CN1618466A - Vaccine for treating hepatitis B, and its prepn. method - Google Patents
Vaccine for treating hepatitis B, and its prepn. method Download PDFInfo
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- CN1618466A CN1618466A CNA2003101137709A CN200310113770A CN1618466A CN 1618466 A CN1618466 A CN 1618466A CN A2003101137709 A CNA2003101137709 A CN A2003101137709A CN 200310113770 A CN200310113770 A CN 200310113770A CN 1618466 A CN1618466 A CN 1618466A
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Abstract
A therapeutic vaccine of hepatitis B is prepared from the surface antigen of hepatitis B and a protein carrier through chemical covalent coupling. It can induce body to generate the cell immune reaction and body immune reaction, which are specific to the surface antigen of hepatitis B, so preventing and treating hepatitis B.
Description
Technical field
The present invention relates to a kind for the treatment of hepatitis B vaccine.Particularly, the present invention relates to the link coupled treatment vaccine of an a kind of hbs antigen and a protein carrier.More specifically, the present invention relates to a kind of conjugate that hbs antigen and tetanus toxoid form through chemical coupling that contains, become the treatment vaccine through suitable preparation again.The invention still further relates to the preparation method of this treatment vaccine.
Technical background
Hepatitis B is one of infectious disease that has a strong impact on human health, and the whole world has 3.5 hundred million people to be chronic carrier (Lee, people such as W.M., N Engl J Med, 337:1733-45,1997) approximately.China is hepatitis B popular district occurred frequently, has 100,000,000 populations to carry hepatitis B surface antigen approximately, and the chronic viral hepatitis B patient about 2000～3,000 ten thousand.Chronic viral hepatitis B is the main reason (Wright, people such as T.L., Lancet, 342:1340-4,1993) that causes liver cirrhosis, hepatocarcinoma.So far, still do not have effective medicine and treatment means, hepatitis B becomes the important social hygienic issues.
Antiviral therapy is to remove hepatitis B virus, reduces complication, prevents one of measure of hepatic fibrosis.At present the antiviral drugs curative effect is clear and definite interferon and lamivudine, and other drug such as thymosin, il-1 2, famciclovir etc. also have certain antivirus action.Interferon can suppress virus replication, can regulate host immune function again.But the continuous and effective rate of interferon only is 20%, and the medical expense costliness, is difficult to apply, especially to the normal patient's effect of ALT relatively poor (Hoonfnagle, people such as J.H., N Engl J Med, 336:347-356,1997).The lamivudine oral absorption is good, phosphoric acid turns to the triphosphoric acid lamivudine in hepatocyte, play antivirus action by the activity that suppresses HBV-DNA reverse transcriptase and DNA polymerase, but after the lamivudine drug withdrawal behind resume combustion and the long-term prescription appearance of persister be anxious problem (Dienstag to be solved, J.L. wait the people, N Engl J Med, 333:1657-1661,1995).
Hepatitis B chronicity mechanism is very complicated, and wherein immunologic tolerance is one of mechanism that forms chronicity.Chronic HBV antigen and the infection host results of interaction of turning to of hepatitis B, specific active immunotherapy is one of method of breaking immunologic tolerance.Therapeutic hepatitis B vaccine can activate the immunne response of tolerance body, thereby break immunologic tolerance by changing angtigen presentation and processing approach, changes the clinical course of chronic viral hepatitis B.Studies show that Hepatitis B virus vaccine or Hepatitis B virus vaccine share the immunologic tolerance that immunomodulator can be broken transgenic mice, have the prospect for the treatment of preferably (people such as Mancini.M., J Med Virol, 39:67-74,1993).
At present, all existing therapeutic vaccine of company such as GlaxoSmithKline PLC company and U.S. Cytel enters the clinical examination stage.In recent years, domestic therapeutic hepatitis B vaccine development is rapid, hepatitis B surface antigen and anti-hepatis B immunoglobulin complex formulation, and clinical proof safety of I phase is good, is carrying out II phase clinical examination; It is clinical that polypeptide epitope vaccine approved enters the I phase, and the 60 μ g agent Hepatitis B virus vaccines that use as immunomodulator also enter the stage of evaluating in addition.
Hepatitis B antigen is as the main component of therapeutic hepatitis B vaccine, has good prospect in clinical practice: the tool practicality, accumulated abundant clinical experience for many years, the proof safety is good, cheap, therapeutic scheme is more easily accepted, but curative effect does not still have final conclusion, may be relevant with the factors such as selection of kind, dosage, adjuvant composition and the treatment target of selecting vaccine.Research direction should be from the increase of vaccine dose, the cooperation of immunomodulator, route of administration, or with the further investigation of aspects such as interferon anti-reflecting virus medication combined application, particularly new vaccine adjuvant.
Hepatitis B lacks animal model, and pharmacodynamics is estimated difficulty.Single be difficult for breaking immunologic tolerance, must assist increasing the cell immune response immunostimulant of Hepatitis B virus vaccine, and how to select and use immunostimulant be the difficult point that therapeutic hepatitis B vaccine is studied with Hepatitis B virus vaccine.Recent research shows that the cell effect of the specific TH1 class of early evoking is the key of treatment hepatitis B, and therefore the emphasis of research should be immunostimulant or the analog (adjuvant) that searching can increase specific cell immunoreaction.
Immunity facts have proved; the bacterial capsule polysaccharide belongs to T cell dependent/non-dependent antigen; its immune effect is obviously relevant with the age of object of inoculation; the immune system of infant is grown not perfect; can not effectively activate t helper cell and T memory cell behind the inoculation polysaccharide vaccine, can not induce to produce immune anamnesis reaction, thereby; the immunoprotection time is of short duration, and immunity inoculation can not produce the booster immunization effect once more.For example, produce IgM and IgG antibody though meningococcal polysacharide antigen can be induced, the human IgG that is produced mainly is: the IgG2 subclass, and the appearance of human serum IgG 2 subclass antibody is slower, generally will arrive 8～12 years old and just can rise to adult's level.This may be relevant with the bone-marrow-derived lymphocyte hypoevolutism of IgG secretion 2 subclass antibody.So behind the infant inoculation polysaccharide vaccine, it is main that generation has only the IgM antibody of of short duration effect.For improving the immune effect of polysaccharide vaccine, main way is that polysaccharide antigen is combined with protein carrier, and making polysaccharide antigen is T cell dependence antigen by T cell dependent/non-dependent antigenic shift.Studies show that polysaccharide is with after protein carrier combines, the T cell can identification carrier, stimulate the B cell that polysaccharide is produced and replys, and immune anamnesis reaction that can the inducing T cell mediation makes more B cell produce specific antibody.
Have been found that the effect after a bacterial polysaccharides and the protein carrier coupling has obtained checking in Hib (b type hemophilus influenza) combined vaccine and C group meningitis cocci combined vaccine.After polysaccharide and the protein carrier coupling, T cell energy identification carrier stimulates the B cell that polysaccharide is produced antibody response, and can the reaction of inducing T cell immunological memory.Above-mentioned combined vaccine all can produce enough. high-caliber anti-pod membrane IgG antibody and the B cell that immunological memory is arranged.At present existing 4 kinds of albumen carriers are used for combined vaccine, tetanus toxoid, the nontoxic diphtheria toxin, diphtherotoxin (CRM197) that suddenlys change, diphtheria toxoid and B group meningitis cocci outer membrane protein.
The tetanus toxin that is produced in the process of clostridium tetani growth and breeding on appropriate media is that a kind of that be made up of 1,315 aminoacid, molecular weight is 150,700 simple protein.Tetanus toxin is a kind of extracellular toxin, is at first to form the single polypeptide chain in thalline, just forms the polypeptide chain that links to each other with cystine linkage with light chain and heavy chain after discharging from thalline.Tetanus toxin is transformed into toxoid after the formaldehyde detoxification treatment, still keep good tetanus immunogenicity of antigens, and its virulence reduction does not cause corresponding symptom (Gapta, R.K.et al, J.of Biol.Stand., 1985,13:355～359).
In view of the foregoing, the present invention adopts protein carrier and hepatitis B surface antigen through chemical covalent coupling.This protein carrier is induced the cell immune response of body generation to hepatitis B surface antigen as immunostimulant, thereby reaches the immunologic tolerance of breaking the hepatitis B patient, changes the purpose of the clinical course of chronic viral hepatitis B.
The most frequently used vaccine for man adjuvant still is aluminium hydroxide and aluminum phosphate at present.And APC adjuvant, T cell adjuvant, alternative inducing T cell immunity, effectively pathogen and tumor cell in control and the killer cell are so be the emphasis of studying.In recent years, the research of TH1 polarization adjuvant has become the research focus.Some adjuvant prescriptions comprise that microbe-derived FCA, MDP, MPL, cytokine such as IL-12, r-IFN, liposome etc. all can stimulate mice to produce TH1 and reply, and are expected to become the new vaccine adjuvant or the immunomodulator that are used for immunization therapy.
In view of the foregoing, the present invention is the conjugate that the protein carrier of representative becomes through the chemical coupling row with hepatitis B surface antigen and tetanus toxoid, as adjuvant, becomes the treating hepatitis B vaccine through suitable preparation with the bacillus pyocyaneus preparation again.This treatment vaccine is used for treating hepatitis B patient clinically and healthy people prevents hepatitis b virus infected.
Summary of the invention
In order to overcome the deficiencies in the prior art part, the object of the present invention is to provide a kind of new vaccine, particularly, the invention provides the treating hepatitis B vaccine that an a kind of hbs antigen and protein carrier chemistry covalent coupling forms.Preferably, contain with the tetanus toxoid be carrier and with hbs antigen be the treating hepatitis B vaccine of Main Ingredients and Appearance through the covalent conjunct agent that chemical coupling forms, in order to treat hepatitis B patient clinically and healthy people prevents hepatitis b virus infected.
The present invention also provides a kind of method for preparing the treating hepatitis B vaccine, and this method comprises:
(A) with the protein carrier coupling of 2.5～90 microgram hbs antigenes and 5～60 micrograms ground one conjugate;
(B) with the conjugate of hbs antigen and protein carrier through formulated a kind for the treatment of hepatitis B vaccine.
Wherein said protein carrier is be selected from tetanus toxoid, diphtheria toxoid, avirulence diphtheria variant CRM197 toxin and B group meningitis cocci outer membrane protein group any.Preferably, described protein carrier is a tetanus toxoid.Vaccine of the present invention can be prepared with aluminium adjuvant and/or buffer saline.
In the present invention, hbs antigen can form covalent conjunct agent through chemical coupling with the tetanus toxoid of making carrier, this covalent conjunct agent is mixed with a kind of method of new treating hepatitis B vaccine again.
The used hbs antigen of the present invention is commercial genetic engineering hepatitis B antigen, and its source comprises yeast cells and Chinese hamster ovary cell expression systems such as (CHO).The preparation method of hbs antigen is seen " Chinese biological goods goods rules " (Chemical Industry Press,, 175～183 pages in 2000).
The used tetanus toxoid of the present invention is meant the commercialization tetanus toxoid, as tetanus toxoid purified.The preparation method of tetanus toxoid is seen " Chinese biological goods goods rules " (Chemical Industry Press,, 213～218 pages in 2000)
In one embodiment of the invention, as the albumen of carrier can be tetanus toxoid, in the nontoxic diphtheria toxin, diphtherotoxin (CRM197) that suddenlys change, diphtheria toxoid and the B group meningitis cocci outer membrane protein any one.Tetanus toxoid given here only is for illustrative the present invention, also can adopt other protein carrier if satisfy the demand.
The present invention adopts Bromine cyanide. (CNBr) activation hbs antigen, is difunctional nucleophilic base at interval with brave platinum acid imide (SPDP), the formation hbs antigen that is tiger platinum imide derivative.Further, obtain hbs antigen-tetanus toxoid coupling conjugate by the condensation and the tetanus toxoid covalent bond of carbodiimide (EDAC) mediation.Through ultrafiltration and concentration, the macromolecule coupling conjugate that column chromatography purification obtains becomes the treatment vaccine through aseptic filtration, preparation, dilution packing again.
As mentioned above, form in the process of conjugate, have a plurality of sites simultaneously in conjunction with a plurality of tetanus toxoid after can be by single hbs antigen activated at hbs antigen and tetanus toxoid.Equally also can be by a plurality of sites of the single tetanus toxoid after the activation simultaneously in conjunction with a plurality of hbs antigenes.The combination of hbs antigen and tetanus toxoid is not limited to 1: 1, just can coupling on hbs antigen molecule on 1 or a plurality of tetanus toxoid molecule; Similarly, 1 or a plurality of hbs antigen molecule on can coupling on tetanus toxoid molecule.In the coupling of hbs antigen and tetanus toxoid, the combination one to one of hbs antigen and tetanus toxoid preferably takes place.But as mentioned above, the various combinations of hbs antigen and tetanus toxoid are not as long as influence the effect that treatment vaccine of the present invention can reach, and the variations in detail in all these couplings all should be included within the scope of the invention so.
The coupling method of hbs antigen of the present invention and tetanus toxoid is only used for explanation, so, can adopt the various methods that are equal to realize coupling process of the present invention, therefore any link coupled method of protein molecular and technology or process of being used for all might be used to implement the present invention.
In treatment vaccine of the present invention, every dose of immunizing dose of hbs antigen is between 2.5～90 micrograms, protein carrier, tetanus toxoid for example, content is between 5～60 micrograms, as for the upper limit of consumption, those skilled in the art can grasp flexibly according to the conventional practice of the clinical use of treatment vaccine.
Can further add the bacillus pyocyaneus preparation in the treatment vaccine of the present invention as adjuvant, strengthen the immunne response level of treatment vaccine.
In treatment vaccine of the present invention, can further add absorption adjuvant, for example aluminium adjuvant such as aluminium hydroxide and/or aluminum phosphate.During use, can dilute treatment vaccine of the present invention, the example that does not limit of suitable diluent has the buffer saline diluent.
In a word, also there is not a kind of new treatment vaccine that utilizes hbs antigen and a kind of protein carrier to form at present through chemical coupling.The present invention as adjuvant, obtains novel treating hepatitis B vaccine with the bacillus pyocyaneus preparation by hbs antigen and protein carrier are carried out chemical coupling.This treatment vaccine can specific inducing cell immunity and humoral immunization, is used for treating hepatitis B patient clinically and healthy people prevents hepatitis b virus infected.
Another emphasis of the present invention is that adopting the bacillus pyocyaneus preparation is adjuvant, preferably with the Pseudomonos aeruginosa MSHA fimbria strain preparation.The present invention adds Pseudomonos aeruginosa MSHA fimbria strain preparation (PA) in the treating hepatitis B vaccine as adjuvant first.Bacillus pyocyaneus preparation with the preparation of MSHA pilus strain goes on the market as the medicine of treatment tumor, these goods can be adjusted the unbalanced state of human body fluid immunity and cellular immunization, increase macrophage and NK cell activity, keep the quantity and the ratio of T cell, regulate synergism (the Chinese biological goods rules of interleukin II, interferon and antibody, Chemical Industry Press, 2000,265 pages).
Therapeutic hepatitis B vaccine of the present invention has higher safety, the reasons are as follows:
1. hepatitis B surface antigen: worldwide through several hundred million person-times, nearly 20 years extensive application, effect has fully proved its safety to hepatitis B surface antigen as the main component of preventative vaccine.
2. tetanus toxoid: tetanus toxoid uses in worldwide as conventional vaccine, and its good safety and protection effect are known already.
3. the bacillus pyocyaneus preparation safety of using as adjuvant: the bacillus pyocyaneus preparation is the listing kind of the treatment tumor of Chinese food Drug Administration approval, and it is made vertification regulation and lists " Chinese biological goods goods rules " (version in 2000) in.The safety of bacillus pyocyaneus preparation clinical practice is proved.
4. the safety after hepatitis B surface antigen and the tetanus toxin coupling: in whole coupling process, adopted aggregate measures such as dialysis, ultrafiltration, column chromatography, removed the residue and the small molecular weight material of chemical reagent effectively, guaranteed the safety of goods.
5. the safety verification for the treatment of hepatitis B vaccine: good safety is arranged in animal body through undue toxicity's experiment confirm treating hepatitis B vaccine.
Description of drawings
Fig. 1: immune mouse humoral immune reaction after hepatitis B antigen and the TT coupling, wherein: ED50 unit is a dilution factor, HbsAg+TT: hepatitis B antigen and TT coupling, HbsAg+AL-TT: Hepatitis B virus vaccine (containing aluminium adjuvant) mixes with TT, HbsAg+Al: Hepatitis B virus vaccine (containing aluminium adjuvant), HbsAg-TT: hepatitis B antigen mixes with TT.
Fig. 2: hbs antigen coupling TT with induce humoral immune reaction with the PA combined group, wherein, HBsAg+TT: hepatitis B antigen and TT coupling, HBsAg+TT+PA: hepatitis B antigen and TT coupling mixing PA, HBsAg+Al: Hepatitis B virus vaccine (containing aluminium adjuvant).
Fig. 3: the IGg2a/IGg1 ratio of the vaccine-induced antibody for the treatment of hepatitis B, wherein G1: hbs antigen coupling TT and PA combined group; G2: Hepatitis B virus vaccine group; G3: hbs antigen coupling TT group.
Fig. 4: therapeutic hepatitis B vaccine immune mouse spleen cell ELISPOT detects γ-IFN result.
Fig. 5 therapeutic hepatitis B vaccine immune mouse spleen cell ELISPOT detects IL-2 result.
Fig. 6 therapeutic hepatitis B vaccine various dose HBsAg immune mouse spleen cell ELISPOT detects γ-IFN result.
The specific embodiment
Describing the present invention below in conjunction with drawings and Examples is that protein carrier and hbs antigen form covalent conjunct agent by chemical coupling with the tetanus toxoid, through being mixed with the treatment vaccine, and prove that through animal experiment this treatment vaccine has reliable safety and significant immunogenicity.
The preparation of hbs antigen (HBsAg) and tetanus toxoid purified (TT) covalent coupling thing
Get the HBsAg that concentration is 1～2mg/ml, add 0.5～1.0mg/ml Bromine cyanide. (CNBr) activation, act on 0.5～1 hour down, keep PH8.2～10 at 23 ± 3 ℃.Add N-amber platinum acid imide-3-(2-sulfo-pyridine) propanoic acid (SPDP) 2.5～4mg/ml activator, keep PH8.2～10.0, act on 10～30 minutes.Stirred dialysis removal small-molecule substance 10～20 hours in 2～8 ℃.Adding concentration is the equal-volume TT mix homogeneously of 2～4mg/ml, adds carbodiimide (EDAC) 15～25mg/ml mixture, and 5～15 ℃ act on 0.5～1 hour, transfer PH5～7.Coupling stock solution was dialysed 12～15 hours at 0.2M NaCl solution through the 300KD dialyzer.Collect dialysis back conjugate,,, collect V with 0.15～0.2M NaCl eluting through Sephacryl S 400 column chromatographies
0Near the high molecular conjugate peak.Asepticly after the aseptic filtration be stored in 2～8 ℃.
The covalent conjunct agent column chromatography figure that Fig. 1, hbs antigen and tetanus toxoid form through chemical coupling.Last range upon range of mountains among the figure: the hbs antigen cutting edge of a knife or a sword, after tetanus toxoid combined, molecular weight increased, with V
0Near high-molecular weight compounds is main; Following range upon range of mountains: the tetanus toxoid peak, after hbs antigen combined, molecular weight increased thereupon, with V
0Near high-molecular weight compounds is main.
Preparation of embodiment 2 treating hepatitis B vaccines and dilution packing (no adjuvant)
According to hbs antigen assay result in hbs antigen and the tetanus toxoid conjugate,, make every dose of treatment vaccine be with the aseptic dilution packing of buffer saline; 〉=0.5ml branch loading amount, contain hbs antigen 2.5～90 micrograms, contain tetanus toxoid 5～60 micrograms, pH 5.5～7.2, thimerosal 30～70 micrograms.
Preparation for the treatment of hepatitis B vaccine and dilution packing (containing adjuvant)
According to hbs antigen assay result in hbs antigen and the tetanus toxoid conjugate,, make every dose of treatment vaccine be with the aseptic dilution packing of aluminium hydroxide diluent; 〉=0.5ml branch loading amount, contain hbs antigen 2.5～90 micrograms, contain tetanus toxoid 5～60 micrograms, 0.35～0.70 milligram of pH 5.5～7.2, thimerosal 30～70 micrograms, aluminium hydroxide.
Treating hepatitis B vaccine safety Journal of Sex Research:
Safety to the treating hepatitis B vaccine is examined, and adopts mice, Cavia porcellus abnormal toxicity test method respectively, examines its safety.
Select Cavia porcellus, abdominal cavity inoculation 5ml treating hepatitis B vaccine for use, preceding respectively at exempting from, exempt to weigh in back 7 days, and observe reaction of inoculation every day.
Select SPF level Balb/C mice for use, abdominal cavity inoculation 0.5ml treating hepatitis B vaccine, preceding respectively at exempting from, exempt to weigh in back 7 days, and observe reaction of inoculation every day.
Experimental result is listed in table 1, table 2.
Table 1. treating hepatitis B vaccine Cavia porcellus safety testing
| The vaccine lot number | The inoculation precursor | Inoculate back 7 days body weight | The observation period reaction of inoculation | Conclusion |
| Heavy (gram) | (gram) | |||
| HB200201 | ????268 ????272 | ????355 ????343 | Be good for and deposit the no abnormal reaction of weight increase | Qualified |
| HB200202 | ????302 ????298 | ????378 ????369 | Be good for and deposit the no abnormal reaction of weight increase | Qualified |
| HB200203 | ????290 ????285 | ????360 ????349 | Be good for and deposit the no abnormal reaction of weight increase | Qualified |
Table 2. treating hepatitis B vaccine mice safety testing
| The vaccine lot number | Body weight (gram) before the inoculation | Inoculate back 7 days body weight (gram) | The observation period reaction of inoculation | Conclusion |
| HB200201 | ?19.5 ?18.8 ?19.3 ?19.0 ?20.5 | ?24.0 ?23.8 ?23.3 ?23.7 ?24.0 | Be good for and deposit the no abnormal reaction of weight increase | Qualified |
| HB200202 | ?18.7 ?20.3 ?20.5 ?21.0 ?19.1 | ?24.5 ?25.3 ?24.6 ?25.4 ?24.7 | Be good for and deposit the no abnormal reaction of weight increase | Qualified |
| HB200203 | ?20.9 ?19.6 ?18.6 ?19.7 ?19.9 | ?25.0 ?23.9 ?24.1 ?25.1 ?24.5 | Be good for and deposit the no abnormal reaction of weight increase | Qualified |
Abnormal toxicity test shows that the treating hepatitis B vaccine has reliable safety.
Research-the ED of hbs antigen coupling TT induced animal humoral immunization
50Measure
With hepatitis B surface antigen and tetanus toxoid conjugate, hepatitis B surface antigen and tetanus toxoid (TT) mixture, after Hepatitis B virus vaccine and tetanus toxoid mixture and Hepatitis B virus vaccine dilute 4,16,64,256 times respectively, the lumbar injection BALB/c mouse, former times of antigen is for containing 10 μ g HbsAg, every group of 10 mices.Serum is gathered during 4 weeks in the immunity back, uses RIA reagent and detects hepatitis B surface antibody.Use the Reed-Munch method and calculate ED50.Hepatitis B virus vaccine-TT coupling group antibody is renderd a service the highest (6.68) as seen from Figure 1, is significantly higher than Hepatitis B virus vaccine and TT combined group (2.59), Hepatitis B virus vaccine and TT combined group (0) and Hepatitis B virus vaccine group (4) (P＜0.01).Secondly be Hepatitis B virus vaccine and TT combined group and Hepatitis B virus vaccine group, and detect less than antibody response behind hepatitis B surface antigen and the TT combined group immune mouse.
Hbs antigen coupling TT mixes the research of inducing humoral immunization with bacillus pyocyaneus preparation (PA):
Hbs antigen coupling TT with after PA mixes, is diluted 4,16,64,256 times of pneumoretroperitoneums injection BALB/c mouse.Every group 10, matched group is a surface antigen mixing PA group, the aluminum hydroxide adjuvant vaccine group.Immunity, collection serum, detection hepatitis B surface antibody and ED50 statistical method are the same.
Hepatitis B virus vaccine-TT coupling group and Hepatitis B virus vaccine-TT coupling mixing PA group antibody is renderd a service and is significantly higher than Hepatitis B virus vaccine group (19.0 as seen from Figure 2; 28.9; 5.0, P＜0.01); And Hepatitis B virus vaccine-TT coupling mixing PA group induces antibody response apparently higher than Hepatitis B virus vaccine-TT coupling group, and prompting mixes the antibody response that PA can further increase hbs antigen coupling TT.
Hbs antigen coupling TT and PA combined group are induced the research of body fluid immune antibody hypotype:
With hbs antigen coupling TT group and hbs antigen coupling TT and PA combined group, dilute 4,16,64,256 times of pneumoretroperitoneum injection BALB/c mouse.Every group 10, matched group is the aluminum hydroxide adjuvant vaccine group.Methods such as immunity, collection serum are the same, use enzyme-linked immunoassay method and detect anti--HBs IgG, IGg2a and IGg1 respectively.The results are shown in Figure 3.
Therapeutic hepatitis B vaccine group immune mouse induces IGg2a/IGg1 ratio (1.1412) to be significantly higher than hbs antigen coupling TT group (0.421) and Hepatitis B virus vaccine group (0.235) (P＜0.01) as seen from Figure 3, show that therapeutic hepatitis B vaccine can induce high-level IGg2a, can induce stronger TH1 cell effect.
Hbs antigen coupling tetanus toxoid (TT) and PA combined group inducing specific Study of cytokines:
With hbs antigen coupling TT with after PA mixes, the subcutaneous immune mouse in back, wherein the content of HBsAg only is 3 μ g/, presses every group of 10 mices of experimental group and matched group respectively, totally 5 groups.Respectively at the 5th, 10,15 sacrificed by exsanguination.Prepare the spleen suspension routinely and isolate mononuclearcell (MNC), adjust cell concentration to 2 * 10 with complete medium (the RPMI-1640 culture fluid that contains 15% hyclone) through lymphocyte separation medium
6Ml
-1,, every hole adds 1000 these suspensions of μ l on 24 well culture plates, 200 μ l HBsAg polypeptide, and the final concentration of HBsAg is 50 μ gml
-1, culture plate is put 37 ℃, 5%CO
2Cultivated 5 days under the condition, collect supernatant.Use ELISPOT kit detection cell factor gamma-IFN and IL-2 respectively.
By Fig. 4,5,6 as seen, exempts from the back in the time of 5,10 and 20 days mice, and the ability of inducing CTL effector lymphocyte factor gamma-IFN or IL-2 to produce is all the highest with therapeutic hepatitis B vaccine and PA combined group, is the therapeutic hepatitis B vaccine group secondly, all is significantly higher than the vaccine matched group.
Application contains therapeutic hepatitis B vaccine and PA combined group, therapeutic hepatitis B vaccine group, the vaccine matched group of 1,3 and 6 μ g HBsAg and distinguishes immune mouse, puts to death when exempting from back 10 days, uses the ELISPOT test kit and detects γ-IFN.The result shows with immune HBsAg content to be increased, therapeutic hepatitis B vaccine group and therapeutic hepatitis B vaccine and PA combined group induce γ-IFN cell number obviously to raise, and the therapeutic hepatitis B vaccine group of 3 and 6 μ g HBsAg reaches with the PA combined group and is significantly higher than 1 μ g group (P＜0.05); And 6 μ g therapeutic hepatitis B vaccine groups and organize with PA combined group and 3 μ g between do not have significant difference (P＞0.05).
Embodiment 9
The HBsAg transgenic mice is broken the research of immunologic tolerance
Use hbs antigen coupling TT and PA combined group, therapeutic hepatitis B vaccine group, Hepatitis B virus vaccine matched group respectively, immune HBsAg transgenic mice, wherein HbsAg content is 3ug, and in 1 when week, put to death after the immunity 3 times, detects HBsAg, anti--HBs.
The research of breaking immunologic tolerance behind the table 3 therapeutic hepatitis B vaccine immune transgenic mice
| Group | Exempt from preceding number positive | Exempt from the back number positive | ||
| ??HBsAg | Anti--HBs | ?HBsAg | Anti--HBs | |
| Hbs antigen coupling TT and PA combined group | ????10 | ????0 | ????3 | ????7 |
| Hbs antigen coupling TT | ????10 | ????0 | ????5 | ????5 |
| The Hepatitis B virus vaccine group | ????10 | ????0 | ????9 | ????1 |
Referring to table 3, the result shows, hbs antigen coupling TT group and hbs antigen coupling TT and PA combined group all can significantly strengthen the immunologic tolerance of breaking transgenic mice, and behind contrast Hepatitis B virus vaccine group 10 mouse immunes 3 times, still have 9 HBsAg positives, the treatment prospect of prompting therapeutic hepatitis B vaccine.
To sum up: from inducing humoral immunization, antibody subtype; The aspects such as research that specific CTL effector lymphocyte factor determination, transgenic mice are broken immunologic tolerance have proved the therapeutical effect of hbs antigen coupling TT (HBsAg+TT); Discover that simultaneously hbs antigen coupling TT and PA combined group (HBsAg+TT+PA) group immune effect are more excellent.
Claims (14)
1. a treating hepatitis B vaccine is formed by hbs antigen and protein carrier chemistry covalent coupling.
2. treating hepatitis B vaccine according to claim 1 is characterized in that, described hbs antigen is the genetic engineering hbs antigen.
3. treating hepatitis B vaccine according to claim 2 is characterized in that, described genetic engineering hbs antigen source comprises the hbs antigen of yeast cells and the preparation of Chinese hamster ovary cell (CHO) expression system.
4. treating hepatitis B vaccine according to claim 1, it is characterized in that described protein carrier is be selected from tetanus toxoid, diphtheria toxoid, avirulence diphtheria variant CRM197 toxin and B group meningitis cocci outer membrane protein group any.
5. treating hepatitis B vaccine according to claim 1 is characterized in that, realize in conjunction with a plurality of tetanus toxoid simultaneously in a plurality of sites after activated by single hbs antigen in described chemical coupling.
6. treating hepatitis B vaccine according to claim 1 is characterized in that, realize in conjunction with a plurality of hbs antigenes simultaneously in a plurality of sites after activated by single tetanus toxoid in described chemical coupling.
7. treating hepatitis B vaccine according to claim 1 is characterized in that, the content of described hbs antigen is between every milliliter 2.5～90 microgram, and protein carrier content is between 5～60 micrograms.
8. according to the described treating hepatitis B vaccine of one of claim 1-7, it is characterized in that, can prepare with aluminium adjuvant and/or buffer saline.
9. according to the described treating hepatitis B vaccine of one of claim 1-7, it is characterized in that, is adjuvant with the bacillus pyocyaneus preparation.
10. treating hepatitis B vaccine according to claim 9 is characterized in that, described bacillus pyocyaneus preparation is the Pseudomonos aeruginosa MSHA fimbria strain preparation.
11., it is characterized in that its preparation comprises the preparation of liposome, nanoparticles (nano-scale particle) and their lyophilized powder form according to the described treating hepatitis B vaccine of one of claim 1-7.
12. a method for preparing the treating hepatitis B vaccine, this method comprises:
(A) with the protein carrier coupling of 2.5～90 microgram hbs antigenes and 5～60 micrograms ground one conjugate;
(B) with the conjugate of hbs antigen and protein carrier through formulated a kind for the treatment of hepatitis B vaccine.
13. the method for the treatment of hepatitis B vaccine according to claim 12, it is characterized in that described protein carrier is be selected from tetanus toxoid, diphtheria toxoid, avirulence diphtheria variant CRM197 toxin and B group meningitis cocci outer membrane protein group any.
14. the method for the treatment of hepatitis B vaccine according to claim 13 is characterized in that, described protein carrier is to be selected from tetanus toxoid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100388953C (en) * | 2006-06-15 | 2008-05-21 | 南京农业大学 | Composite mucosa immunoadjuvant for oral vaccine |
| CN113423423A (en) * | 2018-11-13 | 2021-09-21 | 变异生物技术公司 | Immunogenic compositions for the treatment of hepatitis B |
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| WO1994019011A1 (en) * | 1993-02-26 | 1994-09-01 | The Scripps Research Institute | Peptides for inducing cytotoxic t lymphocyte responses to hepatitis b virus |
| BR9907855A (en) * | 1998-02-12 | 2001-04-24 | Immune Complex Corp | Strategically modified hepatitis b core protein conjugate, protein particle, protein particle conjugate, immunogenic fusion protein conjugate, inoculum, and, process to induce antibodies in an animal host |
| CN1270772C (en) * | 2002-12-30 | 2006-08-23 | 苏盛 | Anti-hepatitis-B-virus therapeutic vaccine and its adjuvant |
| CN1424110A (en) * | 2002-12-30 | 2003-06-18 | 北京市希波医学技术开发公司 | Vaccine adjuvant against hepatitis B |
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| CN100388953C (en) * | 2006-06-15 | 2008-05-21 | 南京农业大学 | Composite mucosa immunoadjuvant for oral vaccine |
| CN113423423A (en) * | 2018-11-13 | 2021-09-21 | 变异生物技术公司 | Immunogenic compositions for the treatment of hepatitis B |
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