CN100409896C - Senile dementia vaccinum and preparing method thereof - Google Patents

Abstract

The present invention relates to a senile dementia vaccine and a preparing method thereof. The senile dementia vaccine comprises at least one artificially synthesized polypeptide which is coupled to carrier protein or carrier polypeptide to form a coupled object, wherein the polypeptide contains A beta-42 neutralizing epitope. The preparing method comprises the following steps: (1) at least one polypeptide comprising A beta-42 neutralizing epitope is artificially synthesized; (2) the polypeptide is respectively coupled to the carrier protein or the carrier polypeptide to form the coupled object which is incubated and aged; (3) the coupled object is matched with acceptable adjuvants to prepare A beta-42 subunit vaccine for treating senile dementia. The senile dementia vaccine removes common toxicity fragments, so that the vaccine structure is improved, the immunogenicity is enhanced, and effective antigenic determinants are retained.

Description

A kind of alzheimer disease vaccine and preparation method thereof

Technical field

The present invention relates to vaccine with the biotechnology preparation and preparation method thereof, particularly a kind of alzheimer disease vaccine and preparation method thereof.

Background technology

Alzheimer disease (Alzheimer ' s disease, AD) be a kind of neurocyte degenerative disease, clinical is feature with memory of carrying out property and the forfeiture of acquired knowledge, to the last patient loses viability.Along with the increase of number of the infected, AD brings white elephant for family and society, is an important society and health care problem.About the treatment of AD, still there is not desirable method at present, existing therapeutic strategy mainly is conceived to relief of symptoms, does not stop the pathology process of AD, and its clinical efficacy is unsatisfactory.A β is the main component that alzheimer disease pathology changes senile plaque, and it is made up of 39～42 aminoacid.Studies show that in recent years, the infringement of the neuron that gathers and deposit and follow of A β is the key link of alzheimer disease pathology mechanism, therefore, is suggested in succession at therapeutic strategy and the method for A β.Studies show that, Schenk reported first in 1999 Immunization with amyloid-beta attenuatesAlzheimer-disease-like pathology in the PDAPP mouse[see comments] and .Nature, 1999Jul 8; 400 (6740): 173-7} adopts immunological method that transgenic AD model mouse (PDAPP Mus) is treated; At present, external alzheimer disease vaccine has entered the IIa clinical trial, because inoculation back syndrome has appearred in the part test patient, comprise aseptic meningitis, therefore experiment stops (Schenk D.Amyloid-β immunotherapy forAlzheimer ' s disease:the end of the beginning.Nature Reviews, 2002,3:824～828).

Use A β ₄₂Complete fragment as immunogen, have following problem: (1) A β ₄₂Peptide contains A β toxic fragment (25～35 amino acids), and it is under suspicion as immunogenic safety; (2) the β lamellar structure of A β is the important form that the A beta polypeptides exists in interior A β deposit of brain and the senile plaque, and the A β of synthetic ₄₂Need just have better immunogenicity through allosteric; (3) A β ₄₂Peptide molecular weight＜10000, immunogenicity is poor.

Summary of the invention

Purpose of the present invention is used A β in order to overcome exactly ₄₂Complete fragment as the above-mentioned shortcoming of immunogen preparing alzheimer disease vaccine, a kind of alzheimer disease vaccine that a kind of toxicity is little, immunogenicity is good is provided.Another object of the present invention is the method for producing this vaccine in order to provide.

For achieving the above object, the present invention takes following design: a kind of synthetic peptide A β ₄₂Subunit vaccine, it comprise at least one be coupled to that carrier protein or carrier polypeptide form coupling matter contain A β ₄₂The polypeptide of synthetic of neutralizing epitope.Wherein, the polypeptide of the synthetic of described neutralizing epitope can be one of following seven kinds or wherein two or more kinds of:

(1)Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln(Aβ _1～15)

(2)Val-Gly-Gly-Val-Val-Ile-Ala(Aβ _36～42)

(3)Phe-Arg-His-Asp-Ser-Gly-Tyr(Aβ _4～10)

(4)Glu-Phe-Arg-His(Aβ _3～6)

(5)Asp-Ala-Glu-Phe-Arg(Aβ _1～5)

(6)Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu(Aβ _1～11)

(7)Glu-Asp-Val-Gly-Ser-Asn-Lys(Aβ _22～28)

Wherein adopt a kind of single subunit vaccine that is, adopt two or more the multi-joint subunit vaccine that is.

Wherein, carrier can be carrier protein bovine serum albumin (BSA), or (the multiple antigens peptide system of carrier polypeptide multiple antigenic peptide system, MAPs), oligomerization lysine is an ideal MAPs core frame, as seven polylysines (heptalysine), because the no antigen of seven polylysines own, the anaphylaxis of having avoided carrier protein such as BSA to bring makes the immunoreation specificity stronger, and safety is better; Simultaneously,, be positioned at the core of macromolecular complex, activity that can the blocking antigen epi-position because itself molecule is little.Three polylysines and for example, 12 polylysines etc.

In order to be suitable for injection, also should comprise acceptable medicinal adjuvant in the vaccine.As MF ₅₉(approval in 1997 of Italian government is used for the adjuvant of human body influenza vaccines) adjuvant, aluminium adjuvant etc.

The present invention prepares synthetic peptide A β ₄₂The preparation method of subunit vaccine may further comprise the steps:

What (1) synthetic at least one can be coupled to that carrier protein or carrier polypeptide form coupling matter comprises A β ₄₂The polypeptide of synthetic of neutralizing epitope;

(2) aforementioned polypeptides is coupled to respectively on carrier protein or the carrier polypeptide forms coupling matter;

(3) above-mentioned coupling matter is equipped with acceptable adjuvant and is prepared into senile dementia A β ₄₂Subunit vaccine.

Wherein the method for artificial synthetic polypeptide is generally synthetic at (aminoterminal) to the N end from C end (c-terminus).Polypeptide in the past is synthetic to carry out in solution.Most now solid-phase synthesis that adopt are selected the solid-state synthesizer of polypeptide for use, and are synthetic on synthetic post, thereby alleviate the difficulty that product is purified greatly.In order to prevent the generation of side reaction, synthetic post and the amino acid whose side chain of interpolation are protected.C-terminus is free, and must activation before reaction.The chemosynthesis fado adopts the Fmoc method.

The crosslinked common method that can adopt this area, butanimide-M-dimaleoyl imino benzoate (MBS) is crosslinked as adopting cross-linking agent N-, adopts preparation high performance liquid chromatogram (HPLC) column purification, through mass spectrograph (MS) Analysis and Identification.

The present invention selects A β ₄₂Some fragment as immunogen, so both removed total toxicity segment, thereby improved vaccine construct, enhance immunity originality kept effective antigenic determinant again.

The specific embodiment

The invention will be further described below in conjunction with embodiment.

Embodiment 1 preparation is based on the synthetic peptide A β of neutralizing epitope 1 ₄₂Subunit vaccine may further comprise the steps:

(1) utilizes the polypeptide of the solid-state synthesizer synthetic of polypeptide neutralizing epitope 1

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln (A β _1～15), cross more than the column purification to 95%;

(2) utilize cross-linking agent MBS (M-maleimidobenzoyl-N-hydroxy succinimideester), with the side chain coupling connection of polypeptide and carrier seven polylysines (heptalysine), and through hatching; More than HPLC chromatogram purification to 95%; 0.01mol/L PBS (pH7.4) dilution antigen with sterilization;

(3) coupling matter is equipped with isopyknic MF59 adjuvant,, is prepared into vaccine with the about 30min of vortex agitator mixing.

Embodiment 2 preparations are based on the synthetic peptide A β of neutralizing epitope 2 ₄₂Subunit vaccine may further comprise the steps:

(1) polypeptide of synthetic neutralizing epitope 2

Val-Gly-Gly-Val-Val-lle-Ala(Aβ _36～42)

(2) utilize cross-linking agent MBS (M-maleimidobenzoyl-N-hydroxy succinimideester), with the side chain coupling connection of polypeptide and carrier seven polylysines (heptalysine), and through hatching;

(3) coupling matter is equipped with aluminium adjuvant and is prepared into vaccine.

Embodiment 3 preparations are based on the synthetic peptide A β of neutralizing epitope 3 ₄₂Subunit vaccine may further comprise the steps:

(1) polypeptide of synthetic neutralizing epitope 3

Phe-Arg-His-Asp-Ser-Gly-Tyr

(2) utilize cross-linking agent MBS (M-maleimidobenzoyl-N-hydroxy succinimideester), with the side chain coupling connection of polypeptide and carrier seven polylysines (heptalysine), and through hatching;

(3) coupling matter is equipped with the MF59 adjuvant and is prepared into vaccine.

Embodiment 4 preparations are based on the synthetic peptide A β of neutralizing epitope 4 ₄₂Subunit vaccine may further comprise the steps:

(1) polypeptide of synthetic neutralizing epitope 4

Glu-Phe-Arg-His

(2) utilize cross-linking agent MBS (M-maleimidobenzoyl-N-hydroxy succinimideester), with the side chain coupling connection of polypeptide and carrier seven polylysines (heptalysine), and through hatching;

(3) coupling matter is equipped with the MF59 adjuvant and is prepared into vaccine.

Embodiment 5 preparations are based on the synthetic peptide A β of neutralizing epitope 5 ₄₂Subunit vaccine may further comprise the steps:

(1) polypeptide of synthetic neutralizing epitope 5

Asp-Ala-Glu-Phe-Arg

(2) utilize cross-linking agent MBS (M-maleimidobenzoyl-N-hydroxy succinimideester), with the side chain coupling connection of polypeptide and carrier seven polylysines (heptalysine), and through hatching;

(3) coupling matter is equipped with aluminium adjuvant and is prepared into vaccine.

Embodiment 6 preparations are based on the synthetic peptide A β of neutralizing epitope 6 ₄₂Subunit vaccine may further comprise the steps:

(1) polypeptide of synthetic neutralizing epitope 6

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu

(2) utilize cross-linking agent MBS (M-maleimidobenzoyl-N-hydroxy succinimideester), with the side chain coupling connection of polypeptide and carrier seven polylysines (heptalysine), and through hatching;

(3) coupling matter is equipped with the MF59 adjuvant and is prepared into vaccine.

Embodiment 7 preparations are based on the synthetic peptide A β of neutralizing epitope 7 ₄₂Subunit vaccine may further comprise the steps:

(1) polypeptide of synthetic neutralizing epitope 7

Glu-Asp-Val-Gly-Ser-Asn-Lys

(2) utilize cross-linking agent MBS (M-maleimidobenzoyl-N-hydroxy succinimideester), with the side chain coupling connection of polypeptide and carrier seven polylysines (heptalysine), and through hatching;

(3) coupling matter is equipped with aluminium adjuvant and is prepared into vaccine.

Embodiment 8 preparations are with A β _1-15With A β _36-42Synthetic peptide multiple vaccines for the basis may further comprise the steps:

(1) polypeptide of synthetic neutralizing epitope 1

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln

(2) utilize cross-linking agent MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester),, and, form coupling matter I through hatching with the side chain coupling connection of polypeptide and carrier seven polylysines (heptalysine);

(3) polypeptide of synthetic neutralizing epitope 2

Val-Gly-Gly-Val-Val-Ile-Ala

(4) utilize cross-linking agent MBS (M-maleimidobenzoyl-N-hydroxy succinimideester),, and, form coupling matter II through hatching with the side chain coupling connection of polypeptide and carrier seven polylysines (heptalysine);

(5) coupling matter I and II are pressed 1: 1 or 1: 2 mixed in molar ratio.

(6) mixture is equipped with the MF59 adjuvant and is prepared into vaccine.

Embodiment 9 changes the carrier of embodiment 2 into bovine serum albumin by seven polylysines, all the other embodiments 2.

Behavioristics's viewing test after embodiment 10 embodiment 2 and the embodiment 8 vaccination normal SD rats.

1 materials and methods

1.1 the 3 monthly age male SD rats (SPF level, available from No.1 Military Medical Univ.'s Experimental Animal Center, animal quality quality certification number is Guangdong probatio inspectionem pecuoarem word 2001A051 number) of growing up of 32 health of laboratory animal, body weight 180-200g.After raising a week, observe normally, be divided into 4 groups then at random: 2 groups of embodiment (MAP group), 9 groups of embodiment (BSA group), A β ₄₂Full peptide group and matched group, every group each 8.

1.2 be fixed on the platform behind the immunity inoculation rat anesthesia.The skin of sterilization rat back center line both sides, the skin test pin extraction antigen with 1.00ml carries out multiple spot (15-20 point) subcutaneous injection along center line both sides, back.The booster injection second time were carried out in 2 weeks in the inoculation back first, carried out booster injection later on every 1 month, inoculated altogether 4 times, and the cumulative volume of every subcutaneous rat inoculation is 0.40ml.

1.3 indirect elisa method detects in the serum and anti-A β in the brain homogenate ₄₂Titre

1.4 it is all normal that action and the reaction of SD rat in the whole experiment are observed by behavioristics.One week of last inoculation back adopts Morris water maze method to detect orientation navigation and the space exploration ability of SD rat.Orientation navigation experiment: test makes it be familiar with the labyrinth environment SD rat free swimming 2min that (do not contain platform) in the pond the previous day.Test lasts 5 days altogether, divides two time periods of upper and lower noon (blocks) every day, each time period training 4 times, and every day is totally 8 times (trials).Platform places the EN quadrant during test, Mus respectively from east, south, west, north (E, S, W, N) four quadrants puts into the pond in the face of pool wall, write down this Mus and find the used time of platform (escape latency, escape latenkcky).Rat is found behind the platform or 120s can not find platform and then by the experimenter it drawn upper mounting plate (being designated as 120s incubation period), behind rest 30s on the platform, and row test next time again.Continuous four days, totally eight time periods.The space exploration experiment: the 5th time (being right after the 4th time) in the 5th day removes platform, and an optional place of entry is put into water with rat towards pool wall, the swimming route of rat in the record 2min.In whole test, peripheral pool object of reference (clue outside the labyrinth) comprises experimenter's invariant position, disturbs the moderate white noise of indoor lasting broadcast intensity for eliminating external sound simultaneously.The swimming distance of main relatively each experimental group escape latency and platform quadrant accounts for the percentage ratio of total swimming distance.

2 results

2.1 ordinary circumstance

All inoculation rat outward appearances are normal in the nursing process, and hair does not have change, and skin does not have ulcer, and action and behavior reflex are all no abnormal, and diet, drinking-water and sleep are all normal, and body weight all increases.The anti-A β of the 4th inoculation back experimental group rat ₄₂Antibody all reaches more than 1: 10000, and matched group is 1: 100.Weigh through dissecting rapidly, the weight in wet base of the main organs of brain, liver,spleen,kidney and HE morphological observation are not found to change.Its weight in wet base and matched group compare, no significance difference.

Western Blotting experiment shows that the antibody in enforcement 2 and embodiment 8 serum is specific anti-A β really ₄₂Antibody.

Immunohistochemical experiment shows embodiment 2, embodiment 8 and A β ₄₂Antibody capable combines with the senile plaque specificity of alzheimer disease patient's brain section in the serum of group.

2.2 the antibody titer in serum and the brain homogenate supernatant

The 1st time each experimental group of inoculation back all fails to detect specific anti-A β ₄₂Antibody, the 2nd time each experimental group serum of inoculation back begins to have anti-A β ₄₂Antibody produces, and with the increasing of inoculation times, antibody titer all increases gradually; In the 2nd the gained serum, MAP group and A β ₄₂It is 1: 12800 that group respectively has a titre; In the 3rd the gained serum, the MAP group has 3, and the BSA group has 4, A β ₄₂It is 1: 12800 that group has 6 titre.Each average titer of organizing the antibody of rat blood serum and supernatant sees Table 1.

Anti-A β in serum of inoculation SD rat and the brain homogenate supernatant after table 1 vaccination ₄₂The average titer of antibody

Compare with matched group, ^*P＜0.05, ^*P＜0.01; Compare △ P＜0.05, △ △ P＜0.01 with the BSA group; Compare ##P＜0.01 with the MAP group;

2.3Morris water maze test result

The escape latency curve of each inoculation group rat is all on a declining curve, shows that the SD Mus seeks platform after training time all shortens.To total escape latency of each inoculation group, compare difference that there are no significant (P＞0.05) respectively in twos; Separately its preceding 5 escape latency and back 3 escape latency are compared in twos difference that similarly there are no significant (P＞0.05) again.(the concrete numerical value of the average escape latency of each inoculation group is referring to table 2)

The average escape latency (second) of table 2 inoculation rat (S, X ± SD)

Compare in twos between group, be P＞0.05.

The percentage ratio that accounts for total swimming distance with the swimming of platform quadrant distance is judged the space learning memory ability of rat, MAP group, BSA group, A β ₄₂Group, the percentage ratio of matched group (%) is respectively 30.40 ± 16.19,31.58 ± 7.75,19.38 ± 9.00,23.80 ± 8.45.Difference (P＞0.05) that relatively there are no significant in twos.

From serum and the brain homogenate supernatant result who detects, A β ₄₂Subunit (A β _36～42) vaccine and A β ₄₂Full peptide vaccine is the same, can produce the anti-A β of high titre ₄₂Antibody, and under the prerequisite of the specific antibody that produces high titre, study and the cognitive behavior of the rat of medication group are normal.And our experimental result shows that brain, liver, spleen and kidney do not find that pathologic changes.Show A β ₄₂Subunit (A β _36-42) potential clinical value arranged.

The test of embodiment 1,3～8 shows, embodiment 1,3～8 has the result similar to embodiment 2,9.Multiple vaccines of the present invention can adopt any two, two associating or more multi-joint modes of closing, and is not limited to embodiment 8; In fact, experiment shows, the multiple vaccines that adopts associating in twos or more multi-joint mode of closing to make, and the vaccine effect that more single subunit is made is better.

The aminoacid tabulation:

(1)Asp?Ala?Glu?Phe?Arg?His?Asp?Ser?Gly?Tyr?Glu?Val?His?His?Gln

1 5 10 15

(2)Val?Gly?Gly?Val?Val?Ile?Ala

1 5

(3)Phe?Arg?His?Asp?Ser?Gly?Tyr

1 5

(4)Glu?Phe?Arg?His

1

(5)Asp?Ala?Glu?Phe?Arg

1 5

(6)Asp?Ala?Glu?Phe?Arg?His?Asp?Ser?Gly?Tyr?Glu

1 5 10

(7)Glu?Asp?Val?Gly?Ser?Asn?Lys

1 5

Claims (2)

1. the preparation method of an alzheimer disease vaccine may further comprise the steps:

(1) utilize the solid-state synthesizer synthetic of polypeptide to contain A β ₄₂The polypeptide of neutralizing epitope, cross more than the column purification to 95% the wherein said A β that contains ₄₂The polypeptide of neutralizing epitope be Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln (A β _1～15),

(2) utilize cross-linking agent MBS with the side chain coupling of polypeptide and carrier seven polylysines connection, and through hatching, more than HPLC chromatogram purification to 95%, with the PBS dilution antigen of the 0.01mol/L pH7.4 of sterilization,

(3) coupling matter is equipped with isopyknic MF59 adjuvant,, is prepared into vaccine with the about 30min of vortex agitator mixing.

2. alzheimer disease vaccine that method according to claim 1 prepares.