CN102190707A

CN102190707A - Dendritic cell (DC)-based hepatitis B virus T epitope peptide

Info

Publication number: CN102190707A
Application number: CN2011100738623A
Authority: CN
Inventors: 罗进; 闫小君
Original assignee: Individual
Current assignee: JIANGSU ANTAE BIOGENE TECHNOLOGY Co Ltd
Priority date: 2011-03-25
Filing date: 2011-03-25
Publication date: 2011-09-21

Abstract

The invention relates to the fields of protein engineering, gene engineering and biomedicines, in particular to a dendritic cell (DC)-based hepatitis B virus T epitope peptide. The technical scheme is that: the DC-based hepatitis B virus T epitope peptide is characterized by consisting of nine amino acid residues in the amino acid sequence of QLFHLCLII. The basic amino acid is taken as a framework, same preset amino acid sequences are simultaneously synthesized on the framework by a solid-phase synthesis technology, a hepatitis B virus is taken as a design target, and the DC-based hepatitis B virus specific epitope peptide is produced by the synthesis technology; the DC is loaded with the epitope peptide in vitro, and the mature DC is injected into bodies of a specific group to specifically generate effects of removing in-vive virus and improving clinical symptoms aiming at hepatitis B virus infection, particularly chronic hepatitis.

Description

A kind of hepatitis B virus T epitope peptide based on the DC cell

Technical field

The present invention relates to protein engineering, genetically engineered and biomedicine field, be specifically related to a kind of hepatitis B virus T epitope peptide based on the DC cell.

Background technology

There are 400,000,000 people's hepatitis b virus infections of surpassing in the whole world, and has every year 1 million people to die from the infection syndromes, comprises liver cirrhosis, hepatocellular carcinoma or other complication.Prior art adopts Chinese medicine or western medicine hepatitis B mostly, though certain curative effect is arranged, but can not effect a radical cure hepatitis B or other complication, especially can not effectively block because of the hepatitis b virus infected hepatic fibrosis that causes and sclerosis, inhibition of hepatitis b virus and give breastfeeding newborn infant by milk-born infection for fetus, inhibition of hepatitis b virus by placental infection.

The peptide synthetic technology has some mechanisms all to have at home and abroad to carry out at present, and the I of histocompatibility complex (MHC) class restrictive cell toxicity T cell (CTL) plays a part very important in control intra-cellular pathogens and growth of tumour cell.Analyze by the protein essential property, hydrophobicity analysis is striden the prediction of film district, signal peptide prediction, and the Subcellular Localization prediction, the antigenic site prediction can be made preliminary judgement and prediction to the character of gene coded protein.Especially by hydrophobicity analysis with whether stride film district prediction can predicted gene be membranin.Protein fragments in these 212 amino acid and the ncbi database is carried out Blast, and the result shows that 100 peptides and target peptide have homology, and wherein 16 100%, 84 of homologys are 99%, and these 100 peptides are the precore/core albumen of HBV.Utilize CLC Protein Workbench software to these 212 amino acid whose analyses, can obtain the general biological characteristics of this protein fragments.

Cell immune response plays a part crucial in the immunne response process.After exogenous antigen is caught by antigen presenting cell, proteantigen is degraded to polypeptide fragment in the endocytosis system, immunogenic polypeptide wherein is that t cell epitope combines with MHC I quasi-molecule, be transported to the APC surface for TXi Baoshouti identification, thereby activated cell immunne response, the performance immunoregulation effect, the design that will help the subunit vaccine molecule to the understanding of its aminoacid sequence feature is with development and increase immune further understanding.Therefore, to the prediction of t cell epitope and carry out structure of modification on this basis and have that important theory is worth and practical significance.

Summary of the invention:

In order to overcome the defective of prior art, the purpose of this invention is to provide and a kind ofly can effectively treat hepatitis B and various complication thereof, the blocking-up inhibition of hepatitis b virus gives breastfeeding neonatal a kind of hepatitis B virus T epitope peptide and analogue thereof based on the DC cell by milk-born infection for fetus, inhibition of hepatitis b virus by placental infection.

For achieving the above object, the technical solution used in the present invention is:

A kind of hepatitis B virus T epitope peptide based on the DC cell, it is characterized in that: described hepatitis B virus T epitope peptide based on the DC cell is made up of 9 amino-acid residues, and its aminoacid sequence is: QLFHLCLII(glutamine-Ile-Phe-HIS-LEU-halfcystine-leucine-Isoleucine-Isoleucine).Select hepatitis B virus core protein as purpose antigen.

With respect to prior art, the present invention is a skeleton with the basic aminoacids, takes solid phase synthesis technique, synchronously synthetic simultaneously identical default aminoacid sequence on this skeleton, and be the design target with the hepatitis B virus, and adopt the hepatitis B virus specificity epitope peptide of synthetic technology production based on the DC cell; By this epitope peptide of DC cells in vitro load, and with in the sophisticated DC cell infusion specific crowd body, specific generation is at the harm that hepatitis B virus infection brought, especially at the removing body inner virus that is produced of chronic viral hepatitis B with improve the effect of clinical symptom; Blocking-up is because of hepatitis b virus infected hepatic fibrosis that causes and sclerosis; Inhibition of hepatitis b virus is given fetus by placental infection; Inhibition of hepatitis b virus is given breastfeeding newborn infant by milk-born infection.

Description of drawings

Fig. 1 is the mass spectrum of epitope peptide,

Fig. 2 be in the nonapeptide each site amino-acid residue in conjunction with HLA-A2 molecule priority,

Fig. 3 be in the nonapeptide with in conjunction with or the relevant amino-acid residue of debond,

Fig. 4 is system of polynomials numerical table (HLA-A2),

Fig. 5 is the patient data table,

Fig. 6 is the patient data table,

Fig. 7 is the monocytic phenotype of chronic viral hepatitis B patient,

Fig. 8 cultivates fate,

Fig. 9 is the change (A-F) that peptide impacts back dendritic cell form, the negative contrast of G,

Figure 10 is a patient treatment effect table,

Figure 11 is the level of treatment 48 all blood plasma HBV DNA,

Figure 12 is the level of treatment 48 all plasma A LT.

Embodiment

Hepatitis B virus T epitope peptide based on the DC cell of the present invention, its aminoacid sequence is: QLFHLCLII, molecular weight are 1150.

Epitope peptide adopts standard Fmoc scheme to synthesize, and behind the HPLC purifying, its purity is greater than 98%, and referring to Fig. 1, mass spectroscopy also confirms that its molecular weight meets theoretical value.

The synthetic method that the present invention is based on the hepatitis B virus T epitope peptide of DC cell is:

One, the design of epitope peptide and screening

1, hepatitis B virus antigen

The antigenic cAg of B virus is the major antigen that hepatitis B virus causes ctl response in vivo, nucleotides sequence is classified 639bp as, proteins encoded is 212 amino acid, MQLFHLCLIISCSCPTVQASKLCLGWLWGMDIDPYKEFGASVELLSFLPSDFFPSI RDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLAT W VGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIR TPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQ

2, homology analysis

Protein fragments in these 212 amino acid and the ncbi database is carried out Blast, to determine the proteinic specificity of this section.

3, the general characteristic of HBV cAg prediction

Application software CLC Protein Workbench3 version and Signal P3.0 software are predicted the coded protein of HBV cAg, comprise hydrophobicity, wetting ability, antigen

Property, membrane spaning domain, iso-electric point and the potential cleavage site of signal peptide.

4, utilize SYFPEITHI to carry out the tentative prediction of CTL epi-position

5, PREDEPP program prediction

6, motif method

(1) different MHCI class has different requirements to the composition of institute's bonded peptide, but wherein play a major role so to peptide chain and MHCI quasi-molecule what be combined with material impact is two main anchor point residues of peptide chain, they polypeptide be essential during the high-affinity of MHCI quasi-molecule combines.For HLA-A2, two anchor points of nonapeptide lay respectively at the 2nd amino acids residue and the C-terminal (the 9th amino acids residue) of peptide chain; And the 2nd amino acids residue should be leucine (L) or methionine(Met) (M), and the 9th amino acids residue should be Xie Ansuan (V), different bright amino acid (I) or L.Referring to Fig. 2, the possibility that meets the polypeptide of these conditions and HLA-A2 restricted CTL epitope is bigger.

(2) effect of secondary anchor point residue is except the essential factor of two main anchor point residues, and other amino-acid residues in the polypeptide chain combine with the high or medium avidity of HLA-A2 molecule polypeptide also very important influence.Referring to Fig. 3, on other positions of nonapeptide, the different aminoacids residue has different influences to polypeptide with the combination of HLA-A2 molecule, and the amino-acid residue that has helps combination, and what have then is unfavorable for combination.Ideal and HLA-A2 bonded CTL epi-position (promptly having high or medium avidity) should not comprise and be unfavorable for the bonded amino-acid residue, or help the bonded residue more than being unfavorable for the bonded residue.

7, polynomial expression program prediction epitope peptide

The polynomial expression scheme is meant when certain residue R appears at the I position of certain peptide, does not consider under the situation of other peptide sequences, and this residue provides a constant Ri for whole peptide with the free energy that combines of MHCI quasi-molecule, is used in the IC that the I position is a large amount of polypeptide of residue R ₅₀The mean value of negative log10 value estimate this constant.IC ₅₀Be defined as peptide to be measured and 50% control peptide in control peptide-MHC mixture replaced the concentration of getting off.20 seed amino acid residues are listed in the given a certain nonapeptide of Fig. 4 in the Ri of 1-9 position value respectively, and with the Ri value addition of its all amino-acid residue correspondences, selecting score value is the possible back peptide that selects greater than the antigen peptide of selected threshold value.Referring to Fig. 4, the present invention selects 22.

Two, the evaluation of epitope peptide, modification and synthetic

From 5 CTL epitope peptides of software prediction, search out and have the antigens with higher bonding properties; and it is used the B cell epitope; Th epi-position and palmitoyl groups are modified, and the Fmoc scheme is synthetic, lay the foundation for the exogenous small molecules epitope peptide of further discussion enters the reaction of cell activation MHC1 class.The solid state chemistry of polypeptide is synthetic to mainly contain two kinds of strategies: Boc solid phase synthesis strategy and Fmoc solid phase synthesis strategy.Amino acid whose alpha-amino blocking group is selected the weak acid sensitive group in the Boc method, and the blocking group of side chain active group is selected the strong acid sensitive group for use.Amino acid whose alpha-amino blocking group is selected the weak base sensitive group in the Fmoc method, and the blocking group of side chain active group is selected the weak acid sensitive group for use.

1, the Fmoc method solid phase synthesis of antigen peptide and mark (sequence according to antigen peptide is synthetic successively to the ammonia end from the carboxylic end).

(1) being connected of first amino acid and resin: 2-Chlorotrityl Chloride Resin resin 1g is placed the synthetic post of peptide of dried and clean, add 8mlDCM, swelling 5min, vacuum is inhaled and is abandoned solvent.Get 2 mmol Fmoc amino acid and 5 mmol DIEA respectively, be dissolved in 8 ml DCM, and be incorporated in the resin, room temperature is shaken reaction 60 min gently.Vacuum is inhaled and is abandoned solvent.With 10 ml DMF washing resins 2 times, each 2 min.Add 10 ml DCM/MeOH/DIEA (80:15:5), shake reaction 10min gently, vacuum is inhaled and is abandoned solvent.Repeat once.With 10 ml DMF washing resins 3 times, each 2 min.Vacuum is inhaled and is abandoned solvent, N ₂Dry up.

(2) the coupling rate of first amino acid and resin is measured: accurately take by weighing 2mg exsiccant Fmoc amino acid-resin and place cuvette, add 3ml 20% piperidines/DMF, shake reaction 10min gently, return to zero the 290nm absorbance value of ultraviolet spectrophotometer working sample as blank with 20% piperidines/DMF.Replication 2 times is averaged.The coupling rate is calculated by following formula:

Coupling rate (mmol/g)=(Abs sample)/(example weight mg * 1.75) ... formula (1)

(3) deprotection of Fmoc group: adding 10 ml deprotection (DEBLOCK) reagent in resin (25% piperidines/DMF), mixing, room temperature is shaken reaction 5 min gently.Abandon solvent, with 10 mlDMF washing resins 3 times, each 2 min.With 6 ml washed with isopropyl alcohol resins 3 times, each 5 min.With 6 ml hexane wash resins 3 times, each 5 min.Vacuum is inhaled and is abandoned solvent.Resin sample takes a morsel; with the free amine group content on ninhydrin (Kaiser method) the rapid determination resin: with resin 2 ml with washing with alcohol 3 times; add 2 5% triketohydrindene hydrates, 80% phenol and KCN(2 ml 0.001MKCN:98 ml piperidines respectively); abundant mixing; 120 ℃ of heating 6 min, the protective reaction degree of judgement Fmoc group.

(4) second amino acid whose linked reactions: the in-situ activation method is adopted in second amino acid whose connection, get the Fmoc amino acid of 2 mmol, the TBTU of 4.0mmol and the HOBT of 4.0mmol, the DIEA that adds minimum DMF dissolving back adding 5 mmol, fully behind the mixing, be added in the resin that takes off the Fmoc group.Room temperature is shaken reaction 60 min gently.Vacuum is inhaled and is abandoned solvent.With 5 ml methanol wash resins 3 times, each 5 min.With 10 ml DMF washing resins 3 times, each 2 min.Vacuum is inhaled and is abandoned solvent.The resin sample that takes a morsel is carried out the triketohydrindene hydrate chromogenic assay.Measure the coupling rate.

(5) extension of peptide chain: slough the Fmoc blocking group of a last amino acid N end with 10 ml DEBLOCK reagent, 10 ml DMF washing resins 3 times, vacuum is inhaled and is abandoned solvent.The resin sample that takes a morsel is carried out the triketohydrindene hydrate chromogenic assay.According to the next amino acid of (3) method coupling.Repeat Fmoc blocking group deprotection and amino acid linked reaction, obtain required polypeptide chain until coupling.

(6) side chain of peptide chain goes to protect and the cutting from the resin: will synthesize the resin of good whole aminoacid sequences, and use 6 ml washed with isopropyl alcohol resins 3 times again, each 5 min after washing with 10 ml DMF.With 6 ml hexane wash resins 3 times, each 5 min.Vacuum is inhaled and to be abandoned N behind the solvent ₂Dry up, put into the cracking container.The 1g resin adds 25 ml cutting reagents (TFA: thio phenyl methyl ether: water: phenol: EDT=82.5:5:5:5:2.5 v/v), room temperature cleavage reaction 2 h shake mixing frequently, and reacted mixed solution filters resin through glass filter, collect the cleavage reaction mixed solution, use TFA washing resin 3 times.Reaction mixture is shifted in a round-bottomed flask, use the ether of equal-volume precooling to wash collecting precipitation 4 times.Promptly obtain the synthetic antigenic peptide crude product after the drying.

(7) desalination of synthetic antigenic peptide: antigen peptide crude product adding distil water is dissolved.Take by weighing AmershamG-25 gel 15 g, adorn post after the swelling, the pillar after installing with 50ml distilled water balance, after balance is good, is gone up sample 3-5 ml earlier at every turn, carries out wash-out with distilled water, and the uv-spectrophotometric instrument detects 220 nm place uv-absorbing, presses the peak and collects antigen peptide.

(8) HPLC purifying antigen peptide: the waters4000 preparation HPLC high performance chromatograph separation and purification antigen peptide of using waters company.Chromatographic column is the chromatographic column of radially pressurizeing (25 * 100,15 μ m, DELTA PAK C18 filler), and elution system is: A liquid: 5% acetonitrile solution (containing 0.1%TFA); B liquid: 95% acetonitrile solution (containing 0.08%TFA).Hand sampling, each sample introduction 1 ml, flow velocity 4 ml/min, linear gradient, in the 45min, B liquid is raised to 50% from 5%, is raised to 95%B liquid then in 5 min and does last wash-out.Uv-absorbing is detected at 220 nm places, presses the peak and collects component, is used for mass spectrometric detection.The component that molecular weight detection is correct is collected, and vacuum-freeze-dry becomes the pure product that need, and is standby.

(9) mass spectrum is identified antigen peptide: adopt laser to resolve mass spectrum and identify antigen peptide, data analysis software be G2025A Software A.02.01, method is carried out in strict accordance with operational manual, detects the molecular weight of synthetic antigenic peptide.

2, the modification of epitope peptide

Add that at the N-of t cell epitope end glycan molecule, lipopeptid or palmitoyl can increase the location of small-molecular peptides on cytolemma, stride film and penetrativity.And helper T cell epi-position or B cell epitope help to strengthen the biological function of t cell epitope, cause the ability of ctl response in order to search out best t cell epitope by extrinsic pathway, we selected Th, B and three kinds of modification groups of palmityl Serine with collaborative, coupling and-multiple scheme that AAA-flexibly connects carries out the modification of t cell epitope peptide.

3, epitope peptide is synthetic

According to synthetic above-mentioned 9 peptides of peptide synthetic step, synthetic peptide is a lyophilized powder, the 100mg/ bar once more.Synthetic antigenic peptide is all through HPLC purifying (purity＞98%), and the mass spectrum of going simultaneously checks order.The freeze-drying polypeptide is positioned over-20 ℃ of preservations.

Three, treatment chronic viral hepatitis B patient's effect

The antigenic autologous dendritic cell of load (antigen presenting cell) (ADCs) has been used for immunotherapy.We adopt ADCs to treat 380 routine chronic viral hepatitis B patients, in treatment 48 all backs each patient's virusology, biochemical index and serological reaction are assessed, report shows: the HBeAg negative patient of treatment end back 46.36% and 3.13% HBeAg positive patient HBV-DNA detection by quantitative result are negative; HBeAg negative patient (P=0.007) and HBeAg positive patient (P=0.003) gpt transfer to normally entirely.Results suggest: the ADC vaccine is to chronic viral hepatitis B patient immune function's reconstruction, and the improvement of virusology, biochemistry and serological index is extremely effective.

1, patient's situation

380 routine patients among the present invention, the 185 routine HBeAg positives wherein, 195 routine HBeAg feminine genders, nobody carried out antiviral or the immunoregulation druge treatment in 6 months before the present invention.Patient data is seen Fig. 5 and Fig. 6.

Referring to Fig. 6, the 185 routine HBeAg positives are arranged, 195 routine HBeAg negative patients.185 routine HBeAg male patients: 160 routine HBV-DNA 〉=1000 copies/mL(73 example ALT 〉=40 IU wherein, 87 routine ALT＜40 IU), 20 routine HBV-DNA＜1000 copies/mL(22 example ALT 〉=40 IU, 3 routine ALT＜40 IU).195 routine HBeAg negative patients: 110 routine HBV-DNA 〉=1000 copies/mL(43 example ALT 〉=40 IU wherein, 67 routine ALT＜40 IU), 85 routine HBV-DNA＜1000 copies/mL(55 example ALT 〉=40 IU, 28 routine ALT＜40 IU).

2, the propagation of the selection of CD14+ and DC

Referring to Fig. 7, adopt leukapheresis to isolate patient's peripheral blood lymphocytes, the purity that the CD14+ cell is selected reaches the scope of 93%(91 – 95%) (Fig. 7 b1 – b3). after through GM-CSF and IL-4 processing, cell becomes tangible dendron shape, at the 10th day, most of cell expressing costimulatory molecules mark CD86, CD40, CD80 and HLA-DR, MHC-I molecule (Fig. 7, c1 – c5).Referring to Fig. 8, fresh DC cell and the immature cell that thaws are detected, the differential expression of mark is not remarkable.The level of costimulatory molecules mark CD86, CD40, CD80 and HLA-DR, HLA – I all increased in 10 days, and still, the level of all molecular marked compounds was promptly impacted in later 12 hours with peptide and reduced fast at the 11st day.

Cultivate fate

Referring to Fig. 8, the 11 day, negative Quality Control kept low value.At the 9th day, cytomorphosis, molecule marker CD14 reduces fast, and other molecule marker is as CD40, CD80, CD86, HLA-I, HLA-Dr be in the increase state at preceding 9 days, but at the 10th day, the 11st day peptide impacted and begins later to reduce.

Referring to Fig. 8, we to peptide-labeled, carry out cellular localization with FITC, adopt the burnt microscopy of copolymerization to determine that the DC cell is to HBV peptide absorbing state.Analyze and show: peptide will be absorbed by the DC cell in 30 minutes.At first, can see fluorescence at cell paste, nucleus be can't see fluorescence; After 2 hours, peptide can be diffused into dendron via cell space; After 12 hours, detecting fluorescence at dendron and cell space can disappear gradually.Do not observe fluorescence at nucleus.

Referring to Fig. 9, peptide impacts the change (A-F) of back dendritic cell form, the negative contrast of G.A-F is respectively with peptide and impacted 30 minutes, and 1 hour, 2 hours, 4 hours, the change of 6 hours and 12 hours dendritic cell forms.

3, serological reaction

Among the 185 routine HBeAg male patients, during 24 weeks, 22.16% (41/185) routine HBeAg turns out cloudy in treatment, and 16.21% (30/185) serology conversion (producing HBeAb) has taken place; Treatment is during 48 weeks, and 29.73% (55/185) routine HBeAg turns out cloudy, and 21.62% (40/185) serology conversion (producing HBeAb) has taken place.

In the 195 routine HBeAg negative patients, during 24 weeks, 8.21% (16/195) routine HBsAg turns out cloudy in treatment, and HBsAg serology transformation efficiency (HBsAg turns out cloudy or HbsAb occurs) is 1.03% (2/195); Treatment is during 48 weeks, and 10.26% (20/195) routine HBsAg turns out cloudy, and HBsAg serology transformation efficiency (HBsAg turns out cloudy or HbsAb occurs) is 2.56% (5/195) (Table 2).

4, virusology reaction

HBeAg male patient, in 24 whens week of treatment, real-time fluorescence quantitative PCR detects the HBV-DNA level 0.62 log copies/mL that on average descends, and 8.13% (13/160) patient HBV-DNA is undetected, but HBV-DNA is all negative before these patient treatments; In 48 whens week of treatment, the HBV-DNA level 0.88 log copies/mL that on average descends, 3.13% (5/160) patient HBV-DNA is undetected.In 24 whens week of treatment, 20% (32/160) the patient HBV-DNA level 2log10 that descended, in treatment during 48 weeks, 44.38% (71/160) patient HBV-DNA level, 2 log10 that descended.

Referring to Figure 11, the HBeAg negative patients, in 24 whens week of treatment, the HBV-DNA level 3.24 log copies/mL that descended, 64.55% (71/110) patient HBV-DNA is undetected; In 48 whens week of treatment, the HBV-DNA level 3.24 log copies/mL that descended, 50% (55/110) patient HBV-DNA is undetected.Show thus: the HBeAg negative patients is bigger rapider than the decline of HBeAg male patient HBV-DNA level.

All numbers of treatment

Referring to Figure 11, treat the level of 48 all blood plasma HBV DNA.Be limited to 1000copies/ml under detecting.

The conversion of biochemical indicator

Referring to Figure 12,195 routine ALT levels are higher than 40 IU/mL patients among the present invention, and during 48 weeks, its ALT of 92 routine patients (50.26%) recovers normal in treatment; 185 routine ALT levels are lower than 40 IU/mL patients, during 8 weeks, have 7 examples to be higher than normal value in treatment, and 12 weeks were not passed through any treatment afterwards, and its ALT level recovers normal again.During treating, there is the normal patient ALT of 33 examples (16.92%) ALT the property a crossed rising to occur among the 195 routine patients, finish to recover normal again to treatment.

Referring to Figure 10, after 48 weeks of treatment, HBeAg negative patient (P=0.007) and HBeAg positive patient (P=0.003) ALT are tending towards normally, and be evident in efficacy, has 69%HBeAg negative patient ALT to recover normal, and it is normal that 30.5% HBeAg positive patient ALT recovers.

All numbers of treatment

Referring to Figure 12, by the treatment in 48 weeks, the level of plasma A LT.Detect and be limited to 40 IU/L under minimum.

Association response

During 48 weeks, have 25 examples (13.51%) to reach complete reaction (CR) in the 185 routine HBeAg positive patients: HBV-DNA is negative, and ALT is normal in treatment, and HBeAg serology occurs and transforms.135 examples (72.97%) patient reaches partial reaction (PR): HBV-DNA is negative, and ALT is normal, and HBeAg serology do not occur and transforms.25 examples (13.51%) patient no response (not reaching above standard).In addition, 76 examples (47.51%) patient virusology occurred and has replied, and 55 examples (29.73%) the HBeAg serology occurs and transform, and biochemical responses appears in 29 examples (30.5%).

Referring to Figure 10,195 routine HBeAg negative patients, CR appears in 105 examples (53.85%), and PR appears in 53 examples (27.18%), 37 examples (18.97%) no response.In addition, 37 examples (18.97%) patient virusology occurs and replys, and 20 examples (10.26%) patient the HBsAg serology occurs and transforms, and biochemical responses appears in 69 examples (69%).

It is to treat (wherein: 85 routine HBeAg feminine genders, the 25 routine HBeAg positives) first that 110 examples are arranged among the 380 routine patients, and 270 routine patients accepted other treatment (wherein: 115 routine HBeAg feminine genders, the 155 routine HBeAg positives) in the past.

Drug resistance analysis

The patient carries out gene mutation analysis in treatment during 48 weeks, finds not have new point mutation to take place.

Security, side effect and adverse drug reaction

To not finding any allergic phenomena during the patient monitoring of all registrations, do not observe fash or expiratory dyspnea, the low fever appears in 10 routine patients, but can both self-healing in 24 hours.

Statistical study shows: the removing of HBeAg male patient virus during the DC vaccine therapy is not significantly (P=0.273) statistically, on the HBeAg negative patients statistics (P=0.001) evident in efficacy.The HBeAg negative patients HBV-DNA level 3.24 log copies/mL(virus index that descended descends fast, and the HBeAg male patient HBV-DNA 0.88 log copies/mL that only descended).For the HBeAg negative patients, after 24 weeks of treatment, during 48 weeks, the negative conversion rate of HBV-DNA reaches 50%; And the HBeAg negative patients is adopting IFN-α treatment in the time of 6 months, and the negative conversion rate of HBV-DNA is 37%, and keeping of such negative conversion rate only is 24%; Adopt the adefovir ester treatment after 1 year, the negative conversion rate of HBV-DNA is 28%.The old report that waits, HBeAg male patient, before and after treatment, the HBV-DNA 1.771 ± 2.39copies that descends.Compare with present pharmacological agent, the DC vaccine is in that to fall virus load quicker, stable and lasting.Though HBeAg is positive and negative patients virus sweep rate is different,, the DC vaccine can trigger a kind of powerful the CD8+CTL(HBeAg positive and negative patients).HBeAg male patient, duplicating of body inner virus can influence the effect that the DC vaccine is removed virus, presents the low reaction state.Therefore, in order to strengthen the effect of DC vaccine therapy, other antiviral treatment of HBeAg male patient best incorporated.HBeAg negative patients among the present invention, wherein 58 routine DNA〉104 copies/mL, 52 routine DNA are at 103 –, 104 copies/mL, remaining 85 routine DNA＜103/mL.Negative or " immune control " state that has the patient of HBsAb all to belong to of all DNA＜103/mLHBsAg.The HBeAg feminine gender of 20 routine HBV-DNA＜1000/mL or HBeAb negative patients have 20% antibody to occur.The positive HBV-DNA of 55 routine HBeAg feminine genders or HBeAb〉patient of 1000 copies/mL, have 45.45% (15/55) biochemical responses to occur, 45.45% (15/55) has virusology to reply, and 27.27% (9/55) occurs replying fully.

It is an important indicator that suppresses hbv replication that the HBeAg serology transforms.380 routine patients among the present invention, after 48 weeks of treatment, the ratio of the disappearance of HBeAg and generation serology transformation efficiency is respectively 29.73% (55/185) and 21.62% (40/185), has 10 routine patient HBeAg disappearance HBeAb to occur.Adopt the DC vaccine therapy, the transformation efficiency of HBeAg is higher than the other drug treatment: IFN-α (25%), lamivudine (16%) and adefovir ester (12%).The negative conversion rate of HBeAg negative patients HBsAg is 10.26% (20/195), and IFN-α treatment only is 7.8%.The seroconversion of HBsAg (HBsAg disappears, and HbsAb occurs) is 2.56% (5/195).The above Notes of Key Data: the T cell activation patient's that the DC vaccine that stimulates through specific activation peptide can be by exciting CD4+ humoral immunization, rebuild body's immunity.

For the HBeAg negative patients, the present invention has an important breakthrough.We assess 195 routine HBeAg negative patients, finish back clinical efficacy significantly (P=0.001) in the research of 48 weeks.We show data: evident in efficacy for the HBeAg negative patients based on target autogenous cell vaccine.We adopt flow cytometry to assess the level of CD3+, CD4+ and CD8+ of 28 routine HBeAg positive patients, and (these patients' HBV-DNA level is all at 105-108 copies/mL, the ALT level is at 80-200IU/L), after through 2 months DC vaccine therapy, the quantity of CD8+ cell has increased by 10% at least, significant difference (P＜0.05), CD3+ and CD4+ cell difference be (P〉0.05) not significantly.

Claims

1. hepatitis B virus T epitope peptide based on the DC cell, it is characterized in that: described hepatitis B virus T epitope peptide based on the DC cell is made up of 9 amino-acid residues, and its aminoacid sequence is: QLFHLCLII.