CN100562338C

CN100562338C - Amyloid-beta-analog-T-cell epitope vaccine

Info

Publication number: CN100562338C
Application number: CNB028163265A
Authority: CN
Inventors: 彼得·比尔克·拉斯穆森; 马丁·罗兰·詹森; 克劳斯·格雷戈留斯·尼尔森; 彼得·克费欧德; 弗洛伦斯·达尔·德甘
Original assignee: Affitech AS
Current assignee: H Lundbeck AS
Priority date: 2001-08-20
Filing date: 2002-08-20
Publication date: 2009-11-25
Anticipated expiration: 2022-08-20
Also published as: NO335602B1; WO2002066056A2; IL157475A0; JP2004529881A; ZA200400895B; AU2002233166B2; WO2002066056A8; WO2002066056A3; CA2440197A1; CN1893970A; NO20040431L; NZ527720A; EP1363664A2

Abstract

Having invented opposing is the new method of the disease of feature with the amyloid beta deposition.Put it briefly, this method is based on the immunity inoculation of anti-amyloid precursor (APP) or amyloid beta (A β).Preferably immunity inoculation plays a role from source APP or A β analog by administration, and described analog can be induced and be produced anti-antibody from the source amyloid polypeptide.Particularly preferably be, with introduce one or several external source by modification immunodominant mix the type t cell epitope from source A β as immunogen.Also invented the nucleic acid immunization inoculation of anti-APP or A β in addition and used the immunity inoculation of live vaccine, and in immunity inoculation used method and mode.These methods and mode comprise the method for preparing analog and pharmaceutical preparation and nucleic acid fragment, carrier, transformant, polypeptide and drug component.

Description

Amyloid-beta-analog-T-cell epitope vaccine

Invention field

The present invention relates to improve alzheimer's disease (AD) and other are feature with the amyloid beta deposition, for example in central nervous system (CNS), be deposited as the treatment of diseases and the prevention of feature with amyloid.More particularly, the invention provides a kind of by suffer or risky patient with the disease that relates to the amyloid beta deposition pathological characters in produce the method that decorrelation albumen (APP or A β) or the antibody of its component are reduced (unwanted) amyloid beta deposition.The present invention also provides the production method and the modified polypeptides like this of used polypeptide in the method.The present invention also comprises the nucleic acid fragment of the modified polypeptide of encoding and the carrier of integrating these nucleic acid fragments, reaches host cell and cell line with this transfection.At last, the present invention also provides a kind of novel conjugate peptide based immunogens.

Background of invention

Amyloidosis is the fibriilar extracellular of insoluble protein deposition (Pepys, 1996 that cause tissue injury and disease; Tan etc., 1995; Kelly, 1996).When normal soluble protein and peptide carried out self-crosslinking with anomalous mode, fibril formed (Kelly, 1997).

Amyloid and serious disease, comprise that the Transmissible spongiform encephalopathy (itch of people's Kuru disease and Creutzfeldt-Jakob disease and sheep and cattle) that system's amyloidosis, AD, period of maturation outbreak diabetes (maturity onset diabetes), parkinson disease, Huntington Chorea, frontotemporal dementia (fronto-temporal dementia) and Protein virus be correlated with is relevant, as if the formation of amyloid plaque in alzheimer's disease for example the most relevant with people's disease progression.Be presented at the albumen of finding in the deposit, as the overexpression of amyloid-beta in animal model or the various symptoms of the induced expression disease of modified forms, for example alzheimer's disease sample symptom.Do not have specific treatment at present, and these diseases are normally fatal at amyloid beta deposition.

The fibriilar subunit of amyloid can be albumen wild type, variation or truncate, external, can form similar fibril (Bradbury etc., 1960 from oligopeptide and Denatured protein; Filshie etc., 1964; Burke﹠amp; Rougvie, 1972).The feature of the character decision amyloidosis of fibril polypeptide fractions.Although the size of amyloid, natural structure and function difference are very big, all amyloid fibrils all are that branch indefinite length, no, diameter are 70-

Congo red is shown characteristic dyeing (Pepys, 1996).They are characterised in that the beta structure (Pauling﹠amp of intersection; Corey, 1951), wherein polypeptide chain is organized into the beta sheet form.Though amyloid has very different front body structures, they all can experience a kind of structure conversion, perhaps along similar approach, convert the misfolding form of beta sheet spiral fibril member to.

The fiber pattern of this uniqueness causes being called β-fibrillar amyloidosis, and (b), the fibrillin of AD is called beta-protein (Glenner﹠amp before its secondary structure is found for Glenner, 1980a; Wong, 1984).At present, crosslinked β-the diffraction pattern of characteristic becomes the diagnostic flag of acceptable amyloid with fibriilar appearance and tinctorial property, and hints, though fibril forms from very different amyloid protein precursors, but no matter the character of its precursor protein how, they have structural similarity to a certain extent, and comprise a kind of structure superfamily (SundeM, Serpell LC, Bartlam M, Fraser PE, Pepys MB, Blake CCFJ Mol Biol 1997Oct 31; 273 (3): 729-739).

AD is the most extensive and one of disease of knowing, and the amyloid beta deposition thing hint that is present in the central nervous system in the described disease plays central role in disease progression.

AD

Alzheimer's disease (AD) is a kind of irreversible gradual disease of brain, and it takes place gradually, and causes the loss of memory, behavior and personality change and intelligence to descend.These forfeitures decline dead with brain cell and that connect between them is relevant.This sick origin cause of formation varies with each individual, as the speed that descends.On average, though should disease is sustainable reach 20 years, the AD patient after making a definite diagnosis only lived 8 to 10 years.

AD develops by stages, from slightly forgeing in early days the serious forfeiture of moral function.This forfeiture is known as dementia.Suffer among the people of AD at majority, symptom at first showed later at 60 years old, but outbreak more early is unrare.The symptom of earliest period generally includes recent memory forfeiture, misjudge and personality change.Usually, the people's thinking that is in the AD initial stage is more unintelligible, forgets the title of familiar people and general object.In the terminal stage of a disease, they in addition may forget how to do simple thing.At last, the people who suffers from AD loses all inferential capabilities, becomes to rely on other people treatment to its daily life.Finally, the deterioration patient is unable to leave the bed to such an extent as to this pathological changes gets very, and may develop other diseases and infection.The most common ground, the people who suffers from AD dies of pneumonia.

Though the danger that AD takes place increased with the age, AD and dementia symptom are not the part of usual aging.AD and other dementia diseases are to be caused by the disease that influences brain.In usual aging, the neurocyte in the brain is not lost in a large number.On the contrary, AD destroys three critical process: neurocyte communication, metabolism and reparation.This destruction finally causes many neurocytes to stop function, and forfeiture is got in touch with other neurocytes, and dead.

At first, the AD brain that destroys the control memory divides the neuron in the particularly Hippocampus and dependency structure.Because neurocyte in Hippocampus stops function fully, thus short term memory loss caused, and also usually the people does ability easy and that be familiar with thing and begins decline.AD also attacks cerebral cortex, particularly is responsible for the zone of language and reasoning.At last, other zones of many brains also participate in into, all these brain district atrophys (contraction), AD patient become be unable to leave the bed, incontinence, helpless fully, do not react (source: National Institute on Aging Progress Report onAlzheimer ' s Disease, 1999) to external world.

The impact of AD

AD is the common reason of 65 years old and above philtrum dementia.Because its enormous impact to individual, family, healthcare network and entire society, AD has become a major health.Scientist estimates, current have surpass 400 ten thousand people and suffer this disease, and this trend was doubled in the people of over-65s in per 5 years.(sickness rate (incidence)) takes place to estimate also that in addition 360,000 new cases are arranged about every year, should numeral increase (Brookmeyer etc., 1998) with population ages.

AD causes serious financial burden to society.A current research that carries out in the U.S. is estimated, is used for an AD patient's nurse expense every year, concerning a patient who suffers from mild AD, and Shi $18,408, to suffering from patient's Shi $30 of moderate AD, 096, to suffering from patient's Shi $36 of serious AD, 132.In the U.S., estimate annual country cost Lve Gaoyu $500 hundred million (Leon etc., 1998) that are used to nurse AD patient.

About 4,000,000 Americans are 85 years old or older, and in most of industrialized countries, this age cohort is a fastest-rising part in the population.Estimation is in the U.S., and to the year two thousand thirty, this colony will be near 8,500,000; The expert of some research population trends hints this numeral even may be bigger.Along with increasing people live longer, be subjected to diseases of aging, comprise AD, the number of influence will continue growth.For example, some studies show that, the age be 85 years old and older philtrum nearly half suffer from the dementia (National Institute on Aging Progress Report on Alzheimer ' s Disease, 1999) of certain form.

The principal character of AD

The sign of AD is two anomalous structurees in the brain: amyloid plaque and neurofibrillary tangles (NFT).Speckle is intensive, is a large amount of insoluble deposit of and peripheral protein and cellular material outside in brain neuron.Entanglement is the fiber in the insoluble entanglement of inside neurons formation.

Have two types AD: familial AD (FAD) and sporadic AD, familial AD follow a definite hereditary pattern, and do not observe the obvious genetic pattern in sporadic AD.Because the difference of age of onset, AD further can be described as early onset thereof type (taking place) or late onset type (taking place) in 65 years old and older people in less than 65 years old people.Early onset thereof type AD is rare (nearly 10% case), influences the 30-60 people in year usually.The early onset thereof type AD of some form is hereditary, and is popular in family.Early onset thereof type AD is also often developing faster than more general late onset type.

All hitherto known FADs have early onset thereof, and the known 50%FAD of having case is caused by the defective that is positioned at three genes on three coloured differently bodies now.They are sudden changes of app gene on the chromosome 21; Gene mutation that is called presenilin (presenilin) 1 on the chromosome 14; With gene mutation that is called presenilin 2 on the chromosome 1.Yet also there is not evidence to prove in these sudden changes any one in more general sporadic or non-familial late onset AD, play a major role (National Institute on Aging Progress Report on Alzheimer ' s Disease, 1999) yet.

Amyloid plaque

In AD, amyloid plaque at first takes place in the brain zone that is used for remembering with other cognitive functions.They comprise a large amount of insoluble deposit of amyloid beta (called after A β after this), described amyloid beta for be called amyloid precursor protein (APP, its aminoacid sequence list among the SEQ ID NO:2) than the protein fragments-mixing of large protein with partial nerve unit and non-neurocyte such as microglia and astrocyte.Whether do not know at present that amyloid plaque self constitutes the main cause of AD or they are not by-products of AD process.Certainly, the proteic change of APP can cause AD, as in the genotype AD that causes by app gene sudden change, and the formation of A β speckle as if with human disease's development be closely related (Lippa C.F. etc., 1998).

APP

APP is in many protein relevant with cell membrane.After it was synthetic, APP was embedded in the cell membrane of neurocyte, and part is in cell, and part is in the extracellular.Recently studies show that with transgenic mice as if APP play an important role in neuronic growth with in surviving.For example, the APP of a definite form and quantity can neuroprotective unit avoid short-term and long-term damage, and makes the neuron of damage can repair oneself better, and behind brain injury, can help the growth of partial nerve unit.

When APP was embedded in the cell membrane, protease acted on the specific site of APP, is cut into protein fragments.A kind of protease helps the APP cutting is formed A β, and another kind of protease cuts APP at amyloid fragment middle part, thereby A β can not be formed.The A β that forms has two kinds of different lengths, the short individual amino acid whose A β in 40 (or 41), and it is soluble relatively and assembles slowly, a little long, 42 amino acid whose " viscosity " A β, it can the insoluble agglomerate of very fast formation.When forming A β, also imprecise know it how by or center on neurocyte and move.In the final stage of this process, " viscosity " A β is gathered into long filament in the extracellular, and forms speckle as AD feature in the cerebral tissue with the in heaven and neuron fragment of checkmating and microglia and astrocyte.

Some exist evidence to show, the sudden change among the APP is given and made A β shear from the APP precursor, thereby produce more total A β or more relatively " viscosity " form offers additional possibilities.It shows that also the sudden change of presenilin gene can cause neuronic degeneration at least in two ways: by modifying that A β produces or the more directly death by trigger cell.The

presenilin

1 and 2 of other researcher prompting sudden changes may participate in quickening apoptosis speed.

Expection will produce increasing scab along with the carrying out of disease, fills increasing brain district.Research prompting A β may assemble and depolymerization simultaneously with a kind of form of dynamic equilibrium.This brings following hope to us, promptly even might be again to its degrade (National Institute on Aging Progress Report on Alzheimer ' s Disease, 1999) after speckle forms.

Think that A β is virose to neuron.In Study on tissue culture, researcher is found, compares with the neuron of overexpression normal person APP, is transformed into dead increase (Luo etc., 1999) of Hippocampal Neuron Cells of the people APP of overexpression mutant form.

In addition, show in animal model that a of overexpression or expression modified forms is induced alzheimer's disease symptom (Hsiao K. etc., 1998).

Suppose that the neurotoxicity that A β produce to increase, it is polymerized to speckle and cause thus may cause AD, so investigation can reduce or even blocking-up A beta peptide aggregation be that the condition of speckle has the treatment meaning.

Presenilin

Sudden change in the presenilin-1 (S-180) accounts for 50% of all early onset thereof familial AD (FAD) cases greatly.About 30 sudden changes that cause AD have been identified.The outbreak of AD is different with sudden change.Sudden change in the presenilin-2 accounts for the more much smaller part of FAD case, but still is a key factor.Do not know also at present whether presenilin participates in sporadic non-familial AD.The function of presenilin is unknown, but since in having the AD patient of presenilin sudden change A β-42 level increase, as if so their processing of participating in APP is to produce A β-42 (peptide of long sticking form, among the SEQ ID NO:2, residue 673-714).Do not know whether presenilin also has effect in NFT ' s produces.Some prompting presenilins have more direct effect in deterioration of neurons and neuronal death.Presenilin-1 is positioned on the chromosome 14, and presenilin-2 is on chromosome 1.If people has in these genes only mutant form, he or she almost is bound to take place early onset thereof type AD.

About presenilin-1 whether with consistent some uncertainties (Naruse etc., 1998) that still exist of gamma-secretase of the participation APP course of processing of hypothesis.

Apo E

Apo E is usually relevant with cholesterol, but at the scab of AD brain with also discovery existence in tangling.Although as if allele 1-3 do not participate in AD, APOE-ε 4 allelic existence and late period AD development important relationship (Strittmatter etc., 1993) is arranged.Yet it is a risk factor, be not and presenilin and the same immediate cause of APP sudden change situation, and it is not limited among the familial AD.

The also imprecise approach of knowing APOE ε 4 albumen increase generation AD probability, but a possible theory is, it promotes the structure of A β, this causes the reduction of AD age of onset, perhaps exist or do not exist APOE allele to affect the nerves mode (Buttini etc., 1999) that unit replys damage.

Show that in addition Apo A1 is amyloid originality (amyloigenic).External, complete apo A1 self can form fibril (Am J Pathol147 (2): 238-244 (Aug 1995), the Wisniewski T of the male amyloid sample of Congo red, Golabek AA, Kida E, Wisniewski KE, Frangione B).

The result who has some contradictions, promptly with respect to other allele, APOE-ε 4 allele have a positive acting (Stern, Brandt, 1997, Annalsof Neurology 41) in reducing intelligence forfeiture symptom.

Neurofibrillary tangles

Second AD labelling is included in the unusual set of the silk of the inner entanglement of finding of neurocyte.The main component of tangling is the proteic a kind of form that is called tau (τ).Known in the central nervous system, the microtubule of the member of Protein tau combination and stabilized cell internal support structures or skeleton.But in AD, tau carries out chemical modification, and the tau of this change is stabilize microtubules no longer, but causes their decomposition.Disintegrating of this transportation system at first causes communication failure between neurocyte, may cause neuronal death subsequently.

In AD, the tau of chemical modification tangles and is the paired taenidium-two gang tau that entwines mutually.These are main matter of finding in neurofibrillary tangles.In a nearest research, researcher finds, in the specific part of the Hippocampus of healthy brain, is less than 6% neuron generation neurofibril and changes, then neuronic more than 43% in dying from the people of mild AD for these, neuronic for these in dying from the people of serious AD more than 71%.In neuronic the losing of research, find similar progressive relationship.There is the viewpoint (National Institute on Aging Progress Report onAlzheimer ' s Disease, 1999) of the formation and the loss that neuron develops of entanglement simultaneously in this evidence support along with the process of AD.

Tauopathies and entanglement

Except that AD, several neurodegenerative diseases are characterised in that tau is gathered into insoluble filament in neuron and neuroglia, cause dysfunction and death.Recently, several groups of researchs suffer from the researcher discovery of the dull-witted family of multiple genotype except that AD, first sudden change (Clark etc., 1998 on chromosome 17 of tau gene; Hutton etc., 1998; Poorkaj etc., 1998; Spillantini etc., 1998).In these families, the sudden change of tau gene causes neuronal cell dead and dull-witted.These and AD have some common traits, but several main aspect different disease, be generically and collectively referred to as " frontotemporal dementia and the parkinson disease relevant " (FTDP-17) with chromosome 17.These diseases are that for example amyotrophic lateral sclerosis of parkinson disease, some form (spinal cord) lateral sclerosis (ALS), cortex degeneration (corticobasal degeneration), progressive supranuclear plasy and Pick disease are feature with unusual gathering of Protein tau all.

Other AD sample sacred diseases

AD and other sacred diseases comprise prion disease (as Kuru disease, Creutzfeldt-Jakob disease and bovine spongiform encephalitis), parkinson disease, Huntington Chorea and frontotemporal dementia, have important collimation.All these diseases all relate to the deposition of paraprotein in brain.AD and prion disease cause dementia and death, and the two is all relevant with the fibriilar formation of insoluble starch sample albumen, but differs from one another from memebrane protein.

Second the most general neurodegenerative disease-Parkinsonian scientist finds first gene relevant with this disease after the research AD.Albumen that is called synuclein of this gene code interestedly is that this albumen also is found in (Lavedan C, 1998, Genome Res.8 (9): 871-80) in the amyloid plaque of AD patient's brain.Researcher has been found that, genetic defect in another kind of progressive type neurodegenerative disease-Huntington Chorea causes dementia, cause Huntington protein (Huntington protein) to form being very similar among the AD fibriilar insoluble fibril (the Scherzinger E etc. of albumen in the A β fibril and prion disease, 1999, PNAS U.S.A.96 (8): 4604-9).

Scientist has also had been found that a kind of new gene, when it suddenlys change, causes taking place familial British dementia (FBD), and this is that a kind of rare causing is similar to the serious action obstacle seen and the heredopathia of progressive dementia in AD.In the biochemical analysis of the amyloid plaque in being found in the FBD scab, found a unique peptide (Vidal etc., 1999) that is called ABri.Sudden change at this gene specific site causes producing the longer albumen of the normal Bri albumen of ratio.

Shear deposit the same of ABri peptide that obtains by Bri protein mutation end with the amyloid fibril.Think that these speckles cause the neural machine dysfunction and be the dementia of feature with FBD.

With A β immunity

Immune system participate in to be removed foreign protein and proteinic granule usually in body, but mainly comprises oneself protein with the deposit of above-mentioned disease association, therefore makes the effect of immune system in these diseases of control not too obvious.In addition, deposit is usually located at and the isolating interval of immune system (CNS), and the two all points out any vaccine or immunotherapy method will be invalid.

Yet scientist has attempted with comprising allogenic people A β and the known vaccine that excites immune material mice being carried out immunity (Schenk etc., 1999 and WO 99/27944) recently.The part transgenic AD mouse model that personnel selection APP mutant gene is inserted in the mouse DNA detects vaccine.The APP albumen that mice produce to be modified, and grow up and develop into amyloid plaque with it.Whether the inoculation that this mouse model is used for detecting the anti-transgenic human APP that modifies works in the formation of speckle.In first experiment, one group of transgenic mice is given the vaccine of injection in every month, since 6 ages in week, finishes to November.Second group of transgenic mice organized in contrast and do not carried out injecting.To 13 monthly ages, control group mice has the scab that accounts for brain 2-6%.On the contrary, in fact there is not scab to produce in the immune mouse.

In second experiment, researcher is producing 11st month start injection of some scabs.Through 7 months, scab quantity increased by 17 times in the contrast Transgenic Mice Brain, and those mices of accepting vaccine are compared reduction by 99% with 18 monthly ages contrast transgenic mice.As if in some mices, some already present scab deposits are removed by treatment.And find the damage that other scabs are relevant, decline is all arranged as immune result as inflammation and unusual neurocyte process.

Therefore, more than be the preliminary study in mice, for example, whether scientist need understand the mice of inoculation and keep fit in other respects, and whether the mice of those inoculations remembers maintenance normally.In addition, because mouse model is not the representative fully (animal does not produce neurofibrillary tangles, and its most neurons are not dead yet) of AD, so need carry out other research to determine whether the people has the reaction similar or different with mice.Another problem that need consider is this method possibility " healing " amyloid beta deposition, but may not stop dull-witted development.

Technical problem also has main challenge.For example, can not make the people produce the vaccine of anti-himself proteic antibody with this technology manufacturing.Therefore the problem of many safeties and effectiveness needs philtrum where in office to solve before experimentizing.

Therefore, the work of Schenk etc. shows, if possible to protein deposit among the central nervous system, the scab that forms among the AD for example, in oneself protein produce strong immunne response, then both might stop sedimental formation, might remove established scab again.

Recently, because side effect uses the clinical trial of the A β vaccine of above-mentioned discussion to stop carrying out, described side effect is: may be developed into chronic encephalitis by the experimenter of immunity in a large number owing to produce the uncontrollable autoimmune of A β among the anti-CNS.

Goal of the invention

The purpose of this invention is to provide novel opposing is the disease of feature with the amyloid beta deposition, AD for example, Therapeutic Method.Further purpose is the autovaccine of a kind of anti-amyloid of exploitation, and is a kind of novel to the AD that relates to amyloid beta deposition and the Therapeutic Method of other pathologic conditions to obtain.

Summary of the invention

Described herein is to use a kind of autoimmune technology to produce the intensive APP of other non-immunogenicity and the immunne response of A β of resisting.And described and thisly be used to prevent, may cure or the preparation of this class vaccine of this class disease symptoms that mitigation is relevant with amyloid beta deposition.

Therefore, in its extensive and the most generalized scope, the present invention relates to a kind of animal, comprise the method for reducing amyloid precursor protein (APP) or amyloid-beta (A β) in the human body, this method comprises at least one APP or the A β analog of animal immune system effectively being presented immune effective dose, and described analog is integrated B cell epitope and at least one external source t helper cell epi-position (T of at least one APP and/or A β in same molecule _HEpi-position), thus can induce with this analog immune animal and to produce anti-animal from the APP in source or the antibody of A β.Wherein said analog

A) polyamino acid of forming by the subsequence of the residue 672-714 of at least one copy SEQ ID NO:2, wherein external source T _HEpi-position is introduced into by the mode of aminoacid addition and/or insertion and/or deletion and/or replacement, and wherein subsequence is selected from residue 1-42, residue 1-40, residue 1-39, residue 1-35, residue 1-34, residue 1-28, residue 1-12, residue 1-5, residue 13-28, residue 13-35, residue 17-28, residue 25-35, residue 35-40, residue 36-42 and the residue 35-42 of the aminoacid sequence of being made up of amino acid residue 673-714 among the SEQ ID NO:2; And/or

B) be to comprise external source T _HEpi-position and the APP that interrupts or the polyamino acid of A β sequence so that analog do not comprise any can with the subsequence among the effective bonded SEQ ID NO:2 of MHC II quasi-molecule that causes t cell response; And/or

C) be to comprise external source T _HThe deutero-amino acid whose polyamino acid of epi-position and APP or A β, and comprise an independent methionine residues at the analog C-terminal is wherein among APP or the A β and external source T _HOther methionine residues in the epi-position are substituted or delete, and are preferably replaced by leucine or isoleucine; And/or

D) be a kind of conjugate that comprises the polyhydroxylated polymer skeleton, the polyamino acid and/or the b that on described skeleton, define in difference coupling a)) middle polyamino acid and/or the c that defines) the middle polyamino acid that defines; And/or

E) be a kind of conjugate that comprises the polyhydroxylated polymer skeleton, coupling 1 respectively on described skeleton) external source T _HEpi-position and 2) be selected from following polyamino acid: sequence that APP that defines a) middle subsequence, the b that defines) or A β interrupt and APP or A β are deutero-, C-terminal comprises the aminoacid sequence of an independent methionine residues, wherein among APP or the A β and external source T _HOther methionine residues in the epi-position are substituted or delete, and are preferably replaced by leucine or isoleucine.

This agent had before proposed an international patent application, related to anti-amyloid originality polypeptide, and for example the safe vaccination strategies of APP and A β is consulted WO 01/62284.This application do not announce at the application's date of filing, but also do not comprise the particulars about above-mentioned useful APP and A β analog.

The present invention also relates to APP and A β analog and these the nucleic acid fragment of subclass of encoding.The immunogenic composition that comprises analog or nucleic acid fragment also is a part of the present invention.

The accompanying drawing legend

Fig. 1: derive from amyloid precursor protein from immune variant, with the schematic description figure of the antibody response that produces anti-a A β-43 (or C-100).APP is presented at the top of figure, and all the other schematic component representation pattern epi-position P2 and P30 are substituted or are inserted in the various spliced bodies of APP.In the drawings, black part is divided the signal sequence of expression APP, and bidirectional crossed shade is the extracellular part of APP, dark vertical shade is the membrane spaning domain of APP, the vertical shade of light color is born of the same parents' intracellular domain of APP, and thick cross-hatched is represented the P30 epi-position, and thin cross-hatched is represented the P2 epi-position.Solid box is represented A β-42/43, and solid box and dotted box are represented C-100 together." Abeta " expression A β.

Fig. 2: the schematic description of the embodiment of the synthetic immunogenic conjugates of using usually.Peptide A (any antigen sequence, for example, A β sequence described herein) and peptide B (one section aminoacid sequence that comprises external source t helper cell epi-position) are synthesized and mix.Then, they are contacted with suitable activation polyhydroxylated polymer, peptide A and B by activated group with quantitatively being connected corresponding to these two kinds of material initial ratios in peptide mixer.See embodiment 4 for details.

Detailed Description Of The Invention

Definition

The below will define used in the specification and claims a large amount of terms and explain in detail, to illustrate boundary line of the present invention and scope.

Interchangeable term " amyloid " and " amyloid protein " represent the unbranched fibrillation of the uncertain protein of a class length herein. The amyloid fibrillation dyes to the Congo red characteristic that shows, the beta structure with intersection, and wherein polypeptide chain is organized as beta sheet. Amyloid derives from amyloid protein usually, and they have very different front body structures, becomes the misfolding form but all will experience a kind of Structure Transformation, and this form is the member of β-pleated sheet spiral precursor. Usually, the fibriilar diameter of amyloid arrives approximately about 70

Range changing.

Term " amyloid originality (amyloidogenic) protein " intention expression is by so becoming a sedimental part or participating in forming the polypeptide of amyloid beta deposition thing by becoming a part that causes forming the biosynthesis pathway that deposit forms. Therefore, the example of amyloid originality albumen is APP and A β. But the albumen that participates in these metabolism also may be amyloid originality albumen.

" amyloid polypeptide " intention expression herein comprises polypeptide (or its spliced body of the amino acid sequence that derives from people or other mammiferous amyloid originality protein of above-mentioned discussion, described spliced body and complete amyloid originality protein share many B-cell epitopes), therefore-amyloid originality polypeptide can for example comprise the substantial portion (substantial parts) (for A β, a kind of possible amyloid polypeptide can derive from APP) of amyloid originality polypeptide precursor. Be also included within the term scope from the non-glycosylated form of the amyloid originality polypeptide of prokaryotic system preparation, as the form owing to the various glycosylation patterns that use yeast for example or other non-mammality eukaryotic expression systems to have. Yet should be noted that it is immunogenic to refer to that described polypeptide does not normally have when being applied to animal to be treated when using term " amyloid originality polypeptide ". In other words, amyloid originality polypeptide is the analog of oneself protein or this oneself protein, and they do not cause the immune response of the amyloid originality of anti-described animal usually.

" analog " refers to integrate in the molecular structure APP of one or several change or the molecule that A β derives. This change for example can be, the fusion form of APP or A β polyamino acid and suitable fusion partner (namely, on primary structure, only relate to the change that C-and/or-terminal amino acid residue increase), and/or it can be insertion and/or deletion and/or replacement form in polypeptid acid sequence. This term also comprises the molecule that the APP that derives or A β derive, and consults APP discussed below or A β and modifies. In some cases, can make up analog so that seldom or even can not cause the antibody of anti-normal amyloid precursor protein, thereby avoid (normal on the physiology) the non-aggregated forms as the polypeptide of amyloid protein precursor is produced unnecessary interference.

Should notice it is contemplated that the APP of end user in the people or A β xenogenesis analog (for example, the analog of dog or pig) as vaccine, might produce the immunity of required anti-APP or A β. Therefore to carry out the application of immunity also be a part of the present invention to this xenogenesis analog.

Term herein " polypeptide " refers to the small peptide of from 2 to 10 amino acid residues, the oligopeptides of from 11 to 100 amino acid residues, and more than the polypeptide of 100 amino acid residues. In addition, this term also is intended to comprise albumen,, comprises the functional biological molecule of at least one polypeptide that is; When comprising at least two polypeptide, these can covalently boundly or non-covalent be connected to form complex. Polypeptide in the protein can be glycosylated and/or esterified (lipidated) and/or comprise prothetic group. And term " polyamino acid " is the equivalents of term " polypeptide ".

Term " T lymphocyte " and " T cell " can be used mutually, and the lymphocyte in expression thymus gland source is responsible for various cell-mediated immune responses and assisting in HI. Equally, term " B lymphocyte " and " B cell " can be used mutually, refer to produce the lymphocyte of antibody.

Term " subsequence " refers to be directed to respectively naturally occurring amyloid amino acid sequence or nucleotide sequence, at least 3 amino acid or any one section continuous sequence of at least 3 nucleotides when corresponding.

Term herein " animal " is intention expression animal species (preferred mammal) usually, such as the mankind, Canis domesticus etc., rather than refer to a kind of single animal. Yet this term also represents a colony of this animal species, because have a bit very important, i.e. the individual of the method according to this invention immunity all has identical APP or A β really, thereby allows with identical immunogen immune animal. To be clearly to those of skill in the art, namely herein animal refers to have immune biology. Preferred animal is vertebrate, for example mammal.

Term herein " body of APP or A β in downward modulation " is meant the total amount of the amyloid protein that reduces sedimentary correlation type in live organism (or similarly amyloid).This downward modulation can realize by several mechanism: wherein, simply interfering with to stop mistake to be assembled by antibodies to amyloid is the simplest mode.Yet scope of the present invention comprises that also antibodies causes the removing of scavenger cell (for example macrophage and other phagocyte) to amyloid, and antibody interferes other to cause the amyloid originality polypeptide of amyloid formation.Further probability is the outer A β of antibodies CNS, thereby effectively removes A β by simple mass action principle from CNS.

The immune system of statement " effectively presenting ... to immune system " intention expression animal stands a kind of immunogenicity challenge with controlled manner.Show as following content, this immune challenge can be accomplished in several ways, wherein the most important thing is with the polypeptide that comprises " pharmaccine (pharmaccine) " (that is, by administration with treatment or improve the vaccine of PD) inoculation or nucleic acid " pharmaccine " inoculation.The important results that reaches is that immunologically competent cell is with immune effective means antagonism antigen in the animal, and the accurate mode that reaches this result is not too important for supporting that invention of the present invention imagines.

Term " immunity effectively quantity " has its ordinary meaning in the art, that is, a certain amount of immunogen, it can cause and effectively kills and wounds the immunne response that has the pathogenic agent of identical immunological characteristic with immunogen.

When using statement APP or A β, be meant the chemical modification of on APP or A β, having carried out polypeptide herein by " modification ".For example, this modification can be the derivatization (for example alkylation) of particular amino acid residue in the sequence, still, is understood as described below, and preferred modification comprises the change of aminoacid sequence primary structure.

When " to the tolerance certainly of APP or A β " is discussed, can be regarded as, because polypeptide is an oneself protein matter of waiting to inoculate colony, so the normal individual in the colony does not have the immunne response of anti-this polypeptide; Though can not get rid of the antibody that in animal population individual one can produce anti-natural polypeptides, for example, as the part of auto-immune disease.In any case, animal only produces from tolerance himself APP or A β usually, also may not tolerated by described animal but do not get rid of the analog that derives from other animal species or colony with different phenotypes.

" external source t cell epitope " (or " external source T lymphocyte epitopes ") is a kind ofly can and stimulate the peptide of T cell in the animal species in conjunction with the MHC molecule.Preferred external source t cell epitope is " miscellaneous " epi-position among the present invention, that is, and and with the bonded epi-position of MHC molecule of most of particular category in animal species or the colony.It is known that this miscellaneous t cell epitope only has limited quantity, and they will go through below.Miscellaneous t cell epitope also refers to " general " t cell epitope.Should note, immunogen used according to the invention is effective to most as far as possible animal population, must 1) in same analog, insert several external source t cell epitopes, or 2) prepare several analog, wherein every kind of analog has the different epi-positions that mix of insertion.The notion that also it should be noted that the external source t cell epitope also comprises uses the t cell epitope of hiding, and, derives from the epi-position of oneself protein that is, and it only exists with unpack format, rather than brings into play immune behavior during described oneself protein part.

" external source T assists lymphocyte epitopes " (external source T _HEpi-position) be a kind of external source t cell epitope, it combines with MHC II quasi-molecule, can be presented to and the bonded antigen-presenting cell of MHC II quasi-molecule (APC) surface.

" funtion part " of (biology) molecule refers to be responsible for the biochemistry of at least one this molecule performance or the molecular moiety of physiological effect herein.Well known in the art, many enzymes and other effector molecule have an avtive spot of being responsible for described molecule performance effect.Other parts of this molecule can be used for the purpose of enhanced stability or solubility, and therefore, if in the content of specific embodiments of the invention, these purposes are incoherent, then can omit.For example, may in this case, can provide necessary stability owing to be coupled on APP or the A β, so stability problem be that it doesn't matter with specific cytokine as the part of the modification among APP or the A β (consulting following going through).

Term " adjuvant " has implication common in technical field of vaccines, that is, the compositions of a kind of material or material, it is 1 years old) itself can not cause the immunne response of special anti-vaccine immunogens, but it 2) however can strengthen anti-immunogenic immunne response.Or in other words, do not provide anti-immunogenic immunne response with the adjuvant inoculation separately, can cause or not cause anti-immunogenic immunne response with immunogen inoculation, but with immunogen and the inductive stronger anti-immunogenic immunne response of the independent immunogen of adjuvant simultaneous inoculation induction ratio.

Molecule herein " guiding " intention expression position, in this position, the molecule of importing in animal will preferably occur in particular organization or be preferred relevant with specific cell or cell type.This effect can realize by several different methods, is included in the compositions that promotes guiding the preparation molecule or by introduce the group that is beneficial to guiding in molecule, these problems will go through below.

It is general that " immune stimulation " is meant that the compositions table of a kind of material or material reveals, the non-specific immunostimulating effect.The adjuvant (for example some cytokine) of a large amount of adjuvants and supposition has the ability of stimulating immune system.The result who uses immunostimulant is that immune system improves " warning ", promptly with the immunogen while or when carrying out immunity subsequently, induces than the remarkable more effective immunne response of independent use immunogen.

" effectively combination " is meant that peptide is attached to MHC molecule (I class or II class) and goes up can stimulate the T cell, makes it kill and wound the cell of presenting with the bonded peptide of MHC molecule.For example, if this APC will stimulate and the peptide-bonded T of MHC II quasi-molecule complex that presents _HCell, then with the APC surface on the bonded peptide of mhc class ii molecule be known as effectively bonded.

The preferred embodiment of downward modulation amyloid

Preferred is APP or the A beta molecule of modifying as immunogenic analog in the method for the invention, wherein in the aminoacid sequence of APP or A β, exist at least in one and change, because greatly promoted like that to obtain all important blocking-up from the chance of tolerance-for example, this result who obtains in this paper embodiment 2 can obviously find out, in embodiment 2, immunity of carrying out with wild type A β and the immunity of carrying out with A β metagon have been compared.Show (at Dalum I etc., 1996, J.Immunol.157:4796-4804), in normal individual, there is the potential autoreaction bone-marrow-derived lymphocyte of identification oneself protein on the physiology.Yet, produce the antibody that reacts with relevant oneself protein really in order to induce these bone-marrow-derived lymphocytes, need to produce the auxiliary lymphocyte (T of T of cytokine _HCell and T _HLymphocyte) help.Usually, because when by antigen-presenting cell (APCs) when offering, the general nonrecognition of T lymphocyte derives from the t cell epitope of oneself protein, so this help is not provided.But, when the foreign epitope on the identification APC (for example being initially mononuclear cell), activated the T cell of source element outside the identification in oneself protein by a kind of " foreignness " element (promptly by introducing the important modification of immunity) is provided.Can discern the polyclone bone-marrow-derived lymphocyte (also being APCs) of self epi-position on the oneself protein of modification, also discern (internalise) antigen, and present its external source t cell epitope subsequently, the activated T lymphocyte provides cytokine to assist the polyclone bone-marrow-derived lymphocyte of these id reactions subsequently.Because the different epi-positions on the antibody that is produced by these polyclone bone-marrow-derived lymphocytes and the modified polypeptide comprise those reactions that also exist in natural polypeptides, therefore induced antibody with the oneself protein cross reaction of non-modification.In a word, can cause the T lymphocyte to play a role, polyclone bone-marrow-derived lymphocyte colony has discerned whole exogenous antigen seemingly, and the epi-position of in fact only inserting is an external source to the host.In this manner, induce produced can with the antibody of the autoantigen cross reaction of non-modification.

Being known in the art several modes comes the modified peptides autoantigen with blocking-up tolerance certainly.Yet preferably, analog according to the present invention comprises

-introduce a realization at least decorating molecule is navigated to the first of antigen-presenting cell (APC), and/or

-introduce the second portion of a stimulating immune system at least, and/or

-introduce an optimization analog at least to be presented to immune third part.

Yet all these modifications should be in keeping APP or A β be finished under the situation of the substantial portion of original bone-marrow-derived lymphocyte epi-position, thereby because can strengthen bone-marrow-derived lymphocyte identification self molecule.

In a preferred embodiment, side-chain radical (with the form of external source t cell epitope or above-mentioned first, second and third part) is introduced by mode covalently or non-covalently.That is to say that the one section sequence of amino acid residue that derives from APP or A β is not changing the one-level aminoacid sequence, or carry out derivatization less than introducing under the situation about changing in the peptide bond between the independent aminoacid in chain at least.

Alternative and an embodiment preferred are utilized aminoacid replacement and/or deletion and/or insertion and/or interpolation, and (it can synthesize by recombination form or by means of peptide and realizes; The amino acid whose modification that relates to long one section sequence can produce fused polypeptide).A particularly preferred form of the present embodiment is as WO 95/05849 described technology, it discloses a kind of by carrying out the method that oneself protein is reduced in immunity with the oneself protein analog, many aminoacid sequences are replaced by corresponding many each aminoacid sequences that comprise foreign immunologic dominance t cell epitope in the described analog, and keep the whole tertiary structure of oneself protein simultaneously in analog.Yet for the purpose of the present invention, can produce the external source t cell epitope, in APP or A β, preserve a large amount of B cell epitopes simultaneously if modify (can be to insert, add, delete or replace), so just enough.But, the maximal efficiency of inductive immunne response in order to obtain, the whole tertiary structure of preferred APP or A β remains unchanged in the molecule of modifying.

In some cases, preferred APP or A β or its fragment are suddenlyd change.35 the methionine that particularly preferably is at A β-43 is substituted, preferably replaces with leucine or isoleucine, or the replacement variant of being deleted simply.Preferred especially analog comprises an independent methionine that is positioned at the C-end, or owing to its natural amyloid originality polypeptide or external source T of being present in _HIn the epi-position or since it be inserted into or increase.Therefore, also preferably except have a methionine at the C-terminal position, comprise external source T _HThe analog part of epi-position does not contain methionine.

The main cause of deleting all methionines beyond the methionine is, may prepare the poly analog by reorganization, and it can be cut by Bromine cyanide. subsequently and obtain single analog.Its favourable part is to be to be convenient to like this carry out recombinant production.

In fact, usually preferred all APP used according to the invention or A β analog all have following feature, promptly only comprise an independent methionine as C-end amino acid in the analog, and at amyloid originality polypeptide or external source T _HOther methionine in the epi-position is all deleted or use another kind of aminoacid replacement.

A further interested sudden change is deletion or 19 the phenylalanine that replaces A β-43, and preferred especially this sudden change is to replace this phenylalanine residue with proline.

Other interested polyamino acid that uses in analog is the part of the proteic truncate of A β-43.These also can use in immunogenicity analog according to the present invention.Particularly preferably be truncate A β (1-42), A β (1-40), A β (1-39), A β (1-35), A β (1-34), A β (1-34), A β (1-28), A β (1-12), A β (1-5), A β (13-28), A β (13-35), A β (17-28), A β (25-35), A β (35-40), A β (36-42) and A β (35-42) (numeral in its bracket form associated clip A β-43 aminoacid sequence-for example, A β (35-40) is identical with 706-711 amino acids among the SEQ ID NO:2).All these variants with A β-43 of truncate part can prepare with A β fragment described herein, particularly prepare with the variant of mentioning among the

embodiment

19,10,11,12 and 13.

Following formula is described the molecule construction body that the present invention generally includes:

(MOD ₁) _S1(amyloid _E1) _N1(MOD ₂) _S2(amyloid _E2) _N2(MOD _x) _Sx(amyloid _Ex) _Nx(I)

-amyloid wherein _E1-amyloid _ExBe the xB cell epitope that comprises APP or A β subsequence, they are respectively identical or different, and can comprise or not comprise the external source side-chain radical, x is 〉=and 3 integer, nl-nx is x 〉=0 integer (having 〉=1 at least), MOD ₁-MOD _xBe x the modification of between the B cell epitope that keeps, introducing, s ₁-s _xThe integer that is x 〉=0 is (if at amyloid _ExDo not introduce side-chain radical in the sequence, then have one 〉=1 at least).Therefore, consider the general utility functions restriction of construct on immunogenicity, the present invention allows the conversion (permutation) and the wherein modification of all kinds of all kinds of APP or A β original series.Therefore, what the present invention was included is by the deletion sequence, for example show the part of side effect in vivo or be generally in the born of the same parents partly and therefore can produce non-required immunoreactive part, thus the APP or the A β of the modification that obtains.

When using, a preferred form of above-mentioned construct of illustrating be those B cell epitopes that comprise the amyloid subsequence not born of the same parents be exposed to outward in the precursor polypeptide of this amyloid of deriving.By selecting such epi-position, guarantee not produce and produce the antibody that the cell of precursor reacts, thereby make the immunne response that is produced be limited to anti-non-required amyloid beta deposition thing.For example, in this case, when not having their cell of any generation of coupling, induce anti-ly only to be exposed to cell foreign minister's APP or the immunoreation of A β epi-position is feasible.

Keep the substantial portion of B cell epitope (substantialfraction) as described here or even proteic whole tertiary structure by several method through modifying.A kind of mode is the polyclonal antiserum (antiserum that for example prepares in rabbit) for preparing directly anti-described polypeptide simply, then with the detectable (for example, in competitive ELISA) of this antiserum as the anti-modified protein that produces.Modified forms (analog) with the reaction of the degree identical with APP or A β and antiserum must be considered to have and APP or the identical whole tertiary structure of A β, is considered to keep the substantial portion of original B cell epitope and this antiserum is shown restricted (but still for main with special) analog of reaction.

Alternatively, can preparation and APP or A β on unique epi-position reaction selection monoclonal antibody and as test piece.The advantage of this method is to obtain 1) the epi-position collection of illustrative plates and 2 of APP or A β) collection of illustrative plates of the epi-position that in the analog of preparation, keeps.

Certainly, the third method can be decomposed the three dimensional structure of APP or A β or its biological activity truncate (consulting foregoing), and this structure is compared with the three dimensional structure of the decomposition of the analog of preparation.Three dimensional structure can decompose by X-ray diffraction studies and NMR spectrum analysis.Further information about three dimensional structure can be in a way available from circular Dichroism Studies On, it is advantageous that and only need the useful information of pure polypeptide (and X-ray diffraction need provide the polypeptide of crystallization, NMR need provide the isotope variant of polypeptide) with three dimensional structure that given molecule is provided.But, finally need X-ray diffraction and/or NMR to obtain the concluding data, because circular dichroism only can provide the circumstantial evidence of correct three dimensional structure by the information of secondary structure element.

A preferred embodiment of the present invention is utilized multiple the presenting (that is, having at least a B cell epitope to be present in two positions) of the bone-marrow-derived lymphocyte epi-position of APP or A β in formula I.This effect can be finished by the whole bag of tricks, for example, comprises structure (APP or A β polypeptides derived) by preparing simply _mFused polypeptide, wherein m is 〉=2 integer, introduces modification discussed herein in APP or A β sequence at least one then.The preferred modification of introducing comprises the bone-marrow-derived lymphocyte epi-position of at least one copy and/or introduces a hapten.These multiple embodiments of presenting that comprise selected epi-position are particularly preferred in following situation, promptly in vaccine reagent only the less important part of APP or A β as component.

As mentioned above, can be by introducing at least one aminoacid insertion, interpolation, deletion or replacing the introducing of finishing the external source t cell epitope.Certainly, normal situation is to introduce more than one (for example to change in aminoacid sequence, insert or replace with complete t cell epitope), but the important goal that reaches is, when being added man-hour by antigen-presenting cell (APC), analog will produce this foreign immunologic dominance t cell epitope of being presented by the lip-deep MHC II of APC quasi-molecule.Therefore, if the aminoacid sequence of APP or A β comprises many external source T that also are found in place _HAmino acid residue in the epi-position can provide the remaining amino acid of foreign epitope by aminoacid insertion, interpolation, deletion and replacement so, thereby finish external source T _HThe introducing of epi-position.In other words, promptly needn't introduce complete T by inserting or replacing _HEpi-position is to reach the target of invention.

The quantity that preferred amino acid is inserted, deletes, replaced or adds is 2 at least, for example 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 and 25 insertions, replacement, interpolation or deletions.In addition, preferred insert, replace, add or the amino acid number of deletion is no more than 150, for example at the most 100, at the most 90, at the most 80 and at the most 70.The number that especially preferably replaces, inserts, deletes or add is no more than 60, and particularly this numeral is no more than 50 or even 40.Most preferably be that this numeral is no more than 30.About aminoacid addition, it should be noted that when resulting construct was the fused polypeptide form, these normal considerations were higher than 150.

The preferred embodiments of the invention comprise the modification of being undertaken by the t cell epitope of introducing at least one foreign immunologic dominance.The immundominance problem that is appreciated that t cell epitope depends on described animal species.As used herein, term " immundominance " only refers to produce the epi-position of important immunne response in the individuality/colony of inoculation, but well known fact is, be that immunodominant t cell epitope needn't be immundominance in body/colony one by one in another individuality of the same race, even its may combine with MHC II quasi-molecule in one individuality of back.Therefore, for the purpose of the present invention, immunodominant t cell epitope is when having antigen, can effectively provide the T cell auxiliary t cell epitope.Typically, immunodominant t cell epitope has internal characteristics, always promptly they are offered in fact to combine with MHC II class, and no matter their polypeptide of manifesting wherein.

Another important problem is that the MHC of t cell epitope is restricted.Usually, naturally occurring t cell epitope is that MHC is restrictive, that is, some peptide of forming t cell epitope only with effective a combination of subgroup of MHC II quasi-molecule.This has such effect conversely, promptly in most of the cases, using a kind of specific t cell epitope will cause vaccine component only effective to the part in the colony, and depend on the size of this part, must in same molecular, comprise more t cell epitope, or alternatively prepare multicomponent vaccine, wherein component is according to the character of the t cell epitope of introducing and the mutually APP of difference or the variant of A β.

If the MHC of used T cell is restricted is complete unknown (for example having under the situation of the poor MHC that determines composition the inoculation animal), can be by following formula approximate calculation by the part that specific vaccine combination covered

-p wherein _iBe that i external source t cell epitope produces the frequency of responder in colony in the vaccine combination to being present in, n is the sum of external source t cell epitope in the vaccine combination.Therefore, the response frequency in colony is respectively 0.8,0.7 and 0.6 the vaccine combination that comprises 3 external source t cell epitopes, will provide

1-0.2 x 0.3x 0.4＝0.976

-promptly 97.6% colony produces replying the MHC-II of vaccine mediation on statistical significance.

Above formula can not be applied in the situation of more or less knowing the accurate MHC restriction map of used polypeptide.If, for example certain peptide only combines with the people MHC-II molecule of HLA-DR allele D R1, DR3, DR5 and DR7 coding, uses this peptide and another kind can reach 100% to described colony with remaining bonded peptide of MHC-II molecule by HLA-DR allele coding so simultaneously and covers.Equally, if second kind of peptide only combines with DR3 and DR5, add this peptide and will can not increase coverage rate at all.If the calculating that colony is replied is restricted based on the MHC of t cell epitope in the vaccine purely, then can be determined by following formula by the least part of the colony that specific vaccine combination covered:

-wherein

Be that coding combines with any t cell epitope in the vaccine and belongs to the summation of the allele haplotype of the individual MHC molecule of the j in three known HLA sites (DP, DR and DQ) at colony's medium frequency; In fact, to determine that at first which MHC molecule can discern each t cell epitope in the vaccine, list thereafter and with type (DP, DR and DQ) that the single frequency of the different then allele haplotypes of listing sums up with every type, thereby produce

With

P in formula II _iValue may surpass its corresponding theory value ∏ _i:

π_{i} = 1 - Π_{j = 1}^{3} {(1 - v_{j})}^{2} - - - (IV)

-v wherein _jBe that coding combines with i t cell epitope in the vaccine and belongs to the summation of the allele haplotype of the individual MHC molecule of the j in three known HLA sites (DP, DR and DQ) at colony's medium frequency.This shows the ∏ at 1- _iIn the colony, respondent's frequency is f _{Remaining _ i}=(p _i-∏ _i)/(1-∏ _i).Therefore can adjust formula III to obtain formula V:

-term 1-f wherein _Remaining-iUnder negative situation, be set at 0.It should be noted, formula V need all epi-positions all to mutually on the same group haplotype and by the haplotype collection of illustrative platesization.

Therefore, when the t cell epitope selecting to be incorporated in the analog, comprise that all obtainable following knowledge about this epi-position are very important: 1) respondent in colony to the frequency of each epi-position, 2) the restricted data of MHC and 3) frequency of correlation unit type in colony.

Have many naturally occurring " miscellaneous " t cell epitope, they are active in most of individuality of animal species or animal population, and these epi-positions preferably are introduced in the vaccine, thus be reduced in in a kind of vaccine to the demand of a large amount of different analog.

According to the present invention, mixing epi-position can be naturally occurring human T-cell's epi-position, for example, come from tetanus toxoid (for example P2 and P30 epi-position), diphtheria toxoid, influenza virus hemagglutinin (hemagluttinin) (HA) and the antigenic epi-position of P.falciparum CS.

In recent years, a large amount of other miscellaneous t cell epitopes have been identified.Particularly identified and can carry out bonded peptide with the most of HLA-DR molecule by different HLA-DR allele codings, they are that institute might be incorporated into according to the t cell epitope in the analog of the present invention's use.Referring to below with reference to the epi-position of being discussed in the document, they are therefore by with reference to being incorporated into this: WO98/23635 (Frazer IH etc., assigned to The University of Queensland); Southwood S etc., 1998, J.Immunol.160:3363-3373; Sinigaglia F etc., 1988, Nature 336:778-780; Chicz RM etc., 1993, J.Exp.Med 178:27-47; Hammer J etc., 1993, Cell 74:197-203; With Falk K etc., 1994, Immunogenetics 39:230-242.The list of references of back also relates to HLA-DQ and HLA-DP part.Because all listed epi-positions in these five lists of references all have identical primitive, so they are all corresponding as the natural epi-position that is used for candidate of the present invention.

Alternatively, epi-position can be any artificial t cell epitope, and it can combine with most of MHC II quasi-molecule.At the pan DR epitope peptide (" PADRE ") described in the WO 95/07707 with at pertinent literature Alexander J etc., 1994, (two contents all are hereby expressly incorporated by reference) is the candidate of interesting epi-position used according to the invention among the Immunity 1:751-761.It should be noted that the most effective disclosed PADRE peptide has D-aminoacid at C-and N-end in these articles, the stability during with the raising administration.Yet, main purpose of the present invention is to be, integrates the part of relevant epi-position as analog, and its lysosome at APCs is cut apart inner by enzymatic degradation then, so that present in the scope of MHC-II molecule subsequently, it is not favourable therefore integrating D-aminoacid in the epi-position that the present invention uses.

A particularly preferred PADRE peptide is to have aminoacid sequence AKFVAAWTLKAAA (SEQ ID NO:17) or the effective subsequence of its immunogenicity.This peptide is preferred t cell epitope with other epi-position with the restricted defective of identical MHC, exists in the analog that should use in inventive method.The super epi-position that mixes like this can be used for the simplest embodiment of the present invention, wherein only has an independent analog to be the immune system of the animal of passing inoculation.

As mentioned above, the modification of APP or A β can comprise that also introducing is positioned the amyloid originality polypeptide of modifying the first of APC or bone-marrow-derived lymphocyte.For example, first can be the specific bond gametophyte of special surface antigen of bone-marrow-derived lymphocyte or the special surface antigen of APC.Many so special surface antigens are well known in the art.For example, this part can be a kind of sugar, and its receptor (for example mannan or mannose) is arranged on bone-marrow-derived lymphocyte or APC.Alternatively, second portion can be a hapten.And the antibody fragment of specific recognition APCs or lymphocyte upper surface molecule also can be used as first, and (surface molecular can be, for example, macrophage and monocytic FC γ receptor, for example FC γ RI or, any other special surface marker of selecting is as CD40 or CTLA-4).It should be noted that all these guide molecules of giving an example also can be used as the part of adjuvant, referring to the following stated.

As analog being positioned particular cell types, can increase the immune level of replying by the second portion that comprises above-mentioned stimulating immune system to realize a selection or additional of enhanced immunne response.The exemplary of this second portion is cytokine, heatshock protein or molecular chaperones, with and live part.

Suitable cytokine used according to the invention is those cytokines that also work as adjuvant in vaccine combination usually, that is, for example interferon gamma (IFN-γ), interleukin-11 (IL-1), interleukin-22 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin-11 3 (IL-13), interleukin 15 (IL-15) and granulocyte-macrophage colony stimutaing factor (GM-CSF); Alternatively, the funtion part of cytokine molecule is enough to as second portion.About using this cytokine, referring to following discussion as the adjuvant material.

According to the present invention, suitable heatshock protein or molecular chaperones as second portion can be that HSP70, HSP90, HSC70, GRP94 (also are known as gp96, consult, Wearsch PA etc., 1998, Biochemistry 37:5709-19) and CRT (calprotectin).

Alternatively, second portion can be a kind of toxin, for example the molten born of the same parents' element in Listerella (listeriolycin) (LLO), lipid A and heat-labile enterotoxin.And, many mycobacterium derivants, for example MDP (muramyldipeptide), CFA (complete Freund's adjuvant) and trehalose diester TDM and TDE also are interesting may selecting.

Equally, introduce to strengthen that analog is presented to immune third part is important embodiment of the present invention.This area has represented several embodiment of this principle.For example, knownly utilize palmityl fatization (lipidation) anchor in B. burgdorferi (Borrelia burgdorferi) protein A as if the polypeptide (referring to for example WO 96/40718) of self assisting a ruler in governing a country-albumen of fatization forms the structure of micelle sample to provide, have by the nuclear of the fat anchor of polypeptide part with from the remainder of this molecule that stretches out, thereby cause the multiple of antigenic determinant to be presented.Therefore; this method with (for example use different fat anchors; nutmeg base, nutmeg base, farnesyl-, geranyl-geranyl group, GPI anchor and N-acyl group two glycerol ester groups) the application of correlation technique be the preferred embodiment of the invention; be very direct particularly, only need to use the fusion partner of for example naturally occurring signal sequence as analog owing to providing such fat anchor in the albumen that produces in reorganization.Another kind of probability be to use the C3d fragment of complement factor C3 or C3 itself (referring to, Dempsey etc., 1996, Science 271,348-350 and Lou﹠amp; Kohler, 1998, Nature Biotechnology16,458-462).

The important epi-position district that another is selectable in the present invention, cause preferably presenting the APP of a plurality of (for example, at least 2) copy or A β to immune embodiment be with the analog covalent coupling to specific molecular, that is, and above-mentioned variant d and e.For example, can use polymer, as sugar glucosan for example, referring to, Lees A etc. for example, 1994, Vaccine 12:1160-1166; Lees A etc., 1990, J Immunol.145:3594-3600, and also mannose and mannan are useful alternatives.For example coming from, escherichia coli also are useful binding partners with other bacterial integral membrane albumen.Traditional carrier molecule, for example keyhole limpet hemocyanin (KLH), tetanus toxoid, diphtheria toxoid and bovine serum albumin (BSA) also are preferred and useful binding partners.

With APP or the deutero-material covalent coupling of A β to polyhydroxylated polymer for example the preferred embodiment on the sugar relate to and use at least one APP or the deutero-peptide of A β and at least one external source t helper cell epi-position, they are to be coupled to respectively on the polyhydroxylated polymer (promptly, external source t helper cell epi-position and APP or the deutero-aminoacid sequence of A β do not merge mutually, but are connected to subsequently on the polyhydroxylated polymer as carrier framework).And, when the suitable B cell epitope with APP or the deutero-peptide of A β zone makes up by short peptide sequence, such embodiment be most preferred-this be because this method be a kind of multiple approach of presenting very easily that can in the immunoreagent of gained, realize selected epi-position.But, also may only the analog of herein having described be coupled on the polyhydroxylated polymer skeleton, that is, APP or the deutero-material of A β be free of attachment on the skeleton and with external source T _HEpi-position separately.

Particularly preferably be with external source t helper cell epi-position and APP or the coupling of A β deutero-(many) peptide by the amido link that can be cut by peptidase.This tactful effect is that APCs can absorb conjugate, can process this conjugate simultaneously, presents the external source t cell epitope subsequently in MHC II class scope.

A method finishing peptide coupling (the interested APP of being or deutero-peptide of A β and foreign epitope) is for example to use tresyl (trifluoroethyl sulfonyl) group or other suitable activated groups; dimaleoyl imino (maleimido), p-Nitrophenyl cloroformate (activation OH base forms peptide bond between peptide and polyhydroxylated polymer) and the suitable polyhydroxylated polymer of tosyl (ptoluene-sulfonyl) activation.For example, as WO 00/05316 and US 5,874,469 (the two is hereby expressly incorporated by reference) are described may to prepare activatory polysaccharide, and they are coupled to APP or deutero-peptide of A β or polyamino acid and are coupled on the t cell epitope by traditional solid phase or the preparation of liquid phase peptide synthetic technology.The product that obtains comprises polyhydroxylated polymer skeleton (for example, the glucosan skeleton), and this skeleton partly connects the polyamino acid that derives from APP or A β and derive from the external source t cell epitope by its N end-end or by other available nitrogen.If desired, may synthesize APP or the A β peptide of having protected all available amino ends except that the terminal amino of N-, the protected peptide that will obtain subsequently is connected to the glucosan part of trifluoroethyl sulfonylization, and is last, and the conjugate that obtains is gone protection.The specific embodiment of this method is described in the following embodiments.

Except using WO 00/05316 and US 5,874, the water soluble polysaccharide molecule of instruction in 469, can use crosslinked polysaccharide molecule equally, thereby the particulate conjugate of acquisition between polypeptide and polysaccharide-believe that this will cause improvement that polypeptide is presented to immune system, because reached two targets, promptly can obtain a kind of partial sedimentary effect during this conjugate when injection, and to obtain APCs be the particle of attractive target.Method with such microparticulate systems also describes in detail in an embodiment.

Introducing the preferential points for attention in zone of selecting of modifying in APP or A β is, a) keep B cell epitope known and prediction, b) keep tertiary structure, c) avoid the B cell epitope on " production cell " etc., to present, in any case, as mentioned above, be easy to screen one group of analog of introducing t cell epitope at diverse location.

Because the most preferred embodiment of the present invention is the downward modulation of people A β, so preferably above-mentioned APP or A beta polypeptides are people's APP or A beta polypeptides.In this embodiment, particularly preferably be that APP or A beta polypeptides are by or different length isometric with at least one and comprise external source T _HAt least one aminoacid sequence is modified among the aminoacid sequence replacement SEQ ID NO:2 of epi-position.Amyloid originality APP that modifies and the preferred embodiment of A β are that illustration is shown among Fig. 1 with P2 and P30 epi-position.Gone through the ultimate principle of this construct in an embodiment.

More specifically, comprise the T that (or all) is incorporated into the aminoacid sequence among the SEQ ID NO:2 _HCan in any one aminoacid of SEQ ID NO:2, be introduced into.That is to say, introducing may be at amino acid/11-770 after any one, but preferably after any one of 671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713 and 714 amino acids in SEQ ID NO:2.Can be in conjunction with or all with any one of deletion amino acid/11-671, or aminoacid 715-770 any one or all.In addition, when using replacement technique, combine with introducing, can delete any one in the aminoacid 671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713 and 714 among the SEQ ID NO:2.

Another embodiment of the invention is to present the analog that does not comprise any SEQ ID NO:2 subsequence, described subsequence and effective combination of MHC II quasi-molecule that starts t cell response.

This design immunogen is so that immune system induces the ultimate principle of the strategy that produces anti-A β immunne response as follows: should note, when being enough to destroy strongly in the body when a large amount of oneself proteins in the adjuvant of oneself protein tolerance such as A β carried out immunity with being formulated in, have a kind of risk, promptly immunne response can not be terminated by stopping immunity simply in some inoculation individualities.This is because inductive immunne response is most likely by the natural T of oneself protein in this individuality _HEpi-position is caused, and its side effect is that the oneself protein self of the individuality of inoculation will play a role as immunoreagent: therefore set up one from immune condition.

Preferable methods, it comprises external source T _HThe application of epi-position must be that the inventor understands most, does not also observe this effect, because anti-autoimmune response is by external source T _HEpi-position is inductive, and inventor inductively in fact reducing after stopping immunity of confirming repeatedly to be caused by optimization technique.But it may take place in the minority individuality in theory, and is promptly individual by immunity, also can by relevant oneself protein from source T _HEpi-position causes immunne response-when the autologous protein of considering enriches relatively, A β for example, and then this is more relevant, and the relevant autologous protein of other treatment only exists in the part or quantity is very low in vivo, to such an extent as to " autoimmune effect " can not take place.Therefore, avoiding a plain mode of this effect is to avoid being used as in immunogen T fully _HEpi-position (can not be used as T because be shorter than 9 amino acid whose peptides _HEpi-position is the method for a simple possible so use shorter fragment) the occlusion body of peptide sequence.Therefore, this embodiment of the present invention is used to also guarantee that immunogen does not comprise as " autostimulation T _HEpi-position " target APP or the peptide sequence of A β, described " autostimulation T _HEpi-position " comprise and the sequence of the conservative replacement in the sequence that only is included in target protein make it can be used as T in addition _HEpi-position.

The immune preferred embodiment of presenting APP or A β analog comprise use comprise at least one not with the simulating peptide of effective bonded APP of MHC secondary molecule or A β derived peptide, and at least one external source t helper cell epi-position.In addition, preferably, APP or A β derived peptide have the B cell epitope.If immunogenic analog comprises one or more with continuous sequence or comprise the B cell epitope of the insertion sequence representative of external source t helper cell epi-position, then this analog is very favourable.

And, carry the suitable B cell epitope in APP or A β district when making up by short peptide sequence, when it was never effectively combined with MHC II quasi-molecule, this embodiment was most preferred.Therefore, the epi-position of the B cell epitope of selection or amyloid originality polypeptide should comprise 9 continuous amino acids of SEQ ID NO:2 at the most.Shorter peptide is preferred, and for example those have the peptide of 8,7,6,5,4 or 3 continuous amino acids of amyloid originality polypeptid acid sequence at the most.

Preferably, analog comprises the subsequence of SEQ ID NO:2 at least, makes each at least one such subsequence form by being selected from 9 continuous amino acids, 8 continuous amino acids, 7 continuous amino acids, 6 continuous amino acids, 5 continuous amino acids, 4 continuous amino acids and the APP of 3 continuous amino acids or the aminoacid sequence of A β independently.

Particularly preferably be that continuous amino acid begins at residue 672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713 that is selected from SEQ ID NO:2 and 714 amino acid residue.

Albumen/peptide vaccination; The preparation of analog and administration

When realizing that by the administration animal analog is when passing animal immune system, principle recognized in the art is followed in the preparation of polypeptide.

In the art, very understanding comprises the preparation of peptide sequence as the vaccine of active component, as United States Patent (USP) 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792 and 4,578,770 illustrations, all is hereby expressly incorporated by reference.Typically, this vaccine is with injectable forms, perhaps as liquid solution or suspension form and prepare; Also can prepare the solid form that is suitable for before injection, being dissolved or suspended in the liquid.Also can be with preparation emulsifying.The active immne component usually with medicinal and with the mixed with excipients of active component compatibility.Suitable excipient for example is, water, saline, dextrose, glycerol, ethanol or similar substance, and combination.In addition, if desired, vaccine can comprise minor amounts of auxiliary substances, for example wetting agent or emulsifying agent, pH buffer agent or strengthen the adjuvant of vaccine potency; Referring to going through of, following adjuvant.

Traditionally, vaccine is by injection parenteral, for example subcutaneous, Intradermal or intramuscular injection.The other preparation that is suitable for other administering modes comprises suppository, and in some cases, mouthful, suck preparation in (buccal), Sublingual (sublinqual), intraperitoneal, intravaginal, anus, epidural, spinal column, the skull.For suppository, conventional junction mixture and carrier can comprise, for example, and polyalkylene glycol (polyalkalene glycols) or triglyceride; This suppository can be by comprising 0.5%-10%, and preferably the mixture of 1-2% active component forms.Oral formulations comprises normally used excipient, for example, and the mannitol of pharmaceutically grade, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc.These compositionss adopt solution, suspension, tablet, pill, capsule, extended release preparation or powder type, and comprise the 10-95% active component, are preferably 25-70%.To oral formulations, cholera toxin is interesting preparation gametophyte (also being possible conjugate gametophyte).

Polypeptide can neutrality or salt form be formulated in the vaccine.Pharmaceutical salts comprises acid addition salt (forming with the free amino group of peptide), and it uses mineral acid, for example hydrochloric acid or phosphoric acid, or organic acid, for example formation such as acetic acid, oxalic acid, tartaric acid, mandelic acid.The salt that forms with free carboxyl group also can be from inorganic base, for example, and sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or hydrated ferric oxide., and organic base, for example 2-aminopropane., trimethylamine, 2-ethylaminoethanol, histidine, procaine etc. and derive.

Vaccine carries out administration in the mode consistent with dosage formulation and to treat effectively and to have immunogenic dosage.Dosage depends on experimenter to be treated, for example comprises, the individual immunity system causes the ability of immunne response and the degree of required protection.The suitable dose scope is to inoculate the active component of hundreds of Gamma Magnitude at every turn, preferable range is about 1 μ g-2, the 000 μ g higher amount of 1-10mg scope (even expected), for example, at about 0.5 μ g-1,000 μ g scope is preferably in about 1 μ g-500 μ g scope, especially in about 10 μ g-100 μ g scopes.The appropriate therapies of initial administration and booster shot also is variable, but typically after first administration, inoculates subsequently or other administrations.

Application mode can extensively change.Can use any traditional administration of vaccines method.These be included in the physiologically acceptable substrate of solid or physiologically acceptable dispersion form Orally administered, parenteral injection or similar fashion.Vaccine dose depends on route of administration, and preparation changes according to vaccinate's age and antigen.

In vaccine, the polypeptide of some vaccines has enough immunogenicities, and concerning other, if vaccine further comprises the adjuvant material, then can enhance immunity reply.

The known method that various realizations are arranged the adjuvant effect of vaccine.Ultimate principle and method are at " TheTheory and Practical Application of Adjuvants ", 1995, Duncan E.S.Stewart-Tull (editor), John Wiley﹠amp; Sons Ltd, ISBN 0-471-95170-6 and " Vaccines:New Generationn Immunological Adjuvants ", 1995, Gregoriadis G etc., (editor), Plenum Press, New York, be described in detail among the ISBN 0-306-45283-9, therefore the two all be hereby expressly incorporated by reference.

Particularly preferred being to use proves and is beneficial to the adjuvant of destruction to the self-tolerance of self antigen; In fact, when the triterpenoid polypeptide of the non-modification of use was as active component in autovaccine, this point was very important.The non-limiting example of suitable adjuvant is selected from the immune guiding adjuvant; Immunomodulating adjuvant, for example toxin, cytokine and mycobacterium derivant; Oil formulation; Polymer; Micelle form adjuvant; Saponin; Immunostimulating complex substrate (ISCOM substrate); Granule; DDA; Aluminium adjuvant; The DNA adjuvant; γ-inulin and seal adjuvant (encapsulating adjuvant).Usually it should be noted that to relate at first chemical compound and the reagent that uses that the foregoing of second in analog and third part in addition necessary change also relates to their application in the adjuvant of vaccine of the present invention.

The application of adjuvant comprises use reagent, for example aluminium hydroxide or aluminum phosphate, consumption is a 0.05-0.1% solution in buffer saline usually, with the mixture of sugared synthetic polymer (for example, ), consumption is 0.25% solution, by with 70-101 ℃ of temperature proteic polymer in the vaccine that obtains of 30 seconds-2 minutes time of heat treatment respectively, also may be the polymer that obtains by cross-linking agent.Also can utilize the polymer that obtains by antibody (Fab fragment) reactivate with the antialbumin of pepsin, with the bacterial cell mixture of the lipopolysaccharide component of C.parvum or endotoxin or gram negative bacteria for example, physiologically acceptable oils excipient for example the Emulsion in the mannide monoleate (Aracel A) or with Emulsion as 20% perfluocarbon (Fluosol-DA) solution of blocking-up substitute (block substitute).With oil for example the mixture of zamene and IFA also be preferred.

According to the present invention, DDA (dimethyl two (octadecyl) ammonium bromide) is the same with DNA and γ-inulin, be a kind of interested adjuvant material standed for, and Freund's complete adjuvant and Freund and quillaja saponin, for example QuilA is the same with RIBI with QS21 is interesting.Further probability is monophosphoryl lipid class A (MPL), above-mentioned C3 and C3d and muramyldipeptide (MDP).

Known Liposomal formulation also has adjuvant effect, and therefore according to the present invention, the liposome adjuvant is preferred.

According to the present invention, the immunostimulating complex matrix type ( Substrate) adjuvant also is preferred selection, particularly owing to show that the type adjuvant can raise the expression of MHC II quasi-molecule by APCs.

The substrate origin comes from saponin (triterpenoid compound), cholesterol and the phospholipid of quillaja saponin and forms (optionally fraction).When mixing with immunogenic protein, the resulting granules preparation is and is known as the ISCOM granule, and wherein saponin accounts for the 60-70% w/w, and cholesterol and phospholipid are the 10-15% w/w, and albumen is the 10-15% w/w.Can for example find in the above-mentioned teaching material about the composition of immunostimulating complex and the particulars of application about adjuvant, and, Morein B etc., 1995, Clin.Immunother.3:461-475 and Barr IG and Mitchell GF, 1996, Immunol.and Cell Biol.74:8-25 (the two all is hereby expressly incorporated by reference) provides and has prepared the guidance of immunostimulating complex completely.

The probability of the realization adjuvant effect of another very interesting (being preferred therefore) is to be applied in Gosselin etc., the described technology of 1992 (therefore they are hereby expressly incorporated by reference).Briefly, to related antigen antigenic the presenting among the present invention for example, can go up by the antibody (or the bonded antibody fragment of antigen) that antigen is connected to the Fc γ receptor on the anti-monocyte/macrophage and strengthen.But the conjugate that has proved antigen and anti-Fc γ RI in particular is for inoculation enhance immunity originality.

Other probabilities relate to uses above-mentioned guiding and immune regulator (especially cytokine) candidate as first and second parts in the modified forms of amyloid originality polypeptide.In this connection, synthetic cytokine induction thing as poly I:C, also is a kind of probability.

Two fat that suitable mycobacteria derivant is selected from muramyldipeptide, complete Freund's adjuvant, RIBI and trehalose are TDM and TDE for example.

Suitable immune guiding adjuvant is selected from CD40 part and CD40 antibody or its specific bond fragment (referring to above-mentioned discussion), mannose, Fab fragment and CTLA-4.

The suitable polymers adjuvant selects sugar for example glucosan, PEG, starch, mannan and mannose; Plastic polymer for example; With latex latex beads for example.

Yet the mode of another interested adjusting immunne response is (VLN) (by ImmunoTherapy at " effectively lymph node ", Inc., 360 Lexington Avenue, New York, the private medical treatment device of NY10017-6501 invention) comprises immunogen (randomly with adjuvant and pharmaceutical carrier and vehicle) in.The 26S Proteasome Structure and Function of VLN (a kind of thin tube) simulation lymph node.Can create a cytokine and the surging aseptic inflammation site of chemotactic factor at subcutaneous insertion VLN.Very fast danger signal are produced of T cell and B cell and APCs replied, and finds this inflammation site and in the poroid substrate content accumulation of VLN.Shown when using VLN, caused the required essential antigen dose of antigenic immunne response is descended, and inoculated the immunoprotection that is reached with VLN and surpassed the traditional immune protection that is reached as adjuvant with Ribi.This technology is also at Gelber C etc. especially, 1998, " Elicitation of Robust Cellular and Humoral ImmuneResponses to Small Amounts of Immunogens Using a Novel Medical DeviceDesignated the Virtual LymphNode ",: carried out simple description in " From the Laboratory to theClinic; Book of Abstracts; October, 12-15 1998; Seascape Resort; Aptos, Calfornia ".

Shown that the microparticle formulation of vaccine under many circumstances can increase the immunogenicity of proteantigen, so this also is another preferred embodiment of the present invention.With antigen and polymer, lipid, sugar or other be suitable for preparing particulate molecule altogether-the formulation preparation microgranule, perhaps microgranule can be the homogeneous granule that only comprises antigen itself.

Be based on the granule (Gupta, R.K. etc., 1998) of PLGA and PVP based on the embodiment of polymer particulates, wherein polymer and antigen are condensed into solid particle.Microgranule based on lipid can be prepared into lipid micelle (being also referred to as liposome), and envelope antigen in micelle (Pietrobon, P.J.1995).For example starch or chitosan prepare by suitable degradable sugar typically based on the microgranule of sugar.Sugar and antigen mix, and to be condensed into granule (Kas .1997 such as H.S.) with the similar manufacturing process of polymer beads.

Only comprising antigenic granule can prepare by various sprayings and Freeze Drying Technique.What be particularly suitable for purpose of the present invention is supercritical fluid technology, it can be used to prepare the very homogeneous of controlled size granule (York, P.1999﹠amp; Shekunov .1999 such as B.).

The expection vaccine should annual administration 1-6 time, for example in 1 year to there being the individuality of needs to carry out 1,2,3,4,5 or 6 administration.Showed in the past that it was not nonvolatil using according to the inductive Memorability immunity of preferred autovaccine of the present invention, so immune system need regularly stimulate with the amyloid originality polypeptide of amyloid originality polypeptide or modification.

Because hereditary difference, Different Individual may produce the immunne response of varying strength to the phase homopolypeptide.Therefore, vaccine of the present invention can comprise several different polypeptide replys with enhance immunity, referring to the above selection that imports about importing external source t cell epitope.Vaccine can comprise two or more polypeptide, and wherein all these polypeptide as defined above.

Therefore vaccine can comprise 3-20 different modification or non-modified polypeptides, for example 3-10 different polypeptide.

Nucleic acid vaccination

Alternative as traditional vaccine administration mode based on peptide, nucleic acid vaccination technology (being also referred to as " nucleic acid immunization ", " heredoimmunity " and " genetic immunization ") provides a large amount of attracting features.

At first, with respect to the traditional vaccine mode, nucleic acid vaccination does not need large-scale production to have the resource consumption of immunogenic reagent (for example with plant layout ferment the mode of microorganism of amyloid originality polypeptide that produce to modify).In addition, do not need equipment that immunogen is carried out purification and folding again.At last, because depending on to be equipped by the individual biochemistry of inoculation, nucleic acid vaccination produces the expression of nucleic acids product that imports, so wish to take place the suitableeest translation post-treatment process of expression product; This is a particular importance in autovaccine inoculation situation, because as mentioned above, the live part of original B cell epitope should obtain keeping in decorating molecule, and because the B cell epitope can make up by the part of any (biology) molecule (for example sugar, lipid, albumen etc.) on the principle.Therefore, immunogenic Natively glycosylated extremely important to whole immunogenicity with the fat pattern, preferably guarantee this point by having the immunogenic host of generation.

Therefore, the preferred embodiment of variant a-c of the present invention comprises, and the nucleic acid of coding analog is incorporated into zooblast, thereby obtains to be imported into the expression in vivo of the cell of nucleic acid, thereby realizes analog is presented to immune system.

In this embodiment, the nucleic acid that imports is DNA preferably, its form can be, exposed DNA, DNA with electrically charged or uncharged lipid preparation, be formulated in the DNA in the liposome, be included in the DNA in the viral vector, DNA with albumen that is beneficial to transfection or polypeptide preparation, DNA with guiding protein or polypeptide preparation, DNA with the preparation of calcium deposited reagent, the DNA of coupling inert carrier molecule, be encapsulated in polymer for example the DNA in PLGA (referring to the technology of describing among the WO 98/31398) or chitin or the chitosan and with the DNA of adjuvant preparation.It should be noted that in this article in fact all about the consideration of the adjuvant that in the traditional vaccine preparation, is suitable for using and all can be applicable to the dna vaccination preparation.Therefore, changed about all the elements of in based on the vaccine of polypeptide, using adjuvant herein and be suitable in the nucleic acid vaccination technology, using.

About top route of administration and the dosage regimen based on the vaccine of polypeptide that has described in detail, they also can be applicable in the nucleic acid vaccine of the present invention, and above about the route of administration that is suitable for polypeptide and dosage regimen all are discussed to be changed and are applicable to nucleic acid.What should increase this is that nucleic acid vaccine is suitable for intravenous and intra-arterial administration.In addition, be well known in the art, nucleic acid vaccine can carry out administration by using so-called particle gun, so this mode of administration and equivalents also are parts of the present invention.At last, be reported in the nucleic acid administration and used VLN and obtain good result, therefore this special administering mode is particularly preferred.

In addition, can comprise coding first, second and/or third part as the nucleic acid of immunoreagent, the cytokine of for example above-mentioned immune regulator as discussing as useful adjuvant, the zone.The preferred version of the present embodiment comprises that the coding region of the coding region that makes analog and immunomodifier is in different reading frames or at least under different promoters control.Thereby avoid analog or epi-position to produce as fusion partner for immunomodifier.Alternatively, can use two different nucleotide fragments, but this is not preferred, helps guaranteeing that it carries out coexpression because have the coding region of the two in same molecule.

Therefore, the present invention also relates to a kind of compositions that produces anti-APP or A β antibody of inducing, said composition comprises

-nucleic acid fragment of the present invention or carrier (referring to the argumentation of following carrier) and

-medicinal as discussed above and immune acceptable vehicle and/or carrier and/or the adjuvant.

Under normal circumstances, the nucleic acid of coding variant imports with carrier format, wherein expresses under the control of viral promotors.About more going through of carrier of the present invention referring to following discussion.And can obtain the detailed description of nucleic acid vaccine preparation and application, referring to, Donnelly JJ etc., 1997, Annu.Rev.Immunol.15:617-648 and Donnelly JJ etc., 1997, LifeSciences 60:163-172.These reference papers all are hereby expressly incorporated by reference.

Live vaccine

The 3rd will as defined analog in variant a-c effectively be pass immune alternative be to use the live vaccine technology.In live body inoculation, present by a kind of non-pathogenic microorganism that has transformed the nucleic acid fragment of coding analog or integrated the carrier of this nucleic acid fragment is achieved to animals administer immune.This non-pathogenic microorganism can be any suitable bacterial isolates that weakens (by going down to posterity or removing pathogenic expression product by recombinant DNA technology and weakened), for example, cow mycobacteria BCG., non-pathogenic Streptococcus strain, escherichia coli, Salmonella strain, vibrio cholera, Shigella etc.About the preparation live vaccine state-of-art summary can referring to, as Saliou P, 1995, Rev.Prat.45:1492-1496 and Walker PD, 1992, Vaccine 10:977-990, the two all is hereby expressly incorporated by reference.

About the details of used nucleic acid fragment and carrier in this live vaccine, referring to following discussion.

Alternative as the bacteria in viable vaccine, nucleic acid fragment of the present invention discussed below can be incorporated into nontoxic viral vaccine carrier, for example in cowpox bacterial strain or any other the suitable poxvirus.

Usually, non-pathogenic microorganism or virus are only carried out disposable administration to animal, but under specific circumstances, have in life necessary more than this microorganism of single administration to keep protective immunity.Even the above detailed immunization protocol to peptide vaccination of expection also can be used in live vaccine or the viral vaccine.

Alternatively, live or the viral vaccine inoculation with before or after polypeptide and/or nucleic acid vaccination combine.For example, can produce elementary immunity, carry out enhance immunity with polypeptide or nucleic acid method subsequently with live vaccine or viral vaccine.

Microorganism or virus can be with the nucleic acid that comprises first, second and/or third part zone of coding, for example with above-mentioned immune regulator for example as the form of the cytokine of useful adjuvant, transform.The preferred version of the present embodiment comprises that the coding region of the coding region that makes analog and immunomodifier is in different reading frames or at least under different promoters control.Thereby avoid analog or epi-position as producing for the fusion partner of immunomodifier.Alternatively, can use two kinds of different nucleotide fragments as conversion reagent.Certainly, can be provided in same reading frame have first and/or second and/or third part as expression product, analog of the present invention, according to the present invention, this embodiment is particularly preferred.

The application of method of the present invention in disease treatment

As discussed abovely understand, it is the disease of feature with the amyloid beta deposition thing that method provided by the invention allows control.In this article, AD is the main target of the inventive method, but other are that the disease of feature also is feasible target with the A β that comprises the amyloid beta deposition thing.Therefore, the important embodiment that the present invention reduces the active method of amyloid comprises and treats and/or prevents and/or improve AD or other are the disease of feature with the amyloid beta deposition thing, the method according to this invention, this method comprise APP or A β are adjusted downward to the degree that the amount of amyloid significantly descends.

The decline that particularly preferably is amyloid causes the balance between amyloid formation and the amyloid degraded/removing to be inverted, that is, the speed of amyloid degraded/removing surpasses the speed that amyloid forms.To quantity and the immune effect that needs individual immunity arranged, might obtain a kind of balance along with past time by careful control, it causes the clean decline of amyloid beta deposition thing and does not have extra side effect.

Alternatively, if in body one by one, already present amyloid beta deposition thing can not be removed or reduce to method of the present invention, and method of the present invention can be used for reaching the remarkable reduction clinically that new amyloid forms, thereby the significant prolongation disease symptoms does not have the time of deterioration.Should be by measuring the serum-concentration of amyloid (thinking that it and sedimentary material reach balance), or by utilizing positron emission computerized tomography (positron-emission tomography) (PET) to scan, referring to Small GW, Deng, 1996, Ann N Y Acad Sci 802:70-78 monitors amyloid beta deposition speed.

These means and method can be used for treating in a similar manner or other disease and the symptom improved are mentioned in " background of invention ", or to list in following be the part of title with " other amyloid diseases and associated albumen ".

Peptide, polypeptide and the compositions of invention

As saying from foregoing is obvious, and the present invention is based on following notion, promptly individuality is carried out anti-APP or the immunity of A beta antigen, reduces with the quantity that reaches pathogenic relevant amyloid beta deposition thing.The optimal way that reaches this immunity is to use analog described herein, thereby does not have disclosed molecule before providing in this area.

Think that analog discussed herein self has invention, therefore a pith of the present invention is suitable for aforesaid analog.Therefore, the content about the APP that modifies or A β that set forth in any this place is relevant with the amyloid originality analog of the present invention's description, and any such content is changed the description that is suitable for these analog.

It should be noted that the APP of preferred modification or the modification that the A beta molecule comprises should make polypeptide and APP or A β or have at least the subsequence of 10 amino acid lengths to have at least 70% sequence homology with it.Higher sequence homology is preferred, for example, is at least 75% or even be at least 80,85,90 or 95%.The sequence homology of albumen and nucleic acid can pass through (N _Ref-N _Dif) * 100/N _RefCalculate, wherein N _DifBe the sum of the different residues in when comparison two sequences, wherein N _RefIt is residue number in the sequence.Therefore, DNA sequence AGTCAGTC and sequence A ATCAATC have 75% sequence homology (N _Dif=2 and N _Ref=8).

The present invention also is suitable for use in the compositions that detects the inventive method.Therefore, the present invention also relates to a kind of immune composition that comprises the above-mentioned analog of immune effective dose, described compositions further comprises medicinal and immune acceptable diluent and/or vehicle and/or carrier and/or excipient and the optional adjuvant gone up.In other words, this invention part relates to the preparation of analog, in essence as mentioned above.Modify and the preparation of the amyloid originality polypeptide of non-modification when being used for method of the present invention and reducing APP or A β when relating to, adjuvant, carrier and vectorial selection correspondingly meet above-mentionedly have been discussed.

Prepare polypeptide according to method well known in the art.Longer polypeptide is prepared by recombinant DNA technology usually, comprise that the nucleotide sequence with the coding analog imports suitable carriers, transform proper host cell with this carrier, by this nucleotide sequence of host cell expression, from host cell or its culture supernatant, reclaim expression product, and purification subsequently and randomly further modification, for example, folding again or derivatization.

Preferably short peptide prepares by solid phase or the liquid phase peptide synthetic technology of knowing.Yet this Progress in technique is so that polypeptide and albumen that may be by these method production total lengths recently, so it is also included within the scope of the present invention with by the long construct of synthetic method preparation.

Nucleic acid fragment of the present invention and carrier

Be appreciated that from above content the polyamino acid analog can prepare by recombinant DNA technology, also can be by chemosynthesis or semi-synthetic being prepared; When comprising, modification (for example is coupled to protein carrier, KLH, diphtheria toxoid, tetanus toxoid and BSA) and when non-protein molecular such as glycopolymers, and be included in when increasing side chain or side-chain radical on APP or the deutero-peptide chain of A β when modifying, back two kinds of selections are relevant especially.

For recombinant DNA technology, certainly also for nucleic acid immunization, the nucleic acid fragment of coding analog is very important chemical product.Therefore, a pith of the present invention be suitable for the encoding nucleic acid fragment of analog of the present invention, promptly, described analog is APP or A β polypeptides derived, it comprises the native sequences that is added or inserts fusion partner, perhaps preferably APP or A β polypeptides derived, and wherein it is by by inserting and/or increasing, preferably, introduced the external source t cell epitope by replacing and/or deletion.Nucleic acid fragment of the present invention is DNA or RNA fragment.

Nucleic acid fragment of the present invention is inserted into usually and forms clone or the expression vector that carries nucleic acid fragment of the present invention in the suitable carrier; This novel carriers also is a part of the present invention.Following about being described in detail in of the structure of these carriers of the present invention about discussing in transformant and the microorganism.According to application aims and type, carrier can be plasmid, phage, cosmid, mini-chromosome or viral form, and the naked DNA of transient expression also is a kind of important carrier in specific cells.Preferred clone of the present invention and expression vector can self-replicatings, therefore duplicate and can reach high copy number for high level expression or with the high level of rear clone.

The general profile of carrier of the present invention comprises following feature with 5 ' → 3 ' direction with being operatively connected: one is used to start nucleic acid fragment expression promoter of the present invention, randomly coding makes the polypeptide fragment secretion (to the cell foreign minister, or it is applicable, in pericentral siphon) or be incorporated into the nucleotide sequence of the guiding peptide in the film, the nucleotide sequence of the nucleic acid fragment of the present invention and the terminator of randomly encoding.When the expression vector in production bacterial strain or the cell line was operated, for the hereditary stability of transformant, preferably when importing to host cell, vector integration was on the host cell gene group.On the contrary, and when in animal, realizing expression in vivo with carrier (, when application vector in the DNA inoculation) because security reason, preferably this carrier can not be incorporated in the host cell gene group; Typically, use naked DNA or nonconformity viral vector, its selection is known the people who is familiar with this area.

Carrier of the present invention is used for transformed host cell to produce analog of the present invention.This also is that the transformant of a part of the present invention can be to be used to breed nucleic acid fragment of the present invention and carrier, or is used for the cultured cell or the cell line of recombinant production analog of the present invention.Alternatively, cell transformed can be suitable live vaccine strain, wherein has been inserted into nucleic acid fragment (single or a plurality of copies) to realize the analog secretion or to be incorporated in bacterial cell membrane or the cell wall.

The preferred transformant of the present invention is microorganism, for example, antibacterial (for example, Escherichia strain [for example escherichia coli], bacillus [for example bacillus subtilis], Salmonella or Mycobacterium [preferably non-pathogenic, for example Mycobacterium bovis BCG]), yeast (for example saccharomyces cerevisiae) and protozoacide.Alternatively, transformant derives from multicellular organism, for example fungus, insect cell, plant cell or mammalian cell.Most preferably derive from people's cell, referring to the discussion of following cell line and carrier.Nearest result has shown that use can the commercial Drosophila melanogaster cell line that obtains (Schneider 2 (S that can obtain from Invitrogen ₂) cell line and carrier system) have good prospect at the applicant's laboratory recombinant production polypeptide, therefore, this expression system is particularly preferred.

In order to clone and/or optimization expression, preferred cell transformed can be duplicated nucleic acid fragment of the present invention.The segmental cell of express nucleic acid is the preferred useful embodiment of the present invention; They can be used on a small scale or mass preparation analog of the present invention, or under the non-pathogenic bacteria situation, as the vaccine component of live vaccine.

When producing analog of the present invention by transformant, expression product is transported in the culture medium or is presented in the transformant surface is very easily, is necessary but be far from.

When identifying one when effectively producing cell, preferably based on this, set up one and carry carrier of the present invention, and express the stable cell lines of the nucleic acid fragment of the amyloid originality polypeptide that coding modifies.Preferably, this stable cell line is secreted or is carried analog of the present invention, thereby is beneficial to its purification.

In general, derive from the species of host cell compatibility and comprise replicon and the plasmid vector of regulating and controlling sequence and host unite use.This carrier has replication site usually, and the labelled sequence that phenotypic screen can be provided in transformant.For example, escherichia coli are typically used pBR322, a kind of derive from the escherichia coli kind (referring to, Bolivar etc. for example, 1977) plasmid transform.The pBR322 plasmid comprises ampicillin and tetracycline resistance gene, thereby provides the mode of being easy to come identification of transformed cell.PBR plasmid or other microorganism plasmids or phage also must comprise, or modified and comprise and can be used for the prokaryotic micro-organisms expression promoter.

Those promoteres that are most commonly used to recombinant DNA construction comprise beta-lactamase (penicillinase) and lactose promoter systems (Chang etc., 1978; Itakura etc., 1977; Goeddel etc., 1979) and tryptophan (trp) promoter systems (Goeddel etc., 1979; EP-A-0 036 776).Though these promoteres are the most frequently used, but other microorganism promoteres are found and use, details about its nucleotide sequence are reported, are connected to (Siebwenlist etc., 1980) on the plasmid vector functionally thereby those of skill in the art are had them.Derive from procaryotic some gene and can be in escherichia coli carry out effective expression, thereby get rid of the needs that increase another promoter by manual type from himself promoter sequence.

Except prokaryote, also can use eukaryotic microorganisms, yeast culture for example, promoter should be able to start expression herein.Though can obtain a large amount of other bacterial strains usually, saccharomyces cerevisiae or common be the most frequently used in the eukaryotic microorganisms with the bag yeast.For example, in saccharomyces cerevisiae was expressed, plasmid YRp7 was (Stinchcomb etc., 1979 of using always; Kingsman etc., 1979; Tschemper etc., 1980).This plasmid has comprised the trp1 gene, and it is for lacking the yeast mutant of energy for growth in tryptophan, and for example ATCC No.44076 or PEP4-1 (Jones, 1977) provide a selected marker.By under shortage tryptophan condition, growing, provide the effective environment of a detection conversion subsequently as the trp1 damage of the genomic feature of yeast host.

Suitable initiating sequence comprises glycerol 3-phosphate acid kinase (Hitzman etc., 1980) or other glycolysis enzyme (Hess etc., 1968 in the yeast vector; Holland etc., 1978), the promoter of Enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, G-6-P isomerase, 3-phoshoglyceric acid mutase, pyruvate kinase, triose-phosphate isomerase, glucose phosphate isomerase and glucokinase for example.In making up suitable expression plasmid process, also be connected to polyadenylation and the termination of 3 ' end so that mRNA to be provided of required expressed sequence in the expression vector with the terminator sequence of these gene-correlations.

Other promoteres with added benefit of transcribing of growth conditions regulation and control are, the promoter region of the enzyme of alcohol dehydrogenase 2, different cytochrome reductase C, acid phosphatase, the digestive enzyme relevant with nitrogen metabolism and above-mentioned glyceraldehyde-3-phosphate dehydrogenase and responsible maltose and galactose utilization.Any plasmid vector of promoter, replication origin and the terminator sequence of yeast compatibility that comprises is suitable for.

Except microorganism, the cell culture that derives from multicellular organism also can be used as the host.In principle, no matter from vertebrates or invertebrates culture, any this cell culture is available.But,, and become routine operation program (Tissue Culture, 1973) with the vertebrate propagation of form (tissue culture) of culture in recent years to the interest maximum of vertebrate cells.The example that this useful host cell is is VERO and Hela cell, Chinese hamster ovary (CHO) cell line and W138, BHK, COS-7 293, fall army worm (Spodopterafrugiperda) (SF) cell (as the expressed intact system and commercialization available from, changed (i.a.) Protein Sciences, 1000 Research Parkway, Meriden, CT 06450, U.S.A. and available from Invitrogen), and mdck cell system.In the present invention, particularly preferred cell line is from Invitrogen, PO Box 2312,9704 CH Groningen, the S that The Netherlands obtains ₂

The expression vector of this cell generally includes (if must) replication origin, is positioned at promoter and any necessary ribosome binding site, RNA splice site, polyadenylation site and the tanscription termination subsequence for the treatment of the expressing gene front.

For using in mammalian cell, the adjusting function on the expression vector is provided by viral material usually.For example, usually used promoter derives from polyoma virus, adenovirus 2, more often is simian virus 40 (SV40).Because the early stage and late promoter of SV40 virus is easy to obtain from virus as the fragment that also comprises SV40 virus replication starting point, so the two is useful especially (Fiers etc., 1978).As long as comprise beginning the sequence of extending about 250bp from Hind III site, also can use littler or bigger SV40 fragment to the Bgl I site that is positioned at the virus replication starting point.In addition, also possible, and normally want, promoter that utilization is normally relevant with required gene order or regulating and controlling sequence provide the regulating and controlling sequence with the host cell systems compatibility.

Origin of replication can provide to comprise the external source starting point by vector construction, for example can derive from SV40 or other virus (for example polyoma virus, adenovirus, VSV, BSV), perhaps provides by the host cell chromosome replicanism.If vector integration is in host cell chromosome, the latter is normally enough.

The evaluation of useful analog

Concerning those of skill in the art, know clearly to be not all possible variation of naturally occurring APP or A β or to modify the antibody all can in animal, cause with the native form cross reaction very much.But setting up an amyloid originality molecule to the modification that reaches immunoreactive minimum requirements described herein, to carry out effective standard screening not difficult.Therefore, may utilize a kind of method to identify in animal species, to induce the amyloid originality polypeptide of modification of the antibody of the amyloid originality polypeptide that produces anti-non-modification, wherein the amyloid originality polypeptide of non-modification is (non-immunogenicity) oneself protein, and this method comprises

-synthetic or gene engineering prepares one group of mutually different analog of the present invention by peptide, wherein aminoacid be added to, be inserted into, delete from or substitute onto in the aminoacid sequence of the APP of animal species or A β, thereby producing one group, to comprise animal species be the aminoacid sequence of the t cell epitope of external source, or prepare the nucleic acid fragment of one group of mutually different analog of a group coding

-detect this group analog or nucleic acid fragment the member by animal species induce the ability that produces the APP that resists non-modification or A β antibody and

-evaluation and optional separated are significantly induced the APP of anti-non-modification or the member of this group analog that A β antibody produces in species, perhaps identify and optional separated by member's coding of this group nucleic acid fragment, in animal species, significantly induce the APP of anti-non-modification or the expression of polypeptides product of A β antibody generation.

In this article, " the amyloid originality polypeptide group of mutually different modification " is the set of analog inequality, for example, it can standard according to the above discussion select (for example, the research of analyzing in conjunction with circular dichroism, NMR spectroscopy and/or X-ray diffracting spectrum).This is organized only by the minority member composition, but can expect that this group may comprise a hundreds of member.

Detection to member in the group is finally carried out in vivo, but can use many in vitro testses of dwindling the decorating molecule quantity that is suitable for the object of the invention.

Because introducing the purpose of external source t cell epitope is to support the B cell response by assisting of T cell, so prerequisite is to be bred by the analog inducing T cell.Can detect the T cell proliferation by the in-vitro multiplication of standard.In brief, from the experimenter, obtain to be rich in the sample of T cell and maintenance cultivation subsequently.The T cell of cultivating is contacted with experimenter's APCs, and this APCs has absorbed the molecule of modifying in advance, and handles to present its t cell epitope.The propagation of monitoring T cell, and compare with suitable contrast (for example, the T cell in the culture medium contacts with the APCs of the intact native starch sample albumen originality polypeptide with processing).Alternatively, the concentration of the relevant cell factor that can discharge by the identification of measuring their external source T cell of t cell response is measured propagation.

Given the probability of height, at least one analog that is every type of group can be induced the antibody that produces anti-APP or A β, may prepare a kind of immune composition, it comprises the analog of the antibody of at least a APP that can induce anti-non-modification in animal species or A β, wherein the APP of non-modification or A β are oneself proteins, this method comprises and will significantly induce acceptable carrier and/or vehicle and/or diluent and/or mixed with excipients in the member that produces with the antibody of APP or A beta response and medicinal and the immunity in the group in animal species, randomly in conjunction with at least a medicinal and immune acceptable adjuvant.

The detection of aforementioned polypeptides group can be finished by the following method easily, at first prepare a large amount of mutually different nucleotide sequences of the present invention or carrier, these are inserted in the suitable expression vector, transform proper host cell (or host animal) with carrier, and realize the expression of nucleotide sequence of the present invention.After these steps, carry out the separation of expression product.Preferably, nucleotide sequence and/or carrier be with comprising the molecular cloning technology of carrying out, for example PCR or be prepared by the synthetic method of nucleic acid.

Specific starch sample albumen originality target

Except the most normal albumen relevant with alzheimer's disease, outside APP, ApoE4 and the Tau, other by directly being present in the AD brain scab or tangle in or by danger increase obvious genetic affinity is arranged with development AD, and relevant with AD in some way albumen has very long list.These antigenic major parts if not all, are the target proteins of supposing in the particular of the present invention with above-mentioned A β, APP, presenilin and ApoE4.The target protein of these supposition goes through in WO01/62284.Therefore, the target of these supposition is only briefly mentioned herein, and more detailed background discussion can be found in WO 01/62282, and this article is hereby expressly incorporated by reference:

α 1-chymotrypsin inhibitor (ACT); Alpha2-macroglobulin; ABAD (in conjunction with the alcoholdehydrogenase of A β peptide); APLP1 and-2 (amyloid protein precursor sample albumen 1 and-2); AMY117; Bax; Bcl-2; The bleomycin hydrolytic enzyme; BRI/ABRI; Chromogranin A; Clusterin/ApoJ; CRF (corticotropin-releasing factor) is conjugated protein; EDTF (the toxin factor of endothelium derivation); The heparinoid S-PG; The human brain albumen that declines is replied mediation albumen-2; Huntingtin (Huntington Chorea protein); ICAM-I; IL-6; Lysosome related antigen CD68; P21 ras; PLC-δ 1 (phospholipase C isozyme δ 1); Serum amyloid P component (SAP); Synaptobrevin; Synuclein (α-synuclein or NACP); And TGF-b1 (transforming growth factor b1).

Previously described mode that is used to reduce APP or A β and method can with treatment for example, active specific active immunotherapy is in conjunction with other amyloid originality polypeptide of antagonism any of these.

Except alzheimer's disease, brain amyloid blood vessel disease also is a kind of disease of suitable target of current disclosed technology.

The immunization method that should be taken into account most anti-APP or A β is restricted to the immunity of generation to the antibody of natural A PP or A β cross reaction.Yet, in some cases, the cellular immunization of inducing CTL to reply form resists the cell of offering from the MHC I class epi-position of amyloid originality polypeptide, being interesting--this is favourable in following situation, and the reduction of promptly producing the cell quantity of APP or A β does not cause serious adverse.Reply under the situation at needs CTL, preferably utilize the instruction among the applicant WO00/20027.Therefore the content of these two documents is hereby expressly incorporated by reference.

Immunogenic carrier

As mentioned above, can obtain the APP or the A β peptide, covalently bound to polymer that comprise t helper cell epi-position and representative or comprise the B cell epitope as vectorial non-immunogenicity, on for example polyvalent activatory polyhydroxylated polymer, the molecule that will play a role as the vaccine molecule that only comprises immune relevant portion, and be interested embodiment among above-mentioned disclosed variant d and the e.For example, if the target of vaccine is an autoantigen, for example APP or A β can use miscellaneous or so-called general t helper cell epi-position.In addition, the composition that enhance immunity is replied also can be coupled on the vehicle altogether, thereby as adjuvant.These compositions can be mannose, tuftsin, muramyldipeptide, CpG motif etc.In this case, needn't carry out the adjuvant preparation of vaccine product subsequently, product can be with pure water or brinish form administration.

By with t helper cell epi-position cytotoxic T cell (CTL) epi-position, also may produce and be specific to the antigenic CTL that produces the CTL epi-position.The APC for example promotion product of macrophage absorbs material in the cytosol, and for example mannose also can be coupled on the vehicle altogether with CTL and t helper cell epi-position, and enhanced CT L is replied.

B cell epitope and t helper cell epi-position (P2 and the P30) ratio in end-product changes according to the change of these peptide concentrations in the synthesis step.As mentioned above, immunogenic molecules can be by adding following material in synthesis step in carbonate buffer solution, for example mannose, tuftsin, CpG motif or other immunostimulation materials (described herein) and be labeled, if necessary, use the aminated derivant of these materials to carry out labelling.

As mentioned above, if be connected with the peptide that comprises APP or A β B cell epitope and t helper cell epi-position with insoluble activatory polyhydroxylated polymer, then can be undertaken by solid phase synthesis, the results end-product also passes through washing and filters purification.Can be under low pH with the link coupled composition of the activatory polyhydroxylated polymer of trifluoroethyl sulfonyl (peptide, label etc.), for example pH4-5 joins in the polyhydroxylated polymer, thereby by evenly issuing in passive being diffused in " gel ".Subsequently, rising pH is to pH 9-10, starts the trifluoroethyl sulfonyl radical reaction in the primary amino radical group and polyhydroxylated polymer on peptide and the label.When peptide with after as the coupling of immunostimulation material, gel is ground, form the granule that size is suitable for immunity.

Therefore this immunogen comprises

A) at least one derives from first aminoacid sequence of APP or A β, wherein at least one first aminoacid sequence comprise at least one B cell and/or at least one CTL epi-position and

B) at least one comprises second aminoacid sequence of external source t helper cell epi-position, wherein at least the first and each even-coupling of at least the second aminoacid sequence to medicinal activatory polyhydroxylated polymer carrier.

For aminoacid sequence is coupled on the polyhydroxylated polymer, what the needs use was suitable usually can form the necessary reactive group that is connected " activation " polyhydroxylated polymer with aminoacid sequence.

Term " polyhydroxylated polymer " intention has the implication identical with WO 00/05316, that is, polyhydroxylated polymer has in this application the same characteristic features of instruction in detail really.Therefore, polyhydroxylated polymer can be water miscible or water-insoluble (thereby needing different synthesis steps when the preparation immunogen).Polyhydroxylated polymer can be selected from naturally occurring polyol and synthetic polyol.

Specific and preferred polyhydroxylated polymer is a polysaccharide, be selected from acetan, amylopectin, gum agar-agar, agarose, alginate, Radix Acaciae senegalis, carrageenan (carregeenan), cellulose, cyclodextrin, glucosan, danish agar, galactomannan, gelatin, ghatti, glucosan, glycogen, guar gum, thorn Firmiana platanifolia (Linn. f.) Marsili, konjac/A, locust bean gum, mannan, pectin, Herba Plantaginis, Aureobasidium pullulans polysaccharide, starch, tamarine, Tragacanth, xanthan gum, xylan and xyloglucan, glucosan is particularly preferred.

Yet polyhydroxylated polymer also can be selected from hyperbranched polymine (PEI), tetrathienylene vinylene, fiber B (long-chain of poly-paraphenyl terephtalamide), polyurethane, polysiloxanes, polydimethylsiloxane, siloxanes, polymethyl methacrylate (PMMA), polyvinyl alcohol, polyvinyl pyrrolidone, poly-(2-hydroxymethyl ethyl acrylate), poly-(N-vinyl pyrrolidone), polyvinyl alcohol, polyacrylic acid, polytetrafluoroethylene (PTFE), polyacrylamide, polyethylene-altogether-vinylacetate, Polyethylene Glycol and derivant, polymethylacrylic acid, polyactide (PLA), poly-Acetic acid, hydroxy-, bimol. cyclic ester (PGA), poly-(Acetic acid, hydroxy-, bimol. cyclic ester-altogether-lactide) (PLGA), polyanhydride and poe.

(weight) average molecular weight of described polyhydroxylated polymer (i.e. activation before) typically at least 1,000, for example at least 2,000, preferably 2,500-2,000,000 scope, more preferably 3,000-1,000,000 scope, particularly 5,000-500,000 scope.Show mean molecule quantity in an embodiment 10,000-200, the polyhydroxylated polymer of 000 scope is particularly advantageous.

Preferably, polyhydroxylated polymer water solublity degree at room temperature is at least 10mg/ml, preferably is at least 25mg/ml, for example is at least 50mg/ml, particularly is at least 100mg/ml, for example is at least 150mg/ml.Known glucosan even as herein the activation after, still can reach water miscible requirement.

To some most interested polyhydroxylated polymers, non-activated polyhydroxylated polymer (promptly, natural polyhydroxy polymer before the activation) ratio between C (carbon atom) and the OH group (hydroxyl) is in the scope of 1.3-2.5,1.5-2.3 for example, be preferably 1.6-2.1, particularly in the scope of 1.85-2.05.Not limited by any particular theory, think the favourable level of this C/OH proportional representation hydrophilic height of non-activated polyhydroxylated polymer.Polyvinyl alcohol and polysaccharide are the examples that reaches the polyhydroxylated polymer of this requirement.Think that aforementioned proportion approximately is the same concerning activatory polyhydroxylated polymer, because the activation ratio should be quite low.

The immunogen part that term " polyhydroxylated polymer carrier " refers to carry aminoacid sequence.As universal law, the polyhydroxylated polymer carrier has its outside limits, and aminoacid sequence wherein can for example be processed the peptidase cutting in the immunogenic antigen-presenting cell by peptidase.Therefore, the polyhydroxylated polymer carrier can be the polyhydroxylated polymer with activated group, wherein the key between activated group and the aminoacid sequence can be cut by the peptidase among the APC, perhaps the polyhydroxylated polymer carrier can be to have activated group and for example junctional complex, as single L-aminoacid or the amino acid whose polyhydroxylated polymer of a plurality of D-, wherein the decline of junctional complex can be connected with aminoacid sequence, and is cut by the peptidase among the APC.

As mentioned above, polyhydroxylated polymer has functional group's (activated group), and it promotes peptide is anchored on the carrier.Known many applicable functional groups in this area; for example, tresyl (trifluoroethyl sulfonyl), dimaleoyl imino, p-nitrophenyl cloroformate, Bromine cyanide., tosyl (ptoluene-sulfonyl), triflyl (trifyl), phenyl-pentafluoride sulfonyl and vinyl sulfone group.The preferred embodiment of functional group is trifluoroethyl sulfonyl, dimaleoyl imino, tosyl, trifyl, phenyl-pentafluoride sulfonyl, p-nitrophenyl cloroformate and vinyl sulfone group among the present invention, and trifluoroethyl sulfonyl, dimaleoyl imino and tosyl are relevant especially in them.

As described in the activated dextran among WO 00/05316 embodiment 1 or as Gregorius etc., J.Immunol.Meth.181 (1995) 65-73 is described, the activatory polyhydroxylated polymer of trifluoroethyl sulfonyl can prepare with trifluoroethyl sulfonyl chloride.

The activatory polyhydroxylated polymer of maleimide can be prepared with activated dextran described in right-dimaleoyl imino phenyl isocyanate such as WO 00/05316 embodiment 3.Alternatively, by (being generally H with diamine compound ₂N-C _nH _2n-NH ₂Wherein n is 1-20; be preferably 1-8); for example 1; the 3-diaminopropanes; the activatory polyhydroxylated polymer of trifluoroethyl sulfonyl (for example activatory glucosan of trifluoroethyl sulfonyl (TAD)) of excessively deriving; maleimide base group can be incorporated into polyhydroxylated polymer for example in the glucosan; use reagent subsequently, for example succinimido 4-(N-maleimide ylmethyl) cyclohexane extraction-1-carboxylate (SMCC); sulfo group-succinimido 4-(N-maleimide ylmethyl)-cyclohexane extraction-1-carboxylate (sulfo group-SMCC); sulfo group-succinimido 4-(right-the dimaleoyl imino phenyl) butyrate (SMPB); sulfonic group-succinimido 4-(right-the dimaleoyl imino phenyl) butyrate (sulfo group-SMPB); N-γ-dimaleoyl imino butyryl acyloxy-succinimide ester (GMBS) or N-γ-dimaleoyl imino butyryl acyloxy-sulfosuccinimide ester reacts with the amino that is incorporated among the TAD.Though activatory different reagent and approach can cause the connection between the residue of the functional and parent hydroxy group that carried out of activation of the maleimide of maleimide activation products slightly different, regard " the activatory polyhydroxylated polymer of maleimide " all as.

The activatory polyhydroxylated polymer of tosyl can prepare with the tosyl chloride as described in activated dextran among WO 00/05316 embodiment 2.The activatory analog preparation of trifyl and the activatory polyhydroxylated polymer of phenyl-pentafluoride sulfonyl such as tosyl or trifluoroethyl sulfonyl for example, is used corresponding acid chloride.

The polyhydroxylated polymer of cyanogen bromide-activated can prepare polyhydroxylated polymer and cyanogen bromide reaction by utilizing conventional method.The functional group that obtains normally has the cyanate of the hydroxyl of two polyhydroxylated polymers.

Activation degree can be expressed as the ratio between free hydroxyl and the activated group (that is, functionalized hydroxyl).Think that the free hydroxyl of polyhydroxylated polymer and the ratio between activated group should be between 250: 1 and 4: 1, between the hydrophilic of polyhydroxylated polymer and reactivity, to reach favourable balance.Preferably ratio is between 100: 1 and 6: 1, more preferably is between 60: 1 and 8: 1, particularly between 40: 1 and 10: 1.

According to the present invention; the interested especially activation polyhydroxylated polymer that is used for producing the immunogenic method of common application is the activatory polysaccharide of trifluoroethyl sulfonyl, tosyl and dimaleoyl imino, particularly the activatory glucosan of trifluoroethyl sulfonyl (TAD), the activatory glucosan of tosyl (TosAD) and the activatory glucosan of dimaleoyl imino (MAD).

Preferably, the key between polyhydroxylated polymer carrier and the aminoacid sequence that connected can be cut by peptidase, for example, has active peptidase in APC in the process antigen process.Therefore preferably, at least the first and at least the second aminoacid sequence be coupled on the activatory polyhydroxylated polymer by amido link or peptide bond.Particularly preferably be at least the first and at least the second aminoacid sequence each all supply with the nitrogen part of their amido link separately.

The polyhydroxylated polymer carrier can fully be broken away from amino acid residue, force activated group that peptidase cutting key part is provided, but as mentioned above, carrier also can only comprise a spacer, and described spacer comprises at least one L-aminoacid.Yet, at least the first and at least the second aminoacid sequence usually the nitrogen of the N-end by aminoacid sequence be connected on the activity form of polyhydroxylated polymer.

The immunogen that the invention described above is used usually can used basically as in the immunization method of polypeptide vaccine described herein.That is to say that the description that discussed herein all relate to dosage, administering mode and are used to reduce the polypeptide vaccine preparation of amyloid originality polypeptide is changed is suitable for normally used immunogen.

The conventional safe inoculation technique of using

As mentioned above, the T that can not provide from the immunne response of the caused anti-amyloid originality polypeptide in source is provided a preferred embodiment of the present invention _HThe variant of the amyloid originality polypeptide of epi-position.

Yet the inventor thinks that anti-autovaccine of design and effectively anti-autoimmune strategy are general available technology, and itself has invention.Should prove to be particularly suitable for following situation that promptly its autoantigen of attempting to reduce is abundant in vivo, to such an extent as to may produce the autostimulation of immunne response.Therefore, the anti-autoimmune response that anti-APP or A β are provided that the content of all above the present embodiment scopes relates to is changed the immunity applicable to anti-other self polypeptide, particularly those exist sufficient amount keeping the immunne response of uncontrolled autoimmune disease form, because the T of relevant self polypeptide _HEpi-position causes immunne response.

Embodiment 1

The automatic vaccination method of anti-AD immunity

The a knock-out mice does not show any fact unusual or disadvantageous side effect and shows that the removing of A β or the reduction of quantity are safe, Zheng H. (1996).

That has delivered carries out the immunization experiment prompting of anti-transgenic human a about transgenic animal, if possible destroys self tolerance, then may realize the downward modulation of A β by the antibody of reaction automatically.Further prompting of these experiments, the downward modulation of this A β will block simultaneously potentially scab formation and even from brain, remove established A β scab, referring to (1999) such as Schenk.But can not increase traditionally, the antibody of anti-oneself protein.

Therefore the data of having delivered can not provide the method for blocking-up at the self tolerance of real oneself protein.These data can not provide about how guaranteeing immunoreation only directly or be primarily aimed at A β deposit, and not at the bonded A β of cell membrane precursor protein (APP), are necessary if this point be it seems.May produce immunne response with a kind of non-prosecutor formula with the supposition of immunne response that prior art produced, thereby can produce unnecessary and extra automatic reaction at a part at oneself protein.Therefore, using existing immunization strategy maximum possible is the strong immune response that can not produce at oneself protein, in addition, because the potential strong cross reaction of membrane-bound APP to existing on a large amount of cells among the CNS, so also be unsafe.

The invention provides the method that effectively produces the immunne response of strong regulation and control at real oneself protein, described oneself protein may form scab potentially and cause serious disease at CNS or other zones of body.Go out a kind of safe and effective people's a treatment vaccine by this technological development, be used for the treatment of AD.

In view of the above, may expect AD, a kind of expectation will slacken the disease of healthcare network in the next century, can be cured, or this described vaccine can be set up effective Therapeutic Method of this disease symptoms of treatment and development at least.This technology is set forth the immunization method of amyloid beta deposition in a novel fully blocking-up AD and other sacred diseases.

In following table, shown the construct of 35 expections.The position that provides in all tables is the initial methionine (first aminoacid among the SEQ ID NO:2) with respect to APP, and comprises that initial sum stops aminoacid, and for example the 672-714 fragment comprises aminoacid 672 and 714.The replacement of position epi-position shown in the initial sum final position of P2 and P30 is illustrated in an APP fragment part (two positions include in replacement)-in most of constructs, the epi-position of introducing replaces the fragment of epi-position length.Asterisk in the table has following implication:

^*) only have a P2 and P30 position show epi-position shown in the position be inserted into (epi-position begins at the amino acid whose C-terminal that closes on given position) in the APP derivant.

^*) construct 34 comprises three identical APP fragments that separated by P30 and P2 respectively.

^{* *}) construct 35 comprises nine identical APP fragments that separated by alternative P30 and P2 epi-position.

The most interested APP that its generation is replied partly is 43 amino acid whose A β core peptides (A β-43 is corresponding to residue 672-714 among the SEQ ID NO:2), and it is the key component of amyloid plaque in the AD brain.This APP fragment is the part of all constructs listed above.

Variant

1 and 2 comprises the APP upstream portion of A β-43, has placed pattern epi-position (modelepitopes) P2 and P30 herein.Variant 1 and 3-8 all comprise the C-100 fragment, have shown that it is that neurovirulent-C-100 fragment is corresponding to amino acid residue 714-770 among the SEQ ID NO:2.In variant 3-5, epi-position replaces the segmental part of C-100, and has inserted C-100 in variant 6-8.

Variant 9-35 only comprises core A β-43 albumen.Among the variant 9-13, P2 and P30 merge any end at A β-43; In 14-21, P2 and P30 replace the part of A β-43; In 22-33, P2 and P30 are inserted among the A β-43; 34 comprise three respectively by P30 and P2 identical A β-43 fragment at interval; 35 comprise 9 by alternative P2 and A β-43 repetition at interval of P30 epi-position.

According to the present invention, the proteic truncate part of above-mentioned A β-43 also is applied in the immunogenic analog.Particularly preferably be truncate A β (1-42), A β (1-40), A β (1-39), A β (1-35), A β (1-34), A β (1-34), A β (1-28), A β (1-12), A β (1-5), A β (13-28), A β (13-35), A β (17-28), A β (25-35), A β (35-40), A β (36-42) and A β (35-42) (numeral in its bracket constitutes the aminoacid sequence of the A β-43 of associated clip, one for example A β (35-42) is identical with aminoacid 706-711 among the SEQ ID NO:2).All these have the variant of A β-43 truncate part, available A β produced in fragments described herein, and particularly

variant

9,10,11,12 and 13.

In some cases, preferred A β-43 or its fragment are suddenlyd change.Particularly preferably be the replacement variant, wherein 35 of A β-43 methionine is substituted, preferably replaced by leucine or isoleucine, or simply deleted.Particularly preferably be, because at amyloid originality polypeptide or external source T _HNatural existence in the epi-position perhaps because it is inserted into or adds, and only comprises an analog at the single methionine of C-end.Therefore, except a methionine that may be positioned at the C-end, also preferably include external source T _HThe analog of epi-position does not partly have methionine.

In fact, APP preferred used according to the invention usually or all analog of A β all have following feature, promptly only comprise a single methionine as C-end amino acid in the analog, and other are at amyloid originality polypeptide or external source T _HMethionine in the epi-position is deleted or by another kind of aminoacid replacement.

More interested sudden change is that 19 phenylalanine is deleted or replace among the A β-43, and particularly preferred sudden change is to replace this phenylalanine with proline.

List the truncate of the one group of particularly preferred A of using β-43 or the construct of mutation construction in the following table:

Variant number	The A β fragment of in molecule, using with respect to A β (1-42/43) aa1	A β fragment position with respect to the aa1 of molecule	Position with respect to the P2 epi-position of the aa1 of molecule	Position with respect to the P30 epi-position of the aa1 of molecule	The length overall of molecule (aa)
Variant number		A β fragment position with respect to the aa1 of molecule			The length overall of molecule (aa)	36	1-28	22-49	50-64	1-21	64
37	1-12(a)+13-28(b)	1-12(a)+49-64(b)	34-48	13-33	64	36	1-28	22-49	50-64	1-21	64
37	1-12(a)+13-28(b)	1-12(a)+49-64(b)	34-48	13-33	64	38	1-12(x3)	1-12，34-45，61-72	46-60	13-33	72
39	13-28(x3)	1-16，38-53，69-84	54-68	17-37	84	38	1-12(x3)	1-12，34-45，61-72	46-60	13-33	72
39	13-28(x3)	1-16，38-53，69-84	54-68	17-37	84	40	1-12(a)+13-35(b) +36-42(c)	1-12(a)+34-56(b)+ 72-78(c)	57-71	13-33	78
41	1-28(x3)	1-28，50-77，93-120	78-92	29-49	120	40	1-12(a)+13-35(b) +36-42(c)	1-12(a)+34-56(b)+ 72-78(c)	57-71	13-33	78
41	1-28(x3)	1-28，50-77，93-120	78-92	29-49	120	42	1-43(F19P/M35K)	1-43	65-79	44-64	79

In this table, used A β fragment represents that by the amino acid no with respect to the aa1 of A β (1-42/43) molecule promptly, 1-28 is illustrated in the fragment 1-28 that uses A β (1-42/43) in the molecule in the molecule.If use 2 or a plurality of different fragments, the two all is illustrated in the table, that is, 1-12 (a)+13-28 (b) is illustrated in fragment 1-12 and the fragment 13-28 that uses A β (1-42/43) in the molecule.

And if existence is more than the same clip of 1 copy in construct, it points out that in table promptly, there are 3 copies in the fragment 1-12 of 1-12 (x3) expression A β (1-42/43) in construct.

In addition, the segmental position of A β is by representing with respect to first amino acid whose amino acid position of molecule in the molecule, that is, 22-49 represents that described A β fragment is positioned at from aminoacid 22 to aminoacid 49 position in molecule, comprise the position of the two.Shown in the position of P2 and P30 epi-position is same.If use 2 or a plurality of different A β fragment in molecule, their position all shows, that is, and and the position of 1-12 (a)+49-64 (b) expression fragment (a) aa 1-12 in molecule, and fragment (b) is in the position of aa 49-64.

And if existence is more than the same clip of 1 copy in molecule, the position of all these copies all shows, that is, 1-12,34-45,61-72 are illustrated in the A β fragment that has 3 copies in the molecule, respectively at position 1-12,34-45 and 61-72.

At last, the total length shown in each molecule comprises A β fragment and P2 and P30 epi-position.

Variant 42 comprises 2 aminoacid replacement, and 19 (phenylalanine is replaced by proline) in the position, 35 (methionine is replaced by lysine) in the position are as showing as shown in the segmental row of A β.

About introduce the details of external source t cell epitope at specific site, referring to Fig. 1 and above chart.

A kind of construct of other type is particularly preferred.Because a target of the present invention is to avoid destroying the cell that produces APP, and needs to remove A β, the autovaccine construct that therefore prepares the A beat portion that is not exposed to the cell foreign minister when only comprising in being present in APP is seemingly feasible.Therefore, this construct need comprise the B cell epitope that at least one derives from the determined amino acid fragment of aminoacid 700-714 among the SEQ ID NO:2.Because the polypeptide fragment of this weak point estimates to have only very weak immunogenicity, therefore preferred this autovaccine construct comprises the B cell epitope of several copies, for example, having the construct of structure shown in the formula I in detailed content of the present invention, referring to as mentioned above.In the form of formula I, the term amyloid _E1-amyloid _ExBe meant that x comprises the B cell epitope that derives from the aminoacid sequence of aminoacid 700-714 among the SEQ ID NO:2.The external source t helper cell epi-position that preferred alternatives is above-mentioned amyloid originality (many) peptides and selection is coupled on the polysaccharide carrier molecule through amido link--in this method, may carry out the multiple of epi-position of " weak " formed by aminoacid 700-714 among the SEQID NO:2 and present, and may select the optimal proportion of B cell and t cell epitope.

Embodiment 2

Protein immunization transgenic mice with A β of the present invention and modification

Make up the DNA of coding hA β 43+-34

HA β 43+-34 gene makes up by several steps.At first using primer ME#801 (SEQ IDNO:10) and ME#802 (SEQID NO:11), is that template produces the PCR fragment with primer ME#800 (SEQ ID NO:9).ME#800 coding has people A β-43 fragment of suitable codon of escherichia coli.ME#801 and 802 pairs of fragments increase suitable restriction site.

Purification PCR fragment digests with Nco I and Hind III, carries out purification once more, and is cloned in the pET28b+ coli expression carrier of Nco I-Hind III digestion and purification.The encoding wild type people A β-43 plasmid called after pAB1 that obtains.

In next step, t helper cell epi-position P2 joins the C-end of molecule.Primer ME#806 (SEQ ID NO:12) comprises the sequence of coding P2 epi-position, thereby obtains the fusant of P2 and A β-43 by the PCR reaction.

The clone is by with primer ME#178 (SEQ ID NO:8) and ME#806, and prepares the PCR fragment as template and carry out with pAB1.This fragment of purification digests with Nco I and Hind III, carries out purification once more, and is cloned in the pET28b+ carrier of Nco I-Hind III digestion and purification.The plasmid called after pAB2 that obtains.

In a similar fashion, prepare the plasmid that another has A β-43 coded sequence and another t helper cell epi-position P30 is increased to N-terminal.This is by being that template prepares the PCR fragment and carries out with primer ME#105 (SEQ ID NO:7) and ME#807 (SEQ ID NO:13) and with pAB1.

This fragment of purification digests with Nco I and Hind III, carries out purification once more, and is cloned in the pET28b+ carrier of Nco I-Hind III digestion and purification.The plasmid called after pAB3 that obtains.

In the 3rd step, second A β-43 repeats to join by primer ME#809 (SEQ ID NO:14) C-terminal of the P2 epi-position of plasmid pAB2.ME#809 follows closely after A β-43 repeats simultaneously, creates a BamHI site.The PCR fragment is that template is prepared with pAB2 with primer ME#178 and ME#809.This fragment digests with Nco I and Hind III, and purification also is cloned in the pET28b+ carrier of Nco I-Hind III digestion and purification.The plasmid called after pAB4 that obtains.

At last, be cloned in the pAB4 plasmid from P30 epi-position-A β-43 repetitive sequence of pAB3.This is by being that template prepares the PCR fragment and carries out with pAB3 with primer ME#811 (SEQ ID NO:16) and ME#105.This fragment is purified, and in PCR subsequently with ME#810 (SEQ ID NO:15) as primer, and be that template is carried out PCR with pAB3.The fragment that obtains is carried out purification, digests with BamHI and Hind III, and is cloned in the pAB4 plasmid of BamHI-Hind III digestion and purification.The plasmid pAB5 coding hAB43+-34 molecule that obtains.

All PCR and clone's program be basically as Sambrook, J., Fritsch, E.F.﹠amp; Maniatis, T.1989 " Molecular cloning:a laboratory manual ". the 2nd edition .ColdSpring Harbor Laboratory, N.Y. is described to carry out.

To all clone's programs, use be e. coli k-12 cell, bacterial strain Top-10F ' (Stratagene, the U.S.).The pET28b+ carrier is from Novagen, and the U.S. buys.All primers are at DNA Technology, and Denmark is synthetic.

The expression of hAB43+-34 and purification

As described in the supplier of pET28b+ system (Novagen), by the hAB43+-34 albumen of pAB5 coding at e. coli bl21-Gold Novagen) express in the cell.

The hAB43+-34 albumen of expressing is by the washing occlusion body and use BioCad purification work station subsequently (PerSeptive Biosystems USA) carries out cation-exchange chromatography and is purified to and surpasses 85% purity under the situation that has the 6M urea.Urea is dialysed step by step and is removed to comprise the urea solution that reduces quantity subsequently.Final buffer is 10mM Tris, and pH 8.5.

Immune Research

People APP (alzheimer's disease precursor protein) transgenic mice is used for research.These mices are called TgRND8+, express a kind of mutant form of APP, and it causes the generation (Janus, C etc.) of high concentration A β-40 and A β-42 in mouse brain.

Mice (every group of 8-10 mice) carries out immunity with A β-42 (SEQ ID NO:2, residue 673-714 is tactful synthetic by the Fmoc of standard) or hAB43+-34 variant (construct 34 in embodiment 1 table, reorganization produces), is total to immunity 4 times with 2 weekly intervals.Dosage is 100mg A β or 50mghAB43+-34.Mice carries out blood-letting in the 43rd day (three injection backs) and the 52nd day (four injection backs), measures the level of the special titre of anti-A β-42 by direct A β-42ELISA with serum.

Shown in the following table is on average to resist A β-42 titre mutually.

Obviously, the antibody titer that obtains after three and four immunity when carrying out immunity with hAB43+-34A β variant approximately is respectively 4 times and 7.5 times of the titre that obtains as antigen with unaltered wild type A β-42.When the amount of the variant of considering to be used for immunity only be used for immunity the wild-type sequence amount 50% true the time, this fact is correctly treated.

Embodiment 3

Synthesize A β peptide copolymer vaccine with activatory polyhydroxylated polymer as cross-linking agent

Brief introduction

Traditional conjugate vaccine comprises covalent coupling (many) peptides to the carrier protein.This peptide comprises the B cell epitope, and carrier protein provides the t helper cell epi-position.But the major part of carrier protein is normally irrelevant as the source of t helper cell epi-position, because only there is sub-fraction to comprise relevant t helper cell epi-position in the whole sequence.This epi-position is as peptide, and 12-15 amino acid whose peptide for example is defined and synthesizes.If these peptides are covalently bound to the peptide that comprises the B cell epitope,, can obtain only to comprise the vaccine molecule of relevant portion for example by the activatory polyhydroxylated polymer of multivalence.The protein-polysaccharide conjugate vaccines of optimized proportion between a kind of B of comprising cell and the t cell epitope further may be provided.

Synthesizing of activatory (acticated) polyhydroxylated polymer

Polyhydroxylated polymer; for example glucosan, starch, agarose etc.; available 2; 2; 2-trifluoroethyl sulfonyl chlorine (trifluoroethyl sulfonyl chlorine); perhaps by being dissolved in the homogeneity synthetic (glucosan) in the N-Methyl pyrrolidone (NMP), perhaps activate by heterogeneous the synthesizing in acetone for example (starch, agarose, crosslinked glucosan).

Under drying condition, replenishing the 500ml round-bottomed flask that Magnet is used for stirring, in cryodesiccated water-soluble glucan (4.5g, 83mmol, the clinical grade of using, mean molecule quantity 78000), add the exsiccant N-Methyl pyrrolidone of 225ml (NMP).Be placed on flask in the 60 oil baths and carry out magnetic agitation.Temperature was increased to 92 ℃ in 20 minutes.When glucosan dissolves, immediately flask is removed from oil bath, and oil bath temperature is reduced to 40 ℃.Flask (agaom) is again put into oil bath, still carries out magnetic agitation, and dropwise add trifluoroethyl sulfonyl chlorine (2.764ml, 25mmol).After 15 minutes, dropwise add exsiccant pyridine (anhydrous, 2.020ml, 25mmol).Flask is removed from oil bath, and at room temperature stirred 1 hour.Product (the activatory glucosan of trifluoroethyl sulfonyl, TAD) precipitation in the cold ethanol of 1200ml (99.9%).2000rpm carries out centrifugally in centrifuge, decants supernatant, collecting precipitation in the 50ml polypropylene tube.Resolution of precipitate is in 50ml 0.5% acetic acid, and dialysis is 2 times in 5000ml 0.5% acetic acid, and carries out lyophilization.TAD is stored in-20 ℃ with the lyophilization powder.

Insoluble polyhydroxylated polymer, for example agarose or crosslinked glucosan can be activated by the trifluoroethyl sulfonyl by the suspension of preparation polyhydroxylated polymer in acetone for example, and synthesize with solid phase synthesis technique.Activatory polyhydroxylated polymer can be collected by filtration.Suitable method exists, for example Nilsson K and Mosbach K (1987), Methods in Enzymology135,67 pages and at (1992) such as Hermansson GT, in " Immobilized Affinity LigandTechniques ", Academic Press, Inc., report in 87 pages.

Synthesizing of A β peptide copolymer vaccine

TAD (10mg) is dissolved in 100 μ l H ₂Among the O, and add 1000 μ l carbonate buffer solutions, among the pH 9.6, it contains 5mg A β-42 (SEQ ID NO:2, residue 673-714), 2.5mg P2 (SEQ ID NO:4) and 2.5mg P30 (SEQ ID NO:6).A β-42 and P2 and P30 peptide all comprise protected lysine group: they are lysine groups of 1-(4,4-dimethyl-2,6-dioxy hexamethylene-1-subunit) ethyl (Dde) protection form.By the Fmoc strategy preparation peptide of standard, wherein traditional Fmoc-Lys (Boc)-OH is replaced by Fmoc-Lys (Dde)-OH (available from Novabiochem, catalog number (Cat.No.) 04-12-1121), that is, the epsilon-amino in the lysine replaces Boc with Dde and protects.

Measure pH value and be adjusted to 9.6 with 1M HCl.After at room temperature 2.5 hours, adding 80% hydrazine solution to the final concentration of hydrazine is 8%, and solution is incubation 30 minutes more at room temperature, carries out lyophilization then immediately.Cryodesiccated product is dissolved in the water, and water is dialysed fully before final lyophilization.

The ratio of B cell epitope in the end-product (A β) and t helper cell epi-position (P2 and P30) can change by these peptides that use variable concentrations in synthesis step.In addition, end-product can carry out labelling, for example uses mannose (so that conjugate is navigated on the APCs), realizes by adding aminated mannose at synthesis step in carbonate buffer solution.

If use insoluble activatory polyhydroxylated polymer to connect the peptide that comprises B cell epitope and t helper cell epi-position, can be accomplished to the coupling of polymer with solid phase synthesis, the results end-product also passes through washing and filters purification.

As mentioned in general description, the method that being used for of current description prepares based on the vaccine of peptide can be applicable to any other polypeptide antigen, wherein preparing pure synthetic peptide vaccine is easily, and wherein said polypeptide antigen provides enough immunogenicities with a single peptide.

Embodiment 4

Synthesizing of peptide copolymer vaccine

TAD (10mg) is dissolved in 100 μ l H ₂Among the O, and add 1000 μ l carbonate buffer solutions, among the pH 9.6, contain 1-5mg peptide A (any interested immunogenic peptide that has! ), 1-5mg P2 (diphtheria toxoid P2 epi-position) and 1-5mg P30 (diphtheria toxoid P30 epi-position).Measure pH value and be adjusted to 9.6 with 0.1M HCl.After at room temperature 2.5 hours, solution is immediately carried out lyophilization.Cryodesiccated product is dissolved in the water, and before final lyophilization, water is fully dialysed, or carries out desalination on solvent resistant column.Have in peptide sequence under the situation of lysine, the epsilon-amino of lysine side-chain is used Fmoc-Lys (Dde)-OH derivant and is protected (Gregorius and Theisen 2001 are in the submission) by Dde in synthetic.After the coupling, adding 80% hydrazine solution to the final concentration of hydrazine is 1-20%, and solution is incubation 30 minutes more at room temperature, immediately carry out lyophilization, and before final lyophilization, water is dialysed fully, or carries out desalination on solvent resistant column.This principle is set forth in the sketch map of Fig. 2.

This para-immunity is former to be used by the inventor, with the terminal short-movie section of the C-of B. burgdorferi albumen OspC as " peptide A ", and with diphtheria toxoid (diptheria toxoid) epi-position (P2 or P30) as peptide B.Show to have only the OspC of comprising fragment of the present invention and in these mices, induce the antibody with the OspC reaction to produce with the immune proper energy of the external source diphtheria toxoid epi-position that is complementary with the MHC haplotype of inoculation mice with this antigenic immune Research result.On the contrary, a molecule that only comprises the OspC peptide can not induce antibody to produce, and to 2 kinds of immunogens, one of them comprises OspC, and another comprises epi-position, mixture be so equally.Therefore conclude that the Inclusion in identical polyhydroxylated polymer carrier is preferred, if not essential, to induce anti-small peptide hapten, OspC for example, antibody produce.

List of references

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Sequence table

＜110〉Pharmexa AS

＜120〉new method of downward modulation amyloid

<130>P1014PC1

<140>

<141>

<160>16

<170>PatentIn Ver.3.1

<210>1

<211>2313

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)..(2313)

<220>

<221>misc_feature

<222>(2098)..(2169)

＜223〉coding is striden the nucleotide in film district

<220>

<221>misc_feature

<222>(2014)..(2313)

＜223〉nucleotide of coding C-100

<220>

<221>misc_feature

<222>(2016)..(2144)

<223>(42/43

<220>

<221>misc_feature

<222>(2014)..(2142)

<223>(42/43

<400>1

atg ctg ccc ggt ttg gca ctg ctc ctg ctg gcc gcc tgg acg gct cgg 48

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg

1 5 10 15

gcg ctg gag gta ccc act gat ggt aat gct ggc ctg ctg gct gaa ccc 96

Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro

20 25 30

cag att gcc atg ttc tgt ggc aga ctg aac atg cac atg aat gtc cag 144

Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln

35 40 45

aat ggg aag tgg gat tca gat cca tca ggg acc aaa acc tgc att gat 192

Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp

50 55 60

acc aag gaa ggc atc ctg cag tat tgc caa gaa gtc tac cct gaa ctg 240

Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu

65 70 75 80

cag atc acc aat gtg gta gaa gcc aac caa cca gtg acc atc cag aac 288

Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn

85 90 95

tgg tgc aag cgg ggc cgc aag cag tgc aag acc cat ccc cac ttt gtg 336

Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val

100 105 110

att ccc tac cgc tgc tta gtt ggt gag ttt gta agt gat gcc ctt ctc 384

Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu

115 120 125

gtt cct gac aag tgc aaa ttc tta cac cag gag agg atg gat gtt tgc 432

Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys

130 135 140

gaa act cat ctt cac tgg cac acc gtc gcc aaa gag aca tgc agt gag 480

Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu

145 150 155 160

aag agt acc aac ttg cat gac tac ggc atg ttg ctg ccc tgc gga att 528

Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile

165 170 175

gac aag ttc cga ggg gta gag ttt gtg tgt tgc cca ctg gct gaa gaa 576

Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu

180 185 190

agt gac aat gtg gat tct gct gat gcg gag gag gat gac tcg gat gtc 624

Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val

195 200 205

tgg tgg ggc gga gca gac aca gac tat gca gat ggg agt gaa gac aaa 672

Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys

210 215 220

gta gta gaa gta gca gag gag gaa gaa gtg gct gag gtg gaa gaa gaa 720

Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu

225 230 235 240

gaa gcc gat gat gac gag gac gat gag gat ggt gat gag gta gag gaa 768

Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu

245 250 255

gag gct gag gaa ccc tac gaa gaa gcc aca gag aga acc acc agc att 816

Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile

260 265 270

gcc acc acc acc acc acc acc aca gag tct gtg gaa gag gtg gtt cga 864

Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg

275 280 285

gag gtg tgc tct gaa caa gcc gag acg ggg ccg tgc cga gca atg atc 912

Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile

290 295 300

tcc cgc tgg tac ttt gat gtg act gaa ggg aag tgt gcc cca ttc ttt 960

Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe

305 310 315 320

tac ggc gga tgt ggc ggc aac cgg aac aac ttt gac aca gaa gag tac 1008

Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr

325 330 335

tgc atg gcc gtg tgt ggc agc gcc atg tcc caa agt tta ctc aag act 1056

Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr

340 345 350

acc cag gaa cct ctt gcc cga gat cct gtt aaa ctt cct aca aca gca 1104

Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala

355 360 365

gcc agt acc cct gat gcc gtt gac aag tat ctc gag aca cct ggg gat 1152

Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp

370 375 380

gag aat gaa cat gcc cat ttc cag aaa gcc aaa gag agg ctt gag gcc 1200

Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala

385 390 395 400

aag cac cga gag aga atg tcc cag gtc atg aga gaa tgg gaa gag gca 1248

Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala

405 410 415

gaa cgt caa gca aag aac ttg cct aaa gct gat aag aag gca gtt atc 1296

Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile

420 425 430

cag cat ttc cag gag aaa gtg gaa tct ttg gaa cag gaa gca gcc aac 1344

Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn

435 440 445

gag aga cag cag ctg gtg gag aca cac atg gcc aga gtg gaa gcc atg 1392

Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met

450 455 460

ctc aat gac cgc cgc cgc ctg gcc ctg gag aac tac atc acc gct ctg 1440

Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu

465 470 475 480

cag gct gtt cct cct cgg cct cgt cac gtg ttc aat atg cta aag aag 1488

Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys

485 490 495

tat gtc cgc gca gaa cag aag gac aga cag cac acc cta aag cat ttc 1536

Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe

500 505 510

gag cat gtg cgc atg gtg gat ccc aag aaa gcc gct cag atc cgg tcc 1584

Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser

515 520 525

cag gtt atg aca cac ctc cgt gtg att tat gag cgc atg aat cag tct 1632

Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser

530 535 540

ctc tcc ctg ctc tac aac gtg cct gca gtg gcc gag gag att cag gat 1680

Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp

545 550 555 560

gaa gtt gat gag ctg ctt cag aaa gag caa aac tat tca gat gac gtc 1728

Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val

565 570 575

ttg gcc aac atg att agt gaa cca agg atc agt tac gga aac gat gct 1776

Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala

580 585 590

ctc atg cca tct ttg acc gaa acg aaa acc acc gtg gag ctc ctt ccc 1824

Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro

595 600 605

gtg aat gga gag ttc agc ctg gac gat ctc cag ccg tgg cat tct ttt 1872

Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe

610 615 620

ggg gct gac tct gtg cca gcc aac aca gaa aac gaa gtt gag cct gtt 1920

Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val

625 630 635 640

gat gcc cgc cct gct gcc gac cga gga ctg acc act cga cca ggt tct 1968

Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser

645 650 655

ggg ttg aca aat atc aag acg gag gag atc tct gaa gtg aag atg gat 2016

Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp

660 665 670

gca gaa ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa ttg 2064

Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu

675 680 685

gtg ttc ttt gca gaa gat gtg ggt tca aac aaa ggt gca atc att gga 2112

Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly

690 695 700

ctc atg gtg ggc ggt gtt gtc ata gcg aca gtg atc gtc atc acc ttg 2160

Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu

705 710 715 720

gtg atg ctg aag aag aaa cag tac aca tcc att cat cat ggt gtg gtg 2208

Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val

725 730 735

gag gtt gac gcc gct gtc acc cca gag gag cgc cac ctg tcc aag atg 2256

Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met

740 745 750

cag cag aac ggc tac gaa aat cca acc tac aag ttc ttt gag cag atg 2304

Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met

755 760 765

cag aac tag 2313

Gln Asn

770

<210>2

<211>770

<212>PRT

＜213〉people

<400>2

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg

1 5 10 15

Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro

20 25 30

Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln

35 40 45

Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp

50 55 60

Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu

65 70 75 80

Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn

85 90 95

Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val

100 105 110

Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu

115 120 125

Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys

130 135 140

Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu

145 150 155 160

Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile

165 170 175

Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu

180 185 190

Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val

195 200 205

Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys

210 215 220

Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu

225 230 235 240

Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu

245 250 255

Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile

260 265 270

Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg

275 280 285

Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile

290 295 300

Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe

305 310 315 320

Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr

325 330 335

Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr

340 345 350

Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala

355 360 365

Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp

370 375 380

Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala

385 390 395 400

Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala

405 410 415

Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile

420 425 430

Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn

435 440 445

Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met

450 455 460

Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu

465 470 475 480

Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys

485 490 495

Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe

500 505 510

Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser

515 520 525

Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser

530 535 540

Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp

545 550 555 560

Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val

565 570 575

Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala

580 585 590

Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro

595 600 605

Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe

610 615 620

Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val

625 630 635 640

Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser

645 650 655

Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp

660 665 670

Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu

675 680 685

Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly

690 695 700

Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu

705 710 715 720

Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val

725 730 735

Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met

740 745 750

Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met

755 760 765

Gln Asn

770

<210>3

<211>45

<212>DNA

＜213〉clostridium tetani (Clostridium tetani)

<220>

<221>CDS

<222>(1)..(45)

＜223〉DNA of coding P2 epi-position

<400>3

cag tac atc aaa gct aac tcc aaa ttc atc ggt atc acc gag ctg 45

Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210>4

<211>15

<212>PRT

＜213〉clostridium tetani (Clostridium tetani)

<400>4

Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210>5

<211>63

<212>DNA

＜213〉clostridium tetani (Clostridium tetani)

<220>

<221>CDS

<222>(1)..(63)

＜223〉DNA of coding P30 epi-position

<400>5

ttc aac aac ttc acc gta agc ttc tgg ctg cgt gtt ccg aaa gtt agc 48

Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser

1 5 10 15

gct agc cac ctg gaa 63

Ala Ser His Leu Glu

20

<210>6

<211>21

<212>PRT

＜213〉clostridium tetani (Clostridium tetani)

<400>6

Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser

1 5 10 15

Ala Ser His Leu Glu

20

<210>7

<211>21

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>7

caactcagct tcctttcggg c 21

<210>8

<211>21

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>8

agatctcgat cccgcgaaat t 21

<210>9

<211>135

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>9

atggatgcag aattccgtca cgactccggt tacgaagttc accaccagaa actggttttc 60

ttcgcagaag atgttggttc caacaaaggt gcaatcatcg gtctgatggt tggcggtgtt 120

gttatcgcga cctag 135

<210>10

<211>31

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>10

gccggccatg gatgcagaat tccgtcacga c 31

<210>11

<211>39

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>11

gccggaagct tctaggtcgc gataacaaca ccgccaacc 39

<210>12

<211>84

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>12

ccggcaagct tctacagctc ggtgataccg atgaatttgg agttagcttt gatgtactgg 60

gtcgcgataa caacaccgcc aacc 84

<210>13

<211>101

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>13

gccggccatg ggtttcaaca acttcaccgt tagcttctgg ctgcgtgttc cgaaagttag 60

cgcgagccac ctggaagatg cagaattccg tcacgactcc g 101

<210>14

<211>172

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>14

gggccaagct tggatccggt cgcgataaca acaccgccaa ccatcagacc gatgattgca 60

cctttgttgg aaccaacatc ttctgcgaag aaaaccagtt tctggtggtg aacttcgtaa 120

ccggagtcgt gacggaactc tgcatccagc tcggtgatac cgatgaattt gg 172

<210>15

<211>30

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>15

ctggaagatg cagagttccg tcacgactcc 30

<210>16

<211>35

<212>DNA

＜213〉synthetic

＜223〉synthetic PCR primer

<400>16

gcgccggatc cttcaacaac ttcaccgtta gcttc 35

<210>17

<211>13

<212>PRT

＜213〉synthetic

＜223〉synthetic HLA DR binding sequence

<400>17

Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala

1 5 10

Claims

1. an immunogen is used for treating animal in preparation, prevents or improves application in the pharmaceutical preparation of disease that alzheimer's disease or other are feature with the amyloid beta deposition, and described pharmaceutical preparation comprises described immunogen, described immunogen

A) be a kind of polyamino acid, it has integrated the substantial portion of the B cell antigen epi-position of APP and/or A β in same molecule, make described polyamino acid react and at least one external source T-helper epitopes (T with polyclonal serum with APP or A β equal extent ground and anti-APP or A β generation _HEpi-position), and its comprise at least one external source T _HEpi-position and APP who interrupts or A β sequence make described polyamino acid not comprise subsequence any and the effective bonded SEQ ID NO:2 of MHC II quasi-molecule that causes the T-cell response; Or

B) be a kind of conjugate, it comprises the polyhydroxylated polymer skeleton, the coupling of described skeleton independence such as a) in defined polyamino acid; Or

C) be a kind of nucleic acid, its coding is described as defined polyamino acid in a); Or

D) be a kind of non-pathogenic microorganism or virus, it carries coding and expresses nucleic acid as defined polyamino acid in a).

2. according to the application of claim 1, wherein said immunogen is a kind of conjugate, and described conjugate comprises the polyhydroxylated polymer skeleton, described skeleton independence coupling 1) described at least one external source T _HEpi-position and 2) sequence that interrupts as APP described in a) or A β.

3. according to the application of claim 1, wherein said polyamino acid or conjugate comprise

-at least one first, it navigates to antigen presenting cell (APC) or B-lymphocyte with described polyamino acid, and/or

-at least one second portion, it stimulates described immune system, and/or

-at least one third part, it is optimized described polyamino acid and is presented to described immune system.

4. according to the application of claim 3, wherein said first and/or second and/or third part by covalently or non-covalently being connected on the chemical group suitable in described APP or the A β sequence and be attached as side-chain radical.

5. according to the application of claim 1, wherein said polyamino acid or conjugate comprise fused polypeptide.

6. according to the application of claim 1, wherein polyamino acid or conjugate comprise at least one APP or A β the B cell epitope copy and/or introduce a kind of hapten.

7. according to the application of claim 1, wherein said external source T-cell epitope is immunodominant in described animal.

8. according to the application of claim 1, wherein said external source T-cell epitope is miscellaneous.

9. application according to Claim 8, wherein said external source T-cell epitope are the external source t cell epitopes of selecting from natural miscellaneous t cell epitope and artificial MHC-II peptide binding sequence.

10. according to the application of claim 9, wherein said natural T-cell epitope is selected from tetanus toxoid epi-position, diphtheria toxoid epi-position, influenza virus hemagglutinin epi-position and Plasmodium falciparum (P.falciparum) CS epi-position.

11. according to the application of claim 10, wherein said tetanus toxoid epi-position is P2 or P30.

12. according to the application of claim 1, wherein said polyamino acid or conjugate comprise the B cell epitope, are not exposed to the cell foreign minister when its cell combining form with the precursor polypeptide of A β exists.

13. according to the application of claim 1, wherein said polyamino acid or conjugate lack at least one B cell epitope, are exposed to the cell foreign minister when its cell combining form with the precursor polypeptide of A β exists.

14. according to the application of claim 1, wherein said polyamino acid or conjugate comprise 9 continuous amino acids at the most of SEQID NO:2.

15. according to the application of claim 14, wherein said polyamino acid or conjugate comprise 8 continuous amino acids at the most of SEQID NO:2.

16. according to the application of claim 14, wherein said polyamino acid or conjugate comprise 7 continuous amino acids at the most of SEQID NO:2.

17. according to the application of claim 14, wherein said polyamino acid or conjugate comprise 6 continuous amino acids at the most of SEQID NO:2.

18. according to the application of claim 14, wherein said polyamino acid or conjugate comprise 5 continuous amino acids at the most of SEQID NO:2.

19. according to the application of claim 14, wherein said polyamino acid or conjugate comprise 4 continuous amino acids at the most of SEQID NO:2.

20. according to the application of claim 14, wherein said polyamino acid or conjugate comprise 3 continuous amino acids at the most of SEQID NO:2.

21. application according to claim 14, wherein said polyamino acid or conjugate comprise the subsequence of at least one SEQ ID NO:2, make the subsequence of at least one SEQ ID NO:2 that each is such be made up of aminoacid sequence individually, described aminoacid sequence is selected from 9 continuous amino acids of SEQ ID NO:2,8 continuous amino acids of SEQ ID NO:2,7 continuous amino acids of SEQ ID NO:2,6 continuous amino acids of SEQ ID NO:2,5 continuous amino acids of SEQ ID NO:2,4 continuous amino acids of SEQ ID NO:2 and 3 continuous amino acids of SEQ ID NO:2.

22. according to the application of claim 14 or 21, wherein said successive aminoacid originates in a kind of amino acid residue, it is selected from residue 672,673,674,675,676,677,678,679 and 680.

23. according to the application of claim 14 or 21, wherein said successive aminoacid originates in a kind of amino acid residue, it is selected from residue 681,682,683,684,685,686,687,688 and 689.

24. according to the application of claim 14 or 21, wherein said successive aminoacid originates in a kind of amino acid residue, it is selected from residue 690,691,692,693,694,695,696,697 and 698.

25. according to the application of claim 14 or 21, wherein said successive aminoacid originates in a kind of amino acid residue, it is selected from residue 699,700,701,702,703,704,705,706 and 707.

26. according to the application of claim 14 or 21, wherein said successive aminoacid originates in a kind of amino acid residue, it is selected from residue 708,709,710,711,712,713 and 714.

27. according to the application of claim 1, wherein the described polyamino acid of at least 2 copies or conjugate covalently or non-covalently are connected to and can realize on the carrier molecule that the multicopy antigenic determinant presents.

28. according to the application of claim 1, wherein said polyamino acid is connected on the described polyhydroxylated polymer by amido link.

29. according to the application of claim 2, wherein said T _HEpi-position is connected on the described polyhydroxylated polymer by amido link.

30. according to the application of claim 1, wherein said polyhydroxylated polymer is a polysaccharide.

31. according to the application of claim 1, wherein said polyamino acid or conjugate are prepared with a kind of adjuvant, described adjuvant promotes to destroy the automatic tolerance to autoantigen.

32. according to the application of claim 1, the effective dose of wherein said polyamino acid or conjugate is 0.5 μ g-2,000 μ g.

33. according to the application of claim 1, wherein said pharmaceutical preparation comprises the nucleic acid of the described polyamino acid of encoding, described nucleic acid is directed in the cell of described animal, thereby obtains the cell expression in vivo of the nucleic acid of described importing.

34. according to the application of claim 33, the nucleic acid of wherein said importing is selected from naked DNA, with the DNA of electrically charged or uncharged lipid preparation, be formulated in DNA in the liposome, be included in DNA in the viral vector, with the DNA of albumen that promotes transfection or polypeptide preparation, with the DNA of guiding protein or polypeptide preparation, with the DNA of the reagent preparation of precipitated calcium, be coupled to the inert carrier molecule DNA, be encapsulated in chitin or the chitosan DNA and with the DNA of adjuvant preparation.

35. according to the application of claim 1, wherein said treatment, prevention or improvement need with APP or A β be lowered to the reduction of amyloid total amount or amyloid formation speed has the degree that clinical meaning reduces.

36. according to the application of claim 1, wherein said pharmaceutical preparation comprises a kind of non-pathogenic microorganism or virus, described non-pathogenic microorganism or virus are carried coding and are expressed nucleic acid fragment as defined polyamino acid in a).

A 37. polyamino acid or conjugate that derives from animal APP or A β, wherein introduce and modify, described modification causes inducing the antibody that produces anti-described animal self APP or A β with the described animal of described polyamino acid or conjugate immunity, and wherein said polyamino acid or conjugate such as claim 1-30 each define.

38. one kind comprise immune effective dose according to the polyamino acid of claim 37 or the immune composition of conjugate, described compositions further comprises medicinal and immune acceptable carrier and/or the vehicle of going up.

39. according to the immune composition of claim 38, described compositions further comprises adjuvant.

40. a nucleic acid fragment, the polyamino acid of its coding claim 37.

41. carrier that carries the nucleic acid fragment of claim 40.

42. according to the carrier of claim 41, its be can self-replicating carrier.

43. according to the carrier of claim 41, it is selected from plasmid, phage, cosmid, mini-chromosome and virus.

44. the carrier of claim 41 or 43, in 5 ' → 3 ' direction and can being operatively connected, it comprises a kind of be used to start the nucleic acid fragment expression promoter of claim 40 and the nucleic acid fragment of claim 40.

45. according to the carrier of claim 44, it comprises the nucleotide sequence that coding makes described nucleic acid fragment encoded polypeptides fragment secretion or is incorporated into the guiding peptide in the film in addition.

46. according to the carrier of claim 44, it comprises a kind of terminator in addition.

47. the carrier of claim 41 or 43, in the time of in being incorporated into host cell, it can maybe can not be integrated in the described host cell gene group.

48. the carrier of claim 44, wherein said promoter is enabled in the expression in eukaryotic cell and/or the prokaryotic cell.

49. one kind is carried each the transformant of carrier of claim 41-48, it can duplicate the nucleic acid fragment of claim 40.

50. according to the transformant of claim 49, it is a kind of cell transformed.

51. the transformant of claim 49, it is a kind of be selected from antibacterial, yeast, protozoacide microorganism, or deriving from the cell of multicellular organism, the described cell that derives from multicellular organism is selected from fungus, insect cell, plant cell and mammalian cell.

52. according to the transformant of claim 51, wherein said insect cell is S ₂Or SF cell.

53. the transformant of claim 49 or 51, it expresses the nucleic acid fragment of claim 40.

54. according to the transformant of claim 53, it is a kind of cell transformed, its secretion or carry the polyamino acid or the conjugate of claim 37 on its surface.

55. a compositions of inducing the antibody that produces anti-amyloid, described compositions comprises

The nucleic acid fragment of-claim 40 or claim 41-48 each carrier and

-medicinal and immune acceptable carrier and/or vehicle and/or the adjuvant gone up.

56. a stable cell line, it carries each carrier of claim 41-48, and expresses the nucleic acid fragment of claim 40.

57. according to the cell line of claim 56, its secretion or carry the polyamino acid or the conjugate of claim 37 on its surface.