CN100391536C - Therapeutic vaccine composition for hepatitis B - Google Patents

Abstract

The invention relates to a vaccine composite used to treat hepatitis-B, wherein it contains 10-20 mug recombined HBsAg protein vaccine, 10-20 mug recombined interleukins 12, and 0.9-1.8 million bacillus pyocyaneus, and the left is water in each unit ml. The inventive composite can activate several immunity targets as NKT, cell, NK cell, etc, to break HBV immunity resistance, rebuild cell immunity function, and restrain HBV.

Description

A kind of therapeutic vaccine composition for hepatitis B

Technical field

The present invention is a kind of biological preparation, is meant a kind of therapeutic vaccine composition for hepatitis B that is used for the chronic hepatitis B treatment especially.

Technical background

Chronic hepatitis B is one of common infectious disease of China, and China belongs to the high popular district of hepatitis B virus (HBV), and it is a serious public health problem that HBV infects.2004, CDC shows the blood examination result of Chinese residents nutrition in 2002 and investigation of health conditions, the prevalence rate of HBsAg is up to 9.09% among the general crowd of China, infer in view of the above, China has about 1.2 hundred million people of chronic HBV infection person, wherein about 3,000 ten thousand examples of chronic hepatitis B now.Chronic hepatitis B has brought heavy financial burden for country and individual, and China every year is because of about 9,000 hundred million yuans of chronic hepatitis B direct and consequential damage.Should be understood that aspect treating chronic hepatitis B and do not make a breakthrough as yet.

The overall goal for the treatment of hepatitis is: long term inhibition or eliminate HBV to greatest extent, alleviate necrosis of hepatocyte inflammation and hepatic fibrosis, delay and stop progression of disease, reduce and prevent that liver from losing the generation of compensatory, liver cirrhosis, hepatocarcinoma, thereby make the life better quality and prolong the time-to-live.The chronic hepatitis B treatment mainly comprises antiviral, immunomodulating, fibrosis and symptomatic treatment.

The antiviral drugs that China SFDA approval at present is used for the treatment of chronic hepatitis B has interferon and nucleoside analog (comprising lamivudine, adefovir ester and Entecavir), though these medicines play an important role to suppressing virus replication, exist no thoroughness.Generally acknowledge that at present " virusology that regular antiviral therapy finished after 1 year is replied " is as the major criterion of estimating the chronic hepatitis B therapeutic effect, existing antiviral treatment result effective percentage generally is about 30%, have only small number of patients obtain to virus continue reply.

The influence factor that the treatment of chronic hepatitis B is replied is very complicated, it has related to the age of patient infection HBV, the mode that infects, infect the heterogeneity (different genotype and serotype) of HBV, the carrying capacity of HBV, the replication rate of HBV, the state of immune response of the variation of HBV and the immunosuppressive action of HBV and body be the functional level of dendritic cell and T cell particularly.The use that should be understood that antiviral drugs is the important means of treatment.And the development of nucleoside analog has obtained bigger progress in recent years, but this class medicine can only suppress hbv replication, can't remove HBV in human body completely.We should extremely pay close attention to the important function of immunomodulator in treating chronic hepatitis B, although also lack hepatitis B specific active immunotherapy method at present, but should reasonably use existing immunomodulator, make great efforts to develop new immunoregulation medicament, therapeutic vaccine, new vaccine adjuvant and some cytokine, transferring after all and inducing the immune clearance mechanism of body self is the important channel of treatment chronic hepatitis B.

Inducing in a word and keep the HBV antigen-specific immune responses, break immunologic tolerance and rebuild immunity of organism mechanism, have important treatment meaning, is the most important theories foundation and the approach of the immune modulating treatment that infects of exploitation persistence HBV.The key concept of immune modulating treatment is to attempt to induce in most of HBV chronic infectious patients to keep the HBV antigen-specific immune responses, and this to be antiviral drugs be beyond one's reach target, in all more options of these immune modulating treatment methods, therapeutic hepatitis B vaccine is the emphasis and the focus of research undoubtedly.

The dna vaccination of developments such as development hepatitis b surface antigen protein vaccines such as the therapeutic hepatitis B vaccine of China's development still is in the MAP small peptide vaccine of the TT830HBc 18-27 epi-position composition of developments such as vaccine such as anti-HBs antibody complex such as development HBsAg-such as clinical observation stage: Wen Yumei etc., Wu Yuzhang, Zhang Yijun and Chen Guangming is carrying out I/II phase clinical observation.These different development persons common characteristic are attempt epi-position and glycosyl side chain ornamental equivalent or change vaccine forms by increasing HBV structure or non-structural protein, be aided with approach development novel therapeutic hepatitis B vaccines such as heterogeneity adjuvant, wish to break immunologic tolerance, rebuild immunne response and particularly induce and keep cellullar immunologic response based on the specific CTL of HBV, the clinical efficacy of these therapeutic hepatitis B vaccines awaits more and the clinical observation of long period.

The effect of nonspecific immunity mechanism in viral hepatitis of NK cell and NKT cell in recent years, particularly the liver autarcesiology more and more comes into one's own to the influence that HBV infects, NK cell, NKT cell, gamma delta T cells account for about 2/3 of liver lymph sum in the liver, so huge natural immunity lymphocyte HBV infect and immunity is caused a disease and treatment in effect, cause showing great attention to of scholar.Traditional theory thinks that Th1 cell and CD8+CTL play a leading role in HBV infection, immunologic injury, virus sweep.Hepar damnification and the virus sweep that HBV causes but can this class T cell that only accounts for liver lymphocyte sum 1/3 undertake the whole thing is worth deeply observing.NK cell and NKT cell are proved fully by the secretion of cytokine and the laboratory that is replicated in of cytotoxicity inhibition HBV, should be understood that especially the cytokine of NKT cell generation not only can strengthen the cytotoxicity of NKT cell self, also optionally activates other cells such as NK cell and T cell.The IFN-γ that NKT expresses can activate the Th1 of pathogen specific and the cytotoxicity of CD8+CTL mediation, and visible NKT cell also can activate other cells, particularly the T cell subsequently except that the immunocompetence of self.Therefore be considered to be the bridge between intrinsic and the acquired immunity.The experimental results prompting NKT and NK system have great potentiality as the vaccine composition of anti HBV infecting, by vaccine to the activation of NK and NKT activate pathogen specific and nonspecific immune response be a kind of effectively and wise selection.Visible in sum liver natural immunity cell plays an important role in the body anti HBV infecting, and activation liver natural immunity cell particularly NKT and NK cell may become the brand-new route of removing HBV.The foreign scholar is seeking and is screening the activator of NKT and NK.Discoveries such as Kakimi (can detect IFN-γ and IFN-α/β in the HBV transgenic mice liver in the 24h of α-GalCer), and hbv replication are terminated at injection α-galactose acid anhydride ceramide.In this phenomenon and the liver NKT cell rapidly disappear and activated NK cell is raised of short duration relevantly in liver, this phenomenon shows that α-GalCer activates the antiviral cytokine inhibition of NK emiocytosis hbv replication then by direct activation NKT cell.α-GalCer has hope is used for chronic hepatitis B as NK and NKT cell activator treatment.

Summary of the invention

The technical problem that will solve of the present invention provides a kind of therapeutic vaccine composition for hepatitis B with better therapeutic effect.

The present inventor finds by a large amount of experiments, on the basis of recombinant HBsAg protein vaccine (HBsAg vaccine), add adjuvant recombinant human interleukin-11 2 (rhIL-12) and Pseudomonas Vaccine preparation (PA-MSHA vaccine), can obtain the better therapeutic vaccine composition for hepatitis B of a kind of therapeutic effect.

The present inventor finds that in every milliliter of therapeutic vaccine composition for hepatitis B, each component all has effect preferably in following ranges, wherein the water of HBsAg vaccine 10～20 μ g, rhIL-1210～20 μ g, PA-MSHA vaccine 9～1,800,000,000 and surplus.

Therapeutic vaccine composition for hepatitis B of the present invention is the best with the aqueous solution that every milliliter of compositions contains 10 μ g recombinant HBsAg protein vaccines, 10 μ g recombinant human interleukin-11s, 2,1,800,000,000 Pseudomonas Vaccine preparations and surplus.

The preparation method of therapeutic vaccine composition for hepatitis B of the present invention is also very simple, is HBsAg vaccine, rhIL-12 and PA-MSHA vaccine directly to be mixed make, and preparation is a water preparation.

Key of the present invention is to add adjuvant rhIL-12 and PA-MSHA vaccine on the basis of HBsAg vaccine, utilizes the cooperative effect of rhIL-12 and PA-MSHA vaccine, improves therapeutic effect, and the concrete effect from each component is elaborated below.

The HBsAg vaccine: the prolonged and repeated immunity of HBsAg vaccine of doses can induce body immune system to produce antigen-specific immune responses.Be mainly CD4+T cell and CD8+CTL, raise non-molten cell and cytolytic biologically, suppress duplicating and expressing of HBV.

RhIL-12:rhIL-12 is the important cytokine of occupying core position in immunological network.The main product survivor of IL-12 is the Monocytes to the microorganism stimulation responses, antigen presenting cell (APC), B cell, neutrophil cell and horn cell.Maron thinks that the generation of IL-12 is through 2 different stimulation approach, first approach is to directly act on APC by various antibacterials, fungus, protozoon, virus or their product, induce IL-12 to produce and be called the non-T cell dependent pathway, another approach is to rely on APC and activated T cell interaction and induce and produce IL-12, then is called T cell dependent pathway.IL-12 is the light chain (P35 by 35kd, IL-12 α) and the heterodimer of two chains of the different genes coding formed of the heavy chain of 40kd (P40, IL-12 β), connect by a plurality of disulfide bond, relative molecular weight is 70～75kd, and isoelectric point, IP is between pH3.5～pH5.5.Because two subunits are by different gene codes, the gene transfection result of the test shows to have only the IL-12 that the transfection simultaneously of two subunits could be obtained biologically active, abiology activity during two subunit individualisms.

The biologic activity of IL-12 has specificity, must be mediated by the receptor of high-affinity.IL-12R is made of β 1 and β 2 chains, the affinity of IL-12 and independent β 1 or β 2 chains a little less than, have only β 1 and β 2 coexpressions could form the receptor complex that has high-affinity with IL-12.P40 combines mediation P35 and combines with IL-12R β 1 with IL-12R β 2, the latter brings into play biological effect, and IL-12R is present in activated T cell and static or activatory NK cell.After the T of NK cell, CD4+, CD8+ cell was activated, its cell surface can give expression to the IL-12R of high-affinity.The signal path of IL-12 still imperfectly understands.This process is main relevant with JAK/STAT.Nearest report IL-12R β 1 is that CD4+ effect type T cell is necessary in central type Memorability CD4+T cell transformation.

IL-12 occupies the very core cytokine of critical role in immunological network, can make the activated T cell proliferation and increase its cytotoxic activity, induces the secretion of IFN-γ, regulates the Th1/Th2 cell development, impels it to the Th1 cell differentiation.IL-12 is that the optimum cell factor that induces the CTL immunne response starts cell-mediated immunoreation.Not only can activate the innate immunity cell, promote the APC function, promote its maturation, improve angtigen presentation efficient.Also can be used as the 3rd signal direct activation T cells, make it change T effector cell into.Proved that responsiveness and Memorability CD8+T cell are lacking under the isogeneic stimulation, IL-12, IL-18 can impel it to have the ability of secretion of gamma-IFN.IL-12 still keeps a kind of candidate's factor of Memorability CD4+T cell.Prompting IL-12 works in promoting Memorability cell-stimulating process.Be worth especially pointing out that the not only collaborative IL-18 of IL-12 promotes the propagation activation NK cell of NK cell to produce cytotoxicity, induce the NK cell and produce IFN-γ, and IL-12 is most important to the activation of NKT and the secretion of IFN-γ subsequently, the IL-12 receptor complex that the NKT cellular expression is abundant becomes the primary goal of IL-12 effect.NKT is amplified by IL-12 the faint reaction of self part, thereby starts immunne response, and visible IL-12 replys the important function of removing among the HBV at the startup inherent immunity.

PA-MSHA vaccine preparation: bacillus pyocyaneus PA-MSHA formal name used at school is a Pseudomonas aeruginosa, belongs to gram-negative bacterium, is a kind of conditioned pathogen.Bacillus pyocyaneus can produce the multiple material relevant with virulence, for example endotoxin, exotoxin A, elastoser, collagenase, pancreas peptidase etc.The dead thalline of this inferior discovery bacillus pyocyaneus in 1980 has immunoregulation effect, can excite body fluid and cellular immunization in the dead thalline injection experimental animals, and clinical practice is failed in inducing interferon production, but because thalline toxicity is big, and immunne response is low.Gitboa-Garber in 1981 finds that the functional gene that forms mannose sensitive haemagglutination pilus is arranged on the bacillus pyocyaneus chromosome, but can not can not form mannose sensitive haemagglutination pilus on the thalline surface with the mannose sensitive haemagglutination protein excretion outside thalline.Chinese scholar has been set up pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain, and its principal character is mainly many thin and upright and outspoken pili around thalline.The mannose sensitive haemagglutination test is strong positive.Its composition comprises dna molecular, RNA molecule, protein, lipid molecule etc., and its characteristics are that this bacterial strain toxicity is low, and immunization is strong, has the wide spectrum characteristic.Can activate and regulate body fluid and cellular immunization.

Mechanism about the adjusting of PA-MSHA vaccine immunization.Proved the bacillus pyocyaneus flagellin as a kind of ectogenic part, (expressing TLR-2, TLR-5 and TLR-11 part) can specificly be discerned by Toll sample receptor.The immune system various kinds of cell can be expressed the identification by molecular pattern such as TLRs such as DC, macrophage etc., start very complicated signal transduction pathway, activation T cells and NK cell, thus trigger and activate inherent immunity and the acquired immune response generation immunoregulation effect.

Research work such as Carm show that people's T cell can express the mRNA coding of most of TLRs, and prompting bacillus pyocyaneus flagellin may directly act on the T cell, impels its activation.The part (as the bacillus pyocyaneus flagellin) that recent in addition research also shows TLRs is the activation effect memory t cell directly.The synthesis result prompting PA-MSHA vaccine of these two effects may raise the immunologic function of CD4+T cell and CD8+T cell, promotes the synthetic and secretion of IFN-γ.Our laboratory work shows that the collaborative rhIL-12 of PA-MSHA vaccine can produce a large amount of IFN-γ at external evoked PBMCs, detect through the polychrome flow cytometer, the main product survivor of the IFN-γ that (approximately acts on back 5 hours) in early days is the NK cell, and the main product survivor of (about 48 hours) IFN-γ is CD4+T cell and CD8+T cell in the later stage.

Known NK cellular expression TLR3, TLR7 and TLR8, and flagellin is expressed the part of TLR2, TLR5 and TLR11.Like getting rid of the recognition system direct activation NK cell of flagellin by TLR, but we use the PA-MSHA vaccine and have comprised it being the comprehensive antigenic substance that common I type pili and bacillus pyocyaneus whole body pili and thalline combine, complicated component still can not be got rid of the part that contains TLR3, TLR7 and TLR8 fully.Our experiment shows is using the test in external evoked PBMCs secretion of gamma-IFN of rhIL-12 and PA-MSHA vaccine, proof promptly occurs positive reaction in effect after 5 hours, action time is of short duration, and prompting PA-MSHA vaccine may be also nonessential by the APC mediated pathways.

The performance that the NK cell has secrete cytokines and a this function of cellulotoxic effect mainly relies on the NK cell and can express and multiple MHC or the bonded receptor of non-MHC class part can be arranged.But the receptor of NK cell has multiformity, and is very complicated, found tens of kinds more than, adheres to inhibition receptor and reactivity receptor separately.In the reactivity receptor, NKG2D is the very important non-MHC type reactivity receptor of a class, use a kind of induced adaptation mechanism (inducedfit mechanism) when the identification different ligands, this mechanism can make acceptor molecule can tolerate the height variability of part, therefore has high-affinity.The NK cell starts activation signals by NKG2D, when the cell of viral infection or tumor cell owing to being subjected to environment change and expressing the NKG2D part, then discerned and be activated by NK.Whether the PA-MSHA vaccine can be as the part of NKG2D and is activated the NK cell, and this probability exists.It is expressed to be also pointed out that NKG2D has on many immune effector cells, comprises NK cell, NKT cell, CD8+ α β T cell and gamma delta T cells, these cells all in liver content abundant, activating that these cells act in anti HBV infecting may be very important.

In sum in the therapeutic vaccine composition for hepatitis B of forming by HBsAg vaccine and adjuvant rhIL-12 and PA-MSHA vaccine, the HBsAg vaccine is the auxiliary mediation of passing through the 1st signal and the 3rd signal down at rhIL-12, or induce specific CD4+ of HBV and CD8+CTL to produce IFN-γ to start non-molten cell or molten cell mechanism and suppress or remove duplicating of HBV as entering immune same antigen for the 2nd time, and rhIL-12 can direct activation NKT cell, be rich in the NKT cell in the known liver, account for 30% cell of liver endolymph total cellular score, NKT cell after the activation passes through perforin, granzyme, the cell that Fas and part energy direct killing thereof are infected by HBV, secondly NKT by excretory cytokine particularly IFN-γ further suppress or removing HBV, and the NK cell in the activation liver, and cause a large amount of secretion of gamma-IFN of NK cell and bring into play the effect of anti-HBV, be also pointed out that NKT cell after the activation is by raising the expression of CD40L, adjusted the activation of CD40-CD40L interaction partners DC, thereby promoted that DC begins to secrete IL-12.IL-12 further activates the IFN-γ secretion that NKT excites a new round.And flagellin is as the part of TLR2, TLR5, TLR11, by the mediation activation NK cell of APC, (the NK cell contains TLR7, TLR8 and TLR3 receptor, does not find TLR2, TLR5, TLR11 receptor as yet).The ratio of NK cell in liver lymphocyte is about 30% (and only accounting for 2% in peripheral blood lymphocyte), and the NK cell does not need special antigenic stimulation at the effect of virus infected cell in vivo.Immunoreation takes place rapidly and can peak in a few hours or one day in the NK cell after the activation, the NK cell is secreted a large amount of cytokines by the cytotoxic reaction direct killing simultaneously by the cell of viral infection, as TNF-α, TNF-γ, IL-3, GM-MSF, M-CSF etc.The activation that studies show that NK cell and NKT cell to the HBV transgenic mouse can stop hbv replication.This shows that with rhIL-12 and PA-MASH vaccine be the therapeutic hepatitis B vaccine of adjuvant, inherent immunity (NKT cell and NK cell) can be started and acquired immunity (specific CD4+ of HBV and CD8+CTL) can be activated again, just rhIL-12 and PA-MSHA vaccine are linked up nonspecific immunity and HBV specific cellular immunity as bridge, can activate a plurality of immune target spots such as NKT cell, the NK cell, DC and T cell (CD4+T cell, the CD8+T cell, memory t cell), point out this vaccine to breaking the HBV immunologic tolerance, the reconstituted cell immunologic function will produce good result to suppressing and removing HBV.

The specific embodiment

Below in conjunction with embodiment the present invention is described in further detail, but does not constitute any limitation of the invention.

Embodiment

The HBsAg vaccine is provided by Dalian Chinese letter biological product company limited in the embodiment of the present invention, and the PA-MSHA vaccine is from Hainan microgram west Bioceuticals Inc..

RhIL-12 is developed voluntarily by the present inventor, is positioned on the different chromosomes by the encode gene of P35 and P40 subunit of many heterologous protein dimerizations that disulfide bond is formed by two subunits of P40 and P35 because IL-12 is one.The P35 subunit can not be secreted into the extracellular separately, and the P40 subunit can be secreted into the extracellular separately.Therefore expressing two subunits in same cell inner equilibrium is keys of expressing the IL-12 of biologically active, and therefore the Chinese hamster ovary celI strain that makes up must make two subunit balances of IL-12 express.We insert two carriers that expression efficiency is different respectively with P40cDNA and P35cDNA for this reason, and cotransfection Chinese hamster ovary celI then can make the expression of two different subunits of IL-12 in a basic balance like this.Further improve the expression of P35 subunit in addition by the amplification system on the carrier.So just make the balance of P40 and P35 express further reinforcement.The CHO-SFM-III that successfully constructs the Chinese hamster ovary celI strain application Gibco that expresses IL-12 carries out static cultivation, use Sartorius BiosSTAT plus cell culture jar again and criticize cultivation and continuous culture, cell strain has reached generation more than 30, cell growth state is good, and protein expression is stable, adopt the ELISA method to detect content and the activity of rhIL-12, static cultivation mean concentration is 4 μ g/ml, cell culture jar continuous culture mean concentration is 10 μ g/ml, and the IL-12 determination of activity shows that having good IFN-γ induces ability.

The purification basic skills of rhIL-12 is that the supernatant with culture fluid carries out centrifugal treating, remove cell and fragment thereof, carry out ultrafiltration and concentration again through cation chromatography, anion chromatography, obtain the rhIL-12 of purification at last by molecular sieve, high performance liquid chromatography (HPLC) detects and shows that purity is near 98%.

It below is the preliminary pharmacodynamic experiment of this therapeutic vaccine composition for hepatitis B.

1. use the ELISA method and observe the influence of therapeutic vaccine composition for hepatitis B healthy people and each phase patient P BMC generation IFN-γ of chronic viral hepatitis B

Test kit (Human IFN-gamma ELISA set) is a BD biosciences company product, by the explanation operation.Concise and to the point flow process is as follows: get each phase patient peripheral blood 5ml of healthy people and chronic viral hepatitis B; anticoagulant heparin; the lymphocyte separation medium that uses the Shanghai smart company of China to produce separates mononuclearcell routinely, and the washing back is made into the suspension of 2 * 106/ml with the RPMI-1640 culture medium (production of GIBCO company) that contains 10% hyclone (production of GIBCO company).In 96 hole microwell plate apertures of IFN γ monoclonal antibody bag quilt and blocker sealing already, every hole is got 100ul cell suspension and is gone into 96 well culture plates, add 100ulRPMI-1640 (10%FCS again, room temperature) the good stimulus object of dilution, and establish the negative control hole that does not add stimulus object, same sample is all established multiple hole, put hatch 42～48h in 37 ℃, the incubator (U.S. SHELLAB board CO2 incubator) of 5%CO2 after, do ELISA INF-γ quantitative assay at aseptic condition collecting cell supernatant.Adopt HumanIFN-gamma ELISA set to measure test kit (BD biosciences company product), ELISA detection method operation routinely.Through coated antibody, 4 ℃ are spent the night, and application of sample adds two steps such as anti-, and the colour developing back uses MULTISKAN MK3 microplate reader (U.S. Thermo company) 450nm to measure each hole 0.D value, calculates the acquisition result.

The test blood sampling, healthy people 10 people, chronic viral hepatitis B immunologic tolerance phases 25 people, chronic viral hepatitis B immune clearance phases 18 people, non-activity or low (non-) replicative phase 22 people.Test is divided into blank, and anti--CD3 contrasts (0.2 μ g/ml), HBsAg vaccine (0.1,0.5,1 μ g/ml), rhIL-12 (0.1,1,2ng/ml), and the PA-MSHA vaccine (1: 1000,1: 100,1: 10), HBsAg vaccine (0.1 μ g/ml)+PA-MSHA vaccine (1: 1000)+rhIL-12 (0.1ng/ml), HBsAg (0.5ug/ml)+PA-MSHA vaccine (1: 100)+IL-12 (1ng/ml), HBsAg (1ug/ml)+PA-MSHA vaccine (1: 10)+rhIL-12 (2ng/ml).

The different stimulated source the results are shown in Table 1 to what normal person and chronic viral hepatitis B patient PBMC induced IFN-γ.The result shows that the HBsAg vaccine induces the ability of IFN-γ less than PA-MSHA vaccine and rhIL-12 (P＜0.01) separately.RhIL-12 induces the ability of IFN-γ greater than HBsAg vaccine and PA-MSHA vaccine (P＜0.01) separately.HBsAg vaccine+PA-MSHA vaccine+rhIL-12 induces the ability of IFN-γ and obviously uses separately or two kinds of medicine addition numerical value (P＜0.01) greater than any medicine.The normal person and different developing period the chronic viral hepatitis B patient to above-mentioned different stimulated source separately or unite to use and induce PBMC to produce no significant difference (P＞0.01) between the IFN-γ.

Table 1: the former result who healthy people and chronic viral hepatitis B patient PBMCs is induced IFN-γ of different stimulated

Group	Healthy people	Immunologic tolerance phase patient	Immune clearance phase patient	Non-activity or low (non-) replicative phase patient
					Blank	22.8±6.18	20.32±23.19	34.72±87.44	25.5±37.73
Anti--CD3 contrasts (0.2 μ g/ml)	19461±8739.10	14274.08± 12004.99	15314.28± 9627.91	12936.14±7602.37
					HBsAg vaccine (0.1 μ g/ml)	37.9±22.42	24.12±28.94	12±12.53	32.27±32.51

HBsAg vaccine (0.5 μ g/ml)	32.47±34.55	20.21±17.44	21.26±14.42	32.65±25.89
					HBsAg vaccine (1 μ g/ml)	30.44±19.54	18.41±26.64	26.64±20.05	29.84±16.46
rhIL-12(0.1ng/ml)	256.6±145.21	102.42±82.46	144.54±126.93	202.78±154.68
					rhIL-12(1ng/ml)	1229.6±725.17	786.32±720.20	941.94±780.55	967.9i±918.35
rhIL-12(2ng/ml)	1682.44± 1650.96	1385.78±550.46	1475.62±1066.63	1564.35±1233.26
					PA-MSHA vaccine (1: 10)	212.2±88.52	69.76±57.73	118.61±138.15	133.67±133.35
PA-MSHA vaccine (1: 100)	266.22±104.59	66.23±34.55	130.37±142.96	124.78±77.52
					PA-MSHA vaccine (1: 1000)	78.56±65.54	23.25±15.85	34.55±26.39	45.85±32.74
HBsAg vaccine (0.1 μ g/ml)+PA-MSHA vaccine (1: 1000)+rhIL-12 (0.1ng/ml)	2045.54± 1075.55	1826.56±1442.22	1922.01±1053.02	1865.23±1553.32
					HBsAg vaccine (0.5 μ g/ml)+PA-MSHA vaccine (1: 100)+rhIL-12 (1ng/ml)	14198±3037.31	9922.72±5289.06	11048.72± 4919.42	11340.59±6013.75
HBsAg vaccine (1 μ g/ml)+PA-MSHA vaccine (1: 10)+rhIL-12 (2ng/ml)	18250.33± 7245.32	13362.05± 6235.65	17900.46± 8871.74	17022.93±6627.25

2. use 4 color FCM and detect the influence of therapeutic vaccine composition for hepatitis B IFN-γ+CD4+ cell, IFN-γ+CD8+ cell and IFN-γ+NK cell frequency in 5 chronic viral hepatitis B patient peripheral bloods.

Whole blood is cultivated, matched group adds HBsAg (2 μ g/ml) to stimulate, vaccine group adds HBsAg vaccine (2 μ g/ml), rhIL-12 (1ng/ml) and PA-MSHA vaccine (1: 10) to stimulate, monoclonal antibody with immunofluorescence label after 6 hours dyes, FCM detects T cell, IFN-γ+CD8+T cell and the IFN-γ+NK cell of IFN-γ+CD4+, the result: IFN-γ+CD4+ matched group accounts for 4.42 ± 2.3% of total T, and vaccine group accounts for 14.7 ± 3.3%; IFN-γ+CD8+ matched group accounts for 5.62 ± 2.3% of total T, and vaccine group accounts for 13.8 ± 4.9%; It is 5.96 ± 3.3% that IFN-γ+NK cell accounts for NK sum matched group, and vaccine group accounts for 35.7 ± 3.2%.Show that the Hepatitis B virus vaccine compositions can promote T cell and the CD8+T cell, particularly NK emiocytosis IFN-γ of CD4+, apparently higher than independent use HBsAg vaccine group.

Claims (2)

1. therapeutic vaccine composition for hepatitis B is characterized in that containing in every milliliter of compositions the aqueous solution of 10～20 μ g recombinant HBsAg protein vaccines, 10～20 μ g recombinant human interleukin-11s, 2,9～1,800,000,000 Pseudomonas Vaccine preparations and surplus.

2. therapeutic vaccine composition for hepatitis B according to claim 1 is characterized in that containing in every milliliter of compositions the aqueous solution of 10 μ g recombinant HBsAg protein vaccines, 10 μ g recombinant human interleukin-11s, 2,1,800,000,000 Pseudomonas Vaccine preparations and surplus.