CN1270772C - Anti-hepatitis-B-virus therapeutic vaccine and its adjuvant - Google Patents
Anti-hepatitis-B-virus therapeutic vaccine and its adjuvant Download PDFInfo
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- CN1270772C CN1270772C CN 03104697 CN03104697A CN1270772C CN 1270772 C CN1270772 C CN 1270772C CN 03104697 CN03104697 CN 03104697 CN 03104697 A CN03104697 A CN 03104697A CN 1270772 C CN1270772 C CN 1270772C
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Abstract
The present invention relates to a novel therapeutic vaccine and an adjuvant thereof. The adjuvant is particularly suitable for an anti-HBV therapeutic vaccine. The adjuvant of the vaccine is prepared from interleukin-2 (IL-2), a human granular cell-macrophage colony stimulating factor (GM-CSF) and tetanus toxoid derived polypeptide (TT). The vaccine comprises the adjuvant and hepatitis B virus surface antigens.
Description
The present invention relates to a kind of new therapeutic vaccine, be specifically related to anti-hepatitis B virus (HBV) therapeutic vaccine antigen and adjuvant, and the weight ratio of antigen and adjuvant.
Hepatitis B is by the high disease of the infectious sickness rate of HBV.Antiviral therapy is the main means of treatment chronic HBV infection.
So far develop many nucleoside analog antiviral agents the nineties in 20th century, wherein draw the miaow furan sting (Lamivudine) in 1998 by drugs approved by FDA, be used for the treatment of chronic viral hepatitis B.
In addition, the anti-HBV therapy of a kind of new specificity (anti-HBV therapeutic vaccine) is also developed, and wherein S and S fragment albumen and t cell epitope polypeptide therapeutic vaccine also come out before the HBV, and have been used for clinical treatment chronic HBV infection patient.Calendar year 2001 Merck institute research worker Hilleman on the Vaccine magazine brief overview treat the main Therapeutic Method of chronic HBV infection now in the world.Narrate the main antiviral therapy for the treatment of chronic HBV infection at present in the world in the article and comprised antiviral chemotherapy, interferons antiviral agents, biology therapy, specificity immunology therapy etc.Wherein the antiviral chemotherapy is used the nucleoside analog antiviral agents.The interferons antiviral agents has IFN-α 1,2a and 2b.Biology, therapy was to influence transcription behind the hbv replication, and this therapy is used materials such as cytokine (IFN-γ, TNF-α, IL-2 and IL-12), thymosin (Thymosin α 1), antisense nucleotide and antisensenucleic acids.The specificity immunology therapy is used anti-HBV specific immune globulin, anti-HBV therapeutic vaccine.Existing anti-HBV therapeutic vaccine mainly contains three kinds at present: preceding S and S fragment protein for treatment vaccine, t cell epitope fat polypeptide therapeutic vaccine and DNA plasmid vaccine.The existing clinical practice report of preceding two kinds of anti-HBV therapeutic vaccines, the DNA plasmid vaccine then still is in the external and zooperal stage owing to there is still unsolved safety problem.
According to the HBV amynologic mechanism of present discovery, chronic HBV infection patient's the effect that lapses to antiviral therapy has substantial connection.The only a few case is eliminated with the interior HBV of blood circulation in the liver in the antiviral therapy process, shows as HBsAg, HBeAg and HBV DNA clinically and disappears, even finally produce anti-HBs, obtains the radical treatment effect; Most of case then just during medication hbv replication be suppressed, show as HBeAg and HBV DNA clinically and in circulation blood, reach the level that detection does not go out.Yet recurrence in a period of time after the drug withdrawal turns back to the preceding situation of treatment.This present situation of antiviral therapy curative effect shows that the radical treatment problem of chronic HBV infection also solves far away.
In three kinds of above-mentioned existing anti-HBV therapeutic vaccines, first kind of anti-HBV therapeutic vaccine is made of first HBV specific antigen two kinds of preparations.General in the world HBV S fragment or Pre S and S fragment albumen are the HBV specific antigen; It two is immunological adjuvants.Two kinds of compositions all can influence the clinical efficacy of anti-HBV therapeutic vaccine.
The major issue that chronic HBV infection patient immune system exists is an immunologic tolerance.Because immunologic tolerance makes patient's anti-HBV immunoreation lymphocyte not produce immunne response to HBV and antigen thereof, therefore make HBV be the persistent infection state in vivo.Modern immunologic research prompting, the unique channel that solves this state is to treat by using anti-HBV therapeutic vaccine, breaks immunologic tolerance.So, break chronic HBV infection patient's immunologic tolerance with anti-HBV therapeutic vaccine, rebuild its normal anti-HBV immunne response, be not only current a kind of new treatment approach, and be current up-to-date treatment thinking.
In above-mentioned three kinds of anti-HBV therapeutic vaccines, being considered to safe and effective in cure person now is anti-HBVPre S and S fragment protein for treatment vaccine.In this field, international competition is very fierce.Focusing in the development to this therapeutic vaccine adjuvant of competition.We can say that who works out best adjuvant, who just accounts for the superiors in international competition.
Aspect the research of adjuvant, most people liopopolysaccharides material commonly used or old tuberculin.Because we find in research for many years, why the chronic HBV infection patient is immune tolerance state to HBV, and very one of important reasons is patient's the last immune secondary signal (be called B7.1 and B7.2 or claim CD80 and CD86 polypeptide) that lacks of antigen processing cell (APCs).Just because this immune signal lacks, make two kinds of cells that start anti-HBV immunne response, promptly HBV antigenic specificity cytotoxic T cell (abbreviation specific CTL) (is called for short CD4 with helper T cell
+Cell or HTL) can not activate, and then intravital HBV can not be eliminated.
In view of the foregoing, the emphasis of inventor research is to develop peptide material with the immune secondary signal function adjuvant as anti-HBV therapeutic vaccine.After research for many years, we have had very big discovery, be that gene recombinaton interleukin II (IL-2), gene recombinaton GM-CSF and tetanus toxoid are combined by special ratios, add the Pre S and the S fragment protein vaccine of doses, can show two effects: 1. substitute the immune secondary signal that patient APCs lacks; 2. rebuild the anti-HBV immunne response that the patient loses.Up to the present the new approaches of the anti-HBV therapeutic vaccine adjuvant of this development, and the new adjuvant of developing on this new approaches basis, remain unique in the world.
The purpose of this invention is to provide new adjuvant, the adjuvant that is applicable to anti-HBV therapeutic vaccine particularly is provided.
The purpose of this invention is to provide the new anti-hepatitis B virus that is used for the treatment of chronic HBV infection (HBV) therapeutic vaccine.
One embodiment of the invention provide a kind of new vaccine adjuvant, and wherein said adjuvant is made up of interleukin II (IL-2), human granulocyte-macrophage colony stimulating factor (GM-CSF), tetanus toxoid.IL-2 and GM-CSF can be native proteins, also can be recombiant proteins.IL-2, GM-CSF and tetanus toxoid all have commercially available, and IL-2 can obtain with injection recombinant human interleukin--2's ProductName from Fourth Ring, Beijing bioengineering product factory; GM-CSF can obtain with the trade name of Leucomax from Schering-Plough (Brinny) Co.; Tetanus toxoid can obtain with the trade name of tetanus toxoid from Lanzhou Institute of Biological Products.In addition, the production method of IL-2, GM-CSF and tetanus toxoid also is known in the art, for example referring to wong GG, witek JS, Temple PA, et al, Molecular cloning ofhuman and givvon GM-CSF cDNA ' s and purification of the natural and recombinanthuman proteins.Cancer Cells 1985; 3:235-242.
Another embodiment of the present invention provides the new adjuvant of being made up of interleukin II (IL-2), human granulocyte-macrophage colony stimulating factor (GM-CSF), tetanus toxoid, the ratio of wherein said IL-2, GM-CSF and tetanus toxoid (weight ratio) is 50-600: 10-200: 6-20, and the part by weight of preferred IL-2, GM-CSF and tetanus toxoid is 56: 16: 6.Described three kinds of components contents can not be as the criterion by toxigenicity with the adjuvant of gained.
Adjuvant of the present invention can be used for vaccine, especially for anti-HBV therapeutic vaccine.
Get IL-2, GM-CSF and tetanus toxoid according to the above ratio and mix, promptly can be made into adjuvant of the present invention.According to method well known in the art, make water preparation or lyophilized formulations after said components can being made up in proportion.
Another embodiment of the present invention provides a kind of new anti-HBV therapeutic vaccine.This anti-HBV therapeutic vaccine is made of two kinds of main components.That is: 1.HBV specific antigen protein, the S fragment master albumen (containing 226 aminoacid) or the S fragment master albumen of taking in the HBV gene structure add preceding S
1(containing 108 aminoacid) and preceding S
2The albumen (see figure 1) of (55 aminoacid) gene expression.2. adjuvant: the cytokine adjuvant that adopts inventor's invention.This adjuvant is made up of by certain weight ratio with tetanus toxoid interleukin II, human granulocyte-macrophage colony stimulating factor (GM-CSF) that (the relevant adjuvant patent that the inventor has applied for, the patent No. is: 02159913.0).
S1 and the anterior s2 gene expressed proteins new HBV therapeutic vaccine that above-mentioned cytokine adjuvant is formed for the HBV specific antigen adds before S1 or S fragment master albumen added before another embodiment of the present invention provided and added by S fragment master's albumen in the HBV gene structure or S fragment master albumen.The weight ratio of wherein said antigen and cytokine adjuvant is: S1 fragment and pre-s2 protein before S fragment albumen or S fragment add (being called for short the HBV specific antigen protein): IL-2: GM-CSF: tetanus toxoid=5-100: 50-600: 10-200: 4-10, the weight ratio of preferred HBV specific antigen, IL-2, GM-CSF and tetanus toxoid is: 60: 56: 16: 6.The used hepatitis B virus surface antigen of the present invention can be bought from the market, for example Beijing Tiantan Bio-pharmaceuticals goods company, safe company.
Get HBV specific antigen protein, IL-2, GM-CSF and tetanus toxoid according to the above ratio and mix, promptly can be made into anti-HBV therapeutic vaccine of the present invention.According to method well known in the art, after making up in proportion, said components makes water preparation or lyophilized formulations.
Description of drawings
Fig. 1 represents hepatitis B virus gene group structure, has wherein represented S fragment, preceding S1 fragment and preceding S2 fragment respectively.
Fig. 2 shows the anti-HBV effect of anti-HBV therapeutic vaccine of the present invention in the transgenic Hepar Mus.
The preparation of embodiment 1 adjuvant
Get 56 parts of recombinant il-2s (obtaining), 16 parts of GM-CSF (obtaining) and 6 parts of tetanus toxoid (obtaining) aqueous solution from Lanzhou Institute of Biological Products from Schering-Plough (Brinny) Co. from Fourth Ring, Beijing bioengineering product factory, mix, make lyophilized formulations according to " Chinese biological goods rules " (version in 2000, Chinese biological standard of articles committee compiles) disclosed method.
The preparation of embodiment 2 vaccines
Get 60 parts of above-mentioned HBV specific antigen proteins of weight (supply of Beijing Tiantan Bio-pharmaceuticals goods company), 56 parts of heavy IL-2,16 parts of heavy GM-CSF and 6 parts heavy tetanus toxoid aqueous solutions, make lyophilized formulations according to " Chinese biological goods rules " version disclosed method in 2000 of Chinese biological standard of articles committee establishment.
The preparation of embodiment 3 vaccines
Get 5 parts of above-mentioned HBV specific antigen proteins of weight (supply of Beijing Tiantan Bio-pharmaceuticals goods company), 56 parts of heavy IL-2,16 parts of heavy GM-CSF and 6 parts of tetanus toxoid aqueous solutions, make lyophilized formulations according to " Chinese biological goods rules " version disclosed method in 2000 of Chinese biological standard of articles committee establishment.
The preparation of embodiment 4 vaccines
Get 100 parts of above-mentioned HBV specific antigen proteins of weight (supply of Beijing Tiantan Bio-pharmaceuticals goods company), 56 parts of heavy IL-2,16 parts of heavy GM-CSF and 6 parts heavy tetanus toxoid aqueous solutions, make lyophilized formulations according to " Chinese biological goods rules " version disclosed method in 2000 of Chinese biological standard of articles committee establishment.
The use of embodiment 5 vaccines
When anti-HBV therapeutic vaccine immunity HBV transgenic mouse (1.3.32 Mus) immunity made from foundation the foregoing description 1 finishes Mus is put to death, get Hepar Mus and make leachate, extract total DNA and RNA in the liver by leachate, analyze HBV RC DNA and SS DNA in the liver with Southern blot method, analyze gamma interferon (IFN-γ) and tumor necrosis factor-alpha (TNF-α) RNA with Northen blot method simultaneously.In the experimentation, establish a matched group.Matched group 1.3.32 Mus is used the normal saline immunity.Experimental result such as Fig. 2.As seen from Figure 2, after the treatment group was treated with above-mentioned anti-HBV therapeutic vaccine, HBV RC DNA and SS DNA district band disappeared.High concentration IFN-γ and TNF-α RNA district band appears simultaneously.This description of test, above-mentioned anti-HBV therapeutic vaccine has anti-HBV effect to the HBV transgenic mouse, and it can remove HBV DNA in the HBV transgenic Hepar Mus.After it was removed mechanism and injects anti-HBV therapeutic vaccine, generation high concentration IFN-γ and TNF-α were relevant in the liver.
The use of embodiment 6 vaccines
With anti-HBV therapeutic vaccine 10 μ g immunity HBV transgenic mouse of the foregoing description 3 and the HBV specific cytotoxic t cell responses (abbreviation ctl response) of two kinds of Mus of normal control Mus (B6D2F1) immunity back survey in the 11st day.Survey the preceding ctl response of two kinds of Mus immunity of the same race simultaneously.The result proves that the preceding 90% pair of HBV S fragment albumen of B6D2F1 Mus immunity has ctl response; After the immunity of anti-HBV therapeutic vaccine, its ctl response occurrence rate rises to 100%.And the HBV transgenic mouse before the immunity of anti-HBV therapeutic vaccine, has only 30% ctl response to occur, rises to 93% after the immunity.The overwhelming majority (70%) HBV transgenic mouse has immunologic tolerance (no ctl response) before this description of test, the immunity of anti-HBV therapeutic vaccine.After the immunity of anti-HBV therapeutic vaccine, immunologic tolerance is broken, and 93% ctl response occurs.Therefore the anti-HBV therapeutic vaccine of this description of test has the immunologic tolerance effect of breaking the HBV transgenic mouse.
The testing result that embodiment 7 vaccines use
Give with gene recombinaton HBV S fragment albumen Hepatitis B virus vaccine (Tiantan Bio-pharmaceuticals goods company provides by Beijing) and to inject the normal person that above-mentioned Hepatitis B virus vaccine produces anti--HBs and do intradermal test, can measure 95% normal person occur the delayed intradermoreaction positive (be that erythema appears in the injection site, diameter＞0.5cm).Get erythema part skin and cut into slices, check the skin infiltration cell, prove that soaking into cell is CD8
+(CTL) cell and CD4
+(HTL) cell is main.Illustrate that the intradermoreaction that this intradermal test is drawn belongs to the delayed intradermoreaction, represent CTL and HTL cell the proteic cell immune response of HBV S fragment.Similar to external ctl response.95% has this intradermoreaction positive of anti--HBs normal person, illustrates through hepatitis b vaccine immune to produce anti--HBs normal person, and the overwhelming majority (95%) does not have immunologic tolerance.But check the Chronic HBV carrier with same intradermoreaction, then have only 5% to be positive, 95% is negative.Illustrate that the overwhelming majority (95%) Chronic HBV carrier has immunologic tolerance.With anti-HBV therapeutic vaccine treatment, 1 time every month, inject altogether 12 times, then 90% patient's intradermal test changes the positive into by feminine gender.Prove that anti-HBV therapeutic vaccine has the effect of breaking its immunologic tolerance to Chronic HBV carrier patient.
Claims (5)
1, a kind of anti-hepatitis B virus therapeutic vaccine is made up of HBV specific antigen protein and adjuvant, and wherein said adjuvant contains interleukin II, human granulocyte-macrophage colony stimulating factor, tetanus toxoid.
2, according to the vaccine of claim 1, in the wherein said adjuvant, the part by weight of interleukin II, human granulocyte-macrophage colony stimulating factor, tetanus toxoid is 50-600: 10-200: 6-20.
3, according to the vaccine of claim 1 or 2, in the wherein said adjuvant, the part by weight of interleukin II, human granulocyte-macrophage colony stimulating factor, tetanus toxoid is 56: 16: 6.
4, according to the vaccine of claim 1, the part by weight of wherein said HBV specific antigen protein, interleukin II, human granulocyte-macrophage colony stimulating factor, tetanus toxoid is 5-100: 50-600: 10-200: 4-10.
5, according to the vaccine of claim 4, the part by weight of wherein said HBV specific antigen protein, interleukin II, human granulocyte-macrophage colony stimulating factor, tetanus toxoid is 60: 56: 16: 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03104697 CN1270772C (en) | 2002-12-30 | 2003-02-21 | Anti-hepatitis-B-virus therapeutic vaccine and its adjuvant |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02159913 CN1424110A (en) | 2002-12-30 | 2002-12-30 | Vaccine adjuvant against hepatitis B |
| CN02159913.0 | 2002-12-31 | ||
| CN 03104697 CN1270772C (en) | 2002-12-30 | 2003-02-21 | Anti-hepatitis-B-virus therapeutic vaccine and its adjuvant |
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| CN1454668A CN1454668A (en) | 2003-11-12 |
| CN1270772C true CN1270772C (en) | 2006-08-23 |
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| CN 03104697 Expired - Lifetime CN1270772C (en) | 2002-12-30 | 2003-02-21 | Anti-hepatitis-B-virus therapeutic vaccine and its adjuvant |
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| CN1305527C (en) * | 2003-11-21 | 2007-03-21 | 薛平 | Vaccine for treating hepatitis B, and its prepn. method |
| CN105854009A (en) * | 2016-05-03 | 2016-08-17 | 重庆三杰众鑫生物工程有限公司 | Preparation method for preparing inactivated vaccine by means of Muscovy duck hepatitis virus |
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Owner name: SU SHENG Free format text: FORMER OWNER: XIBO MEDICAL TECHNOLOGY DEVELOPMENT CO., BEIJING Effective date: 20051223 |
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Effective date of registration: 20051223 Address after: 100085, No. 28, Middle Road, Haidian District information industry base, Beijing Applicant after: Su Sheng Address before: 100085, No. 28, Middle Road, Haidian District information industry base, Beijing Applicant before: XIBO MEDICAL TECHNOLOGY DEV Co. |
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Granted publication date: 20060823 |