CN101380466A

CN101380466A - Polypeptide vaccine for treating diseased induced by high pathogenic avian influenza virus H5N1

Info

Publication number: CN101380466A
Application number: CNA2008102328513A
Authority: CN
Inventors: 吕凤林; 田菲菲; 梅虎; 张一�; 李元朝
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2008-10-10
Filing date: 2008-10-10
Publication date: 2009-03-11

Abstract

The invention relates to a polypeptide vaccine for treating diseases caused by highly pathogenic avian influenza virus H5N1, which is characterize in that: the amino acid sequence is as follows: amino terminal-QIGNISIWV-carboxyl terminal; and lysine is adopted as a linker to connect a plurality of amino acid sequences to form a multi-copy series structure. The invention has the advantages of convenient transportation and preservation and automated mass production, and can activate specific cytotoxic T lymphocyte (CTL) which can effectively kill and lyses target cells with the N1 protein of the highly pathogenic avian influenza virus H5N1 without toxicity and side effects.

Description

A kind of polypeptide vaccine for the treatment of diseased induced by high pathogenic avian influenza virus H 5 N 1

(1), technical field

The present invention relates to a kind of biomedical sector, particularly a kind of about high pathogenic avian influenza virus diseases induced polypeptide drugs or vaccine.

(2), background technology

Avian influenza (Avian influenza) is called for short bird flu, is the infectious disease that is extensively taken place by the birds that A type influenza virus (AIV) causes.According to incompletely statistics, global bird flu epidemic situation has involved 47 countries and regions, and Crinis Carbonisatus bird flu case has reached more than 195, death at least 110 people wherein, and mortality rate is up to 56.4%.The bird flu that took place in Hong Kong in 1997 has caused people's morbidity and death, thereby has caused global great attention.AIV is according to its hemagglutinin (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA) antigenic difference can be divided into 16 H hypotypes (H1～H16) and 10 N hypotypes ([Alexander DJ.A review of avian influenza in different birdspecies.Vet Microbiol of N1～N10) at present, 2000,74 (1-2): 3-13].With pathogenic different, it can be divided into highly pathogenic, low pathogenicity and no pathogenicity three classes according to the bird flu virus virulence.Poultry disease has features such as propagation is fast, harm is big, the state of an illness is dangerous due to H7, the H5 hypotype, except that the acute respiratory inflammation, often sexually revise with systemic bleeding, multiple organs failures such as the heart, lung, kidney, mortality rate reaches 100%, so be called high pathogenic avian influenza (highlypathogenic avianinfluenza), at present H5N1 be classified as high pathogenic avian influenza virus.

For human, maximum threat is, if while infected person parainfluenza virus and bird flu virus just might reassemble into the new virus that can propagate mutually in vivo between the mankind, and people almost do not have immunity to this, and that certainly will will cause another flu outbreak.Because aquatic bird and birds are natural hosts of AIV, the long-term existence of this virus will provide condition for it mixes, produces new strain with infected person or other mammiferous influenza virus.Vaccine or medicine at AIV mainly contains following several at present:

1, plasmid vaccine: Lalor PA etc. has contrasted the immune effect of the plasmid vaccine of HA, the NP (nucleoprotein) of structure, M2 gene respectively.Three kinds of vaccines all have the significant protection effect to experimental mouse and ferret, and made up vaccine effect the best [Lalor PA of NP and M2 gene simultaneously, Webby RJ, Morrow J.Plasmid DNA-based vaccines protect mice andferrets against lethal challenge with A/Vietnam/1203/04 (H5N1) influenza virus.J Infect Dis.2008; 197 (12): 1643-1652].Li C etc. has made up the plasmid vaccine after optimizing: H5N1/PR8-5B19.This vaccine comprises the HA and the NA gene of H5N1 virus.And the HA cleavage site has been modified-revised to HA and NA gene make that it is similar with low pathogenicity feelings influenza, the stem district of NA is replaced with the 5B19 epi-position of the S2 glycoprotein of murine hepatitis virus, pathogenic and strengthen its immunogenicity to reduce it.This vaccine shows stronger humoral immunoresponse(HI) at last, experimental chicken has been produced immunoprotection [LiC preferably, Ping J, Jing B.H5N1 influenza marker vaccine for serologicaldifferentiation between vaccinated and infected chickens.Biochem Biophys Res Commun.2008; 372 (2): 293-297.].

2, recombinant vaccine

Wei CJ etc. systematically analyzes the potentiality that several multi-form recombinant HA albumen are applied as vaccine.The H5N1HA albumen of monomer, trimer and oligomer is isolated from cell sub-department, adopts its immunogenicity of neutralizing antibody reaction pair to assess.The result: the antibody response that high-molecular weight oligomer HA protein vaccine causes is the strongest, trimer takes second place, the most weak [the Wei CJ of monomer, Xu L, Kong WP.Comparative efficacy of neutralizing antibodieselicited by recombinant hemagglutinin proteins from avian H5N1influenza virus.J Virol.2008Jul; 82 (13): 6200-6208].

Watanabe etc. discover that behind 11 aminoacid of H5N1 virus M2 hydroxyl terminal excision, this virus is compared with wild type similar energy for growth, but reduces greatly at the intravital duplicating efficiency of mice.The author has made up recombinant virus-VN1203M2del11, and the mutant of the cleavage site of this recombinant virus HA after by 11 aminoacid of excision replaces.The result: this vaccine has produced better protect to mice.M2 kytoplasm afterbody mutant has potentiality [the Watanabe T that develops into the H5N1 virus attenuated live vaccine, Watanabe S, Kim JH.Novel approachto the development of effective H5N1 influenza A virus vaccines:use of M2 cytoplasmic tail mutants. J Virol. 2008Mar; 82 (5): 2486-92].

3, vector-viral vaccine: Rmer etc. utilize Avian pneumo-encephalitis virus to make carrier, express the HA immunogen as vaccine.The result shows: this vaccine (NDVNDVH5m) is once just having effect preferably to chicken after the immunity.The attenuated vaccine of making carrier by Avian pneumo-encephalitis virus to the protection level that infects H5 avian influenza by the homology size decision [Rmer-Oberd of this vaccine with the H5 sequence that infects H5N1 virus, rfer A, Veits J.Level of protection of chickensagainst highly pathogenic H5 avian influenza virus withNewcastle disease virus based live attenuated vector vaccinedepends on homology of H5 sequence between vaccine and challengevirus.Vaccine.2008; 26 (19): 2307-13].

Usefulness HIV virions such as Mar are made carrier, and bag is configured to H5N1 HaNaM-pseudotyped HIV carrier system by the HA of H5N1, NA, M2 gene.This carrier system can be simulated bird flu virus reaction in vivo more accurately, can replace bird flu virus, is used for assessing vaccine, medicine.Special is being that this system of the lower laboratory applications of safety has Practical significance [Mar Ao Z, Patel A, Tran K.Characteri zat ion of atrypsin-dependent avian influenza H5N1-pseudotyped HIV vectorsystem for high throughput screening of inhibitorymolecules.Antiviral Res.2008 Jul; 79 (1): 12-8].

Singh etc. are with the hypotype (BAd3) of the cow adenovirus AD HA gene as vector expression H5N1, be built into the BAd-H5NA vaccine, immune effect [Singh N is preferably arranged, Pandey A, Jayashankar L.Bovine adenoviral vector-based H5N1influenza vaccine overcomes exceptionally high levels ofpre-existing immunity against human adenovirus.Singh Mol Ther.2008; 16 (5): 965-71].

(3), summary of the invention

Purpose of the present invention just provides a kind of polypeptide vaccine for the treatment of diseased induced by high pathogenic avian influenza virus H 5 N 1, and the cell that it can infect at the highly pathogenic bird flu virus H 5 N 1 type specially destroys effectively, and human body is had no side effect.

The objective of the invention is to realize that by such technical scheme its aminoacid sequence is: aminoterminal-QIGNISIWV-c-terminus; With lysine is joint, a plurality of these aminoacid sequences is formed the cascaded structure of multicopy.

Normal human's peripheral blood lymphocytes (being called for short PBMC) does not generally have the molten broken function that is subjected to the cell of highly pathogenic bird flu virus H 5 N 1 infection of specificity, polypeptide provided by the invention is hatched jointly in external peripheral blood lymphocytes with the positive human body of HLA-A*0201, can increase these peripheral blood lymphocytes and be converted into the cytotoxic T lymphocyte quantity 45%-80% of (being called for short CTL), can also induce and activate the molten broken positive cell that infected by highly pathogenic bird flu virus H 5 N 1 of CTL, molten broken rate is 60%-88%.

Basic aminoacid sequence of the present invention derives from the proteic aminoacid sequence of N1 of the highly pathogenic bird flu virus H 5 N 1 of GENBANK report, adopt the principle and the method for protein epitope MOLECULE DESIGN, the N1 protein sequence of highly pathogenic bird flu virus H 5 N 1 carries out the CTL epitope analysis, mainly be based on quantification motif method and determine the proteic CTL epi-position of N1, specific practice is: from N1 albumen the 1st amino acids residue, intercept nonapeptide section or decapeptide section one by one, obtain 6 nonapeptide sections altogether.For each bar peptide section, in associate(d) matrix, search the attachment coefficient of each site amino acid residue, after multiplying each other, this nonapeptide attachment coefficient multiply by typical coefficient again, be the attachment coefficient of this nonapeptide or decapeptide and HLA-A*0201.At last, choose several the highest peptide sections of attachment coefficient as candidate CTL epi-position.Found aminoacid sequence of the present invention with the method, that is: aminoterminal-QIGNISIWV-c-terminus with function.

Synthetic aminoacid sequence method of the present invention all is existing mature technology, and it is made as follows:

Employing standard Fmoc scheme, the initial 0.0125mmol that selects for use, (ABI company produces the PSC resin, lot number A5F013), respectively according to claim 1,2,3 or 4 described sequence signatures, peptide chain is extended to the N end one by one from the C end, and the consumption of each amino acid starting material (production of American AB I company) is 0.1mmol.Each seed amino acid blocking group is: the amino Pmoc of being of each amino acid whose alpha protects all the other side chain protected groups, Arg (Mtr), tyr (tBu), Thr (tBu), Tyr (tBu), Asp (OtBu).Per step condensation all adds the amino acid whose carboxyl of HoBt/Dcc activates relay.Per step condensation is removed the Fmoc protecting group with the nmp solution that contains 20% hexahydropyridine; after peptide chain is synthetic; step according to the recommendation of ABI company; resiniferous peptide chain is added in the mixed reaction solution that is under the condition of ice bath composition of reactant liquor: crystallization benzoic acid 0.75g, ethylenediamine tartrate (EDT) 0.25ml, thioanisole (Thionaisole) 0.5ml, deionized water 0.5ml, trifluoroacetic acid 10ml.Continue to stir at ambient temperature, the response time is 4.5 hours, and peptide chain cracking from the resin is got off, and removes the kinds of protect base simultaneously.Mixed liquor is filtered through the glass filter of 4G, with resin and the blocking group that filters cutting-out, and with trifluoroacetic acid flushing reaction bulb and filter; with filtrate at normal temperatures low pressure be evaporated to 1～2ml, the 50ml that adds diethyl ether makes the polypeptide post precipitation, after the 6G filter filters; lyophilization, gained are peptide products.Above process all is to finish on ABI-431A solid phase automatic peptide synthesizer (produced in USA).

Synthetic polypeptide medicaments that the present invention relates to or vaccine can room temperature preservation, transportation, also can automatic batch production.

Use existing mature technology, the target cell that the N1 transfection of highly pathogenic bird flu virus H 5 N 1 is expressed in preparation is HEK293 cell;

Obtain the peripheral blood lymphocytes (being called for short PBMC) of the positive human body of HLA-A*0201 according to method well known in the art;

Cultivate above-mentioned PBMC cell after 24 hours, get suspension (2 * 10 ⁶/ ml) 4 bottles.Add aminoacid sequence polypeptide of the present invention (20 μ g/ml), add the equivalent complete medium and organize in contrast, 37 ℃, hatch under the 5%CO2 environment.Repeat this stimulating course after 7 days, repetitious stimulation is 3 times altogether.The last time stimulates the back to collect suspension cell on the 3rd day, these cells have the cytotoxicity CTL cell at the proteic target cell of N1 of highly pathogenic bird flu virus H 5 N 1, and count detection is found: use synthetic polypeptide medicaments or the vaccine that the present invention relates to can make the CTL cell quantity increase 45%-88% than matched group.

With transfection the HEK293 cell and the above-mentioned CTL cell of N1 gene of highly pathogenic bird flu virus H 5 N 1 hatch jointly, find this CTL cell can molten broken transfection the HEK293 cell of N1 gene of highly pathogenic bird flu virus H 5 N 1, molten broken rate obtains 60%-88%.

Owing to adopted technique scheme, the present invention has the advantage of being convenient to transport preservation and automatic batch production, it has the ability of activation specificity cell toxicity T lymphocyte (CTL), this Specific CTL Cells have kill and wound, molten broken target cell with N1 gene of highly pathogenic bird flu virus H 5 N 1, effect is obvious, and has no side effect.

(4) specific embodiment

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

Aminoacid sequence of the present invention is: aminoterminal-QIGNISIWV-c-terminus; With lysine is joint, a plurality of these aminoacid sequences is formed the cascaded structure of multicopy.

This polypeptide can stimulated in vitro HLA-A*0201 the peripheral blood lymphocytes of positive human body, can increase the quantity raising 50%-80% that these peripheral blood lymphocytes are converted into cytotoxic T lymphocyte (being called for short CTL), can also activate the cell of the H5 gene of the molten broken highly pathogenic bird flu virus H 5 N 1 of CTL, molten broken rate is 60%-88%.

Embodiment 1: its aminoacid sequence can for:

This polypeptide can stimulated in vitro HLA-A*0201 the peripheral blood lymphocytes of positive human body, can increase the quantity raising 60% that these peripheral blood lymphocytes are converted into cytotoxic T lymphocyte (being called for short CTL), can also activate the molten cell that breaks the H5 gene of highly pathogenic bird flu virus H 5 N 1 of CTL, molten broken rate is 83.10%.

Embodiment 2: its aminoacid sequence also can for:

This polypeptide can stimulated in vitro HLA-A*0201 the peripheral blood lymphocytes of positive human body, can increase the quantity raising 50% that these peripheral blood lymphocytes are converted into cytotoxic T lymphocyte (being called for short CTL), can also activate the molten cell that breaks the H5 gene of highly pathogenic bird flu virus H 5 N 1 of CTL, molten broken rate is 79.0%.

Embodiment 3: its aminoacid sequence can also for:

This polypeptide can stimulated in vitro HLA-A*0201 the peripheral blood lymphocytes of positive human body, can increase the quantity raising 55% that these peripheral blood lymphocytes are converted into cytotoxic T lymphocyte (being called for short CTL), can also activate the molten cell that breaks the H5 gene of highly pathogenic bird flu virus H 5 N 1 of CTL, molten broken rate is 68.0%.

Above-described aminoacid sequence method all is existing mature technology, and it is made as follows:

Below in conjunction with experimental example the present invention is explained as follows:

The proteic target cell model of N1 (HEK293 cell) of highly pathogenic bird flu virus H 5 N 1 is expressed in experimental example 1 preparation

1, key instrument: micro-High speed refrigerated centrifuge; MJ.Research pcr amplification instrument; The gel imaging analysis system; Electrophoresis system; FX-302 type camera bellows formula Ultraviolet Detector; YJ-875 SA type superclean bench; The THZ-C constant temperature oscillator; FA 1004 electronic balances; Health and happiness electric heating constant temperature water bath; ￠ 300 type pH meters.

2, main material: 1) bacterial strain: E.coli DH5 α.2) plasmid: HA/pIRES2-EGFP is the recombiant plasmid that contains the H5 of genes of interest H5N1; PIRES2-EGFP is the carrier for expression of eukaryon that contains the green fluorescent protein reporter gene, contains the Kana resistant gene.Available from Clontech company.

3, main solution preparation:

1) LB culture fluid: tryptone 10g, yeast extract 5g, NaCl 10g is dissolved among the 800mlddH20, adjusts pH to 7.4 with 5M NaOH, is settled to 1000ml, be distributed into 100ml after, autoclaving, 4 ℃ of preservations.

2) LB solid medium: add the 1.8g agar powder in the 100ml LB culture fluid, behind the autoclaving, bed board, 4 ℃ of preservations.

3) LB agar+Amp culture fluid: preparation LB, autoclave sterilization.Be cooled to about 50 ℃, adding Amp, to make final concentration be 100 μ g/ml, 4 ℃ of preservations.

4) LB agar+Amp solid medium: preparation LB solid medium, autoclave sterilization.Be cooled to about 50 ℃, adding Amp, to make final concentration be 100 μ g/ml, bed board, 4 ℃ of preservations.

5) LB agar+Kana culture fluid: preparation LB, autoclave sterilization.Be cooled to about 50 ℃, adding Kana, to make final concentration be 50 μ g/ml, 4 ℃ of preservations.

6) LB agar+Kana solid medium: preparation LB solid medium, autoclave sterilization.Be cooled to about 50 ℃, adding Kana, to make final concentration be 50 μ g/ml, bed board, 4 ℃ of preservations.

7) electrophoretic buffer (50 * TAE): Tris alkali 24.2g, 0.5M EDTA (pH8.0) 10ml, glacial acetic acid 5.71ml adds water to 100ml.Application concentration is 1 * TAE.

8) ethidium bromide (EB): in 20mlH20, dissolve 0.2gEB, keep in Dark Place in 4 ℃ behind the mixing.

9) 0.1M CaCl2:CaCl22H201.47g is dissolved in the 100ml water, autoclave sterilization, 4 ℃ of preservations.

4, reaction system:

P1(10μmol/L) 1μl

P2(10μmol/L) 1μl

dNTP(2.5mM) 1μl

10×Buffer 5μl

MgCl ₂(25mM) 3μl

pBR322/HPV16 1μl

pfu?Taq(5u/μl) 1μl

ddH ₂O?up?to 50μl

5, reaction condition:

lcycle 94℃ 2min

30cycle 94℃ 50sec

54℃ 30sec

72℃ 2min

1cycle 72℃ 10min

4℃ Hold

Get 4 μ l product after reaction finishes, in 1.5% agarose gel, carry out electrophoretic analysis.

6, the cultivation of HEK293 cell:

1) after cell is sent it to, places 37 ℃, 5%CO ₂Cultivate in the incubator, under inverted microscope, observe, when treating cell attachment to 70-80%, the cultivation of going down to posterity

2) discard cell culture fluid, PBS washing 2～3 times

3) contain 0.02%EDTA with 0.25% trypsin) digest and add rapidly 1640 culture medium that contain serum to suspended state and stop digestion

4) the centrifugal 5min of 500rpm/min

5) abandon supernatant, add fresh culture 4mL, the piping and druming re-suspended cell

6) branch is filled in 3 culture bottles, adds the 2mL culture medium in each culture bottle again

7, plasmid DNA transfection HEK293 cell:

Cell is prepared results and is in cell about 1 * 10 exponential phase of growth ⁵Individual cell inoculation makes cell reach 40-60% and merges in 6 porocyte culture plates, places 37 ℃, 5%CO2 incubator, serum-free culture 24 hours.After the transfection 48 hours, 6 well culture plates are directly placed under the fluorescence microscope, under the excitation wavelength of 479nm, excite green fluorescence, observe location and the expression of H5 albumen in cell of green fluorescent protein EGEP and H5N1.

Experimental example 2 external evoked specificity cytotoxic T lymphocytes (CTL)

1, reagent and material

1) contains the RPMI1640 culture medium of 10% hyclone.

2) PBS cleaning mixture.

3) recombination human interleukin (recombinant human IL-2).

4) specification is the measuring pipette of 1 milliliter and 10 milliliters.

5) 75% ethanol, aseptic cotton balls, tweezers.

6) microscope, horizontal centrifuge.

7) the positive blood of human body 20mL (4 * 5mL) of HLA-A*0201.Obtain peripheral blood lymphocytes (PBMC) according to method well known in the art.

2, method:

Cultivate the PBMC cell after 24 hours, get suspension (2 * 10 ⁶/ ml) 4 bottles.Add basic aminoacid sequence polypeptide of the present invention (20 μ g/ml) and equivalent complete medium, adding simultaneously rhIL-2 in the culture bottle, to make final concentration be to hatch under the 50U/mL, 37 ℃, 5%CO2.Repeat this stimulating course after 7 days, stimulate altogether 3 times.Between each the stimulation, add fresh RPMI1640 culture medium according to circumstances, all need blow and beat attached cell when stimulating each time to suspended state, culture bottle is changed in washing.Just can obtain cytotoxicity CTL cell according to this method.

Experimental example 3[ ⁵¹Cr] release test detects the ability of CTL cell lysis HEK293 cell

1, key instrument: YJ-875 SA type superclean bench; The horizontal centrifuge; Health and happiness electric heating constant temperature water bath; The constant temperature incubator; γ-calculating instrument

2, main experiment material: target cell the has been transfection HEK293 cell of H5 gene of H5N1; The effector lymphocyte is that basic aminoacid sequence polypeptide of the present invention and recombinant human il-2 work in coordination with the T lymphocyte that obtains behind the stimulation human peripheral blood mononuclearcell.Radiosiotope Na ₂ ⁵¹Cr O ₄The RPMI1640 culture medium; Hydrochloric acid (HCl); Specification is 6 holes, 96 hole culture bottles.

3, target cell is prepared: results are in the target cell (transfection the HEK293 cell of H5 plasmid of pIRES2-EGFP-H5N1) of exponential phase, and it is 2 * 10 that cell concentration is adjusted in the washing back ⁶/ ml.Get 1 * 10 ⁶Cell (0.5ml) adds Na ₂ ⁵¹Cr O ₄100 μ ci are hatched 2～3h for 37 ℃, and jolting is once gently for every 15min.With the RPMI-1640 washing that contains 10%FCS 3 times, each 800rpm is centrifugal, and 5min abandons supernatant, and re-suspended cell to concentration is 1 * 10 ⁵/ ml.In 96 well culture plates, every hole adds 100 μ l (1 * 10 ⁴Cell), 3 every part multiple hole, totally 42 holes.

4, the adding of CTL cell: every hole adds the CTL cell of the different cell concentrations of 100 μ l, and maximum release group adds the 1%mol/L HCL of 100 μ l, is 100% with this molten broken target cell efficient; Nature release group adds 100 μ l complete mediums, as negative control group.100 μ l supernatants are taken out in every hole, measure the cpm value according to method well known in the art on the γ calculating instrument.

The result shows: this polypeptide can stimulated in vitro HLA-A*0201 the peripheral blood lymphocytes of positive human body, can increase the quantity raising 45%-88% that these peripheral blood lymphocytes are converted into cytotoxic T lymphocyte (being called for short CTL), it is molten broken to the proteic cell of the H5 of H5N1 to activate CTL, and molten broken rate is 60%-88%.

The computer-reader form of the N1 amino acid series table of H5N1

＜110〉Lv Fenglin

＜120〉a kind of polypeptide vaccine for the treatment of diseased induced by high pathogenic avian influenza virus H 5 N 1

＜130〉do not have

＜140〉application number

<141>2008-10-8

<160>3

<170>Patent?In?Version?2.1

<210>1

<211>9

<212>PRT

＜213〉artificial sequence

<400>1

Gln ₁?Ile?Gly?Asn?Ile?Ser?Ile?Trp?Val ₉

<210>2

<211>9

<212>PRT

＜213〉artificial sequence

<400>2

Arg ₁?Ile?Gly?Ser?Gly?Asp?Val?Phe?Val ₉

<210>3

<211>9

<212>PRT

＜213〉artificial sequence

<400>3

Leul?Met?Leu?Gln?Ile?Gly?Asn?Met?Ile ₉

Claims

1. a polypeptide vaccine for the treatment of diseased induced by high pathogenic avian influenza virus H 5 N 1 is characterized in that its aminoacid sequence is: aminoterminal-QIGNISIWV-c-terminus; With lysine is joint, a plurality of these aminoacid sequences is formed the cascaded structure of multicopy.

2. the polypeptide vaccine of treatment diseased induced by high pathogenic avian influenza virus H 5 N 1 as claimed in claim 1 is characterized in that its aminoacid sequence is:

3. the polypeptide vaccine of treatment diseased induced by high pathogenic avian influenza virus H 5 N 1 as claimed in claim 1 is characterized in that its aminoacid sequence is:

4. the polypeptide vaccine of treatment diseased induced by high pathogenic avian influenza virus H 5 N 1 as claimed in claim 1 is characterized in that its aminoacid sequence is: