CN102127162A - Alzheimer's disease dendritic cell (DC) vaccine prepared by stimulation by mutant Abeta polypeptide - Google Patents

Abstract

The invention relates to a vaccine for preventing and/or treating Alzheimer's disease. The vaccine is obtained by using mutant β-amyloid polypeptide and its biological stimulation to sensitize dendritic cells, and the safety of the vaccine is confirmed by animal experiments. ,efficient. The present invention can be used for the prevention and/or treatment of Alzheimer's disease.

Description

Mutant A beta polypeptides stimulates the alzheimer's disease DC vaccine of preparation

Technical field

The present invention be a kind of utilize dendritic cell after mutant amyloid beta polypeptide and the Xing biostimulation sensitization thereof (Dendritic cell, DC) and the alzheimer's disease that obtains (Alzheimer ' s disease, AD) vaccine.

Background technology

AD is a kind of age of onset morning, sickness rate height, very harmful central nervous system degeneration.How in morbidity more than 60 years old, its sickness rate increases with the age and increases.U.S. alzheimer's disease association has reached 5,200,000 according to the statistics report of U.S. CDC by U.S. AD patient in 2008, and global AD patient is near 30,000,000.Have 13% to suffer from this disease among the over-65s old man, 1 new cases was on average just arranged in per 71 seconds.And according to estimates to mid-term in this century, the AD patient in the whole world will reach 100,000,000 people, just will have 1 new cases and occur in average per 33 seconds.Along with the gradually aging of China's population, the sickness rate of AD improves constantly, and seeks active and effective prevention and treatment measure, and for the quality of life that improves elderly population, the economy and the mental burden that alleviate family and society have very important significance.

The characteristic pathological change of AD comprise by amyloid beta (Amyloid beta, A β) senile plaque of formation of deposits in brain essence (Senileplaques, SP) and the neurofibrillary tangles of the tau protein formation of deposits of neurocyte camber phosphorylation.Along with the amyloid theory obtains many-sided experiment confirm and obtains most of people's approvals, people have proposed the various AD treatment plans at A β.Wherein utilizing anti-amyloid beta antibodies to carry out immunotherapy is to promote body to remove the important means of established A β.The experiment in vivo and vitro of many use anti-amyloid beta antibodies confirms that all A β immunotherapy can reduce A β deposition, improves AD model mouse learning and memory function.A β active immunity treatment, i.e. AD vaccine is held time advantage such as long and is subjected to most researchists' favor with its economy, convenient, effect.

So far the AD vaccine the most approaching successful be the AD vaccine AN1792 of Elan company.This vaccine is made of A β 1-42 polypeptide and QS-21 adjuvant.This vaccine has but been stopped by FDA because of the symptom that has 18 patients non-specific meningoencephalitis to occur among 300 triers in the II clinical trial phase.Although this test failure, some of them are accepted the AD patient of vaccine inoculation and are shown good effect.The anti-amyloid beta antibodies that has occurred high titre among wherein many patients serums, its antiserum(antisera) can react with the A β settling in the section of encephalopathic reason.Along with the prolongation of the time of following up a case by regular visits to, its cognitive decrease degree of patient that has produced antibody significantly is lower than the person that do not produce antibody, and antibody titers is high more, and difference is obvious more.In addition, by the postmortem of dying from meningoencephalitis complication patient is found that the A beta plaque obviously is less than nonvaccinated AD patient in its brain, also find to have the infiltration of the activation of microglia and T cell simultaneously to central nervous system.Subsequently the state of an illness of analysis revealed meningoencephalitis that the meningoencephalitis patient is taken place and the degree of antibody response and the specificity of epitope be there is no direct correlation.And meningoencephalitis does not appear in most of patients that anti-amyloid beta antibodies occurs.It is relevant with the T cell autoimmune response at A β that these situations all point out the patient meningoencephalitis to occur, comprising A beta response Th1T cell in the activation of periphery and to the infiltration of A beta plaque.Most scholars think that the serious side reaction in the AN1792 experiment belongs to autoimmunization generation activated sterility immune inflammation, and is relevant with the used QS21 adjuvant of vaccine.This adjuvant produces the pro-inflammatory cytokine of IFN-γ and so on to induce the reaction of Th1 type, stimulates the T cell to produce A beta response cytotoxic T cell.And the generation of protection antibody needs Th2 type reacting activation bone-marrow-derived lymphocyte.

But A β is the proteic meta-bolites of normal neurons cellularstructure, and there is the central immunological tolerance in body to it.Have only by adding limited meanses such as adjuvant to strengthen its antigenicity and could more stably induce generation antibody, do not add adjuvant and strengthen vaccine antigen and just be difficult to guarantee to obtain antibody.How avoiding the Th1 type reaction of adjuvant or induce the reaction of Th2 type under the situation of not using adjuvant, will be the key that the AD vaccine is succeedd thereby obtain A β antibody safely and effectively.Many scholars adopt the several different methods preparations such as Th2 type adjuvant, reduction vaccine t cell epitope antigen, change route of inoculation, preparation DNA or RNA vaccine of improvement not cause the AD vaccine of Th1 type reaction.And the method applied in the present invention is to utilize the antigen presentation ability of dendritic cell (DC), and preparation can cause that body produces the AD vaccine of enough antibody titerss.Preliminary experiment through us confirms this a kind of vaccine safe, effective, economic and easy to use.

Summary of the invention

One of basis of the present invention is to have found that mutant amyloid beta immunogenicity is stronger than wild-type beta amyloid.Through the different mutant amyloid beta immunogenicity of experiment confirm variant (polypeptide 1～8).Therefrom our two mutant polypeptides (polypeptide 1) of having chosen the synthetic that wherein immunogenicity is stronger tentatively reach subsequent experimental.Experiment has also comprised the research (polypeptide 9～11) of different lengths immunogenicity of polypeptides.

Preliminary experiment of the present invention confirms, adopts wild-type beta amyloid sensitization DC can not lure body to produce anti-amyloid beta antibodies, adopts mutant AB polypeptide sensitization DC then can induce body to produce the anti-amyloid beta antibodies of high titre.

On the basis of preliminary experiment, we utilize mutant amyloid beta polypeptide sensitization DC, the PDAPP transgenic mice is tested, adopt liquid-phase chip technology to detect the changing conditions of multiple inflammatory factor in treatment front and back serum and the cerebrospinal fluid, confirm to adopt the method for sensitization DC vaccine can not resemble and caused the inflammatory reaction of Th1 type the Adjuvanted vaccines, thus the serious side reactions such as meningoencephalitis of avoiding common A β vaccine to cause.By study of behaviour after the transgenic mice treatment and pathological examination are confirmed that the prepared DC vaccine of present method can improve cognition and the memory capability of transgenic models mouse, can reduce the A β deposition in the Transgenic Mice Brain.

Embodiment

The present invention gathers in the crops medullary cell in mouse femur and tibial bone pulp cavity, DC substratum (adding penicillin and Streptomycin sulphate to the 0.1%2-mercaptoethanol of each 100U/mL of final concentration, final concentration 0.33% and the RPMI-1640 substratum of final concentration 10% foetal calf serum) with reorganization pearl mouse interleukin 4 (hereinafter to be referred as IL-4) and each 10ng/mL of granulocyte-macrophage colony stimutaing factor (hereinafter to be referred as GM-CSF) is cultivated, discard substratum and wherein not adherent cell after 24 hours, add the DC substratum again by former substratum cytokine concentration.Cultivate to inhale after 72 hours and abandon 1/3 supernatant liquor, replenish and added the DC substratum of same concentrations cytokine.Adding the mutant AB polypeptide select, wild-type AB polypeptide simultaneously cultivates with sensitization DC altogether to final concentration 22.2 μ g/mL, results float on the ripe DC in the cultivation after 72 hours, with Flow Cytometry DC is detected, promptly be used for the immunization experiment after ripening degree is up to standard as the DC vaccine.

C57 and BALB/C mice are adopted in preliminary experiment that the present invention does, divide 5 groups, every group 10, except that with using the DC vaccine of the B-mode amyloid polypeptide of mutant and wild-type sensitization respectively, also comprise the B-mode amyloid polypeptide+Adjuvanted vaccines of wild-type (hereinafter to be referred as Adjuvanted vaccines), the phosphate buffered saline(PBS) (hereinafter to be referred as PBS) of not having the DC cell vaccine of polypeptide sensitization and not having result of treatment merely.Every the equal abdominal cavity inoculation of mouse 0.2ml liquid.Wherein DC vaccine inoculation adopts 1 * 10 ⁵The DC cell suspension is inoculated in PBS.Adjuvanted vaccines inoculation adopts the B-mode amyloid polypeptide of 50 μ g wild-types to be dissolved among the 0.1mlPBS, and with 0.1ml Fu Shi adjuvant mixing, emulsification after inoculate.Per 7 days of inoculation back venous plexus under the cheek of mouse is got blood, detects with ELISA sandwich assay antagonism A β antibody titers, up to inoculating back 35 days.Gained is respectively organized average anti-amyloid beta antibodies titre and is changed as shown in Figure 1.

Follow-up experiment adopts the PDAPP transgenic mice to carry out.Experiment divides 4 groups, and 12 every group, cancelled the DC cell vaccine group of the wild-type A beta polypeptides sensitization in the former preliminary experiment in the grouping because of economic factors, all the other are with the preliminary experiment unanimity.Per 10 days of inoculation back venous plexus under the cheek of mouse is got blood, detects with ELISA sandwich assay antagonism A β antibody titers, detects multiple inflammatory factor concentration in the serum with liquid-phase chip, up to inoculating back 50 days.Inoculate 60 days and begin to carry out the Morris water maze laboratory to check the cognition and the memory function of transgenic mice.Consummatory behavior experiment back anesthetized mice is got its cerebrospinal fluid and is carried out the inflammatory factor detection.Behind the emptying venous blood, the capable again pathological examination of row heart perfusion.Gained is respectively organized average anti-amyloid beta antibodies titre and is changed as shown in Figure 2.Each organizes the average inflammatory factor change in concentration of serum as shown in Figure 3.Each is organized and changes as shown in Figure 4 latent period.Each organizes average spanning platform number of times as shown in Figure 5.Each organizes the average criterion quadrant residence time as shown in Figure 6.

Description of drawings

Figure 1 shows that the C57 mouse is by the anti-AB antibody titers changing conditions in different methods inoculation back.

Figure 2 shows that the PDAPP transgenic mouse is by the anti-AB antibody titers changing conditions in different methods inoculation back.

Figure 3 shows that the PDAPP transgenic mouse is by the multiple level of inflammation changing conditions of different methods inoculation back serum.

Figure 4 shows that the PDAPP transgenic mice is by different methods inoculation back orientation navigation test escape latency changing conditions.

Figure 5 shows that the PDAPP transgenic mice is by spanning platform number of times situation in the different methods inoculation rear space search test.

Figure 6 shows that the PDAPP transgenic mice is by stopping situation at original platform place target quadrant in the different methods inoculation rear space search test.

Sequence table:

Polypeptide 1:DAEFR HDSGY EVHHQ KLVFF GQDVG SNKGA IIGLM VGGVV IA

Polypeptide 2:DAEFR HDSGY EVHHQ KLVFF GEDVG SNKGA IIGLM VGGVV IA

Polypeptide 3:DAEFR HDSGY EVHHQ KLVFF AQDVG SNKGA IIGLM VGGVV IA

Polypeptide 4:DAEFR HDSGY EVHHQ KLVFF AKDVG SNKGA IIGLM VGGVV IA

Polypeptide 5:DAEFR HDSGY EVHHQ KLVFF AGDVG SNKGA IIGLM VGGVV IA

Polypeptide 6:DAEFR HDSGY EVHHQ KLVFF AWDVG SNKGA IIGLM VGGVV IA

Polypeptide 7:DAEFR HDSGY EVHHQ KLVFF AENVG SNKGA IIGLM VGGVV IA

Polypeptide 8:DAEFR RNSGY EVHHQ KLVFF AEDVG SNKGA IIGLM VGGVV IA

Polypeptide 9:DAEFR HDSGY EVHHQ KLVFF GQDVG SNKGA IIGLM

Polypeptide 10:DAEFR HDSGY EVHHQ KLVFF GQDVG SNKGA IIG

Polypeptide 11:DAEFR HDSGY EVHHQ KLVFF GQDVG SNKGA

Claims (6)

1. a peptide species, this polypeptide is artificial synthetic mutant amyloid beta polypeptide.

2. according to the described polypeptide of claim 1, change the novel polypeptide that its amino acid length obtains, polypeptide length does not wait from 25 amino acid to 42 amino acid.

3. according to the described polypeptide of claim 2, change new the mutant amyloid beta polypeptide and the combination thereof of the 21st, 22,23 and the 6th, 7 position amino acid manufacturings in its aminoacid sequence.

4. according to claim 1, claim 2 and the described polypeptide of claim 3, the N-terminal and the C-terminal of peptide sequence carried out the compound that chemically modified obtained.This modification mode meets following condition: R-(P)-R '.Wherein

P is claim 1, claim 2 and the described polypeptide of claim 3.

The group of R for this polypeptide N-terminal is carried out chemically modified, these chemically modified groups comprise following type:

Hydrogen;

Halogen;

Little alkyl group comprises 8 carbon atoms aliphatics or the cyclic group of following (containing 8), can be with or without functional group, as carboxylate salt, sulfonate or phosphoric acid salt;

Aromatic group;

Heterocyclic group;

Carboxyl groups is as alkyl acyl, aryl-acyl, alkylsulfonyl or phosphoryl group.

R ' replaces for C-terminal or the group of chemically modified, and these groups comprise following type:

Hydroxyl;

8 following (containing 8) alkoxyl groups of carbon atom comprise chain type or ring type group;

Aryloxy;

The amide group group.

5. one kind prevents and/or treats the method for being tried body and alzheimer's disease relative disease.Comprise and use claim 1, claim 2, claim 3 and the described polypeptide of claim 4 that the dendritic cell that obtains with gather in the crops cultivation or the conversion of use peripheral blood lymphocytes from marrow is carried out common cultivation, stimulation sensitization.The sensitization dendritic cell that obtained quantitatively is inoculated in after through separation, purifying, counting process tried body.

6. method according to claim 5, it is tried body and is comprised mouse, rat, rabbit, dog, monkey and people.

Images