodiment

The model vaccine box of embodiment 1. tool antigenic structure territories and PAMP domain

In order to produce a model vaccine box of the present invention, we with the molecule pattern (PAMP) of pathogen association be fused to characterization mice antigen E α on.The PAMP that we select is that BLP is known stimulates innate immune system by receptor class Toll receptor-2 (TLR-2).

In the vaccine box, be used for bacterial lipoprotein (BLP) protein sequence of purpose antigen fusion as follows: MKATK LVLGA VILGS TLLAG CSSNA KIDQL SSDVQ TLNAK VDQLS NDVNAMRSDV QAAKD DAARA NQRLD NMATK YRK (SEQ ID NO:2).Targeting sequencing comprises the 1st amino acids to the 20 amino acids of SEQ ID NO:2.First cysteine in antibacterial (the 21st amino acids of SEQ ID NO:2) is a fatization.This fatization that can only take place in antibacterial is basic for BLP by Toll and TLRs identification.Lysine (the 78th amino acids of the SEQID NO:2) sudden change that makes the C-end is to increase the output of recombiant vaccine, because this lysine can form covalent bond with Peptidoglycan.

In order to help antigenic evaluation and purification, in proteinic C-tip designs one six histidine mark.Final construct as shown in Figure 3.

Fused protein is expressed in antibacterial and is induced by IPTG.Protein can also be purified with supersound process in 8M carbamide, 20mM Tris, 20mM NaCl, 2%Triton-X-100, pH 8.0 in cracking.Lysate is also washed with 8M carbamide, 20mM Tris, 20mM NaCl, 0.2%Triton-X-100, the pH 8.0 of 5 times of column volumes by 100ml Q-agarose gel ion exchange column in identical buffer.Protein is by salt gradient eluting (20mM NaCl is to 800mMNaCl).Positive component is identified at the immunoblotting of the antibody of histidine mark by utilization.Collect these components and make its nickel-agarose column by 2ml.With same buffer (10 times of column volumes) thorough washing pillar, the phosphate buffer (20mM) that contains 200mM NaCl, 0.2%Triton-X-100,20mM imidazoles, pH 8.0 with 5 times of column volumes washs then.Protein eluting in 20mM phosphate buffer, 200mM NaCl, 0.1%Triton-X-100,250mM imidazoles of purifying, component is checked protein with immunoblotting once more then.Collect positive component and use the phosphate-buffered salt dialysed overnight that contains 0.1%Triton-X-100 once more.By any contaminated with endotoxins in the polymyxin B pillar removal sample, and centrifugal concentrated in the Amicon concentrator, then with immunoblotting and protein concentration check Protein content.

Embodiment 2. in the RAW cell NF-κ B by the antigenic stimulation of BLP/E α model

Whether can the necessary signal transduction pathway of immune response stimulating for testing model antigen, we have measured the activation of NF-κ B in the external RAW mouse macrophage.We have developed a kind of stable RAW cell line that contains NF-κ B-dependent form firefly luciferase gene.The activator of NF-κ B causes the generation of luciferase to the stimulation of these cells, and this luciferase can be measured in cell lysate by using luminometer.BLP/E α with indicatrix stimulated 5 hours with cell, and results are used for the luciferase measurement then.

In contrast, the RAW cell is stimulated with LPS when existing and not having polymyxin B (PmB).PmB can make the endotoxin inactivation, and as expected, the active activation of NF-κ B reduces 98% in the LPS+PmB sample.BLP/E α also can activate NF-κ B in dose dependent mode as shown in Figure 4, yet, can not make stimulation with statistically evident degree inactivation with the PmB processing.These results hint BLP/E α to the activation of NF-κ B not owing to the pollution of endotoxin to preparation.

Embodiment 3.BLP/E α model vaccine is external evoked, and dendritic cell are produced IL-6

Effectively vaccine must be able to stimulate the ripe and antigen-presenting of dendritic cell (DC).In order to check BLP/E α whether can induce the DC function, we have checked the ability of producing IL-6 derived from the DC of bone marrow after stimulated in vitro.Separate the bone marrow dendritic cell, and it was grown in culture 5 days.After 5 days, cell is forwarded in the ware in 96-hole with 250,000 cells/well, and handle with E α peptide (0.3 μ g/ml), LPS (100ng/ml)+E α peptide (0.3 μ g/ml) or BLP/E α.BLP/E α can be in the production of these cell moderate stimulations IL-6, as measured in sandwich ELISA (Fig. 5).

Embodiment 4.BLP/E α has stimulated the maturation of immature dendritic cell

In order to determine whether BLP/E α vaccine can and be presented by dendritic cell processing, we with boosting vaccine dendritic cell and checked B7.2 and be attached to E α peptide in the MHC Type II in the expression on their surfaces.The dendritic cell of cultivating derived from bone marrow (5 days) are stimulated with E α peptide or BLP/E α, and the Yae antibody staining that is attached to the E α peptide in the MHC Type II with the antibody and/or the identification of B7.2 costimulatory molecules.Analyze (Fig. 6) by FACS.

The specific T-cell of embodiment 5.BLP/E α model vaccine stimulated in vitro

We analyze next step the BLP/E α that is processed and presented by DC whether can be in stimulated in vitro antigen specific T-proliferation of cells.Cultivate in containing the culture medium of 1%GM-CSF with the mice DC separation of bone marrow derived and with 750,000 cells/well.Cultured cell was collected DC, wash, count after 6 days, forwarded in the ware in 96-hole with 250,000 cells/well then.Cell is stimulated with the antigen of pointing out above, place its maturation of 3 angels.After 3 days, that DC is resuspended and cultivate in the ware in 96-hole with 5,000 or 10,000 cells/well.To cultivate on DC with 100,000 cells/well from the T-cell of 1H3.1 TCR transgenic mice (1H3.1 TCR is specific to E α peptide) lymph node.Cell placed in culture medium used 0.5 μ Ci/ hole in 3 days then ³The H-thymidine carries out " pulse ".Harvesting after 24 hours, and the integration (T-proliferation of cells) of measuring thymidine with cpm is (Fig. 7).

Embodiment 6.BLP/E α activates specific T-cell in vivo

In order to estimate that vaccine produces the ability of specific T-cell effect in vivo, we to injected in mice fused protein.Three injected in mice are as follows:

Mice #	The sample of injection	The lymph-node cell number
Mice #	The sample of injection	The lymph-node cell number	1	E α peptide 30 μ g in PBS	1.9×10 ⁶
2	E α peptide 30 μ g in CFA*	3.29×10 ⁷	1	E α peptide 30 μ g in PBS	1.9×10 ⁶
2	E α peptide 30 μ g in CFA*	3.29×10 ⁷	3	BLP/Eα100μg	5.2×10 ⁶

* complete Freund's adjuvant

The palmula of the injection of mice #2 is swelling considerably during Therapy lasted, and the palmula of mice #1 and #3 is acted normally.After 6 days, mice is painless deadly, gather in the crops related draining (draining) lymph node and be used for T-hyperplasia mensuration.The T-cell is cultivated in the plate in 96-hole with 400,000 cells/well, and stimulated once more with E α peptide or BLP/E α with described dosage.Cell placed made it begin hypertrophy in 48 hours, with 0.5 μ Ci/ hole in culture medium ³The H-thymidine carries out pulse and results after 16 hours.The integration of thymidine is measured (beta-plate reader) (Fig. 8) with β-dull and stereotyped reader.

Embodiment 7. has the relevant antigenic model vaccine box of allergen

The program of illustrating in producing BLP/E α model antigen above utilizing is used the former Ra5G of pollen allergy from giant ragweed (Ambrosia trifida (Ambrosia trifida)) to make and is had the relevant antigenic model vaccine box of allergen.The aminoacid sequence of Ra5G is as follows:

MKNIF?MLTLF?ILIIT?STIKA?IGSTN?EVDEI?KQEDD?GLCYE?GTNCG?KVGKYCCSPI?GKYCV?CYDSK?AICNK?NCT(SEQ?ID?NO：9)。

The aminoacid sequence of this allergen can merge with the aminoacid sequence (SEQ ID NO:1) of BLP to generate the BLP/Ra5G fused protein.The recombiant vaccine of gained places the IL-12 inducement signal with allergen as a result, and the PAMP in this situation is BLP.

In the time of in being incorporated into object, this vaccine will generate the specific T-cell effect of allergen, and this reaction will be because IL-12 will be divided into the Th1 reaction by inducing of BLP in dendritic cell and macrophage.

Embodiment 8. has the relevant antigenic model vaccine box of tumor

The program of illustrating in producing BLP/E α model antigen above utilizing has made with the relevant protein 2 (TRP-2) of model tumor antigen tryrosinase and to have had the antigenic model vaccine box that tumor is correlated with.The nucleotide sequence of TRP-2 and amino acid sequence corresponding provide in SEQ ID NO:10 (as shown in figure 20) and SEQ ID NO:11 (as shown in figure 21) respectively.The zone that is used for the BLP fusion comprises that SEQ ID NO:10 nucleotide numbering 840 is to nucleotide numbering 1040.T-cellular antigens decision position comprises that nucleotide numbering 945 is to nucleotide numbering 968 among the SEQ ID NO:10.

Can be used to TRP-2 regional as follows of vaccine construction:

LDLAK?KSIHP?DYVIT?TQHWL?GLLGP?NGTQP?QIANC?SVYDF?FVWLH?YYSVRDTLLG?PGRPY?KAIDF?SHQ(SEQ?ID?NO：12)。

The T-cellular antigens decision position of SEQ ID NO:12 is VYDFFVWL (SEQ ID NO:13).

Embodiment 9.CpG immunostimulation

Identified that recently TLRs family is basis (people (1999) the Science 284:1313-1318 such as Hoffmann of innate immunity identification in fruit bat and the mammalian organism; People such as Imler (2000) Curr.Opin.Microbiol.3:16-22).Fruit bat Toll surveys fungal infection and anti-fungus peptide drosomycin to induce necessary people (1996) Cell86:973-983 such as () Lemaitre.In mice, shown that TLR2 and TLR4 mediate the identification of antibacterial PGN and LPS (people (1999) Immunity11:443-451 such as Takeuchi) respectively.The function of other members in fruit bat and the mammal Toll family is on the knees of the gods at present, although expection at least some in them also relate to innate immunity identification.

Result described herein shows that jointly CpG-DNA is by the signal path mediation of MyD88 to the immunostimulation of three class professional antigen presenting cells (DC, macrophage and B cell).MyD88 relates to the signal transduction pathway by Toll and IL-1 receptor family.Cytokine IL-1 family comprises that the activation of IL-1 and IL-18 depends on the processing of being undertaken by caspase-1, but in all experiments of describing herein, disappearance caspase-1 is to not influence of the inductive cell effect of CpG-DNA people (1999) j.Clin.Immunol.19:1-11 such as () Fantuzzi.

We have checked TLR2 and TLR4 whether to relate to the identification of CpG-DNA, and discovery does not relate to, at least based on analysis provided herein.Therefore, we believe that CpG-DNA is by Toll receptor rather than TLR2 and TLR4 identification.The TLR1 of expression endogenous or transfection does not respond (data do not show) to CpG-DNA to the cell line of TLR6, and some other members of this hint Toll family can mediate the identification of CpG-DNA.

Though cause the characteristic of Toll receptor of reason of CpG-DNA identification still unknown this moment, CpG-DNA must internalization with the fact of bringing into play its stimulation (people (1995) Nature374:546-549 such as Krieg; People such as Stacey (1996) J.Immunol.157:2116-2122) TLR of hint mediation identification can be expressed in intracellular region chamber such as endosome in late period (late endosome), phagosome or lysosome.

Embodiment 10.CpG and B-cell-stimulating

To from spleen, kill (complementkill) CD4 from the B-cell of described mouse species by complement ⁺, CD8 ⁺Purify with macrophage.When not commensurability zest CpG-DNA (thiophosphate modify 5 '-TCCAT GACGT TCCTG ACGTT-3 ' (SEQ ID NO:8)) exists or does not exist with 1 * 10 ⁶The concentration of cells/ml is cultivated the Abherent cell.After 48 hours, cell is used [ ³H] (pulse NEN) was carried out 16 hours in 0.5 μ Ci/ hole to thymidine, and processing is to be used for β (beta) numeration.

Result shown in Fig. 9 A is the representative of three independent experiments.In response to the hypertrophy of CpG and wild-type cell quite (Fig. 9 A), the effect of hint MyD88 disappearance is not owing to the defective of the signal transmission of IL-1/IL-18 mediation derived from the B-lymphocyte of caspase-1 knock out mice.This result shows that CpG-DNA transmits signal by the receptor of Toll family.From the B-cell of two kinds of obtainable TLR deficient mice strains all have with wild-type cell similarly in response to the hypertrophy (Fig. 9 A) of CpG, mouse species wherein is to have C57BL/10ScCr strain (people (1998) the Science 282:2085-2088 such as Poltorak that spontaneously lacks the TLR4 gene; People J.Exp.Med.1999 such as Qureshi, 189:615-625) and TLR2 knock out mice people (1999) Immunity11:443-451 such as () Takeuchi.The normal reaction of this result and caspase-1 deficient cells hints that together the member (rather than TLR2 or TLR4) of Toll family relates to the identification of CpG-DNA.

The CpG of embodiment 11.CD86 and MHC Type II and B-cellular expression

The expression of the inductive CD86 of CpG in the B cell and the up regulation of MHC Type II molecule are tested to determine what whether these processes were mediated by the MyD88 signal path.Stimulate from MyD88 knock out mice and wild type littermate control mice and from the B-lymphocyte of TLR4-deficient mice with CpG-DNA.CD86 and MHC Type II are at the expression facs analysis of cell surface.

The B-cell with as mentioned above the preparation and when being with or without 10mM CpG with 3 * 10 ⁶Cells/ml was cultivated 12 hours.After the stimulation, with the surface expression of flow cytometry analysis CD86 and MHC Type II.Result shown in Fig. 9 B represents the B-cell of gate.Stimulated cells is represented in the shadow region, and non-hatched area is represented untreated contrast.As shown in Fig. 9 B, CpG-DNA has induced CD86 and the expression of MHC Type II on wild type and TLR4 deficient mice B cell consumingly.On the contrary, this is induced in MyD88 defective B-lymphocyte and is not taken place fully.

Embodiment 12. Salmonella typhimuriums (Salmonella Tymphimurium) flagellum egg White and the proteic clone of coli flagellum

With total length Salmonella typhimurium (Salmonella Typhimurium) flagellin and coli flagellum albumen is cloned from genomic DNA separately and in escherichia coli with the recombinant protein formal representation.Flagellin is expressed separately, or with expressing as fused protein from tryrosinase-2 protein (TRP2) of Mus B16 cell or the C-terminal fragment that contains the I-E alpha protein of E α epitope from the antigenic epitopes (SIINFEKL) of ovalbumin, clone.In addition, all recombinant proteins contain the 6x-histidine repetition of a C-end to help purification.

Inductive antibacterial is being contained Triton-X 100, glycerol, imidazoles, NaCl and Tris, and cracking is to keep proteinic native conformation in the gentle lysate of pH=8.0.By make filtering lysate by a nickel-NTA agarose column and subsequently in several buffer that contain imidazoles thorough washing with the purification fused protein.Protein eluting in the 250mM imidazoles of purifying, in the polymyxin B post through twice to remove the lipopolysaccharide that pollutes, then in 4 ℃ of abundant dialysed overnight in PBS.The protein of the purification of gained is highly stable as a result, can keep activity to reach at least one month at 4 ℃.

Embodiment 13. flagellin external tests

External test is to carry out with following process with the flagellin fused protein of purifying:

People's 293 cell lines and Mus RAW cell line are carried out transfection (this construct is called as " pBIIxluc ") with the reporter gene that contains the Ig κ NF-kB site that drives two copies that luciferase transcribes.The cell line of gained (293LUC and RAWkb) as a result cultivated handling with flagellin fused protein or control protein (lacZ) on the ware in 24-hole and after 24 hours, this control protein is to prepare in identical carrier and use with the identical mode of flagellin and purify.After handling 5 hours, obtain cell pyrolysis liquid, and check uciferase activity the inducing of this lysate with demonstration NF-κ B.In this analysis, flagellin has been induced NF-κ B significantly, especially in 293 cells, and to not influence of control protein, shown in Figure 12 and 13.Believe that this induces not the pollution owing to LPS, this is that polymyxin B does not suppress the activation in the RAW κ B cell because shown in Figure 12 and 14, and the 293LUC cell is not in response to LPS but be in response to flagellin.

The result of external test proves that the flagellin fused protein has kept them to stimulate the ability of class Toll receptor, and therefore can be used for generating reorganization flagellin-antigen coalescence protein matter to inoculate.In flagellin-antigen coalescence protein matter, believe that flagellin stimulates innate immune system by triggering class Toll receptor, and the antigen that is fused to flagellin provides the epitope by T and bone-marrow-derived lymphocyte identification.

The production of embodiment 14. CpG and IL-6 in macrophage

The PE attached cell (PECs) that will cause from the TGA of described mouse species was handled 24 hours with different stimulus object.IL-6 is to analyze with the enzyme-linked immunosorbent assay (ELISA) of the anti-mice IL-6 of specific use monoclonal antibody to the release of supernatant.Owing to also show CpG-DNA macrophage had significant stimulation (people (2000) Curr.Top.Microbiol.Immunol.247:41-58 such as Stacey; People such as Lipford (1998) TrendsMicrobiol.6:496-500; People such as Stacey (1996) J.Immunol.157:2116-2122), checked that in the macrophage of defective IL-6 is by the inductive expression of CpG among wild type and the MyD88.Be used as the inductive contrast to IL-6 of IL-1 mediation derived from the cell of caspase-1 knock out mice.The IL-6 that stimulates in response to CpG be created in MyD88-/-macrophage in complete obiteration, but be normal (Figure 10 A) in caspase-1, TLR2-and TLR4-deficient cells.The oligonucleotide of being made up of reverse CpG sequence (GpC) is used as contrast, and as what expect, it does not induce the amount (Figure 10 A) of detectable IL-6.

The degraded of the inductive I κ of embodiment 15.CpG-DNA B α

We next step checked the NF-B signal path activation MyD88-/-whether be defective in the macrophage.Peritoneal macrophages is stimulated 0,10,20,60 and 90 minute with in contrast with CpG-DNA or LPS, and in cracking thereafter.To each time point, the 30mg gross protein is carried out the SDS-PAGE electrophoresis and analyzes I κ B alpha protein (Figure 10 B) with immunoblotting.In wild-type cell, LPS and CpG-DNA the two can induce NF-κ B to activate, and this degraded by I kB protein matter is confirmed (Figure 10 B).MyD88-/-macrophage in, though the kinetics that delays is arranged, LPS still induces I κ B degraded, this is consistent with the observations of delivering people (1999) Immunity11:115-122 such as () Kawai.Yet, different with LPS, CpG-DNA not MyD88-/-induce I κ B degraded (Figure 10 B) in the macrophage.Therefore, though LPS and CpG-DNA both are transmitted signal by MyD88, by these stimulus object initial signal path be inequality, reflect that different TLRs can activate crossover but the probability of different signal path.

Embodiment 16.CpG and the IL-2 production in dendritic cell

Shown that CpG-DNA is the activated effective inducer of DC (people (1998) Eur.J.Immunol.28:2045-2054 such as Sparwasser).DC in adaptive immunity reaction initial, play a crucial role people (1998) Nature 392:245-252 such as () Banchereau.When interacting with the deutero-product of microorganism (PAMPs) in perienchyma, DC has experienced and has been called sophisticated growth and changes people (1998) Nature 392:245-252 such as () Banchereau.The sophisticated characteristics of DC are CD80 and CD86 molecule the inducing of cell surface expression, and to the production (people (1998) Nature 392:245-252 such as Banchereau) of adenoid transfer and cytokine such as IL-12.Therefore, we have checked CpG-DNA whether sophisticated the inducing of DC is mediated by the MyD88 signal path.MyD88-/-animal generation IL-12 when stimulating with the CpG oligonucleotide.Wild type, B10/ScCr and MyD88-/-bone marrow DC is from the bone marrow suspension preparation of cultivating 5 days DC growth medium (RPMI 5%FC+1%GM-CSF), and with 10mm CpG or the stimulation of 10mm GpC oligonucleotide or do not handle.After stimulating 24 hours and 48 hours, obtain supernatant and analyze IL-12 with ELISA with specific capture antibodies and detection antibody.

Result as shown in figure 11 is from one of three experiments of independently carrying out.Consistent with the report of delivering, CpG-DNA has induced the secretion of a large amount of IL-12 in wild-type mice DC.Yet, in DC, do not have detectable IL-12 to produce (Figure 11) for the stimulation of CpG derived from the MyD88 knock out mice.As expected, produced the IL-12 (Figure 11) of wild type level in response to CpG-DNA from the DC of TLR4 deficient mice.

Embodiment 17.CpG/E α chimeric construct body

According at U.S. Patent number 6,060, the method described in 056, the copolymer that is CpG by PEG polymer connector and/or D-lysine and D-glutamic acid with the PAMP of a nonprotein is conjugated on the mice antigen E α of characterization.One comprises CpG ₄₀The CpG-DNA derivant be used as nonprotein PAMP.

Below with reference to all articles, patent and other materials all be specifically to quote as a reference herein.

Though the present invention discloses with reference to particular, obviously others skilled in the art can design other embodiments of the present invention and modification when not deviating from true intention of the present invention and scope.Additional claim should be interpreted as comprising embodiment that all are such and suitable modification.

Sequence table

<110>YALE?UNIVERSITY

＜120〉innate immune system-directed vaccine

<130>044574-5071-WO

＜140〉do not specify as yet

<141>2001-07-31

<150>US?60/222,042

<151>2000-07-31

<160>13

<170>PatentIn?version?3.0

<210>1

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉fat site

<220>

＜221〉modification

<222>(2)..(3)

＜223〉any aminoacid of X=is preferably serine

<400>1

Cys?Xaa?Xaa?Asn

1

<210>2

<211>78

<212>PRT

＜213〉escherichia coli

<220>

<221>misc_feature

<223>BLP

<400>2

Met?Lys?Ala?Thr?Lys?Leu?Val?Leu?Gly?Ala?Val?Ile?Leu?Gly?Ser?Thr

1???????????????5???????????????????10??????????????????15

Leu?Leu?Ala?Gly?Cys?Ser?Ser?Asn?Ala?Lys?Ile?Asp?Gln?Leu?Ser?Ser

20??????????????????25??????????????????30

Asp?Val?Gln?Thr?Leu?Asn?Ala?Lys?Val?Asp?Gln?Leu?Ser?Asn?Asp?Val

35??????????????????40??????????????????45

Asn?Ala?Met?Arg?Ser?Asp?Val?Gln?Ala?Ala?Lys?Asp?Asp?Ala?Ala?Arg

50??????????????????55??????????????????60

Ala?Asn?Gln?Arg?Leu?Asp?Asn?Met?Ala?Thr?Lys?Tyr?Arg?Lys

65??????????????????70??????????????????75

<210>3

<211>20

<212>PRT

＜213〉escherichia coli

<220>

<221>misc_feature

＜223〉BLP targeting sequencing

<400>3

Met?Lys?Ala?Thr?Lys?Leu?Val?Leu?Gly?Ala?Val?Ile?Leu?Gly?Ser?Thr

1???????????????5???????????????????10??????????????????15

Leu?Leu?Ala?Gly

20

<210>4

<211>20

<212>PRT

＜213〉separate starch Erwinia (Erwinia amylovora)

<220>

<221>misc_feature

＜223〉BLP targeting sequencing

<400>4

Met?Asn?Arg?Thr?Lys?Leu?Val?Leu?Gly?Ala?Val?Ile?Leu?Gly?Ser?Thr

1???????????????5???????????????????10??????????????????15

Leu?Leu?Ala?Gly

20

<210>5

<211>19

<212>PRT

＜213〉serratia marcescens (Serratia marcescens)

<220>

<221>misc_feature

＜223〉BLP targeting sequencing

<400>5

Met?Asn?Arg?Thr?Lys?Leu?Val?Leu?Gly?Ala?Val?Ile?Leu?Gly?Ser?His

1???????????????5???????????????????10??????????????????15

Ser?Ala?Gly

<210>6

<211>19

<212>PRT

＜213〉proteus mirabilis (Proteus mirabilis)

<220>

<221>misc_feature

＜223〉BLP targeting sequencing

<400>6

Met?Lys?Ala?Lys?Ile?Val?Leu?Gly?Ala?Val?Ile?Leu?Ala?Ser?Gly?Leu

1???????????????5???????????????????10??????????????????15

Leu?Ala?Gly

<210>7

<211>16

<212>PRT

＜213〉B. burgdorferi (Borrelia burgdorferi)

<220>

<221>misc_feature

＜223〉outer surface proteins A

<400>7

Met?Lys?Lys?Tyr?Leu?Leu?Gly?Ile?Gly?Leu?Ile?Leu?Ala?Leu?Ile?Ala

1???????????????5???????????????????10??????????????????15

<210>8

<211>20

<212>DNA

＜213〉artificial

<220>

<223>CpG-DNA

<400>8

tccatgacgt?tcctgacgtt

20

<210>9

<211>73

<212>PRT

＜213〉Ambrosia trifida

<220>

<221>misc_feature

＜223〉Ra5G ragweed pollen allergen

<400>9

Met?Lys?Asn?Ile?Phe?Met?Leu?Thr?Leu?Phe?Ile?Leu?Ile?Ile?Thr?Ser

1???????????????5???????????????????10??????????????????15

Thr?Ile?Lys?Ala?Ile?Gly?Ser?Thr?Asn?Glu?Val?Asp?Glu?Ile?Lys?Gln

20??????????????????25??????????????????30

Glu?Asp?Asp?Gly?Leu?Cys?Tyr?Glu?Gly?Thr?Asn?Cys?Gly?Lys?Val?Gly

35??????????????????40??????????????????45

Lys?Tyr?Cys?Cys?Ser?Pro?Ile?Gly?Lys?Tyr?Cys?Val?Cys?Tyr?Asp?Ser

50??????????????????55??????????????????60

Lys?Ala?Ile?Cys?Asn?Lys?Asn?Cys?Thr

65??????????????????70

<210>10

<211>2182

<212>DNA

<213>Mus?musculus

<220>

<221>misc_feature

<222>(405)..(1958)

<223>Tyrosinase-Related?Kinase?Protein?2??(TRP-2)

<220>

<221>misc_feature

<222>(945)..(968)

＜223〉T cellular antigens decision position

<220>

<221>misc_recomb

<222>(840)..(1040)

＜223〉be connected to BLP to form the zone of fused protein

<400>10

gcagcataat?aagcagtatg?gctggagcac?tctgtaaatt?aactcaatta?gacagagcct

60

gatttaacaa?ggaagactgg?cgagaagctc?ccctcattaa?acctgatgtt?agaggagctt

120

cggatgaaat?taaatcagtg?ttagttgttt?gagtcacata?aaattgcatg?agcgtgtaca

180

catgtgcaca?cgtgtaggct?ctgtgattta?ggtgggaatt?ttgagaggag?aggaaagggc

240

tagaactaaa?cccaaagaaa?aggaaagaag?agaagaggaa?aggaaagaaa?aaagaaaagg

300

caatttgagt?gagtaaaggt?tccagaactc?aggagtggaa?gacaaggagt?aaagtcagac

360

agaaaccagg?tgggacgccg?gccaggcctc?ccaattaaga?aggcatgggc?cttgtgggat

420

gggggcttct?gctgggttgt?ctgggctgcg?gaattctgct?cagagctcgg?gctcagtttc

480

cccgagtctg?catgaccttg?gatggcgtgc?tgaacaagga?atgctgcccg?cctctgggtc

540

ccgaggcaac?caacatctgt?ggatttctag?agggcagggg?gcagtgcgca?gaggtgcaaa

600

cagacaccag?accctggagt?ggcccttata?tccttcgaaa?ccaggatgac?cgtgagcaat

660

ggccgagaaa?attcttcaac?cggacatgca?aatgcacagg?aaactttgct?ggttataatt

720

gtggaggctg?caagttcggc?tggaccggcc?ccgactgtaa?tcggaagaag?ccggccatcc

780

taagacggaa?tatccattcc?ctgactgccc?aggagaggga?gcagttcttg?ggcgccttag

840

acctggccaa?gaagagtatc?catccagact?acgtgatcac?cacgcaacac?tggctggggc

900

tgctcggacc?caacgggacc?cagccccaga?tcgccaactg?cagcgtgtat?gacttttttg

960

tgtggctcca?ttattattct?gttcgagaca?cattattagg?tccaggacgc?ccctataagg

1020

ccattgattt?ctctcaccaa?gggcctgcct?ttgtcacgtg?gcacaggtac?catctgttgt

1080

ggctggaaag?agaactccag?agactcactg?gcaatgagtc?ctttgcgttg?ccctactgga

1140

actttgcaac?cgggaagaac?gagtgtgacg?tgtgcacaga?cgactggctt?ggagcagcaa

1200

gacaagatga?cccaacgctg?attagtcgga?actcgagatt?ctctacctgg?gagattgtgt

1260

gcgacagctt?ggatgactac?aaccgccggg?tcacactgtg?taatggaacc?tatgaaggtt

1320

tgctgagaag?aaacaaagta?ggcagaaata?atgagaaact?gccaacctta?aaaaatgtgc

1380

aagattgcct?gtctctccag?aagtttgaca?gccctccctt?cttccagaac?tctaccttca

1440

gcttcaggaa?tgcactggaa?gggtttgata?aagcagacgg?aacactggac?tctcaagtca

1500

tgaaccttca?taacttggct?cactccttcc?tgaatgggac?caatgccttg?ccacactcag

1560

cagccaacga?ccctgtgttt?gtggtcctcc?actcttttac?agacgccatc?tttgatgagt

1620

ggctgaagag?aaacaaccct?tccacagatg?cctggcctca?ggaactggca?cccattggtc

1680

acaaccgaat?gtataacatg?gtccccttct?tcccaccggt?gactaatgag?gagctcttcc

1740

taaccgcaga?gcaacttggc?tacaattacg?ccgttgatct?gtcagaggaa?gaagctccag

1800

tttggtccac?aactctctca?gtggtcattg?gaatcctggg?agctttcgtc?ttgctcttgg

1860

ggttgctggc?ttttcttcaa?tacagaaggc?ttcgcaaagg?ctatgcgccc?ttaatggaga

1920

caggtctcag?cagcaagaga?tacacggagg?aagcctagca?tgctcctacc?tggcctgacc

1980

tgggtagtaa?ctaattacac?cgtcgctcat?cttgagacag?gtggaactct?tcagcgtgtg

2040

ctctttagta?gtgatgatga?tgatgcctta?gcaatgacaa?ttatctctag?ttgctgcttt

2100

gcttattgta?cacagacaaa?atgcttgggt?cattcaccac?ggtcaaagta?aggtgtggct

2160

agtatatgtg?acctttgatt?ag

2182

<210>11

<211>517

<212>PRT

<213>Mus?musculus

<220>

<221>misc_feature

<222>(181)..(188)

＜223〉T cellular antigens decision position

<220>

<221>misc_recomb

<222>(146)..(212)

＜223〉be connected to BLP to form the zone of fused protein

<400>11

Met?Gly?Leu?Val?Gly?Trp?Gly?Leu?Leu?Leu?Gly?Cys?Leu?Gly?Cys?Gly

1???????????????5???????????????????10??????????????????15

Ile?Leu?Leu?Arg?Ala?Arg?Ala?Gln?Phe?Pro?Arg?Val?Cys?Met?Thr?Leu

20??????????????????25??????????????????30

Asp?Gly?Val?Leu?Asn?Lys?Glu?Cys?Cys?Pro?Pro?Leu?Gly?Pro?Glu?Ala

35??????????????????40??????????????????45

Thr?Asn?Ile?Cys?Gly?Phe?Leu?Glu?Gly?Arg?Gly?Gln?Cys?Ala?Glu?Val

50??????????????????55??????????????????60

Gln?Thr?Asp?Thr?Arg?Pro?Trp?Ser?Gly?Pro?Tyr?Ile?Leu?Arg?Asn?Gln

65??????????????????70??????????????????75??????????????????80

Asp?Asp?Arg?Glu?Gln?Trp?Pro?Arg?Lys?Phe?Phe?Asn?Arg?Thr?Cys?Lys

85??????????????????90??????????????????95

Cys?Thr?Gly?Asn?Phe?Ala?Gly?Tyr?Asn?Cys?Gly?Gly?Cys?Lys?Phe?Gly

100?????????????????105?????????????????110

Trp?Thr?Gly?Pro?Asp?Cys?Asn?Arg?Lys?Lys?Pro?Ala?Ile?Leu?Arg?Arg

115?????????????????120?????????????????125

Asn?Ile?His?Ser?Leu?Thr?Ala?Gln?Glu?Arg?Glu?Gln?Phe?Leu?Gly?Ala

130?????????????????135?????????????????140

Leu?Asp?Leu?Ala?Lys?Lys?Ser?Ile?His?Pro?Asp?Tyr?Val?Ile?Thr?Thr

145?????????????????150?????????????????155?????????????????160

Gln?His?Trp?Leu?Gly?Leu?Leu?Gly?Pro?Asn?Gly?Thr?G1n?Pro?Gln?Ile

165?????????????????170?????????????????175

Ala?Asn?Cys?Ser?Val?Tyr?Asp?Phe?Phe?Val?Trp?Leu?His?Tyr?Tyr?Ser

180?????????????????185?????????????????190

Val?Arg?Asp?Thr?Leu?Leu?Gly?Pro?Gly?Arg?Pro?Tyr?Lys?Ala?Ile?Asp

195?????????????????200?????????????????205

Phe?Ser?His?Gln?Gly?Pro?Ala?Phe?Val?Thr?Trp?His?Arg?Tyr?His?Leu

210?????????????????215?????????????????220

Leu?Trp?Leu?Glu?Arg?Glu?Leu?Gln?Arg?Leu?Thr?Gly?Asn?Glu?Ser?Phe

225?????????????????230?????????????????235?????????????????240

Ala?Leu?Pro?Tyr?Trp?Asn?Phe?Ala?Thr?Gly?Lys?Asn?Glu?Cys?Asp?Val

245?????????????????250?????????????????255

Cys?Thr?Asp?Asp?Trp?Leu?Gly?Ala?Ala?Arg?Gln?Asp?Asp?Pro?Thr?Leu

260?????????????????265?????????????????270

Ile?Ser?Arg?Asn?Ser?Arg?Phe?Ser?Thr?Trp?Glu?Ile?Val?Cys?Asp?Ser

275?????????????????280?????????????????285

Leu?Asp?Asp?Tyr?Asn?Arg?Arg?Val?Thr?Leu?Cys?Asn?Gly?Thr?Thr?Glu

290?????????????????295?????????????????300

Gly?Leu?Leu?Arg?Arg?Asn?Lys?Val?Gly?Arg?Asn?Asn?Glu?Lys?Leu?Pro

305?????????????????310?????????????????315?????????????????320

Thr?Leu?Lys?Asn?Val?Gln?Asp?Cys?Leu?Ser?Leu?Gln?Lys?Phe?Asp?Ser

325?????????????????330?????????????????335

Pro?Pro?Phe?Phe?Gln?Asn?Ser?Thr?Phe?Ser?Phe?Arg?Asn?Ala?Leu?Glu

340?????????????????345?????????????????350

Gly?Phe?Asp?Lys?Ala?Asp?Gly?Thr?Leu?Asp?Ser?Gln?Val?Met?Asn?Leu

355?????????????????360?????????????????365

His?Asn?Leu?Ala?His?Ser?Phe?Leu?Asn?Gly?Thr?Asn?Ala?Leu?Pro?His

370?????????????????375?????????????????380

Ser?Ala?Ala?Asn?Asp?Pro?Val?Phe?Val?Val?Leu?His?Ser?Phe?Thr?Asp

385?????????????????390?????????????????395?????????????????400

Ala?Ile?Phe?Asp?Glu?Trp?Leu?Lys?Arg?Asn?Asn?Pro?Ser?Thr?Asp?Ala

405?????????????????410?????????????????415

Trp?Pro?Gln?Glu?Leu?Ala?Pro?Ile?Gly?His?Asn?Arg?Met?Tyr?Asn?Met

420?????????????????425?????????????????430

Val?Pro?Phe?Phe?Pro?Pro?Val?Thr?Asn?Glu?Glu?Leu?Phe?Leu?Thr?Ala

435?????????????????440?????????????????445

Glu?Gln?Leu?Gly?Tyr?Asn?Tyr?Ala?Val?Asp?Leu?Ser?Glu?Glu?Glu?Ala

450?????????????????455?????????????????460

Pro?Val?Trp?Ser?Thr?Thr?Leu?Ser?Val?Val?Ile?Gly?Ile?Leu?Gly?Ala

465?????????????????470?????????????????475?????????????????480

Phe?Val?Leu?Leu?Leu?Gly?Leu?Leu?Ala?Phe?Leu?Gln?Tyr?Arg?Arg?Leu

485?????????????????490?????????????????495

Arg?Lys?Gly?Tyr?Ala?Pro?Leu?Met?Glu?Thr?Gly?Leu?Ser?Ser?Lys?Arg

500?????????????????505?????????????????510

Tyr?Thr?Glu?Glu?Ala

515

<210>12

<211>68

<212>PRT

<213>Mus?musculus

<220>

<221>SITE

<222>(37)..(44)

＜223〉T cellular antigens decision position

<220>

<221>misc_feature

<223>TRP-2

<400>12

Leu?Asp?Leu?Ala?Lys?Lys?Ser?Ile?His?Pro?Asp?Tyr?Val?Ile?Thr?Thr

1???????????????5???????????????????10??????????????????15

Gln?His?Trp?Leu?Gly?Leu?Leu?Gly?Pro?Asn?Gly?Thr?Gln?Pro?Gln?Ile

20??????????????????25??????????????????30

Ala?Asn?Cys?Ser?Val?Tyr?Asp?Phe?Phe?Val?Trp?Leu?His?Tyr?Tyr?Ser

35??????????????????40??????????????????45

Val?Arg?Asp?Thr?Leu?Leu?Gly?Pro?Gly?Arg?Pro?Tyr?Lys?Ala?Ile?Asp

50??????????????????55??????????????????60

Phe?Ser?His?Gln

65

<210>13

<211>8

<212>PRT

<213>Mus?musculus

<220>

<221>SITE

<222>(1)..(8)

＜223〉T cellular antigens decision position

<400>13

Val?Tyr?Asp?Phe?Phe?Val?Trp?Leu

1???????????????5

Claims

1. fused protein that comprises isolating PAMP or its immunostimulation part or its immunostimulation derivant and antigen or its immunogenicity part or its immunogenic derivatives.

2. the fused protein of claim 1, wherein PAMP is selected from peptide, protein, lipoprotein and glycoprotein.

3. the fused protein of claim 1, wherein PAMP is the part of PRR.

4. the fused protein of claim 1, wherein antigen can obtain from the source that is selected from antibacterial, virus, fungus, yeast, protozoacide, metazoa, tumor, malignant cell, unusual neurocyte, arthritis focus, cardiovascular focus, plant, animal, people, allergen and hormone.

5. the fused protein of claim 1, antigen wherein are that microorganism is relevant, allergen is relevant or relevant with unusual human or animal's cell.

6. the fused protein of claim 1, wherein PAMP is connected by chemical connector with antigen.

7. the fused protein of claim 1, wherein fused protein further comprises one or more other PAMPs or its immunostimulation part or its immunostimulation derivant, and the PAMPs of fused protein, immunostimulation part or immunostimulation derivant or identical or different.

8. the fused protein of claim 1, wherein vaccine further comprises one or more other antigens or its immunogenicity part or its immunogenic derivatives, and the antigen of fused protein, immunogenicity part or immunogenic derivatives or identical or different.

9. the fused protein of claim 1, wherein fused protein further comprises one or more other PAMPs or its immunostimulation part or its immunostimulation derivant and one or more other antigen or its immunogenicity part or its immunogenic derivatives, and wherein antigen, immunogenicity part or the immunogenic derivatives of PAMPs, its immunostimulation part or its immunostimulation derivant and/or fused protein or identical or different.

10. the fused protein of claim 1, wherein fused protein further comprises one or more carrier proteins.

11. the fused protein of claim 1, wherein PAMP and antigen are separated by spacer.

12. the fused protein of claim 1, PAMP wherein is BLP.

13. the fused protein of claim 12, BLP wherein are the aminoacid sequences of SEQ ID NO:2.

14. the fused protein of claim 1, antigen wherein are selected from amyloid-β peptide, the molten born of the same parents' element in Listerella, HIV gp120 and p24, Ra5G and TRP-2, EGFR, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), Her-2neu, SPAS-1, TRP-1, tryrosinase, Melan A/Mart-1, gp100, BAGE, GAGE, the GM2 ganglioside, kinesin 2, the TATA element regulation factor 1, oncoprotein matter D52, MAGED, ING2, HIP-55, the TGF-1 anti-apoptotic factor, MAGE-1, HOM-Mel-40/SSX2, NY-ESO-1EGFR, CEA, MAGE D, Her-2neu, NY-ESO-1, glycoprotein MUC1 and MUC10 mucin, p53, EGFR, CDC27, triose-phosphate isomerase, HLA-DRB1, HLA-DR1, HLA-DR6 B1, CD11a, LFA-1, IFN γ, IL-10, the TCR analog, the IgR analog, 21-hydroxylase, the calcium sensory receptors, tryrosinase, ldl receptor, glutamate decarboxylase (GAD), insulin B chain, PC-1, IA-2, IA-2b, GLIMA-38 and NMDA.

15. the fused protein of claim 1, wherein PAMP is that peptide mimics and/or the antigen of a kind of nonprotein PAMP are the antigenic peptide mimicses of a kind of nonprotein.

16. a fused protein that comprises targeting sequencing, concensus sequence and antigen sequence, concensus sequence wherein are glycosylation or fat concensus sequence.

17. the fused protein of claim 16, concensus sequence wherein are glycosylation or fat concensus sequence.

18. the fused protein of claim 16, targeting sequencing wherein provides signal for the post-translational glycosylation or the fatization of concensus sequence.

19. the fused protein of claim 18, leader peptide wherein is selected from:

A) aminoacid sequence of SEQ ID NO:3;

B) aminoacid sequence of SEQ ID NO:4;

C) aminoacid sequence of SEQ ID NO:5;

D) aminoacid sequence of SEQ ID NO:6; With

E) aminoacid sequence of SEQ ID NO:7.

20. the fused protein of claim 16, concensus sequence wherein are CXXN (SEQ IDNO:1).

21. the fused protein of claim 17, concensus sequence wherein are CXXN (SEQ IDNO:1).

22. the fused protein of claim 16, antigen wherein can obtain from the source that is selected from antibacterial, virus, fungus, yeast, protozoacide, metazoa, tumor, malignant cell, unusual neurocyte, arthritis focus, cardiovascular focus, plant, animal, people, allergen and hormone.

23. the fused protein of claim 16, antigen wherein are that microorganism is relevant, allergen is relevant or relevant with unusual human or animal's cell.

24. recombinant vector that contains the nucleotide of coding claim 1 or 16 fused protein.

25. contain the host cell of the recombinant vector of claim 24.

26. the host cell of claim 25, host cell wherein are the cells that is selected from antibacterial, yeast, plant, animal and these hosts of insecticide.

27. the host cell of claim 25, host cell wherein are the antibacterials that produces PAMP natively.

28. the host cell of claim 25, host cell wherein are the antibacterials that makes PAMP fatization.

29. a production comprises the method for the fused protein of PAMP or its immunostimulation part or its immunostimulation derivant and antigen or its immunogenicity part or its immunogenic derivatives, this method comprises the cell of cultivating claim 16 and the fused protein of isolated cell production.

30. one kind comprises the fused protein of claim 1 or claim 16 and the vaccine of pharmaceutically acceptable carrier.

31. the vaccine of claim 30, antigen wherein joins with disease association.

32. the vaccine of claim 30, antigen wherein are that allergy is correlated with or relevant with unusual human or animal's cell.

33. the vaccine of claim 30, antigen wherein is hormone.

34. the vaccine of claim 30, antigen wherein are amyloid-β peptides.

35. the vaccine of claim 30, PAMP wherein are the peptide mimicses of a kind of nonprotein PAMP.

36. the vaccine of claim 30, antigen wherein are the antigenic peptide mimicses of a kind of nonprotein.

37. method that makes animal immune that comprises the step of the vaccine that gives animal right requirement 30.

38. method that makes mammalian immune that comprises the step of the vaccine that gives mammal claim 30.

39. the method for claim 38, mammal wherein is the people.

40. the method for claim 37, vaccine wherein be that parenteral gives, intravenous gives, orally give, utilize suppository to give, or give by mucomembranous surface.

41. the method for claim 39, antigen wherein are amyloid-β peptide or its immunogenicity part.

42. the method for claim 39, wherein fused protein is the people who is diagnosed as Alzheimer disease.

43. the method for a treatment target, comprise the step that gives this object antibody or activated immunocyte and give a kind of vaccine of the fused protein that comprises claim 1 or claim 16, wherein antibody or activated immunocyte are antigenic at fused protein.

44. the method for claim 43, antibody wherein is monoclonal.

45. the method for a treatment target comprises and gives a kind of vaccine and a kind of step that is selected from the reagent of chemotherapeutics and antiangiogenic agent that comprises the fused protein of claim 1 or claim 16.

46. the method for claim 45, chemotherapeutics wherein are a kind of antitumor and anticancer agents.

47. the method for a treatment target comprises the vaccine that gives a kind of fused protein that comprises claim 1 or claim 16 and in conjunction with surgery or radiotherapy.

48. one kind comprises isolating PAMP and antigenic fused protein, antigen wherein is autoantigen.

49. the fused protein of claim 48, antigen wherein are selected from amyloid-β peptide, TRP-2, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), Her-2neu, SPAS-1, TRP-1, tryrosinase, Melan A/Mart-1, gp100, BAGE, GAGE, the GM2 ganglioside, kinesin 2, the TATA element regulation factor 1, oncoprotein matter D52, MAGE D, ING2, HIP-55, the TGF-1 anti-apoptotic factor, MAGE-1, HOM-Mel-40/SSX2, NY-ESO-1EGFR, CEA, MAGE D, Her-2neu, NY-ESO-1, glycoprotein MUC1 and MUC10 mucin, p53, EGFR, CDC27, triose-phosphate isomerase, HLA-DRB1, HLA-DR1, HLA-DR6 B1, CD11a, LFA-1, IFN γ, IL-10, the TCR analog, the IgR analog, 21-hydroxylase, the calcium sensory receptors, tryrosinase, ldl receptor, glutamate decarboxylase (GAD), insulin B chain, PC-1, IA-2, IA-2b, GLIMA-38 and NMDA.

50. the fused protein of claim 48, PAMP wherein is selected from peptide, protein, lipoprotein and glycoprotein.

51. the fused protein of claim 48, PAMP wherein are the parts of PRR.

52. the fused protein of claim 48, PAMP wherein is a fatization.

53. the fused protein of claim 48, antigen wherein can obtain from the source that is selected from tumor, malignant cell, unusual neurocyte, arthritis focus, cardiovascular focus.

54. the fused protein of claim 48, antigen wherein is relevant with unusual human or animal's cell.

55. the fused protein of claim 48, PAMP wherein is connected by chemical connector with antigen.

56. the fused protein of claim 48, fused protein wherein further comprise one or more other PAMPs, and PAMPs wherein is identical or different.

57. the fused protein of claim 48, fused protein wherein further comprise one or more other antigen, and antigen wherein is identical or different.

58. the fused protein of claim 48, fused protein wherein further comprise one or more other PAMPs and one or more other antigens, and PAMPs wherein and/or antigen are identical or different.

59. the fused protein of claim 48, fused protein wherein further comprises one or more carrier proteins.

60. the fused protein of claim 48, PAMP wherein and antigen are separated by spacer.

61. the fused protein of claim 48, PAMP wherein are BLP, OMP, OSP, flagellin or porin.

62. the fused protein of claim 61, PAMP wherein are the BLP with aminoacid sequence of SEQ ID NO:2.

63. the fused protein of claim 48, wherein PAMP is that peptide mimics and/or the antigen of a kind of nonprotein PAMP are the antigenic peptide mimicses of a kind of nonprotein.

Thereby 64. one kind in animal moderate stimulation innate immunity reaction and strengthen method to the adaptive immunity reaction of external source or autoantigen, comprise with external source or autoantigen and give a kind of PAMP jointly.

65. the method for claim 64, wherein the innate immunity reaction stimulates by activating one or more classes Toll receptor.

66. the method for claim 65, animal wherein are a kind of mammals.

67. the method for claim 66, the reaction of wherein adaptive immunity are by because the activation of the APCs that the activation of one or more classes Toll receptor causes comes enhanced.

68. the method for claim 67, antigen wherein are antibacterial, virus, protozoacide, metazoa or originated from fungus.

69. being the forms with fused protein, the method for claim 68, PAMP wherein and antigen give jointly.

70. the method for claim 69, PAMP wherein is selected from bacterial lipoprotein, bacterial outer membrane albumen, antibacterial outer surface proteins, flagellin or porin.

71. the method for claim 70, PAMP wherein is selected from the tetrapeptide and the Klebsiella ompA of the fatization of Borrelia ospA, Borrelia ospB, Borrelia ospC, bacterial lipoprotein.

72. the method for claim 71, PAMP wherein is the tetrapeptide of the fatization of bacterial lipoprotein.

73. the method for claim 70, autoantigen wherein are selected from amyloid-β peptide, TRP-2, EGFR, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), Her-2neu, SPAS-1, TRP-1, tryrosinase, Melan A/Mart-1, gp100, BAGE, GAGE, the GM2 ganglioside, kinesin 2, the TATA element regulation factor 1, oncoprotein matter D52, MAGE D, ING2, HIP-55, the TGF-1 anti-apoptotic factor, MAGE-1, HOM-Mel-40/SSX2, NY-ESO-1EGFR, CEA, MAGE D, Her-2neu, NY-ESO-1, glycoprotein MUC1 and MUC10 mucin, p53, EGFR, CDC27, triose-phosphate isomerase, HLA-DRB1, HLA-DR1, HLA-DR6 B1, CD11a, LFA-1, IFN γ, IL-10, the TCR analog, the IgR analog, 21-hydroxylase, the calcium sensory receptors, tryrosinase, ldl receptor, glutamate decarboxylase (GAD), insulin B chain, PC-1, IA-2, IA-2b, GLIMA-38 and NMDA.

74. the method for claim 67, antigen wherein are a kind of autoantigens.

75. being the modes with fused protein, the method for claim 73, PAMP wherein and antigen give jointly.

76. the method for claim 74, PAMP wherein is selected from bacterial lipoprotein, bacterial outer membrane albumen, antibacterial outer surface proteins, flagellin or porin.

77. the method for claim 75, PAMP wherein is selected from the tetrapeptide and the Klebsiella ompA of the fatization of Borrelia ospA, Borrelia ospB, Borrelia ospC, bacterial lipoprotein.

78. the method for claim 77, PAMP wherein is the tetrapeptide of the fatization of bacterial lipoprotein.

79. the method for claim 75, autoantigen wherein are selected from amyloid-β peptide, TRP-2, EGFR, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), Her-2neu, SPAS-1, TRP-1, tryrosinase, Melan A/Mart-1, gp100, BAGE, GAGE, the GM2 ganglioside, kinesin 2, the TATA element regulation factor 1, oncoprotein matter D52, MAGE D, ING2, HIP-55, the TGF-1 anti-apoptotic factor, MAGE-1, HOM-Mel-40/SSX2, NY-ESO-1EGFR, CEA, MAGE D, Her-2neu, NY-ESO-1, glycoprotein MUC1 and MUC10 mucin, p53, EGFR, CDC27, triose-phosphate isomerase, HLA-DRB1, HLA-DR1, HLA-DR6 B1, CD11a, LFA-1, IFN γ, IL-10, the TCR analog, the IgR analog, 21-hydroxylase, the calcium sensory receptors, tryrosinase, ldl receptor, glutamate decarboxylase (GAD), insulin B chain, PC-1, IA-2, IA-2b, GLIMA-38 and NMDA.

80. the method for claim 69, fused protein wherein and pharmaceutically acceptable adjuvant are together prepared.

81. the fused protein of claim 48, antigen wherein is selected from VEGF, vascular endothelial growth factor receptor, fibroblast growth factor and fibroblast growth factor acceptor.

82. a vaccine that comprises the PAMP that puts together with external source or autoantigen, thereby this vaccine strengthens the adaptive immunity reaction of external source or autoantigen in animal moderate stimulation innate immunity reaction but does not cause unwanted level of inflammation.

83. vaccine that comprises the PAMP that puts together with external source or autoantigen, when giving with therapeutic activity dosage, thereby this vaccine can and strengthen the adaptive immunity reaction of external source or autoantigen but not cause unwanted level of inflammation in animal moderate stimulation innate immunity reaction.

84. Therapeutic Method, comprise and control the step of giving individual vaccine, this vaccine contains the PAMP that puts together with external source or autoantigen, thereby and can and increase the adaptive immunity reaction of external source or autoantigen but do not cause unwanted level of inflammation in animal moderate stimulation innate immunity reaction.