CN1167491A - Synthetic IgE membrane anchor peptide immunogens for the treatment of allergy - Google Patents

Abstract

The present invention relates to a method for eliciting the production in healthy mammals, including humans, of high titer antibodies specific for sites on the extracellular segment of the anchor domain of the membrane-bound epsilon heavy chain of B cell-expressed humain IgE by the use of a composition comprising a synthetic peptide immunogen containing extracellular membrane anchor sites, to reduce IgE-secreting B leukocytes and allergen-induced IgE production. It also relates to the use of optimally designed, carrier protein free, IgE epsilon -chain related immunogens as key components in a synthetic vaccine to provide an immunotherapy for the treatment of allergy. The subject peptides contain immune stimulator sequences, including a tandemly linked helper T cell epitope, to aid in stimulating the immune response towards the mIgE membrane anchor domain.

Description

For treating the synthesis IgE membrane anchor peptide immunogens of allergy

Field that the present invention belongs to

The present invention relates to a kind of purposes of synthetic peptide based immunogens composition, which includes the target antigen epitope being covalently attached in the form of linear series and helper T cell epitope.More specifically, the present invention relates to cause the purposes for generating the composition of high titre antibody in the healthy mammal for including the mankind, site on the ε heavy chain for the people IgE that the antibody combines the film that B cell is expressed, i.e. film combine people ε-chain anchor domain extracellular segment on site be it is specific, the present invention also relates to the composition provided as vaccine treat allergy immunotherapy purposes.

Background of invention

Immunotherapy for preventing the allergy (such as asthma and hay fever) of IgE mediation known and practiced if early in the century, and this method involves the desensitization and hyposensitization that the effect for then contacting that allergen (1) is reduced by the allergen to patient's application amount of gradually increasing.The limitation of this immunotherapy based on immunogene includes the difficulty for identifying the allergen being related to and the side reaction due to using the allergen (1) of identification often to generate.Other processing methods for mitigating allergy block the cascade of the cell event to work in allergy using therapeutic compounds.These compounds include antihistamine, decongestant, β₂Excitant and corticosteroid.Antihistamine, decongestant and β₂Excitant is acted in allergia cascade reaction in IgE downstream events, carries out palliative treatment, and this treatment points out allergic symptoms rather than must act upon the preventative process for being closer to the event of allergic starting reaction of IgE mediation.These palliative treatments provide short-term and partial mitigation, but are often accompanied with unfavorable side effect.For example, antihistamine can lead to uneasy or not calm, β₂Excitant is sometimes related with the increase of asthma patient's morbidity.

Compared with antihistamine, decongestant, β₂Excitant and corticosteroid are strong immunosuppressor, are very effective to the processing of allergic symptoms.However, they generate unfavorable hormonal activity and can lead to unwanted wide scope immunosupress.The shortcomings that avoid these therapeutic compounds, it is desirable to which the inhibition by being selectively aligning with IgE prevents allergy in IgE level.This can be by directly or indirectly inhibiting the synthesis of IgE to complete.Inhibit to complete by the mediator for the IgE synthesis (3) for desensitizing or inhibiting IL-4 and other T cells to generate indirectly.As shown directly to inhibit to complete by being specifically directed at the B cell of IgE generation with antibodies selective as Chang etc. (4).

Chang etc. (4-8) and other people (9) once studied mankind ε-chain and corresponding antibody and coding ε-chain gene and mRNAs.They have illustrated the molecular basis of two class IgE expression: the B cell secreted form and film combining form of IgE synthesis are carried out by being integrated to.The film combining form of IgE (mIgE) can be distinguished by additional film anchor domain and secreted form, this structural domain is stretched from the C-terminal of heavy chain and the CH with IgE₄Rock-steady structure domain is adjacent.Film combining form be integrated to that carry out the B cell surface that synthesizes IgE significantly different.This kind of cell is positioned by the specific antibody of the extracellular segment of that anchor domain with contact, this kind of cell can be removed or inactivate.The cytotoxicity (ADCC) of antibody-dependant and the cell dissolution (6,7) of complement-mediated can be passed through by the mechanism that this kind of antibody removes IgE secretory cell.Circulation IgE and IgE expression cell is reduced in Mice Body by anti-IgE antibodies it has been proved that being by the way that in vivo passively the inhibition of cutaneous anaphylaxis proves in rat model.This point is also proven in people's IgE secretory cell system, and anti-IgE shows the reduction for causing cell to grow, the accumulation for reducing IgE and the cell dissolution in cell dissolution measurement (1) complement-mediated and that ADCC is mediated in this cell line.The nucleotide sequence of the appropriate segment of the mRNA of B cell is expressed by measurement human genome DNA and people mIgE to predict the amino acid sequence of anchor domain extracellular portion.The presence in these sites and accessibility specific antibody (4,8) confirmation specific and that they are to antibody.It is derivative personal ε chain relevant " peptide carrier protein conjugate " (4,8) immune antibody with monoclonal antibody that these are polyclonal.Carrier protein is keyhole limpet hemacyanin (KLH), it is known that it has the ability to stimulate the antibody response to target peptide, thus is useful.This method can be used for proving availability of the mIgE specific peptide immunogene for the immunotherapy of allergic disease, this immunotherapy with monoclonal antibody passive immunity or by active immunity (6,7) by carrying out.

The feasibility of the patient's offer immunization therapy for the sensibility for having IgE to mediate is given to report via Stanworth etc. using different ε chain peptides using this kind of peptide KLH vaccine^(12,13).It is discharged from rat peritoneum mast cell in a manner of titre dependence selected from the histamine for reducing induction from the rabbit-anti peptide serum in the blood that repeatedly immune (can produce and be higher than average anti-peptide titre) obtains.This inhibitory activity is further confirmed by reacting progress live test in (PCA) model in models of passive skin irritability of rats.This measurement evaluation for acting through dye blue area domain for anaphylactoid rabbit-anti peptide serum.When it is assumed that rat caused allergic reaction with allergen before first pass through a variety of allergens application sensitization when can be by estimated color intensity evaluation.These results tentatively show the feasibility using the vaccine therapy allergy based on peptide.However, this peptide conjugation strategy encounters many difficulties.For example, it has been found that the clone of formation and the antiserum with the generation of this kind of conjugate include on the direct anti-protein carrier KLH rather than antibody of the epitope in target peptide (14).Another major defect of protein carrier conjugate vaccine includes: less than optimal immunostimulatory potency, since the production of the extremely limited composition of carrier protein is difficult and the inhomogeneity of conjugation reaction.

The conjugated protein carrier of the peptide based immunogens of the usually used synthesis of those skilled in the art, because small peptide is bad immunogene.In order to make these peptides become immunogene, usually they chemically or by the way that gene is warm are conjugated on big carrier protein.However, these processes can cause the uncertain conformational change of peptide.Moreover, immune response is often misled into carry out advantage immune carrier reaction.Thus, the exploitation for providing the efficient vaccine of prevention allergy lasting for a long time needs further lmmunogen design.It is still to be done carefully to probe " optimal immunogene " design (including attempting sufficiently to confirm with comprehensive test).

Bibliography

1.Noon " pollinosis immunization campaign " Lancet.i:1572-1573 (1991)

2. world health organization and international federation, immunology association working group report: allergen is immune

The current state of therapy, Lancet, i:259-261 (1989)

3.Pene waiting, Proc. Natl. Acad. Sci.USA, 85:6880-6884 (1988)

4.Dayis, Gosset and Chang, biotechnology, 9:53-56 (1991)

5.Sun, Lieu, Sun, Gosset etc., Journal of Immunology, 146:199-205 (1991)

6.Chang. is present in B cell rather than the epitope of the IgE on basocyte surface, the U.S. are special

Benefit number 5,091,313 (1992)

The mankind's ε immunoglobulin peptide and related product that 7.Chang. identifies recently, United States Patent (USP)

5,274,075(1993).

8.Peng, Dayis, Sun etc., Journal of Immunology, 148:129-136 (1992)

9.Saxon, Kurbe-Leamer etc., Journal of Immunology, 147:4000-4006 (1991)

10.Haba and Nisonoff, Proc. Natl. Acad. Sci.USA, 87:3363-3367 (1990)

11.Davis, Gosset, Pinkston, Lieu etc., Sprinser Semin.Immunopath,

15:51-73 (1993)

12.Stanworth, Jones, Lewin and Nayyar, Lancet, 336:1279-

1281(1990).

13.Stanwortb, Lewin, Nayyax and Jones, immunoreactive peptide and antibody and it

Purposes .EPO 403 312 Al (1990) in antiallergic treatment

14.Stanworth and Burt, molecular immunology, 23:1231-1235 (1986)

15.Brett etc., European Journal of Immunology, 23:1608-1614 (1993)

16.Weismuller etc., international peptide study magazine, 40:255-260 (1992)

17.Celis etc., Journal of Immunology, 140:1608-1815 (1988)

18.Demotz etc., Journal of Immunology, 142:394-402 (1989)

19.Chong etc., immunology of infection, 60:4640-4647 (1992)

20.Grant, ed. synthetic peptide instruction, W.H.Freeman&Co., New York, NY,

(1992), the of page 382

21.O ' Hagan etc., vaccine, 9:768-771 (1991)

22.Eldridge etc., molecular immunology, 28:287-294 (1991)

Goal of the invention

It is an object of the present invention to use one group of chemically synthesized IgE (mIgE) ε chain peptide based immunogens in conjunction with the film of T auxiliary epitope peptide linear series, wherein, the high titre antibody that peptide site is set for the dew of people's mIgE film anchor domain can be caused when T auxiliary epitope peptide is imported into the mammal including the mankind.

Another target is the peptide based immunogens of design optimization, this peptide based immunogens, which have, is derived from mankind's mIgE heavy chain film anchor domain (SEQ ID NOS:1,2) specific amino acid and being in a certain direction connected on the peptide comprising the people's helper T cell epitope mixed, generation when this amino acid sequence imports in mammal (including mankind), by stimulation to the effective antibody in people's mIgE anchor domain site.The reduction for the lymphocyte that these antibody can cause IgE to generate, it thus can be with: weakening the generation of the IgE of allergen induction, the activity of mast cell is limited by IgE allergen compound, the subsequent release for generating the chemical mediator (such as histamine) of allergic symptoms is reduced, the passive cutaneous anaphylaxis for inhibiting IgE to mediate reacts (PCA).It is expected that final result is the symptom for reducing allergy.

Another target is the vaccine developed effectively based on mIgE ε chain, provides immunotherapy using the composition comprising this kind of linear peptide based immunogens for the treatment of allergy.

Summary of the invention

According to the present invention, a series of linearly aligned synthetic peptides (a part that this synthetic peptide includes one of two kinds of peptide sequences or their immunogenic analogue and helper T cell epitope (Th epitope) that moiety site (SEQ ID NO:1,2) is set corresponding to people mIgE film anchor domain dew) pass through synthesis in solid state preparation.Composition of the invention is used for immune health mammal, such as generates to mIgE anchor film site (SEQ ID NO:1,2) specificity and without irrelevant antibody the sero-fast rat of high titre and the mankind.

Vaccine according to the invention comprising synthetic peptide combinations as its critical immune original the linear synthetic peptide of immunological effective amount can also be prepared in the presence of adjuvant appropriate and/or application carrier.It is expected that such vaccine composition causes the anti-IgE peptide more concentrated reaction than the peptide carrier protein conjugate currently used by Chang etc. (4,6-8), the better immunotherapy for the treatment of allergy is provided in this way.

Detailed description of the invention

The present invention includes the purposes of one group of new immunogene based on peptide, this immunogene for generating mIgE anchor film site (the SEQ ID NO:1 of people IgE ε heavy chain in mammals (including humans), 2) high titre antibody, the allergic disease that this immunogene is mediated eventually for treatment IgE.

Table I shows arrangement and the amino acid sequence of the film anchor domain of the ε heavy chain of people film combination IgE (mIgE), which is that the nucleotide sequence derivation of the dominant species of the mRNA of the ε chain (7,8) combined from coding film is got.Region on the ε chain-ordering of the peptide used as target immunogene of the present invention, which is drawn, underscore: SEQ ID NO:1's draws single line, and SEQ ID NO:2's draws two-wire.

Table I CH4 structural domain | film anchor domain/extracellular segment 0...ValAsnPro | GlyLeuAlaGlyGlySerAlaGlnSerGlnArgAlaProAspArgValLeuCysHi sSerGlyGlnGlnGlnGlyLeuProArgAlaAlaGlyGlySerValProHisPro

/ extracellular ArgCysHisCysGlyAlaGlyArgAlaAspTrpProGlyProProGluLeuAspVa lCysVal segment 1/cross-film anchors is broken GluGluAlaGluGlyGluAlaProTrpThrTrpThrGlyLeuCysIlePheAlaAl aLeuPhe

/ cytoplasm anchors section LeuLeuSerValSerTyrSerAlaAlaLeuThrLeuLeuMetValGlnArgPheLe uSerAlaThrArgGlnGlyArgProGlnThrSerLeuAspTyrThrAsnValLeuG lnProHisAla

Usual those skilled in the art recognize: allergic symptoms (hypersensitive direct result that IgE is relied on) are caused by the chemical mediator discharged as mast cell and basocyte.When the secretory IgE on the receptor for being connected to mast cell or basocyte is combined by the allergen of the IgE specificity combined to receptor, this release is triggered.In the interaction of antigen-antibody type, after the Fab segment portion that allergen is integrated to the IgE of surface combination, starting triggering reaction.The IgE that allergen/antibody combination cross-linked bivalent surface combines, while the conformation change in the distal side area FC of IgE is induced, this region IgE is connected directly with the acceptor site in mast cell/basocyte cell surface high-affinity Fc receptor and cell surface.Due to not still being so far accurately to understand to mechanism, conformation change activating cell-IgE- allergen compound, while from chemical mediator of the cell release including histamine, cause allergic symptoms and the further secretion of IgE.The secretion IgE of mediate allergic reaction is generated in allergen reaction by the B cell of terminal differentiation.

In addition to secretion is integrated to the circulation IgE of mast cell and basocyte, the B cell for carrying out IgE synthesis also shows the IgE (mIgE) that membrane combines on their surfaces.MIgE molecule is allergic effect original receptor, is considered being played regulatory role in the activation of allergen specific T cell in the maturation and B cell of B cell.MIgE can pass through film anchors section and secretion IgE difference, C-side extension of this film anchors section from the heavy chain for being used to mIgE to be connected to cell membrane.The nucleotide sequence of the appropriate segment of the mRNA of the B cell of mIgE, the amino acid sequence (SEQ ID NO:1,2) of two immunogenic sites of deducibility anchor domain extracellular portion are expressed by measurement human genome DNA and people.These sites exist with the alternative form derived from the mIgE of different mRNA engagement reaction.The direction that two kinds of sites are shown with Table I exists in the dominant species for the ε chain that film combines.The presence in these sites and proximity specific antibody (4,8) confirmation specific and that they are to antibody.Directly resist the presence of the anti-IgE antibodies in this kind of specificity site mIgE, it can lead to the adjoint reduction of the B cell number reduction for generating IgE and circulation IgE by being actively or passively immunized, what this lysis removing that may be by the cell (10,11) of expression IgE reached.Moreover, being desirable to the anti-IgE antibodies of film anchor domain, because structural domain is the surface marker to the cell-specific of expression IgE.There is no this target site on secretory IgE.In this way, this kind of anti-mIgE cannot combine and be crosslinked the IgE being integrated on mast cell and basocyte receptor, and itself it cannot cause histamine release.The cell of the expression IgE of host by allergy is removed the negative regulator that IgE can be caused to generate, and there is the result for the treatment of.

Pass through the use of specific anti-IgE antibodies, this kind of intervention used in treatment allergy, i.e. a kind of immunotherapy, can be passively by being reached with " direct anti-site " the antibody prevention processing of the specificity of IgE or more preferably by initiatively the vaccine being made of the peptide based immunogens in direct anti-site is provided to host with causing host itself to generate the anti-IgE antibodies in directly anti-site.It is believed that active immunity will provide the protection of more effective and longer-term persistent forms.

Film anchor domain (the SEQ ID NO:1 arranged on the IgE that the film shown such as Table I combines, 2) it has confirmed as immunogenic sites in the site on extracellular segment, this immunogenic sites can pass through the close with antibody cross reaction of the IgE secretory cell surface of antibody, antibody therein, which passes through, is immunized animal generation with the mIgE anchor film peptide for being coupled to carrier protein keyhole limpet hemacyanin (KLH) (4,8).

The major defect of these prototype " mIgE peptide " vaccines is their weak immunogene and intrinsic problem relevant to almost all of autoantigen.In the present invention, provide specific embodiment: the immunostimulation unit of synthesis is connected to mIgE peptide (SEQ ID NOS:1 in a certain direction, 2), so that the efficient antibody in these sites on direct anti-mIgE can generate in the host individual of genetic diversity.Conversely, these antibody may cause the Leukopenia of expression IgE, reduce the level of circulation IgE and the reaction of reduced IgE mediation, this creates the terminal a kind of effective treatments of allergic disease for preventing IgE mediation.

While peptide based immunogens of the invention cannot substantially mediate non-cytolytic histamine release, but it can cause antibody with cross reaction is carried out with the target amino acid sequence of the extracellular regions of people's mIgE film anchor domain (SEQ ID NOS:1,2) in serology.

Predose (for example, 0.2-2.5mg；Preferably lmg) immunogene pass through injection (preferably passing through intramuscular injection) application, then repeated drug taking (booster immunization).As known to the vaccine and therapy field, dose-dependant is in the age of patient, weight and general health status.

The present invention does not limit the mammal for providing antibody preparation specifically, it is generally preferred that uses mouse, rabbit, cavy, pig, goat, rat or sheep etc. as host.

For active immunization, term " immunogene " as referred to herein relates to induce the antibody of anti-mIgE film anchor domain (SEQ ID NO:1,2) and leads to the synthetic peptide of the degradation particles of the basocyte for inhibiting IgE to mediate and mast cell.Immunogene of the invention includes linear synthetic peptide, this peptide includes the helper T cell epitope (Th epitope) mixed.To cause efficient antibody response, Th peptide by interval be covalently attached to mIgE film anchor domain peptide (SEQ ID NO:1,2) so as to film anchor peptide N-terminal or C-terminal it is neighbouring.Immunogene also may include the immunologic stimulant amino acid sequence of the invasin protein domain of several kinds (15) corresponding to bacterium Yersinia.Invasin structural domain is connected on Th peptide by interval.

Immunogene of the invention reduces the generation of uncorrelated antibody to cause the immune response more concentrated to " target sequence ", it is i.e. required to mIgE film anchor position point (SEQ ID NOS:1,2) reaction, without generating the unwanted side effect that the immunization therapy process for treating allergy can be made to complicate.However, as required target sequence (mIgE peptide (the SEQ ID NO:1 of 26 and 17 amino acid such as of the invention very in short-term, 2)), due to this kind of short peptide antigens be usually T cell rely on antigen (i.e., these antigens are made to become the presence for needing T auxiliary epitope of immunogenicity), it is a kind of to face another induction to antibody.To provide the t cell epitope of function, it is necessary to make great efforts linear synthetic immunogen of the design comprising short mIgE anchor film peptide (SEQ ID NOS:l, 2).

Peptide of the invention is represented by formula (A) n- (Th) m- (B) o- (mIgE peptide),

Wherein:

A is amino acid, α-NH₂, fatty acid or their derivative or invasin structural domain；

B is amino acid；

Th is helper T cell epitope or its immune enhancing analog or their segment；

MIgE peptide is:

Gly-Leu-Ala-Gly-Gly-Ser-Ala-Gln-Ser-Gln-Arg-Ala-

Pro-Asp-Arg-Val-Leu-Cys-His-Ser-Gly-Gln-Gln-Gln-

Gly-Leu (SEQ ID NO:1)；

Or

Pro-Glu-Leu-Asp-Val-Cys-Val-Glu-Glu-Ala-Glu-Gly-

Glu-Ala-Pro-Trp-Thr (SEQ ID NO:2)；

Or their immunogenic analogue；

N is from 1 to about 10；

M is from 1 to about 4；

O is from 0 to about 10.

Peptide based immunogens of the invention include about 20 to about 100 amino acid residues, preferably from about 20 to about 50 amino acid residues, more preferably from about 20 to about 35 amino acid residues.

When A is amino acid, any non-naturally occurring or any naturally occurring amino acid can be.Non-naturally occurring amino acid includes but is not limited to Beta-alanine, ornithine, nor-leucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulling and analog.Naturally occurring amino acid includes alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.In addition, when m is greater than 1, and when two or more of A group are amino acid, each amino acid can be independently identical or different.

When A is fatty acid (such as stearic acid, palmitinic acid) or derivative of fatty acid [such as S-N- palmityl-(R)-cysteine] (Pam3Cys), it can be used as adjuvant by improving the immunostimulatory properties of Th epitope (16).Fatty acid part or derivatives thereof is preferably positioned as the amino terminal of mIgE peptide.When A is fatty acid or derivatives thereof, peptide based immunogens include other 2 or 3 kind be amino acid part A.Fatty acid used herein is selected from the hydrocarbon chain group with 8-24 carbon atom.Hydrocarbon chain can be saturated or unsaturated.

When A is invasin structural domain, it is the immunostimulation epitope for infecting cellulose protein of Yersinia spp.This immunostimulatory properties are generated since this invasin has the ability with the beta 1 integrin interaction of molecules being present in T cell (the especially T cell or memory T cell of activated immune).It was found that with beta 1 integrin interaction invasin structural domain specific sequence by Brett etc.⁽¹⁵⁾Description, refers to together herein.In a preferred embodiment, the sequence for being connected to the invasin structural domain (Inv) of Promiscuous Th epitopes is:

Thr-Ala-Lys-Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala-

The immunostimulation analog of Thr-Tyr-Gln-Phe (SEQ ID NO:3) or its corresponding region that cellulose protein is infected derived from another Yersinia spp.Thus, if this analog retains immunostimulatory properties, it can be comprising replacing, lacking or be inserted into amino acid residue to accommodate the variation between bacterial strain.

In one embodiment, it is α-NH that n, which is 1, A,₂.In another embodiment, it is α-NH that n, which is 4, A,₂, the sequence of invasin structural domain (Inv) is Gly-Gly.

B is interval, and is naturally occurring or non-naturally occurring amino acid as described above.Every kind of B is independently identical or different.In addition, the amino acid of B can form flexible hinge or interval to improve the immune response to Th epitope and mIgE peptide and its immunogenic analogue.The example for encoding the sequence of flexible hinge is found in heavy chain immunoglobulin hinge area.The frequent Pro-rich of flexible hinge sequence.A kind of flexible hinge being particularly useful is provided by sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ IDNO:4), and wherein Xaa is arbitrary amino acid, preferably aspartic acid.

Immunogenicity can also pass through the additional enhancing of the spacer residue (such as Gly-Gly) between the Th epitope and mIgE film anchor peptide (SEQ ID NOS:1,2) and their immunogenic analogue mixed.In addition to being physically separated Th epitope (i.e. mIgE film anchor peptide (SEQ ID NO:1 from B cell epitope, 2) and their immunology analog) except, interval glycine residue can destroy any by mIgE film anchor peptide (SEQ ID NO:1,2) and the artificial secondary structure of its immunogenic analogue and Th epitope connecting structure, thus the interference between T and/or B cell reaction can be removed.In this way, the separation of conformation between the immunogene and suitable Th and B cell illustrated so that more effectively interact between auxiliary cell and the antibody for causing structural domain.

Th is amino acid (natural or non-natural amino acid) sequence comprising Th epitope.Th epitope can be made of continuous or discontinuous epitope.It therefore be not all amino acid of Th is all the required part of epitope.Thus, Th epitope (analog and segment including Th epitope) can improve or stimulate the immune response to mIgE film anchor peptide (SEQ ID NO:1,2) and its immunogenic analogue.The Th epitope that advantage is immunized and mixes highly is reacted extensively in the animals and humans with extensive divergence MHC type (17-19).The Th structural domain of peptide of the present invention has about 10 to about 50 amino acid, it is therefore preferred to have about 10 to 30 amino acid.When multiple Th epitopes have (i.e. m >=2), each Th epitope is independently identical or different.

Th epitope analogs include replacing, being additional, lacking and being inserted into 1 to about 10 amino acid residues in Th epitope.Th segment is the adjacent part of Th epitope, this Th epitope is enough to improve or stimulate the immune response to mIgE film anchor peptide (SEQ ID NO:1,2) and its immunogenic analogue.

Th epitope of the invention includes Hepatitis B Surface and core antigen helper T cell epitope (HB_sTh and HB_cTh), pertussis toxin helper T cell epitope (PT Th), tetanus toxin helper T cell epitope (TT Th), measles virus F protein matter helper T cell epitope (MV_FTh), Major Outer Membrane Protein of Chla mydia trachomatis matter helper T cell epitope (CT Th), diphtheria toxin helper T cell epitope (DT Th), keyhole limpet hemacyanin, plasmodium falciparum ring falciform bodi2s (circumsporozoite) helper T cell epitope (PF Th), schistosoma mansoni phosphotriose isomerase helper T cell epitope (SM Th), any segment of Escherichia coli TraT helper T cell epitope (TraT Th) and immune enhancing analog and these Th epitopes.Following Table II gives the example of Th epitope sequences:

Table II HB_sTh:Phe-Phe-Leu-Leu-Thr-Arg-Ile-Leu-Thr-Ile-Pro-Gln-

Ser-Leu-Asp (SEQ ID NO:5) PT₁Th:Lys-Lys-Leu-Arg-Arg-Leu-Leu-Tyr-Met-Ile-Tyr-Met-

      Ser-Gly-Leu-Ala-Val-Arg-Val-His-Val-Ser-Lys-Glu-

Glu-Gln-Tyr-Tyr-Asp-Tyr (SEQ ID NO:6) TT₁Th:Lys-Lys-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-

Gly-Ile-Thr-Glu-Leu (SEQ ID NO:7) TT₂Th:Lys-Lys-Phe-Asn-Asn-phe-Thr-Val-Ser-Phe-Trp-Leu-

      Arg-Val-Pro-Lys-Val-Ser-Ala-Ser-His-Leu

(SEQ ID NO:8) PT_1ATh:Tyr-Met-Ser-Gly-Leu-Ala-Val-Arg-Val-His-Val-Ser-

Lys-Glu-Glu (SEQ ID NO:9) TT₃Th:Tyr-Asp-Pro-Asn-Tyr-Leu-Arg-Thr-Asp-Ser-Asp-Lys-

      Asp-Arg-Phe-Leu-Gln-Thr-Met-Val-Lys-Leu-Phe-Asn-

Arg-Ile-Lys (SEQ ID No:10) PT₂Th:Gly-Ala-Try-Ala-Arg-Cys-Pro-Asn-Gly-Thr-Arg-Ala-

      Leu-Thr-Val-Ala-Glu-Leu-Arg-Gly-Asn-Ala-Glu-Leu

(SEQ ID N0:11) MV_F1Th:Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-His-Arg-Leu-

Glu-Gly-Val (SEO ID NO:12) HB_cTh:Val-Ser-Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala-

      Tyr-Arg-Pro-Pro-Asn-Ala-Pro-Ile-Leu

(SEQ ID N0:13) MV_F2Th:Gly-Ile-Leu-Glu-Ser-Arg-Gly-Ile-Lys-Ala-Arg-Ile-

Thr-His-Val-Asp-Thr-Glu-Ser-Tyr (SEQ ID N0:14) TT₄Th:Trp-Val-Arg-Asp-Ile-Ile-Asp-Asp-Phe-Thr-Asn-Glu-

Ser-Ser-Gln-Lys-Thr (SEQ ID N0:15) TT₅Th:Asp-Val-Ser-Thr-Ile-Val-Pro-Tyr-Ile-Gly-Pro-Ala-

Leu-Asn-Ile-Val (SEQ ID NO:16) CT Th:Ala-Leu-Asn-Ile-Trp-Asp-Arg-Phe-Asp-Val-Phe-Cys-

        Thr-Leu-Gly-Ala-Thr-Thr-Gly-Tyr-Leu-Lys-Gly-Asn-

Ser (SEQ ID NO:17) DT₁Th:Asp-Ser-Glu-Thr-Ala-Asp-Asn-Leu-Glu-Lys-Thr-Val-

        Ala-Ala-Leu-Ser-Ile-Leu-Pro-Gly-Ile-Gly-Cys

(SEQ ID NO:18) DT₂Th:Glu-Glu-Ile-Val-Ala-Gln-Ser-Ile-Ala-Leu-Ser-Ser-

        Leu-Met-Val-Ala-Gin-Ala-Ile-Pro-Leu-Val-Gly-Glu-

        Leu-Val-Asp-1Ile-Gly-Phe-Ala-Ala-Thr-Asn-Phe-Val-

Glu-Ser-Cys (SEQ ID NO:19) PF Th:Asp-Ile-Glu-Lys-Lys-Ile-Ala-Lys-Met-Glu-Lys-Ala-

        Ser-Ser-Val-Phe-Asn-Val-Val-Asn-Ser

(SEQ ID NO:20) SM Th:Lys-Trp-Phe-Lys-Thr-Asn-Ala-Pro-Asn-Gly-Val-Asp-

Glu-Lys-Ile-Arg-Ile (SEQ ID NO:21) TraT₁Th:Gly-Leu-Gln-Gly-Lys-His-Ala-Asp-Ala-Val-Lys-Ala-

Lys-Gly (SEQ fD NO:22) TraT₂Th:Gly-Leu-Ala-Ala-Gly-Leu-Val-Gly-Met-Ala-Ala-Asp-

Ala-Met-Val-Glu-Asp-Val-Asn (SEQ ID NO:23) TraT₃Th:Ser-Thr-Glu-Thr-Gly-Asn-Gln-His-His-Tyr-Gln-Thr-

Arg-Val-Val-Ser-Asn-Ala-Asn-Lys (SEQ ID NO:24)

The linear synthetic peptide of the invention as described in formula (A) n- (Th)-(B) o- (mIgE peptide) or (mIgE peptide)-(B) o- (Th) m- (A) n has the Th epitope being covalently attached on mIgE film anchor peptide (SEQID NOS:1,2) and their any N-terminal of immunogenic analogue by interval B.In a preferred embodiment, Th epitope is HB₅、Th、pT₁ Th、PT₂ Th、TT₁ Th、TT₃Th or MV_F1 Th。

The sequence of mIgE film anchor peptide includes:

Gly-Leu-Ala-Gly-Gly-Ser-Ala-Gln-Ser-Gln-

Arg-Ala-Pro-Asp-Arg-Val-Leu-Cys-His-Ser-

Gly-Gln-Gln-Gln-Gly-Leu (SEQ ID NO:1)；

Or

Pro-Glu-Leu-Asp-Val-Cys-Val-Glu-Glu-Ala-

Glu-Gly-Glu-Ala-Pro-Trp-Thr (SEQ ID NO:2)

According to mIgE anchor peptide (SEQ ID NO:1 of the present invention, 2) immunogenicity peptide analogues may further include substitution, addition, missing, from 1 to about 4 amino acid residue of insertion (if peptide analogues can cause with the cross-immune reaction of mIgE film anchor peptide (SEQ ID NOS:1,2)).Replace, addition and insertion can be completed with natural or unnatural amino acid as defined herein.

Therefore, currently preferred peptide based immunogens are the linear synthetic peptides comprising mIgE film anchor peptide (SEQ ID NOS:1,2) or its immunogenic analogue and Th.Preferred peptide based immunogens are linear constructs, these constructs include mIgE film anchor peptide (SEQ ID NOS:1,2) or its immunogenic analogue；It is spaced (such as Gly-Gly)；Selected from HB₅、Th、PT₁、Th、PT₂、Th、TT₁、Th、TT₃, Th and MV_F1The Th epitope of Th (respectively SEQ ID NOS:5,6,11,7,10,12) and dispensable Inv structural domain (SEQ ID NO:3) or its analog.

Peptide based immunogens of the invention can be prepared by the chemical synthesis being well known for ordinary skill in the art.See, e.g., the synthetic peptide of Grant ed.⁽²⁰⁾.Therefore, the automatic Merrifield technology synthesis of synthesis in solid state can be used in peptide, and this synthesis passes through the α-NH of T-Boc or F-moc chemoproection using the amino acid of side chain protection₂It carries out (as using Biosystems Peptide Synthesizer 430A or 431 models).

When A is fatty acid, can be easily added on the N-terminal amino group of synthetic peptide with well known dicyclohexylcarbodiimide coupling method.

[2,3- bis- (palm acyl-oxygen)-(2R)-propyl-N- palmityl-(R)-cysteines can also be synthesized chemically by Pam3Cys, lipoamino acid S-.Such as Pam3Cys can be coupled to the N-terminal of mIgE peptide by synthesis in solid state, and the Pam3Cys-OH for lipoamino acid being connected to the mIgE peptide chain of binding resin has been used in last coupling step.For the dissolubility for improving the lipopeptide product being finally coupled, solid-phase peptide can have other serine and lysine residue to extend in N-terminal.

After required immunogene is assembled completely, resin is handled with the cleavage of peptide from resin according to standard method and makes function base deblocking on amino acid side chain.Free peptide is purified by HPLC, and determines its feature by biochemical method, for example, can be carried out by amino acid analysis or sequencing.The purifying of peptide and qualitative method are well known to those of ordinary skill in the art.

The other chemical methodes for generating the linear peptidic constructs comprising the site mIgE and Th of the invention include that haloacetylating and cysteinylated peptide passes through the connection that " thioether " key is formed.For example, cysteine can be attached to the C-terminal of the peptide comprising Th, the sulfydryl of cysteine can be used for and electrophilic group (such as N^αα-or the ε-NH of the lysine residue of mIgE film anchor peptide (SEQ ID NOS:1 or 2) or the N-terminal of its immunogenic analogue are connected to derived from chloracetyl is modified or maleimide₂Base) form covalent bond.In this way, the construct with Th- (mIgE peptide) or its opposite sequence (mIgE peptide)-Th can be obtained.

Immunogene of the invention can also polymerize to be formed.Such as-NH of the common method by glutaraldehyde and lysine residue can be used in polymerization reaction₂Reaction between base is completed.By another method, linear " interval A-Th--(mIgE peptide) " or " (mIgE peptide)-interval-(Th) " immunogene can be formed by polymerization or combined polymerization and (be attached to linear " interval A-Th--(mIgE peptide) " or " (mIgE peptide)-interval-(Th) m- (A) n " immunogene N-terminal using other cysteine).α-or the ε-NH of lysine residue derived from the amino acid or maleimide that the sulfydryl of N-terminal cysteine is used to modify with halogen acetyl₂Base forms " thioether " key, and wherein lysine residue is connected to the N-terminal of the poly- lysyl core element of branch (for example, K₂K, K₄K₂K or K₈K₄K₂K)。

In addition, longer linear peptide based immunogens are able to use known recombinant DNA technology synthesis.The standard manual of any DNA technique all provides the detailed scheme for generating peptide of the invention.The gene of peptide of the invention is encoded for building, it is nucleic acid sequence that the optimal codon reverse translation in organism expressed by gene, which is preferably used, in amino acid sequence.Then, typically via synthesis overlapping oligonucleotide construction synthesis gene, the oligonucleotides used codified peptide and any adjusting unit when necessary.Synthesis gene can be plugged into suitable cloning vector, obtains recombinant and determines its feature.Then, peptide is expressed under conditions of being suitable for expression system and the host of selection.The method of peptide standard purifies and determines its feature.

The efficiency of immunogene of the invention can be by injecting to animal (for example, rat), and the monitoring being then described in detail such as embodiment determines the humoral immune reaction of mIgE film anchor peptide (SEQ ID NO:1,2) and its immunogenic analogue.

Another aspect of the present invention provides a kind of vaccine composition, and this vaccine composition includes one or more immunogenes of immunological effective amount of the invention, and the immunogene is pharmaceutically in acceptable delivery system.This kind of vaccine composition is used for the prevention of the atopic allergology including allergic rhinitis, food allergy allergy, asthma, allergic reaction and others the IgE allergic reaction (asthma of such as virus induction) mediated.

Therefore, subject of the present invention peptide can be used other conventional constituents used in adjuvant, pharmaceutically acceptable carrier or vaccine composition and be configured to vaccine composition.This kind of preparation is easy to be determined by those skilled in the art, and including discharging at once and/or sustained release and inducible system is immune and/or the preparation of induction local mucosal immunity, for example, this can contain immunogene completion by particle.This vaccine can be applied by any convenient route (including subcutaneous, oral, intramuscular or other parenterals or intestines channel).Similarly, vaccine can be applied as single dose or multi-agent.Immune programme table is easy to be determined by those of ordinary skill.Adjuvant or emulsifier for use in the present invention include alum, incomplete Freund's adjuvant, liposyn, saponin(e, L121, emulsigen and ISA720 and other effective adjuvants and emulsifier.

Vaccine composition of the invention includes a effective amount of one or more immunogenes and pharmaceutically acceptable carrier of the invention.This kind of composition of suitable dosage unit form generally comprises about 0.5 μ g to about 1mg immunogene/kg body weight.When being applied with confection, it is can easily be divided into amount appropriate with every dosage unit.

Immunizing potency can be improved in wider group comprising the vaccine of this immunogenic compounds with two or more Th epitopes, which provides the immune responses of the enhancing to mIgE film anchor peptide (SEQ ID NO:1,2).For example, the mixture of the 7-10 peptide of No. 1-4 of embodiment 1 and embodiment 4 is useful.MIgE anchor peptide immunogens based on other immunologic stimulant synthetic peptides become lipopeptid (such as Pam3Cys) through modification, to provide a kind of adjuvant of insertion to efficient vaccine.The immune response of synthesis mIgE anchor peptide comprising immunogene can be applied by retention in the biodegradable particle by descriptions such as O ' Hagan or thereon to improve.This immunogene can be encapsulated with or without adjuvant, and includes the Pam3Cys of covalent linkage, this based fine particles can carry immunostimulation adjuvant (such as incomplete Freund's adjuvant or alum).The function of particle is the immune response of booster immunization original, the local mucosal immunity including being particularly suitable for mucous membrane part allergy, and provides time controlled released for lasting or periodic reaction, and can be used for being administered orally and local application (21,22).

Specific peptide immunogene and composition are provided in the examples below that illustrate the present invention.Peptide based immunogens of the invention are useful to the allergic disorder that IgE is mediated is improved.

Embodiment 1

1-4 peptide based immunogens " interval Th--(mIgE peptide) " and

The synthesis of " (mIgE peptide)-interval-Th "

Immunogene 1-4 (Table III) passes through synthesis in solid state according to the guidance of manufacturer in application Biosystems Peptide Synthesizer 430A or 431 models using F-moc chemical method.After peptide assembles completely, with the cleavage of peptide from resin and the functional group on amino acid side chain is deprotected according to standard method processing resin.Then free peptide is purified by HPLC, and the feature of amino acid content and sequence is determined with biochemical method.

The structure feature of 1-4 peptide based immunogens (from aminoterminal to c-terminus) is A-Th-B- (mIgE peptide) or (mIgE peptide)-B-Th-A, wherein " A " is Nh₂, " B " is the interval Gly-Gly, and " Th " is measles virus F1 helper T cell epitope MV_F1Th (SEQ ID NO:12), " (mIgE peptide) " are the mIgE peptides of SEQ ID NO:1 or SEQ ID NO:2.In this way, for example, No. 1 peptide can be used as " MV_F1Th-GG- (mIgE1) " is explicitly described, and No. 2 peptides can be used as " (mIgE2)-GG-MV_F1Th " is illustrated.The real sequence of peptide 1-4 (SEQ ID NO:25-28) is displayed in Table 2.

Table 2

The amino acid sequence of 1-4 peptide

Peptide	Sequence
		1.MVF1Th-GG-mIgE1	Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val- His-Arg-Leu-Glu-Gly-Val-Gly-Gly-Leu- Ala-Gly-Gly-Ser-Ala-Gln-Ser-Gin-Arg- Ala-Pro-Asp-Arg-Val-Leu-Cys-His-Ser- Gly-Gln-Gln-Gln-Gly-Leu (SEQ ID No:25)
2.mIgE1-GG-MVF1Th	Leu-Ala-Gly-Gly-Ser-Ala-Gln-Ser-Gln- Arg-Ala-Pro-Asp-Arg-Val-Leu-Cys-His- Ser-Gly-Gln-Gln-Gln-Gly-Leu-Gly-Gly- Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val- His-Arg-Leu-Glu-Gly-Val (SEQ ID No:26)

3.MVF1Th-GG-mIgE2S	Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val- His-Arg-Leu-Glu-Gly-Val-Gly-Gly-Pro- Glu-Leu-Asp-Val-CLs-Val-Glu-Glu-Ala- Glu-Gly-Glu-Ala-Pro-Trp-Thr (SEQ ID No:27)
		4.mIgE2-GG-MVF1Th	Pro-Glu-Leu-Asp-Val-Cys-Val-Glu-Glu- Ala-Glu-Gly-Glu-Ala-Pro-Trp-Thr-Gly- Gly-Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile- Val-His-Arg-Leu-Glu-Gly-Val (SEQ ID No:28)

Embodiment 2

With " interval Th--(mlgE peptide) " and " (mIgE peptide)-interval-Th "

Linear construct and 1-4 peptide immune rat simultaneously evaluate its immunogenicity by ELISA

The efficiency of 1-4 peptide based immunogens is evaluated in the group of 5 rats by the serological analysis of experiment immunization scheme outlined below and measurement immunogenicity.

Experimental design:

Immunogene: 1-4 peptide (every group 1)

Dosage: immune 100 μ g every time

Route: intramuscular

Adjuvant: Freund completely/Freund's incomplete adjuvant

Dose schedule: the 0th week (FCA), 3 weeks and 6 weeks (IFA)

Blood sampling time table: the 0th, 3,6,8,10 week

Species: Sprague-Dawley rat

The size of group: 5

Measurement: the ELISAs measurement of antipeptide activity.

Before by being titrated with target peptide (SEQ ID NO:1,2) through ELISA, blood is collected, serum is processed into and saves.

Anti-peptide antibody activity is measured using 96 hole flat-bottom microtiter plates by ELISA (enzyme linked immunosorbent assay (ELISA)), and titer plate is used as respective target mIgE peptide epitopes (the No.5 peptide of the anchor position the mIgE point of SEQ ID NO:1 description or No. 6 peptides of SEQ ID NO:2 description) coating of immunosorbent.The aliquot (100 μ L) of the peptide based immunogens solution of 5 μ g/ml concentration incubates 1 hour at 37 DEG C.Plate is closed by incubating 1 hour again at 37 DEG C with 3% gelatin/PBS solution.Then closed plate is dry and for measuring.The aliquot (100 μ L) of the rat blood serum of test starts then to be serially diluted, be then added on the plate of peptide coating with 10 times with 1: 10 dilution in sample dilution buffer.Plate incubates 1 hour at 37 DEG C.

Plate is washed six times with 0.05%PBS/Tween buffer.The goat anti-rat antibody of 100 μ L horseradish peroxidase-labeleds is added in conjugate dilution buffer (phosphate buffer comprising 0.5MNaCl and normal lowlenthal serum) with 1: 1,000 dilution.Plate incubates 1 hour at 37 DEG C before as above washing.Then the aliquot (100 μ L) of oxygen o-phenylene diamine substrate solution is added.Colour developing 5-15 minutes, then by the way that 50 μ L 2N H2SO are added₄Stop enzyme-catalytic chromogenic reaction.The A of each hole content_492nmValue is read in plate reader.

All serum is measured by anti-peptide ELISA, the A of those 1: 100 dilutions_492nmThe sample of value >=0.2 is compareed as seropositivity and is recorded.Normal rat blood serum is used as negative control.As a result also compared with the positive control serum for the rat being immunized with the KLH conjugate of above-described No.5 or 6 peptides, to prove the immunogenicity of the enhancing of peptide of the invention.

Embodiment 3

The rat antisera of 1-4 peptide

The evaluation of functional efficiency in the cell line that people secretes IgE

Peptide based immunogens composition of the invention generates the antibody of the human B cell for secretion IgE, and inhibits the generation of IgE.Not as most of antibody of anti-IgE, the antibody that mIgE peptide generates not can be incorporated into the cell for only generating the IgE of secreted form for being integrated to receptor, thus, it itself cannot trigger the release of chemical mediator in the mast cell of IgE sensitization and the allergy of basocyte.These bioactivity are related with the immunotherapy of allergy, can be observed in the rat antisera of 1-4 peptide based immunogens by measuring following function:

1. combining the antibody of basocyte of the B cell of IgE secretion without combining IgE sensitization.B cell system (such as the myeloma cell line SKO-007 (ATCC that people IgE is generated, Rocky ille MD) or EBV conversion B cell system 8866) incubated with the serial dilutions of the anti-mIgE of rat, the anti-rat IgG that FITC label is used in combination of antibody and pass through the quantitative determination detection of fluorescence stream cytometry.The combination degree of the anti-mIgE antibody of rat and normal person's basocyte evaluates (5) in a similar manner, which prepares from peripheral blood and use secretion IgE loading or sensitization preparation.

2. reducing the accumulation of IgE in the cell of secretion IgE.IgE is accumulated on myeloma cell line SKO-007 and the culture medium of similar IgE secretory cell system.The reduction that can IgE secretory cell cause IgE to be secreted into culture medium is handled with the anti-mIgE serum of the rat generated by 1-4 peptide based immunogens to determine, cell is handled with a series of antibody concentrations, and the IgE level in culture medium at any time monitors (11) by the ELISA of IgE specificity.Effective antibody causes the relevant reduction of medicament for accumulating IgE.

3. the cell of IgE is secreted in antibody (ADCC) cracking of the cytotoxicity mediated by dependent cells.The ability of the cell cracking of various concentration induction SKO-007 cell (or similar cell) of the anti-mIgE serum of rat evaluates it by ADCC activity and cracks percentage, and evaluation is carried out by using the measurement of several donor effector cells⁽¹¹⁾。

4. not can induce histamine to discharge from basocyte.The anti-mIgE serum of rat is used to show the ability that induction histamine related with dosage is discharged from the basocyte of the isolated peripheral blood of IgE sensitization with several concentration.Histamine release is quantitative determined by fluorimetry⁽⁵⁾.The evidence of efficiency and safety that histamine release is peptide based immunogens of the invention is not can induce.

Embodiment 4

Further widen the peptide based immunogens mixture of reaction group

The interval Th--(mIgE peptide) of immunogenicity and the mixture of (mIgE peptide)-interval-Th peptide can be used to the immune response in the multifarious crowd's body of Widening genetic, wherein there is more than one Th epitope by different MHC type identifications.From Table II selection to the Promiscuous Th epitopes widened herd immunity and react useful.The Th epitope useful to these peptide mixers is including but not limited in 1-4 peptide (SEQ IDNOS:25-28) and HB in Table II_sMV used in Th peptide (SEQ ID NO:5)_F1Th peptide (SEO IDNO:12).Include two kinds of anchor film mIgE peptide sequences (SEQ ID NOS:1 or 2) and HB_sThe peptide of one of Th peptide describes in table 1V as 7-10 peptide based immunogens (SEQ ID NOS:29-32).

As described in example 1 above synthesis 7-10 peptide based immunogens, and mutually and mixed with 1-4 peptide based immunogens with identical molar ratio to prepare peptide mixer.The immunizing potency of composition of the invention as mixture preparation is evaluated by scheme described below in rats.

Experimental design:

Immunogene: (1) mixture: No. 1-4 and 7-10 peptide

(2) positive control:

(every group of rat is a kind of for the conjugate that 5 and No. 6 peptides are individually combined with KLH with 1: 1

Immunogene)

Dosage: the molar equivalent or mIgE peptide equivalent of every kind of synthetic peptide based immunogens are equal to every time immune

100 μ g are total or every kind of peptide of 12.5 μ g.

Route: intramuscular (injection)

Adjuvant: (1) Freund completely/Freund's incomplete adjuvant

(2) 0.4% alum (aluminium hydroxide) (any adjuvant of every group of every kind of immunogene) dose schedule: 0,2 and 4 week

CFA/IFA group receives IFA on the 2nd and 4 week in the 0th week reception CFA.Alum group connects

By the Alum formulations of all 3 kinds of dosage.Blood sampling time table: 0,3,6 and 8 week

Species: the size of Sprague-Dawley rat group: 5,4 groups

Measurement: measuring the ELISA of antipeptide activity twice, solid substrate, 5 and No. 6 peptides (SEQ IDNOS:1,2).

Blood is collected, be processed into serum before carrying out seroconversion measurement by two kinds as described in Example 2 anti-peptide ELISAs and is saved.

It designs this experiment and is to prove immunogenicity that the application of peptide of the present invention improves, and illustrate the efficiency of the composition of the preparation of the invention with pharmaceutically acceptable adjuvant (alum).

Table IV

The amino acid sequence of 7-10 peptide

Peptide	Sequence
		7.HBsTh-GG-    mIgE1	Phe-phe-Leu-Leu-Thr-Arg-Ile-Leu- Thr-Ile-Pro-Gln-Ser-Leu-Asp-Gly- Gly-Leu-Ala-Gly-Gly-Ser-Ala-Gln- Ser-Gln-Arg-Ala-Pro-Asp-Arg-Val- Leu-Cys-His-Ser-Gly-Gln-Gln-Gln- Gly-Leu (SEQ ID No:29)
8.mIgE1-GG-    HBsTh	Leu-Ala-Gly-Gly-Ser-Ala-Gln-Ser- Gln-Arg-Ala-Pro-Asp-Arg-Val-Leu- Cys-His-Ser-Gly-Gln-Gln-Gln-Gly- Leu-Gly-Gly-Phe-Phe-Leu-Leu-Thr- Arg-Ile-Leu-Thr-Ile-Pro-Gln-Ser- Leu-Asp (SEQ ID No:30)
		9.HBsTh-GG-    mIgE2	Phe-Phe-Leu-Leu-Thr-Arg-Ile-Leu- Thr-Ile-Pro-Gln-Ser-Leu-Asp-Gly- Gly-Pro-Glu-Leu-Asp-Val-Cys-Val- Glu-Glu-Ala-Glu-Gly-Glu-Ala-Pro- Trp-Thr (SEQ ID No:31)
10.mIgE2-GG-    HBsTh	Pro-Glu-Leu-Asp-Val-Cys-Val-Glu- Glu-Ala-Glu-Gly-Glu-Ala-Pro-Trp- Thr-Gly-Gly-Phe-Phe-Leu-Leu-Thr- Arg-Ile-Leu-Thr-Ile-Pro-Gln-Ser- Leu-Asp (SEQ ID No:32)

Embodiment 5

Prove the clinical test of the therapeutic efficiency of mixture

The single construct for carrying the Th epitope sequences of measles virus F and Hepatitis B Surface antigen is the HLA DR antigen of the multiple-effect for human body mixed, to provide maximum immunogenicity in crowd's body of genetic diversity.Moreover, because these Th epitopes are derived from pediatric vaccines, childhood it is immune be immune crowd Th memory molecule potential source.Therefore, pediatric vaccines have the immunizing potency for the antiallergic action vaccine being made of this kind of Th peptide mixer for providing enhancing.The effect of clinical protocol below is to prove composition of the invention is designed, the composition is prepared as this kind of linear " interval Th--(mIgE peptide) " and " (mIgE peptide)-interval-Th " peptidic constructs and adjuvant (alum) mixture accepted extensively.

Experimental design:

Subject: hay feverite

Season and perdurabgility: hay fever season, 8 weeks

Group: 4 groups, 1 group/immunogene/dosage, every group of N=15, wherein 12 receptions

Immunogene, 3 reception placebos

Immunogene: mixture 1；1-4,7-10 peptide

Adjuvant: 0.2% alum

Dosage: the molar equivalent of every kind of synthetic peptide is equal to that 500 μ g are total or every dosage

Every kind of peptide of 62.5 μ g

Route: intramuscular

Dose schedule: the 0th week and the 4th week

Evaluation time table: the 0th, 4 and 6 week

Blood is collected, serum is processed into before titrating as described in Example 2 through ELISA and is saved.

The efficiency of clinical composition and safety are compared the treated patient of hay fever medication, allergic symptoms and the physical examination of side reaction and access by recording patient and are carried out to the evaluation of institute's medication by serological test, the comparative evaluation of skin test reactions.Serological evaluation includes the ELISAs and measurement histamine levels of above-mentioned anti-peptide titre⁽¹³⁾Reduce and determine that product does not trigger the automatic spectrofluorimeter measurement of standard of histamine release.Skin-test is intracutaneous test, wherein the upper layer of the normalization solution injection skin allergen.The reaction of allergen is quantitative determined by typical case " the pomphus and flush " area that measurement in Skin-test generates in allergen reaction.Expected result include at the end of research allergic symptoms be clearly better, and sign without histamine release.

This experiment proves that pharmaceutically acceptable composition of the invention clinical efficacy and safety.

Sequence table (1) general information:

(i) applicant: United Biomedical company；

Walfield, Alan M.；Wang, Chang Yi

(ii) denomination of invention: for treating the synthesis IgE membrane anchor peptide immunogens of allergy

(iiii) sequence number: 32

(iV) address:

(A) receiver: Maria C. H Lin

(B) street: 345 park roads

(C) city: New York

(D) state: New York

(E) national: the U.S.

(F) zip code: 10154

(v) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: WordPerfect 5.1

(Vi) data of earlier application:

(A) application number: 08/328,519

(B) date of application: on October 25th, 1994

(C) classification number:

(vii) data of present application:

(A) apply for quantity: to be specified

(B) applying date: October 25 nineteen ninety-five

(C) classification number:

(viii) lawyer/agent's information:

(A) name: Lin, Maria C.H.

(B) registration number: 29,323

(C) certificate number: 1151-4117

(ix) telecom information:

(A) phone: 212-758-4800

(B) it faxes: 212-751-8849

(C) fax: the information of 421792 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 26 amino acid

(B) type: amino acid

(D) topological structure: linear

(ii) molecule type: peptide

(xi) sequence description: SEQ ID N0:1:

Gly Leu Ala Gly Gly Ser Ale Gln Ser Gln Arg Ala Pro

  1              5                  10

Asp Arg Val Leu Cys His Ser Gly Gln Gln Gln Gly Leu

The information of 15 20 25 (2) SEQ ID NO:2:

(i) sequence signature

(A) length: 17 amino acid

(E) type: amino acid

(D) topological structure: linear

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:2:

Pro Glu Leu Asp Val  Cys Val Glu Glu Ala Glu Gly Glu

 1              5                  10

Ala Pro Trp Thr

The information of 15 (2) SEQ ID NO:3:

(i) sequence signature:

(A) length: 16 amino acid

(B) type: amino acid

(D) topological structure: linear

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:3:

Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala Thr

1              5                  10

Tyr Gln Phe

The information of 15 (2) SEQ ID NO:4:

(i) sequence signature:

(A) length: 6 amino acid

(B) type: amino acid

(D) topological structure: linear

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:4:

Pro Pro Xaa Pro Xaa Pro

The information of 15 (2) SEQ ID NO:5:

(i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological structure: linear

(ii) molecule type: peptide

(xi) sequence description: 15 10 Ser Leu Asp of SEQ ID NO:5:Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gin

The information of 15 (2) SEQ ID NO:6:

(i) sequence signature:

(A) length: 30 amino acid

(B) type: amino acid

(D) topological structure: linear

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:6:

Lys Lys Leu Arg Arg Leu Leu Tyr Met Ile Tyr Met Ser

1              5                   10

Gly Leu Ala Val Arg Val His Val Ser Lye Glu Glu Gin

  15                  20             25  Tyr Tyr Asp Tyr

The information of 30 (2) SEQ ID NO:7:

(i) sequence signature

(A) length: 17 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: 5 10Ile Thr Glu Leu of SEQ ID NO:7:Lys Lys Gin Tyr Ile Lye Ale Asn Ser Lys Phe Ile Gly1

The information of 15 (2) SEQ ID NO:8: (i) sequence signature:

(A) length: 22 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: 15 10Val Pro Lys Val Ser Ale Ser His Leu of SEQ ID NO:8:Lys Lys Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg

The information of 15 20 (2) SEQ ID NO:9:

(i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: 15 10 Glu Glu of SEQ ID NO:9:Tyr Met Ser GlY Leu Ala Val Arg Val His Val Ser Lys

The information of 15 (2) SEQ ID NO:10:

(i) sequence signature:

(A) length: 27 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:10:Tyr Asp Pro Asn Tyr Leu Arg Thr Asp Ser Asp Lys Asp

1              5                  10  Arg Phe Leu Gin Thr Met Val Lye Leu Phe Asn Arg Ile

The information of 15 20 25 Lys (2) SEQ ID NO:11:

(i) sequence signature:

(A) length: 24 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule-type: peptide (xi) sequence description: 15 10 Thr Val Al a Glu Leu Arg Gly Asn Ala Glu Leu of SEQ ID NO:11:Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu

The information of 15 20 (2) SEQ ID NO:12:

(i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: 15 10Gly Val of SEQ ID NO:12:Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu

The information of 15 (2) SEQ ID NO:13: (i) sequence signature:

(A) length: 21 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: 15 10 Arg Pro Pro Asn Ala Pro Ile Leu of SEQ ID NO:13:Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr

The information of 15 20 (2) SEQ ID NO:14:

(i) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: 15 10 His Val Asp Thr Glu Ser Tyr of SEQ ID NO:14:Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr

The information of 15 20 (2) SEQ ID NO:15:

(i) sequence signature:

(A) length: 17 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:15:Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser

1              5                 10  Ser Gln Lys Thr

The information of 15 (2) SEQ ID N0:16:

(i) sequence signature:

(A) length: 16 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID N0:15:Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu

1              5                  10 Asn  Ile  Val

The information of 15 (2) SEQ ID NO:17:

(i) sequence signature:

(A) length: 25 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) type: peptide (xi) sequence description: SEQ ID NO:17:Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr

1              5                  10  Leu Gly Ala Thr Thr Gly Tyr Leu Lys Gly Asn Ser

The information of 15 20 25 (2) SEQ ID NO:18:

(i) sequence signature:

(A) length: 23 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:18:Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val Ala

1              5                 10  Ala Leu Ser Ile Leu Pro Gly Ile Gly Cys

The information of 15 20 (2) SEQ ID NO:19:

(i) sequence signature:

(A) length: 39 amino

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:19:Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu

1              5                  10   Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val

    15                 20                 25   Asp Ile Gly Phe Ala Ala Thr Asn Phe Val Glu Ser Cys

The information of 30 35 (2) SEQ ID NO:20:

(i) sequence signature:

(A) length: 21 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) type: peptide (xi) sequence description: SEQ ID NO:20:Asp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ale Ser

1              5                 10  Ser Val Phe Asn Val Val Asn Ser

The information of 15 20 (2) SEQ ID NO:21:

(i) sequence signature:

(A) length: 17 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) type: peptide (xi) sequence description: SEQ ID NO:21:Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp Glu

The information of 15 10 (2) SEQ ID NO:22:

(i) sequence signature:

(A) length: 14 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: the information of 15 10 Gly (2) SEQ ID NO:23 of SEQ ID NO:22:Gly Leu Gln Gly Lys His Ala Asp Ala Val Lys Ala Lys: (i) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:23:Gly Leu Ala Ala Gly Leu Val Gly Met Ala Ala Asp Ala

1               5                 10  Met Val Glu Asp Val Asn

The information of 15 (2) SEQ ID NO:24:

(i) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:24:Ser Thr Glu Thr Gly Asn Gln His His Tyr Gln Thr Arg

1               5                 10  Val Val Ser Asn Ala Asn Lys

The information of 15 20 (2) SEQ ID NO:25:

(i) sequence signature:

(A) length: 42 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:25:Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu

1              5                  10  Gly Val Gly Gly Leu Ala Gly Gly Ser Ala Gln Ser Gln

   15                 20                  25  Arg Ala Pro Asp Arg Val Leu Cys His Ser Gly Gln Gln

The information of 30 35 Gln Gly Leu, 40 (2) SEQ ID NO:26:

(i) sequence signature:

(A) length: 43 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:26:Gly Leu Ala Gly Gly Ser Ala Gln Ser Gln Arg Ala Pro

1              5                  10  Asp Arg Val Leu Cys His Ser Gly Gln Gln Gln Gly Leu

   15                  20                  25  Gly Gly Leu Ser Glu Ile Lys Gly Val Ile Val His Arg

The information of 30 35 Leu Glu Gly Val, 40 (2) SEQ ID NO:27:

(i) sequence signature:

(A) length: 34 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:27:

Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu

  1              5                  10

Gly Val Gly Gly Pro Glu Leu Asp Val Cys Val Glu Glu

     15                 20                  25

Ala Glu Gly Glu Ala Pro Trp Thr

The information of 30 (2) SEQ ID NO:28:

(i) sequence signature:

(A) length: 34 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:28:Pro Gly Leu Asp Val Cys Val Glu Glu Ala Glu Gly Glu

1               5                 10  Ala Pro Trp Thr Gly Gly Leu Ser Glu Ile Lys Gly Val

   15                 20                  25  Ile Val His Arg Leu Glu Gly Val

The information of 30 (2) SEQ ID NO:29:

(i) sequence signature:

(A) length: 42 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:29:Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser

1              5                  10  Leu Asp Gly Gly Leu Ala Gly Gly Ser Ala Gln Ser Gln

  15                  20                  25  Arg Ala Pro Asp Arg Val Leu Cys His Ser Gly Gln Gln

The information of 30 35 Gln Gly Leu, 40 (2) SEQ ID NO:30:

(i) sequence signature:

(A) length: 43 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:30:Gly Leu Ala Gly Gly Ser Ala Gln Ser Gln Arg Ala Pro

1          5                      10  Asp Arg Val Leu Cys His Ser Gly Gln Gln Gln Gly Leu

   15                 20                  25  Gly Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro

The information of 30 35 Gln Ser Leu Asp, 40 (2) SEQ ID NO:31:

(i) sequence signature

(A) length: 34 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:31:Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser

1               5                 10  Leu Asp Gly Gly Pro Glu Leu Asp Val Cys Val Glu Glu

   15                 20                  25  Ala Glu Gly Glu Ala Pro Trp Thr

The information of 30 (2) SEQ ID NO:32:

(i) sequence signature:

(A) length: 34 amino acid

(B) type: amino acid

(D) topological structure: linear (ii) molecule type: peptide (xi) sequence description: SEQ ID NO:32:Pro Glu Leu Asp Val Cys Val Glu Glu Ala Glu Gly Glu

1             5                 l0  Ala Pro Trp Thr Gly Gly Phe Phe Leu Leu Thr Arg Ile

   15                 20                  25  Leu Thr Ile Pro Gln Ser Leu Asp

          30

Claims (27)

1. a kind of peptide based immunogens selected from the group below: (A) n- (Th) m- (B) o- (mIgE peptide) or (mIgE peptide)-(B) o- (Th) m- (A) n, in which: A is amino acid, α-NH₂, fatty acid or their derivative, or the invasin structural domain with immunostimulatory properties；

B is amino acid；

Th is helper T cell epitope or its immune enhancing analog or segment；

MIgE peptide is SEQ ID NO:1, SEQ ID NO:2 or their immunogenic analogue；

N is from 1 to about 10；

M is from 1 to about 4；

O is from 0 to about 10.

2. the peptide based immunogens of claim 1, wherein described Th has the amino acid sequence selected from SEQ ID NOS:5-24 or their immunogenic analogue or segment.

3. the peptide based immunogens of claim 1 have and are selected from SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:27, SEQ ID NO:28, the amino acid sequence of SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

4. the peptide based immunogens of claim 2 also include fatty acid.

5. the peptide based immunogens of claim 2 also include derivative of fatty acid.

6. the peptide based immunogens of claim 5, wherein described derivative of fatty acid is Pam3Cys.

7. the peptide based immunogens of claim 3 also include fatty acid.

8. the peptide based immunogens of claim 3 also include derivative of fatty acid.

9. the peptide based immunogens of claim 8, wherein the derivative of described fatty acid is Pam3Cys.

10. a kind of vaccine composition, the composition includes peptide based immunogens and pharmaceutically acceptable conveyer system in a effective amount of claim 1.

11. a kind of vaccine composition, peptide based immunogens of the composition comprising a effective amount of claim 2 and pharmaceutically acceptable conveyer system.

12. a kind of vaccine composition, peptide based immunogens of the composition comprising a effective amount of claim 3 and pharmaceutically acceptable conveyer system.

13. a kind of vaccine composition, peptide based immunogens of the composition comprising a effective amount of claim 4 and pharmaceutically acceptable conveyer system.

14. a kind of vaccine composition, peptide based immunogens of the composition comprising a effective amount of claim 5 and pharmaceutically acceptable conveyer system.

15. a kind of vaccine composition, peptide based immunogens of the composition comprising a effective amount of claim 6 and pharmaceutically acceptable conveyer system.

16. a kind of vaccine composition, peptide based immunogens of the composition comprising a effective amount of claim 7 and pharmaceutically acceptable conveyer system.

17. a kind of vaccine composition, peptide based immunogens of the composition comprising a effective amount of claim 8 and pharmaceutically acceptable conveyer system.

18. a kind of vaccine composition, peptide based immunogens of the composition comprising a effective amount of claim 9 and pharmaceutically acceptable conveyer system.

19. a kind of by applying the method that a effective amount of composition according to claim 1 treats allergy to patient.

20. a kind of by applying the method that a effective amount of composition according to claim 2 treats allergy to patient.

21. a kind of by applying the method that a effective amount of composition according to claim 3 treats allergy to patient.

22. a kind of by applying the method that a effective amount of composition according to claim 4 treats allergy to patient.

23. a kind of by applying the method that a effective amount of composition according to claim 5 treats allergy to patient.

24. a kind of by applying the method that a effective amount of composition according to claim 6 treats allergy to patient.

25. a kind of by applying the method that a effective amount of composition according to claim 7 treats allergy to patient.

26. a kind of by applying the method that a effective amount of composition according to claim 8 treats allergy to patient.

27. a kind of by applying the method that a effective amount of composition according to claim 9 treats allergy to patient.