CN109876140A - A kind of vaccine and its preparation method and application for treating chronic hepatitis B - Google Patents
A kind of vaccine and its preparation method and application for treating chronic hepatitis B Download PDFInfo
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- CN109876140A CN109876140A CN201910206739.0A CN201910206739A CN109876140A CN 109876140 A CN109876140 A CN 109876140A CN 201910206739 A CN201910206739 A CN 201910206739A CN 109876140 A CN109876140 A CN 109876140A
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract
The present invention relates to a kind of vaccines and its preparation method and application for treating chronic hepatitis B, and the antigen by hepatitis B is formulated with the composite adjuvant containing molecule adjuvant.The vaccine for the treatment of chronic hepatitis B of the invention can not only induce very strong antibody response, can also induce very strong cell immune response.Antigen very effective can be transmitted to lymphatic system by the carrier for the composite adjuvant that the present invention uses.Molecule adjuvant can activate T cell subgroup, the effective level and persistence for improving antibody and cell immune response.Antibody caused by after immune for antigen of hepatitis B virus can eliminate blood circulation then virus and antigen.The vaccine for the treatment of chronic hepatitis B of the invention cracking can reduce the antigen for even being eliminated virus in blood, eliminate hepatitis B in liver cell, make patient's no longer Hepatitis B carrier.
Description
Technical field
The invention belongs to biological and chemical pharmaceutical technology field, it is related to a kind of vaccine for treating chronic hepatitis B and its preparation side
Method and application.
Background technique
Hepatitis B is the chronic hepatitis as caused by HBV hepatitis type B virus (HBV).According to the statistics of the World Health Organization,
Global about 2,000,000,000 people once infected HBV, wherein 3.5 hundred million people are Patients with Chronic HBV Infection, there are about 1,000,000 people to die of HBV infection every year
Caused hepatic failure, cirrhosis and primary hepatoma.China belongs to the high Endemic Area of HBV infection, and the HBsAg of general population is positive
Rate is 9%.Inoculation and the HBsAg positive rate of non-Hepatitis B Immunization group are respectively 4.51% and 9.51%.China there are about
1.2 hundred million HBV carrier, the one third of Zhan Quanqiu.Chronic hepatitis B (HBV) infection can lead to chronic hepatitis, cirrhosis, very
To liver cancer.
What HB infected diagnoses can include by what the horizontal of detection HBV antigen or antibody coarse evaluation virus often be surveyed
Surface antigen (HBsAg), surface antibody (anti-HBsAg), e antigen (HBeAg), e antibody (anti-HBeAg) and core antibody are (anti-
The five indices such as HBcAg).It can also judge whether virus is replicating by detecting HBV DNA.
Hepatitis B vaccination is the necessary means that effective control HBV is propagated, and China carries out newborn's pressure plan and exempts from present
Epidemic disease, once HB vaccination of being born.There is the protecting effect of antibody responder generally 12 years at least sustainable after vaccine inoculation.
Antiviral therapy is the basic treatment method of chronic hepatitis B, and common drug includes interferon, Lamivudine, A Defu
Wei ester, Sebivo, Entecavir, tenofovir disoproxil etc..But current treatment method can not eliminate virus from internal,
It cannot heal the sick.
Have with vaccine therapy chronic hepatitis B or hepatitis carrier at low cost, validity preferably may.But mesh
Preceding therapeutic vaccine not yet.Main challenge is often to contain a large amount of B-type hepatitis in the blood of hepatitis carrier
Poison antigen, lead to the tolerance to antigen of hepatitis B virus, from be unable to vaccine occur well immune response.Patient is to one
As vaccine immune response it is all very poor.Second the reason is that the virus of hepatitis B is often present in liver cell in a manner of cccDNA
Interior, therapeutic vaccine needs to induce very strong cellular immunity that can have therapeutic effect.It is generally acknowledged that only induction of antibodies is anti-
The vaccine therapy effect answered is bad.
The vaccine of clinical phase exploitation includes the subunit vaccine and antigen-antibody complex of synthesis polypeptide vaccine, high dose.
There is presently no the data of validity.
Synthesis polypeptide vaccine being capable of induction of antibodies reaction.But clinical 3 phases test proves no therapeutic effect.
The composition of the subunit vaccine of high dose is consistent with preventative hepatitis B vaccine, that is, has the surface antigen of hepatitis B
(HBsAg) it is formulated with aluminium hydroxide.The vaccine prevented contains the HBsA of 10-20 microgram, and clinic is in the therapeutic epidemic disease ground
Seedling has used the HBsAg of 60 micrograms.Due to having used aluminium hydroxide, the vaccine is estimated to be merely able to induction of antibodies reaction in vivo,
It is unable to inducing cellular immune reaction.Its validity is proved currently without any clinical data.
Antigen-antibody complex is that HBsAg is naturally combined with the specific antibody of HBsAg.The end Fc of antibody
It can be in conjunction with the Fc receptor of the antigen presenting cell of immune system, so as to promote and improve the presentation effect of antigen.It is theoretical
On say be possible to break immune tolerance status, and induction of antibodies react.Currently without being capable of inducing cellular immune about the technology
The data of reaction.This technology proves in clinical test effective not yet at present.
Theoretically, the antibody of hepatitis B can reduce the viral antigen in blood circulation, but antibody can not
It enters in the liver cell of infection, it can not virus in scavenger-cell.Therapeutic hepatitis B vaccine induction of antibodies react while,
It also needs to induce very strong cellular immunity.Cellular immunity is hidden by generating interferon and can invade to kill into the cell dry
Intracellular virus, or directly kill the stem cell of infection hepatitis B.Be possible to cure the patient of infection in this way.
The technology for capableing of inducing cellular immune includes live vector and nucleic acid vaccine (DNA or RNA).These technologies
Defect is not can induce good antibody response generally.There is presently no the Clinical efficacy data of any of these technologies.
The patent of invention that number of patent application is 03123562.X discloses a kind of vaccine preparation of therapeutic hepatitis B, its system
Preparation Method and application thereof specifically discloses a kind of vaccine preparation of therapeutic hepatitis B, comprising: recombination (yeast) hepatitis B vaccine
(HBsAg): 40~80 μ g/ml, 0.5mg/ml containing aluminium.The patent also discloses a kind of vaccine preparation of therapeutic hepatitis B
Preparation method and its usage, and the composition containing hepatitis B vaccines.Said preparation can induce cellullar immunologic response
And advantageous body removes HBV, can increase the cellular immune function of Balb/c mouse, promotees Th1 type cytokines high level expression, mentions
The level of high T proliferation, and promote specific CTL activity raising.But the result that the patent is reported is not inconsistent with document.Aluminium adjuvant
In the extensive use of vaccine for man, it was demonstrated that CTL cell effect can not be promoted.Faced with the polypeptide hepatitis B vaccine that aluminium adjuvant is prepared
Bed proves no therapeutic effect.
The patent of invention that number of patent application is 200510033869 disclose a kind of hepatitis B nucleic acid vaccine, preparation method and its
Using using pharmaceutically acceptable eukaryon expression plasmid, selection encodes multiple T cell antigen determinants and B cell
The gene order of antigenic determinant constructs multi-epitope gene vaccine.The hepatitis B nucleic acid vaccine of the invention can be used for preparing prevention, control
The preparation of hepatitis B vaccine is treated, but the patent uses genetic engineering means, preparation method is complicated and there is no associated treatment effects
Clinical data.Meanwhile induction of antibodies does not react the technology, many document reports think that therapeutic hepatitis B vaccine needs to induce
Induction of antibodies reaction simultaneously and cell immune response.
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provide a kind of vaccine for treating chronic hepatitis B and preparation method thereof and
It is prepared using the antigen of hepatitis B virus and cellular immunity adjuvant of, use, it, can in mouse model for therapeutic vaccine
Very strong antibody response and cell immune response are induced, has good therapeutic effect.
The present invention provides the following technical solutions:
A kind of vaccine for treating chronic hepatitis B, antigen by hepatitis B and the composite adjuvant containing molecule adjuvant prepare and
At.
The present invention also provides a kind of preparation methods of the vaccine of above-mentioned treatment chronic hepatitis B, comprising the following steps:
(1), the antigen of hepatitis B is prepared；
(2), composite adjuvant is prepared；
(3), the antigen of hepatitis B is mixed with composite adjuvant, and mixing is configured to vaccine.
Preferably, antigen of hepatitis B virus includes surface antigen and nuclear antigen in the step (1).
Hepatitis b virus surface antigen gene can encode 3 kinds of protein.This 3 kinds of protein are close from different startings
Numeral translation, but they share common a reading frame and terminator codon.The surface that preventative vaccine uses
Antigen (HBsAg) contains 226 amino acid, is the main component of peplos.PreS 2 antigen (pre-S2) is the N in HBsAg
End increases 55 amino acid.Pre S 1 antigen (pre-S1) is to increase 119 amino acid in the N-terminal of HBsAg.Due to
Pre-s1 plays an important role in Receptor recognition, and pre-s2 plays an important role in virus into host cell transfer.So
HBsAg, pre-s1 and pre-s2 are potential vaccine antigens.
Hepatis B core antigen can be by inducing cellular immune come direct elimination virus or the stem cell of virus infection.
Antigen of hepatitis B virus includes surface antigen, Pre-S1, Pr-S2 and nuclear antigen, can pass through eukaryon or prokaryotic expression
System production.Common eukaryotic expression system includes Chinese hamster ovary celI, insect baculovirus expression system, yeast expression system etc..Often
Prokaryotic expression system includes Escherichia coli and other expression systems.
The HBsAg Yeast expression that the present invention uses, nuclear antigen are by expressing cho cell and to purify.
Treat chronic hepatitis B vaccine can be used any hepatitis B virus surface antigen (HBsAg, pre-S1, or
) or the mixture of any hepatitis B virus surface antigen and nuclear antigen Pre-S2.
Any of the above-described scheme is preferably, in the step (1) antigen include HBsAg or Pre-S1 containing HBsAg or
Pre-S2。
Any of the above-described scheme is preferably, in the step (1) antigen include HBsAg or Pre-S1 containing HBsAg or
The mixture of Pre-S2 and nuclear antigen.
Any of the above-described scheme is preferably, and antigen dosage is 10-100 microgram in the step (1).
Any of the above-described scheme is preferably, and antigen dosage is 10 micrograms in the step (1).
Any of the above-described scheme is preferably, and antigen dosage is 50 micrograms in the step (1).
Any of the above-described scheme is preferably, and antigen dosage is 100 micrograms in the step (1).
Any of the above-described scheme is preferably, and composite adjuvant includes point of carrier and inducing cellular immune in the step (2)
Sub- adjuvant.
Any of the above-described scheme is preferably, and composite adjuvant contains a carrier and at least one induction in the step (2)
The molecule adjuvant of cellular immunity.
It is capable of the important composition of the vaccine of the adjuvant treatment chronic hepatitis B of inducing cellular immune.There is no the case where adjuvant
Under, usual induction of antibodies reaction of subunit antigen and Th2 reaction.Common aluminium adjuvant can only often improve the reaction of Th2 class, no
The cell immune response of Th1 class can be induced.So successfully exploitation therapeutic hepatitis B vaccine needs to come using novel adjuvant
Enhance antibody response and cell immune response.
There are many immunologic adjuvant compositions being capable of oneself intracorporal cell immune response in document
The chemical component of Toll-like Receptor 3 ligand (TLR3L) receptor activation object is the ribose core of double-strand
Acid, specific TLR3 activator include Poly IC, TLR3 ligand, poly IC (polymer formed with kanamycins),
TLR3 ligand LC (polymer with polylysine) etc..TLR3 receptor activation object can be integrated to intracellular receptor
TLR3, stimulator antigen are in delivery cell and antigen presentation, inducing T cell reaction.
Toll-like Receptor 4 (TLR4) receptor activation object is detoxification endotoxin (Monophosphoryl-lipid
A, abbreviation MPL or 3DMPL are to extract from the cell membrane of Salmonella and go its toxicity by chemical modification but do not destroy
The effect of its adjuvant.In addition to the above-mentioned MPL proposed from microorganism, there are also the molecules of artificial synthesized congenerous, including pyrans
Glucosyl group rouge A (glucopyranosyl lipid A), artificial synthesized lipoprotein A (synthetic-lipid A) these
Molecule can be integrated to the TLR4 receptor of Antigen Presenting Cell surface, activate the generation of the cytositimulation inducing T cell.
QS21 is the saponin(e substance extracted from the Quillaia saponaria of South America, the activity with surfactant, can be in human body
The reaction of inducing T cell.Its specific works mechanism is simultaneously indefinite.Similar molecule includes extracting from ginseng or its cauline leaf
Saponin(e molecule.
Toll-like Receptor 7 ligand (TLR7L) receptor activation object activator includes Lei Ximode
(Resiqimod), imiquimod (Imiqimod), 3M-052 etc..These molecules can be integrated to Antigen Presenting Cell surface
TLR7 activates its antigen presentation, the reaction of inducing T cell.
9 ligand of Toll-like receptor (TLR9L) is the CpG DNA of short chain.Its sequence has very
It is a variety of in conjunction with intracellular TLR9 receptor after, can activation antigen be in delivery cell, the reaction of inducing T cell.
But in our experiment, it has been found that TLR3 ligand activator, TLR7 receptor activation object, MPL, QS-21 have
Very strong immunostimulating effect,
These molecular immune adjuvants are needed with the immune effect of the composite adjuvant of the carrier compatibility of a nanometer than immune point
The effect that sub- adjuvant itself or nano-carrier adjuvant are used alone will be got well.
The dosage of molecule adjuvant is generally 1 microgram-to 1 milligram.Different adjuvant dosages is different.
Any of the above-described scheme is preferably, and the carrier is nano-carrier.
Any of the above-described scheme is preferably, and the carrier is nanoemulsion, liposome, any one in Alum adjuvant.
Nanoemulsion is oil-in-water nanoemulsion.
Any of the above-described scheme is preferably, and liposome is made of cholesterol and phosphatide, especially phosphatidyl choline, egg
The spherical vesicles for the double-layer of lipoid that the compositions such as phosphatidyl-ethanolamine are prepared.
Nano-carrier can adsorb or binding molecule adjuvant, on the one hand it can be transmitted to lymphatic system, even carefully
It is intracellular, on the other hand it is that molecule adjuvant is avoided to be diffused into blood, causes side reaction caused by cytokine release.
Any of the above-described scheme is preferably, and the Alum adjuvant includes aluminium hydroxide, aluminum phosphate, any one in calcium phosphate
Kind.Alum adjuvant is very widely used in vaccine.
Any of the above-described scheme is preferably, and the nanoemulsion is oil-in-water emulsion.Nanoemulsion is by immunostimulation
Various oily (such as squalenes, saualane etc.) and surfactant formulatory of function form.
Any of the above-described scheme is preferably, the nanoemulsion include MF59, Stable emulsion, AS04, AF03 or
Any one in similar products.
Any of the above-described scheme is preferably, and the liposome is hollow liposome, has single layer or bilayer structure capsule
Family name's film, diameter 25-1000nm.
Any of the above-described scheme is preferably, and the liposome is the sky as made from cholesterol, lecithin and ceramide etc.
Cardiolipin body.Liposome is the vaccine and drug delivery system prepared by phosphatide, the biomaterial of the lipids such as cholesterol.
Any of the above-described scheme is preferably, the molecule adjuvant of the inducing cellular immune be QS-21, TLR3ligand,
Any one in TLR4 ligand, TLR-7/8ligand, TLR9 ligand or agonist.
Any of the above-described scheme is preferably, and composite adjuvant is using agonist and nanoemulsion, lipid in the step (2)
At least one of body, Alum adjuvant are formulated.
Any of the above-described scheme is preferably, and nanoemulsion is that squalene, Tween 80 and span 85 are formulated.
Any of the above-described scheme is preferably, and nanoemulsion is the 3.5-4.5% squalene by immunostimulation function, 0.5-
1.0% Tween 80 and 0.5-1.0% span 85 are formulated by high pressure homogenization.
Any of the above-described scheme is preferably, range of the average grain diameter of nanoemulsion at 130-170 nanometers.
Any of the above-described scheme is preferably, and composite adjuvant is the hydroxide for having adsorbed TLR3 ligand in the step (2)
Aluminium, the nanoemulsion of agonist containing TLR-7/8, agonist containing TLR-7/8 nanoemulsion in any one.
Any of the above-described scheme is preferably, and the composite adjuvant is the nanoemulsion of the agonist containing TLR-7/8.
Any of the above-described scheme is preferably, and the dosage of the agonist containing TLR-7/8 is 1-100 microgram.
Any of the above-described scheme is preferably, and the dosage of the agonist containing TLR-7/8 is 1 microgram.
Any of the above-described scheme is preferably, and the dosage of the agonist containing TLR-7/8 is 50 micrograms.
Any of the above-described scheme is preferably, and the dosage of the agonist containing TLR-7/8 is 100 micrograms.
Any of the above-described scheme is preferably, and the dosage of the nanoemulsion is 0.5-2 milliliters.
Any of the above-described scheme is preferably, and the dosage of the nanoemulsion is 0.5 milliliter.
Any of the above-described scheme is preferably, and the dosage of the nanoemulsion is 1 milliliter.
Any of the above-described scheme is preferably, and the dosage of the nanoemulsion is 2 milliliters.
Any of the above-described scheme is preferably, and composite adjuvant is adsorbed onto hydroxide using TLR3 ligand in the step (2)
Aluminium is formulated.
Any of the above-described scheme is preferably, and the aluminium hydroxide dosage is 0.1-1 milligrams, the TLR3 of aluminium hydroxide absorption
The amount of ligand (TLR3L) is 10-1000 microgram.
Any of the above-described scheme is preferably, and the aluminium hydroxide dosage is 0.1 milligram.
Any of the above-described scheme is preferably, and the aluminium hydroxide dosage is 0.5 milligram.
Any of the above-described scheme is preferably, and the aluminium hydroxide dosage is 1 milligram.
Any of the above-described scheme is preferably, and the dosage of the TLR3 ligand (TLR3L) of the aluminium hydroxide absorption is 100-
500 micrograms.
Any of the above-described scheme is preferably, and the amount of the TLR3 ligand (TLR3L) of aluminium hydroxide absorption is 10 micrograms.
Any of the above-described scheme is preferably, and the amount of the TLR3 ligand (TLR3L) of aluminium hydroxide absorption is 100 micrograms.
Any of the above-described scheme is preferably, and the amount of the TLR3 ligand (TLR3L) of aluminium hydroxide absorption is 1000 micrograms.
Any of the above-described scheme is preferably, and the agonist is that the agonist of TOLL-7/8 receptor, TOLL-4 receptor swash
Move the agonist of agent, TLR-9 receptor.
Any of the above-described scheme is preferably, in the step (2) composite adjuvant using TOLL-7/8 receptor agonist with
Nanoemulsion is formulated.
Any of the above-described scheme is preferably, in the step (2) composite adjuvant using TOLL-7/8 receptor agonist with
Liposome formulation forms
Any of the above-described scheme is preferably, and composite adjuvant uses agonist, the QS21 of TOLL-4 receptor in the step (2)
It is formed with liposome formulation.
Any of the above-described scheme is preferably, and composite adjuvant is QS-21 and any one TLR9 in the step (2)
The mixture of ligand.
Any of the above-described scheme is preferably, and composite adjuvant is liposome and QS-21 and TLR4 in the step (2)
The mixture of ligand.
Any of the above-described scheme is preferably, and the dosage of the liposome and QS-21 are 2-100 microgram.
Any of the above-described scheme is preferably, and the dosage of the liposome and QS-21 are 2 micrograms.
Any of the above-described scheme is preferably, and the dosage of the liposome and QS-21 are 50 micrograms.
Any of the above-described scheme is preferably, and the dosage of the liposome and QS-21 are 100 micrograms.
Any of the above-described scheme is preferably, and the TLR4 ligand dosage is 2-100 microgram.
Any of the above-described scheme is preferably, and the TLR4 ligand dosage is 2 micrograms.
Any of the above-described scheme is preferably, and the TLR4 ligand dosage is 50 micrograms.
Any of the above-described scheme is preferably, and the TLR4 ligand dosage is 100 micrograms.
Any of the above-described scheme is preferably, and the vaccine is the vaccine of agonist containing TLR4 and the configuration of QS-21 composite adjuvant,
Preparation method the following steps are included:
(1), aqueous trehalose is preheated, takes aqueous trehalose, QS21 and antigen are added in centrifuge tube, stirring；
(2), it draws respectively and is dissolved in alcohol-chloroform MPLA, DOPC and cholesterol in centrifuge tube, vibrate, MPLA, DOPC
Film is formed in tube bottom with cholesterol；
(3) solution of antigen and QS-21 is poured into the cillin bottle of MPLA, DOPC and cholesterol, ultrasound is after ice-water bath
At.
Any of the above-described scheme is preferably, and the vaccine is the vaccine that agonist containing TLR7/8-nanoemulsion is prepared, preparation
Method the following steps are included:
(1) nanoemulsion is formulated by the squalene, Tween 80 and span 85 of immunostimulation function by high pressure homogenization,
In the process for preparation of nanoemulsion, agonist is added in squalene；
(2) physiological saline, nuclear antigen and surface antigen are added in cillin bottle, after mixing, nanoemulsion and gently is added
Shake up mixing.
Any of the above-described scheme is preferably, 3.5-4.5% squalene of the nanoemulsion by immunostimulation function, 0.5-
1.0% Tween 80 and 0.5-1.0% span 85 are formulated by high pressure homogenization, will in the process for preparation of nanoemulsion
TLR7/8 agonist is added in squalene.
Any of the above-described scheme is preferably, and hepatitis B nuclear antigen is expressing cho cell, and HBsAg is the virus-like of Yeast expression
Particle.
Any of the above-described scheme is preferably, and the vaccine is the vaccine that the composite adjuvant of aluminium hydroxide containing PolyIC- is prepared, system
Preparation Method the following steps are included:
(1) physiological saline, PolyIC and aluminium hydroxide are added in cillin bottle, after shaking up, nuclear antigen is added and surface is anti-
It is former.
(2) it shakes up overnight.
Any of the above-described scheme is preferably, and the vaccine of every 0.5 milliliter of dosage contains the aluminium of 500 micrograms, 200 micrograms
The hepatitis B nuclear antigen of PolyIC, the HBsAg of 20 micrograms and 10 micrograms.
Any of the above-described scheme is preferably, and HBsAg is the virus-like particle of Yeast expression, and hepatitis B nuclear antigen is Chinese hamster ovary celI
Expression.
The present invention also provides the vaccine of above-mentioned treatment chronic hepatitis B preparation for prevent or treat chronic hepatitis B drug and/
Or the application in liver-cancer medicine caused by hepatitis B virus infection.
Beneficial effect
(1) different from other technologies, the vaccine for the treatment of chronic hepatitis B of the invention can not only induce very strong antibody anti-
It answers, very strong cell immune response can also be induced；
(2) antigen, very effective can be transmitted to lymphatic system by the carrier for the composite adjuvant that the present invention uses.Point
Sub- adjuvant can activate T cell subgroup, the effective level and persistence for improving antibody and cell immune response；
(3) antibody caused by after being immunized for antigen of hepatitis B virus can eliminate blood circulation then virus and antigen,
Including surface antigen (HBsAg), e antigen (HBeAg) and nuclear antigen (HBcAg).Killer T cell caused by after immune can kill
The liver cell of dead infection hepatitis B.Cell factor caused by immune t-cell, including gamma- interferon can penetrate into
The liver cell of virus infection, effectively kills hepatitis B, prevents and treats its further activation and diffusion.
The vaccine for the treatment of chronic hepatitis B of the invention cracking can reduce the antigen for even being eliminated virus in blood.This hair
The vaccine of bright treatment chronic hepatitis B can eliminate hepatitis B in liver cell, can cure hepatitis B infected, make patient no longer
Hepatitis B carrier.
Detailed description of the invention
The surface antigen of the mouse for the vaccine immunity that Fig. 1 .TLR4 agonist-QS-21- liposome composite adjuvant is prepared
(HBAg) Conversion rate of antibody；
The e antigen (HBeAg) of the mouse for the vaccine immunity that Fig. 2 .LR4 agonist-QS-21- liposome composite adjuvant is prepared
The Conversion rate of antibody；
R- interference in the serum of the mouse for the vaccine immunity that Fig. 3 .LR4 agonist-QS-21- liposome composite adjuvant is prepared
The concentration of element；
In the protective effect-acceleration blood for the vaccine that Fig. 4 .TLR4 agonist-QS-21- liposome composite adjuvant is prepared
The removing of HBsAg；
In the protective effect-acceleration blood for the vaccine that Fig. 5 .TLR4 agonist-QS-21- liposome composite adjuvant is prepared
The removing of HBeAg；
The sun of surface antigen (HBAg) antibody of the mouse for the vaccine immunity that Fig. 6 .TLR7/8 agonist-nanoemulsion is prepared
Rate of rotation；
E antigen (HBeAg) antibody of the immune mouse for the vaccine immunity that Fig. 7 .TLR7/8 agonist-nanoemulsion is prepared
Conversion rate；
R-interferon is dense in the serum of the epidemic disease mouse for the vaccine immunity that Fig. 8 .TLR7/8 agonist-nanoemulsion is prepared
Degree；
In the protective effect-acceleration blood for the therapeutic hepatitis B vaccine that Fig. 9 .TLR7/8 agonist-nanoemulsion is prepared
The removing of HBsAg；
In the protective effect-acceleration blood for the therapeutic hepatitis B vaccine that Figure 10 .TLR7/8 agonist-nanoemulsion is prepared
The removing of HBeAg；
Surface antigen (HBAg) antibody of the mouse for the vaccine immunity that Figure 11 .TPolyIC- aluminium hydroxide composite adjuvant is prepared
Conversion rate；
E antigen (HBeAg) antibody of the mouse for the vaccine immunity that Figure 12 .PolyIC- aluminium hydroxide composite adjuvant is prepared
Conversion rate.
R-interferon is dense in the serum of the mouse for the vaccine immunity that Figure 13 .PolyIC- aluminium hydroxide composite adjuvant is prepared
Degree；
Protective effect-acceleration blood of the therapeutic hepatitis B vaccine of the preparation of Figure 14 .PolyIC- aluminium hydroxide composite adjuvant
The removing of middle HBsAg；
Protective effect-acceleration blood of the therapeutic hepatitis B vaccine of the preparation of Figure 15 .PolyIC- aluminium hydroxide composite adjuvant
The removing of middle HBsAg.
Specific embodiment
In order to further appreciate that technical characteristic of the invention, the present invention is explained in detail combined with specific embodiments below
It states.
1 high pressure tail vein injection paav-HBV1.2 of examples of implementation establishes hepatitis B mouse model
High pressure water dynamic method establishes the therapeutic effect of HBV mouse nuclei evaluation therapeutic hepatitis B vaccine.High pressure tail is quiet
Arteries and veins injects hepatitis B plasmid (paav-HBV1.2), infects C57 mouse, detects within sustainable 1-2 months the table of hepatitis B
It reaches.Therapeutic hepatitis B vaccine is subcutaneously injected, therapy intervention is carried out to infecting mouse, and is compareed with control group mice.It detects small
The amount of HBsAg and HBeAg can reflect the curative effect of therapeutic vaccine in mouse blood.Confirmed in stem cell by immunohistochemical staining
The presence of immunocyte and the hepatitis B presence or absence in liver cell.
C57 (about 6~7 weeks) starts to carry out experiment after adaptable fed 7 days.The weight of animals selected is consistent, on 20 grams of left sides
It is right.
The paav-HBV1.2 plasmid of hepatitis B is expanded and is purified with Escherichia coli.First plasmid is diluted with physiological saline
At 5ug/ml.Then the plasmid of 8% weight volume is injected according to the average weight of animal.I.e. every mouse injects 1.6ml volume
Dilution plasmid.Vascular compression stops blooding 10 seconds after the completion of injecting.The permeability of duration high pressure change liver cell.Matter is injected
It after grain, in the 3rd to 60 day, takes weekly blood (EDTA-K2 is anticoagulant), 1700g is centrifuged 5min, collects the anti-of supernatant detection hepatitis B
Ultimate constituent, including HBsAg and HBeAg.
As a result it proves within the time of infection 3 days to 45 days, viral antigen in 7 mouse whole bloods of inoculation
(HBsAg and HBeAg) detection is positive, i.e., successful infecting mouse.After 45th day, positive mouse gradually switchs to feminine gender, it was demonstrated that
Mouse can remove hepatitis B automatically.
We also use immunofluorescence dyeing hepatic tissue section simultaneously, confirm virus infection.Hepatic tissue is fixed, paraffin packet
It buries, slice dewaxing, antigen retrieval adds the antibody of anti-hepatitis virus HBsAg or HBcAg, and second along with solidifying signal is anti-
Body is redyed using DAPI, mounting microscopy.HBcAg in blood plasma can be disposed of, but liver cell (uncracked) meeting infected
Continuous expression HBcAg.Hepatocyte infection rate by the liver cell of detection infection, after reaction is immune.
The result shows that within the time of infection 3 days to 45 days, the mouse liver of high pressure tail vein injection hepatitis B plasmid
The HBsAg and HBcAg of middle hepatitis B are the positive.After 45 days, positive mouse gradually switchs to feminine gender.
The preparation of the various compositions of the preparation of 2 agonist containing TLR4 of embodiment and QS-21 liposome composite adjuvant vaccine:
Monophosphoryl lipid A, Monophosphoryl Lipid A (MPLA) is bought by market, after being dissolved in 100% alcohol
Concentration is 1.0 milligrams every milliliter, and QS-21 is bought by market, and the concentration of aqueous solution is 1.0 milligrams of milliliter.
Cholesterol 0.0489g is weighed, chloroform 4.89ml is measured in vent cabinet and is added in the EP pipe equipped with cholesterol, liquid relief
Device makes it dissolve, and mixes.
Weigh 0.0676g dioleyl lecithin 1,2-Dioleoyl-sn-glycero-3-phosphocholine
(DOPC), 1.352ml chloroform is drawn in vent cabinet, is added in DOPC, is mixed with pipettor, perform 4 DEG C of label and be kept in dark place.
Antigen prepares: HBsAg is the virus-like particle of Yeast expression, and purity meets the standard of production of vaccine, uses physiology salt
It is 0.2 milligram every milliliter that water, which is diluted to concentration,.Hepatitis B nuclear antigen is expressing cho cell, is every with normal saline dilution to concentration
1.45 milligrams of milliliter；Purity is higher than 98%.
The preparation of vaccine:
Aqueous trehalose is preheated to 37 DEG C, takes 2780ul aqueous trehalose, QS21 the and 200ul antigen of 20ul is added
In 50ml centrifuge tube, rotor is added in 37 DEG C of stirring 30min.
Draw 50 microlitres respectively and be dissolved in alcohol-chloroform MPLA, 210 microlitres of DOPC and 450 microlitre of cholesterol in
50ml centrifuge tube, is placed in draught cupboard and is vibrated with oscillator, finally chloroform is made to volatilize, and MPLA, DOPC and cholesterol are in tube bottom shape
At film.
Antigen and the mixed solution of Q-21 are poured into the cillin bottle of MPLA, DOPC and cholesterol, ice-water bath 180w is super
Sound 4 minutes.
The vaccine of every 0.3 milliliter of dosage contains 2.0 microgram QS-21,5.0 microgram MPLA (TLR4 agonist), 20 micrograms
The hepatitis B nuclear antigen of HBsAg and 10 micrograms.
The immunogenicity for the therapeutic hepatitis B vaccine that 3 TLR4 agonist of embodiment and QS-21 liposome composite adjuvant are prepared
With mouse every group 8 of C57 (about 6~7 weeks).As described in Example 1, before 3 days vaccinated, high pressure tail
It is injected intravenously hepatitis B plasmid (paav-HBV1.2), infects C57 mouse.1st day, 14 days and 28 days respectively in three times to every
0.3 milliliter of mouse subcutaneous injection of vaccine.The preparation method of vaccine is shown in embodiment 2, and a control group not infected is arranged,
The control group of (no adjuvant) is only immunized after one infection but the control group not being immunized and an infection with antigen.
The 10th day (the 24th day tested) after being immunized at second, venous blood collection separate serum, use ELISA method
Detect the antibody titer (the result is shown in Figure 1) of HBsAg.The therapeutic hepatitis B epidemic disease prepared with TLR4 agonist and QS-21 composite adjuvant
The HBsAg antibody positive of the immune mouse 100% of seedling.There was only 40% HBsAg antibody with antigen (no adjuvant) immune mouse
It is positive.Illustrate the immunogenicity for the therapeutic hepatitis B vaccine that composite adjuvant can be improved.
The 10th day (the 31st day tested) after the third immunization, venous blood collection separate serum, use ELISA method
It detects HBeAg antibody titer (result is shown in Fig. 2).The therapeutic second prepared with TLR4 agonist and QS-21 liposome composite adjuvant
The HBeAg antibody positive of the mouse 80% of liver vaccine immunity.With all HBeAg antibody yin of the immune mouse of antigen (no adjuvant)
Property.HBeAg antibody positive general proof does not have virus replication, illustrates that composite adjuvant can be improved the treatment of therapeutic hepatitis B vaccine
Effect.
The 10th day (the 31st day tested) after the third immunization, venous blood collection separate serum, use ELISA method
Detect the concentration of gamma- interferon in serum (result is shown in Fig. 3).The treatment prepared with TLR4 agonist and QS-21 composite adjuvant
The concentration of the serum gamma- interferon of the mouse of property hepatitis b vaccine immune is far longer than the immune mouse of antigen (no adjuvant)
The concentration of serum gamma- interferon.Gamma- interferon is generally positively correlated with the therapeutic effect of hepatitis B vaccine.
The therapeutic hepatitis B vaccine that 4 TLR4 agonist of embodiment and QS-21 liposome composite adjuvant are prepared is in hepatitis B mouse
The therapeutic effect of model
With mouse every group 8 of C57 (about 6~7 weeks).As described in Example 1, before 3 days vaccinated, high pressure
Tail vein injection hepatitis B plasmid (paav-HBV1.2) infects C57 mouse.1st day, 14 days and 28 days are respectively to every small
The vaccine that 0.3 milliliter of sub-cutaneous injections.The preparation method of vaccine is shown in embodiment 2, is arranged a control group not infected, one
Control group only immune with the antigen without adjuvant after infection but the control group not being immunized and an infection.
Progress blood specimen collection in the 3rd day, 10 days, 17 days, 24 days and 31 days before first time is immune and after immune.With
The viral composition of ELISA method detection HBsAg in serum and HBeAg.
There is no HBsAg and HBeAg in the not immune control group serum for not attacking poison.It is not immunized but attacks the control group of poison in 0-17
There are HBsAg and HBeAg in the serum of its all mouse.During the 24-31 days, the accounting of HBsAg and the HBeAg positive is gradually
It reduces.Only with antigen (no adjuvant) immune mouse, it is fast to attack poison group for the removing speed ratio of antigen in serum, still, uses TLR4
The therapeutic hepatitis B vaccine group that agonist and QS-21 liposome composite adjuvant are prepared removes HBsAg in serum (Fig. 4) and HBeAg
(Fig. 5) and removing speed it is most fast.It proves, adjuvant can be improved the protectiveness of vaccine.
A mouse is taken to take liver solid for every group after 5 days immune (the 5th day, 19 days and 33 days after being immunized for the first time) every time
It is fixed, carry out immunohistochemical analysis (table 1).The virus of detection liver changes over time situation.The results show that in hepatitis B plasmid
The mouse the do not treated HBsAg and HBeAg immunofluorescence positive in the 5th day and the 19th day liver cell is infected, at the 33rd day
HBsAg is feminine gender, but HBeAg remains as the positive, illustrates still virulent duplication in liver cell.Only use antigen (no adjuvant)
Immune mouse does not have significant protective effect, consistent with the result of control at all time points, i.e., no any protection.But
It is that the therapeutic hepatitis B vaccine treatment group prepared with TLR4 agonist and QS-21 liposome composite adjuvant (passes through two on the 19th day
It is secondary immune), HBsAg and HBeAg in liver cell all switch to feminine gender, illustrate, hepatitis B is thoroughly removed.Do not exempt from
It is feminine gender that epidemic disease, which does not attack malicious mouse in all time point HBsAg and HBeAg, as shown in table 1.
The immunohistochemistry for the therapeutic hepatitis B vaccine therapeutic effect that the TLR4 agonist of table 1. and QS-21 composite adjuvant are prepared
Analysis
| DAY5 | DAY19 | DAY33 | |
| It is not immune not attack malicious control | HBsAg-, HBeAg- | HBsAg-, HBeAg- | HBsAg-, HBeAg- |
| It is not immune not attack malicious control | HBsAg+, HBeAg+ | HBsAg+, HBeAg+ | HBsAg-, HBeAg+ |
| Antigen is without adjuvant immunity | HBsAg+, HBeAg+ | HBsAg+, HBeAg+ | HBsAg-, HBeAg+ |
| Antigen plus adjuvant is immune | HBsAg+, HBeAg+ | HBsAg-, HBeAg- | HBsAg-, HBeAg- |
"+" represents the positive."-" represents feminine gender.
5 agonist containing TLR7/8 of embodiment-nanoemulsion vaccine formulation
TLR7/8 agonist is bought by market, and concentration is 1 milligram every milliliter.Nanoemulsion is by immunostimulation function
3.5-4.5% squalene, 0.5-1.0% Tween 80 and 0.5-1.0% span 85 are formulated by high pressure homogenization.In nano-emulsion
In the process for preparation of agent, TLR7/8 agonist is added in squalene, final content is that every 0.3 milliliter of nanoemulsion contains 2
The TLR7/8 agonist of microgram.Range of the average grain diameter of nanoemulsion at 130-170 nanometers.0.22 micron pore size can be passed through
Membrane filtration degerming.
HBsAg is the virus-like particle of Yeast expression, and the standard of purity combination vaccine production, concentration is every milliliter 0.2 milli
Gram.Hepatitis B nuclear antigen is expressing cho cell, and concentration is 1.45 milligrams every milliliter, and purity is higher than 98%.
Aseptically, it is added 750 microlitres of physiological saline in cillin bottle, 115 microlitres of nuclear antigen and 1675 micro-
The surface antigen risen.After jog mixes, 2.5 milliliters of nanoemulsion is added and gently shakes up mixing.The epidemic disease of every 0.3 milliliter of dosage
Seedling contain the TLR7/8 agonist of 1.0 micrograms, the HBsAg of 20 micrograms, 10 micrograms hepatitis B nuclear antigen and 0.15 milliliter of nanometer
Emulsion.
The immunogenicity for the therapeutic hepatitis B vaccine that 6 TLR7/8 agonist of embodiment-nanoemulsion is prepared
With mouse every group 8 of C57 (about 6~7 weeks).As described in Example 1, before 3 days vaccinated, high pressure tail
It is injected intravenously hepatitis B plasmid (paav-HBV1.2), infects C57 mouse.Every mouse is given in 1st day, 14 days and 28 days respectively
The vaccine of 0.3 milliliter of subcutaneous injection.The preparation method of vaccine is shown in embodiment 5, if a control group not infected, an infection
But control group only immune with antigen (no adjuvant) after the control group not being immunized and an infection.
The 10th day (the 24th day tested) after being immunized at second, venous blood collection separate serum, use ELISA method
Detect the antibody titer of HBsAg (result is shown in Fig. 6).Exempted from the therapeutic hepatitis B vaccine that TLR7/8 agonist-nanoemulsion is prepared
The HBsAg antibody positive of the mouse 100% of epidemic disease.There was only 40% HBsAg antibody positive with antigen (no adjuvant) immune mouse.
Illustrate the immunogenicity for the therapeutic hepatitis B vaccine that composite adjuvant can be improved.
The 10th day (the 31st day tested) after the third immunization, venous blood collection separate serum, use ELISA method
It detects HBeAg antibody titer (result is shown in Fig. 7).The therapeutic hepatitis B vaccine prepared with TLR7/8 agonist-nanoemulsion is immune
Mouse 80% HBeAg antibody positive.With all HBeAg negative antibodies of mouse that antigen (no adjuvant) is immune.HBeAg is anti-
Body positive general proof does not have virus replication, illustrates the therapeutic effect for the therapeutic hepatitis B vaccine that composite adjuvant can be improved.
The 10th day (the 31st day tested) after the third immunization, venous blood collection separate serum, use ELISA method
Detect the concentration of gamma- interferon in serum (result is shown in Fig. 8).The therapeutic second prepared with TLR7/8 agonist-nanoemulsion
The concentration of the serum gamma- interferon of the mouse of liver vaccine immunity is far longer than the serum of the immune mouse of antigen (no adjuvant)
The concentration of gamma- interferon.Gamma- interferon is generally positively correlated with the therapeutic effect of hepatitis B vaccine.
The controlling in hepatitis B mouse model for the therapeutic hepatitis B vaccine that 7 TLR7/8 agonist of embodiment-nanoemulsion is prepared
Therapeutic effect
With mouse every group 8 of C57 (about 6~7 weeks).As described in Example 1, before 3 days vaccinated, high pressure tail
It is injected intravenously hepatitis B plasmid (paav-HBV1.2), infects C57 mouse.Every mouse is given in 1st day, 14 days and 28 days respectively
The vaccine of 0.3 milliliter of subcutaneous injection.The preparation method of vaccine is shown in example 5, if a control group not infected, one infection but
Control group only immune with antigen (no adjuvant) after the control group not being immunized and an infection.
Progress blood specimen collection in the 3rd day, 10 days, 17 days, 24 days and 31 days before first time is immune and after immune.With
ELISA method detects the HBsAg and HBeAg of hepatitis B in serum.The results show that TLR7/8 agonist-nanoemulsion is prepared
Vaccine, compared with no vehicle control, can accelerate eliminate HBsAg in serum (Fig. 9) and HBeAg (Figure 10) removing speed,
Illustrate that TLR7/8 agonist-nanoemulsion can be improved the protectiveness of vaccine.
A mouse is taken to take liver solid every group of 5 days immune (i.e. immune for the first time after the 5th day, 19 days and 33 days) every time
It is fixed, carry out immunohistochemical analysis.The virus of detection liver changes over time situation.
Table 2 the results show that TLR7/8 agonist-vaccine prepared of nanoemulsion can be in the 33rd day removing liver cell
HBsAg and HBeAg antigen, i.e., virus do not replicating.Its therapeutic effect is better than the antigen without adjuvant.
The immunohistochemical analysis of the therapeutic effect for the vaccine that table 2. is prepared with TLR7/8 agonist-nanoemulsion
| D5 | D19 | D33 | |
| It is not immune not attack malicious control | HBsAg-, HBeAg- | HBsAg-, HBeAg- | HBsAg-, HBeAg- |
| It is not immune not attack malicious control | HBsAg+, HBeAg+ | HBsAg+, HBeAg+ | HBsAg-, HBeAg+ |
| Antigen is without adjuvant immunity | HBsAg+, HBeAg+ | HBsAg+, HBeAg+ | HBsAg-, HBeAg+ |
| Antigen plus adjuvant is immune | HBsAg+, HBeAg+ | HBsAg+, HBeAg+ | HBsAg-, HBeAg- |
"+" represents the positive."-" represents feminine gender.
The preparation of the vaccine of 8. composite adjuvant of aluminium hydroxide containing PolyIC- of embodiment
PolyIC is bought as the agonist of TLR3 receptor by market, and concentration is 1 milligram every milliliter.Aluminium hydroxide is by chlorine
Change aluminium and sodium hydroxide is formulated, concentration is 10 milligrams every milliliter.HBsAg is the virus-like particle of Yeast expression, and purity is multiple
The standard of production of vaccine is closed, concentration is 0.2 milligram every milliliter.Hepatitis B nuclear antigen is expressing cho cell, and concentration is every milliliter 1.45
Milligram；Purity is higher than 98%.
Aseptically, the physiological saline of 2710 microlitres of addition is in cillin bottle in cillin bottle, 333 microlitres of PolyIC
With 167 microlitres of aluminium hydroxides, after gently shaking up, 115 microlitres of nuclear antigen and 1675 microlitres of surface antigen is added, then sufficiently
It mixes.Cillin bottle is covered into rubber plug, is shaken up overnight with 400r/min revolving speed.The vaccine of every 0.5 milliliter of dosage contains 500 micrograms
The hepatitis B nuclear antigen of aluminium, the PolyIC of 200 micrograms, the HBsAg of 20 micrograms and 10 micrograms.
The immunogenicity for the therapeutic hepatitis B vaccine that 9 PolyIC- aluminium hydroxide composite adjuvant of embodiment is prepared
With mouse every group 8 of C57 (about 6~7 weeks).As described in Example 1, in first day high pressure tail vein injection second
Hepatovirus plasmid (paav-HBV1.2) infects C57 mouse.Every mouse subcutaneous injection is given in 4th day, 18 days and 32 days respectively
0.3 milliliter of vaccine.The preparation method of vaccine is shown in example 8.If a control group not infected, an infection but without immune
Control group and an infection after only with antigen (no adjuvant) immune control group.
The 10th day (the 24th day tested) after being immunized at second, venous blood collection separate serum, use ELISA method
Detect the antibody titer (the result is shown in Figure 1 1) of HBsAg.With the therapeutic hepatitis B epidemic disease of the preparation of PolyIC- aluminium hydroxide composite adjuvant
The HBsAg antibody positive of the immune mouse 80% of seedling.There was only 40% HBsAg antibody sun with antigen (no adjuvant) immune mouse
Property.Illustrate the immunogenicity for the therapeutic hepatitis B vaccine that composite adjuvant can be improved.
The 10th day (the 31st day tested) after the third immunization, venous blood collection separate serum, use ELISA method
It detects HBeAg antibody titer (the result is shown in Figure 1 2).Exempted from the therapeutic hepatitis B vaccine that PolyIC- aluminium hydroxide composite adjuvant is prepared
The HBeAg antibody positive of the mouse 80% of epidemic disease.With all HBeAg negative antibodies of mouse that antigen (no adjuvant) is immune.HBeAg
Antibody positive general proof does not have virus replication, illustrates the therapeutic effect for the therapeutic hepatitis B vaccine that composite adjuvant can be improved.
The 10th day (the 31st day tested) after the third immunization, venous blood collection separate serum, use ELISA method
Detect the concentration (the result is shown in Figure 1 3) of gamma- interferon in serum.The treatment prepared with PolyIC- aluminium hydroxide composite adjuvant
The concentration of the serum gamma- interferon of the mouse of property hepatitis b vaccine immune is far longer than the immune mouse of antigen (no adjuvant)
The concentration of serum gamma- interferon.Gamma- interferon is generally positively correlated with the therapeutic effect of hepatitis B vaccine.
10 PolyIC- aluminium hydroxide composite adjuvant of embodiment prepare therapeutic hepatitis B vaccine in hepatitis B mouse model
Therapeutic effect
With mouse every group 8 of C57 (about 6~7 weeks).As described in Example 1, in first day high pressure tail vein injection second
Hepatovirus plasmid (paav-HBV1.2) infects C57 mouse.Every mouse subcutaneous injection 0.3 is given in 4th day, 18 days and 32 days respectively
The vaccine of milliliter.The preparation method of vaccine is shown in example 8, if control group not infected, an infection but not being immunized
Control group only immune with the antigen without adjuvant after control group and an infection.
Progress blood specimen collection in the 3rd day, 10 days, 17 days, 24 days and 31 days before first time is immune and after immune.With
The viral composition of ELISA method detection HBsAg in serum and HBeAg.
The results show that can be accelerated to eliminate HBsAg in serum with the vaccine that PolyIC- aluminium hydroxide composite adjuvant is prepared
The speed that (Figure 14) and HBeAg (Figure 15) are removed.PolyIC- aluminium hydroxide composite adjuvant can be improved therapeutic hepatitis B vaccine
Protectiveness.
A mouse is taken to take liver solid for every group after 5 days immune (the 5th day, 19 days and 33 days after being immunized for the first time) every time
It is fixed, carry out immunohistochemical analysis.The virus of detection liver changes over time situation.
The results show that with the 19th day after the vaccine immunity of PolyIC- aluminium hydroxide composite adjuvant preparation, in liver cell
HBsAg becomes negative, and the 33rd day HBeAg becomes negative (table 3), illustrates the protecting effect immune better than antigen (no adjuvant).
The immunohistochemical analysis of the therapeutic effect for the vaccine that table 3. is prepared with PolyIC- aluminium hydroxide composite adjuvant
| D5 | D19 | D33 | |
| It is not immune not attack malicious control | HBsAg-, HBeAg- | HBsAg-, HBeAg- | HBsAg-, HBeAg- |
| It is not immune not attack malicious control | HBsAg+, HBeAg+ | HBsAg+, HBeAg+ | HBsAg-, HBeAg+ |
| Antigen is without adjuvant immunity | HBsAg+, HBeAg+ | HBsAg+, HBeAg+ | HBsAg-, HBeAg+ |
| Antigen plus adjuvant is immune | HBsAg+, HBeAg+ | HBsAg-, HBeAg+ | HBsAg-, HBeAg- |
"+" represents the positive."-" represents feminine gender.
Above embodiments only have illustrative effect to the present invention, without the effect of any restrictions, this field
The modification of any unsubstantiality made on the basis of the present invention of technical staff, all should belong to protection scope of the present invention.
Claims (10)
1. a kind of vaccine for treating chronic hepatitis B, it is characterised in that: antigen by hepatitis B with contain the compound of molecule adjuvant
Adjuvant is formulated.
2. the preparation method of the vaccine for the treatment of chronic hepatitis B according to claim 1, it is characterised in that: including following step
It is rapid:
(1), the antigen of hepatitis B is prepared；
(2), composite adjuvant is prepared；
(3), the antigen of hepatitis B is mixed with composite adjuvant, and mixing is configured to vaccine.
3. the preparation method of the vaccine for the treatment of chronic hepatitis B according to claim 2, it is characterised in that: the step (1)
Middle antigen includes HBsAg or Pre-S1 or Pre-S2 containing HBsAg.
4. the preparation method of the vaccine for the treatment of chronic hepatitis B according to claim 2, it is characterised in that: the step (1)
Middle antigen includes the mixture of HBsAg or the Pre-S1 containing HBsAg or the Pre-S2 containing HBsAg and nuclear antigen.
5. the preparation method of the vaccine for the treatment of chronic hepatitis B according to claim 2, it is characterised in that: the step (2)
Middle composite adjuvant includes the molecule adjuvant of carrier and inducing cellular immune.
6. the preparation method of the vaccine for the treatment of chronic hepatitis B according to claim 5, it is characterised in that: the carrier is to receive
Rice milk agent, liposome, any one in Alum adjuvant.
7. the preparation method of the vaccine for the treatment of chronic hepatitis B according to claim 6, it is characterised in that: the Alum adjuvant
Including any one in aluminium hydroxide, aluminum phosphate, calcium phosphate.
8. the preparation method of the vaccine for the treatment of chronic hepatitis B according to claim 5, it is characterised in that: the inducing cell
Immune molecule adjuvant be QS-21, TLR3 ligand, TLR4 ligand, TLR-7/8ligand, TLR9 ligand or
Any one in agonist.
9. the preparation method of the vaccine for the treatment of chronic hepatitis B according to claim 2, it is characterised in that: the step (2)
Middle composite adjuvant is formulated using agonist at least one of nanoemulsion, liposome, Alum adjuvant.
10. the vaccine of the preparation of the preparation method according to any one of claim 2-9 is in preparation prevention or treatment chronic
Application in liver-cancer medicine caused by liver drug and/or hepatitis B virus infection.
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| CN102949717A (en) * | 2012-07-06 | 2013-03-06 | 中国疾病预防控制中心病毒病预防控制所 | Novel hepatitis B vaccine preparation containing poly I:C adjuvant |
| CN103566369A (en) * | 2012-08-01 | 2014-02-12 | 中国医学科学院肿瘤医院 | Hepatitis B vaccine for inducing organism to generate specific immunity in state of chronic hepatitis B virus infection |
| CN107281484A (en) * | 2017-05-31 | 2017-10-24 | 深圳大学 | Composition for preparing hepatitis B vaccine and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1404875A (en) * | 2002-11-22 | 2003-03-26 | 北京绿竹生物技术有限责任公司 | B-type hepatitis vaccine |
| CN102949717A (en) * | 2012-07-06 | 2013-03-06 | 中国疾病预防控制中心病毒病预防控制所 | Novel hepatitis B vaccine preparation containing poly I:C adjuvant |
| CN103566369A (en) * | 2012-08-01 | 2014-02-12 | 中国医学科学院肿瘤医院 | Hepatitis B vaccine for inducing organism to generate specific immunity in state of chronic hepatitis B virus infection |
| CN107281484A (en) * | 2017-05-31 | 2017-10-24 | 深圳大学 | Composition for preparing hepatitis B vaccine and its preparation method and application |
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Effective date of registration: 20210929 Address after: 610041 No. 509, floor 5, building 12, No. 88, Keyuan South Road, high tech Zone, Chengdu, Sichuan (self numbering) Applicant after: Chengdu fanyikang Biomedical Technology Co.,Ltd. Address before: 610041 c1-506, No. 88, Keyuan South Road, high tech Zone, Chengdu, Sichuan Applicant before: He Yonggang |
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| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190614 |