CN107812186A - C-type CpG as adjuvant HBV is preventative and therapeutic vaccine in application and preparation method thereof - Google Patents
C-type CpG as adjuvant HBV is preventative and therapeutic vaccine in application and preparation method thereof Download PDFInfo
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Abstract
The invention discloses c-type CpG M362 as adjuvant HBV is preventative and therapeutic vaccine in application and preparation method thereof, the great advantage that HBV preventative vaccines using CpG M362 as adjuvant are different from traditional HBV preventative vaccines is, it can not only induce body to produce high-caliber humoral response, and the cell response of body can be strengthened.Simultaneously, HBV therapeutic vaccines using CpG M362 as adjuvant are applied in clinical HBV therapeutic processes, the immunosupress microenvironment of liver caused by can improving chronic HBV infection, reverse immune tolerance caused by HBV infection, strengthen the cell response and humoral response of body, so as to fully erased HBV.Importantly, using CpG M362 as the vaccine immunization of adjuvant after, body can be induced to form long-term immunological memory, protection body prevents HBV subinfection again, and the treatment of prevention and Chronic HBV for clinical HBV provides a new available strategy.
Description
Technical field
The invention belongs to biotechnology and recombinant vaccine field, and in particular to c-type CpG M362 are as adjuvant in HBV
Application in preventative and therapeutic vaccine and preparation method thereof.
Background technology
HBV infection endangers the health of global human.Although the application of HBV preventative vaccines (aluminium adjuvant vaccine) is suitable
Generally, the population for but still having 5%-10% can not produce effective Anti-HBV activity antibody (anti-HBs), cause the whole world annual about
There are 3,500,000 people to suffer from chronic HBV infection, wherein 25%-30% patient dies from the hepatic sclerosis as caused by Chronic HBV and liver cancer.
At present, IFN-α and nucleic acid analog both drug therapy Chronic HBVs are clinically commonly used, but IFN-α and nucleic acid
The therapeutic effect of analog has certain limitation.Although IFN-α has antiviral effect, but clinical treatment rate only has 20-
40%；And nucleic acid analog can suppress HBV duplication and transcription to a certain extent, but and without HBV specificity, simultaneously
It may result in side effect and its generation of drug resistance.Therefore, a kind of effective Anti-HBV activity vaccine of new type of safe of exploitation is needed badly to be used for
HBV prevention and treatment.Due to lacking preferable adjuvant so that the research of HBV vaccines faces significant challenge.
A kind of immunologic adjuvant, as nonspecific immunopotentiator, thus it is possible to vary the disadvantage of antigen autoantigenic difference
End, it can effectively strengthen the intensity of immune response together with antigen or when being previously implanted body or change the type of immune response.Assistant
The mechanism of action of agent mainly includes following components：Change the physical behavior of antigen, delay the degraded and exclusion of antigen, so as to
Extend antigen holdup time in vivo, avoid frequently injection so as to more effectively stimulating immune system；Stimulate monokaryon-phagocytosis thin
Born of the same parents' system, strengthen its processing and offer the ability of antigen；Hyperplasia and the differentiation of lymphocyte are stimulated, body can be improved for the first time and again
The antibody titer of secondary immune response；Change the generation type of antibody and produce delayed allergy.Therefore, by adjuvant,
The immunogenicity of vaccine can be strengthened, the immune response for increasing body is horizontal.
There are some researches prove the patient's body CD4 of Chronic HBV+T、CD8+T cell immune response afunction, while high table
Up to Treg cells, IL-10 immunosuppression molecules.However, the HBV vaccines of Current commercial are aluminium adjuvant vaccines, body can be induced
Th2 type immune responses are produced, but body can not be stimulated to produce antiviral Th1 types and CTL immune responses.Therefore, find/grind
The vaccine of Cellular Immunity response can effectively be evoked by sending out a kind of, break the immunosuppressive microenvironment of patient with chronic HBV, can just have
The infection of the treatment Chronic HBV of effect.
CpG ODN refer to containing the nucleotides sequence that the non-cytimidine to methylate and guanine dinucleotides are core sequence
Row.According to its bioactivity and the difference of chemical composition, CpG is broadly divided into three classes：A type CpG, Type B CpG, c-type CpG.A types
CpG, such as ODN 1585, ODN2216, ODN2336, primary activation pDC, promote IFN secretion；Type B CpG, such as ODN
1826th, ODN 7909, ODN 2006, primary activation NF- κ B signal paths, the generation of Th1 type responses is promoted；C-type CpG, such as
ODN 2395, ODN M362, compared with Type B CpG ODNs, c-type CpG activation NF- κ B signals paths and generation IL-12 cells
Factor ability is weaker, its can adjuvant preventative as HBV and therapeutic vaccine currently without correlation report.
The content of the invention
For above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of new HBV is preventative and control
Adjuvant of the property treated vaccine and preparation method thereof.
We are by by analyzing c-type CpG M362 to Cellular Immunity response and humoral immune response in our current research
Adjustment effect, cells and molecular mechanism of the CpG M362 as Anti-HBV activity vaccine adjuvant are illustrated, have rated it and helped as HBV vaccines
The feasibility of agent.
To achieve the above object, the technical solution adopted by the present invention is as follows：
The first aspect of the invention, a kind of c-type CpG M362 are provided first and are preparing HBV preventative vaccines as adjuvant
In application.
Secondly, there is provided a kind of HBV preventative vaccines, its active component are hepatitis b virus s antigen (HBsAg), hydrogen
Aluminum oxide and CpG M362.
Further it is provided that the preparation method of above-mentioned HBV preventative vaccines, this method include：Aluminium hydroxide is added dropwise to HBsAg
In solution, mixing of turning upside down, CpG M362 solution is then added, you can obtain HBV preventative vaccines, this vaccine liquid passes through
Packing can be made into the bacterin preparation of the injection of unit dose.
The second aspect of the invention, a kind of c-type CpG M362 is provided first as adjuvant in HBV therapeutic vaccines
Using.
Secondly, there is provided a kind of HBV therapeutic vaccines, its active component are rHBV vaccines and CpG M362.
Further it is provided that the preparation method of above-mentioned HBV therapeutic vaccines, this method include：RHBV vaccines are taken, add CpG
M362 solution, mix, you can obtain HBV therapeutic vaccine liquid, this vaccine liquid can be made into the injection of unit dose by packing
Bacterin preparation.
Compared with prior art, technical scheme has the advantages that：
Applications of the 1.C types CpG as adjuvant in HBV preventative vaccines
(1) the new HBV preventative vaccines that prepared by the present invention, joined using HBsAg, Alhydrogel 2%, CpG M362
Close and use, it is safe and nontoxic.Gained preparation safety, economy, prepare conveniently, be advantageous to the expansion of immunity inoculation.
(2) compared with " HBsAg " negative control group, individually the adjuvant using CpG M362 as HBV preventative vaccines, is exempting from
Body can be induced to produce the anti-HBs of certain level during epidemic disease, but be below " the positives of HBsAg+Alhydrogel 2% "
Control group.
(3) with " individually that CpG M362 is preventative as HBV compared with the positive controls of HBsAg+Alhydrogel 2% "
The adjuvant of vaccine, the activation of T cell can be promoted, strengthen CD4+The level of T cell secrete cytokines IFN-γ.
(4) with " individually that CpG M362 is preventative as HBV compared with the positive controls of HBsAg+Alhydrogel 2% "
The adjuvant of vaccine, body can be induced to produce the CD8 of antigentic specificity+T cell, promote CD8+The end last Memorability point of T cell
Change.
(5) with " individually that CpG M362 is preventative as HBV compared with the positive controls of HBsAg+Alhydrogel 2% "
The adjuvant of vaccine, body can be protected to prevent HBV infection, while induce the anti-HBs of body generation protectiveness.
(6) with " compared with the positive controls of HBsAg+Alhydrogel 2% ", Alhydrogel 2% and CpG M362 being joined
The adjuvant of HBV preventative vaccines is used as, body can be induced to produce the anti-HBs suitable with positive controls, while can lure
The cell response that body produces antigentic specificity is led, protection body prevents HBV infection.
Applications of the 2.C types CpG as adjuvant in HBV therapeutic vaccines
(1) the new HBV therapeutic vaccines that prepared by the present invention, using preventative the rHBV vaccines and CpG of clinic
M362 is used in combination, safe and nontoxic.Gained preparation safety, economy, prepare conveniently, be advantageous to the expansion of immunity inoculation.
(2), can be safely and effectively clear in Mice Body it is experimentally confirmed that using CpG M362 as the adjuvant of HBV therapeutic vaccines
Except the HBV in HBV-carrier mouse.
(3) mouse In vivo study, the adjuvant using CpG M362 as HBV therapeutic vaccines, can promote peripheral blood, spleen,
Lymph node position DC recruitment, maturation and differentiation；Meanwhile experiment in vitro also confirms that, under CpG M362 stimulations, BMDC is improved
Swallow the ability of antigen, the expression increase of costimulatory molecules CD80, CD86 and MHC-II molecule.
(4) the adjuvant using CpG M362 as HBV therapeutic vaccines, the microenvironment of immunity of organism suppression can be improved, reversed
The CD8 of liver, spleen antigentic specificity+The exhaustion of T cell, reduce its surface immunosuppression molecule LAG-3, TIGIT, Tim-3,
CTLA-4 expression, reduce and negative tune cell T is immunized in liverregS ratio, reduce the level of TGF-β in serum；Increase antigen
Specific CD8+T cell breeds the expression of nuclear antigen.
(5) the adjuvant using CpG M362 as HBV therapeutic vaccines, can strengthen CD8+The activation of T cell and CD107a
Expression；Body is induced to produce IFN-γ；Increase the CD8 of antigentic specificity+The ratio increase of T cell, promotes body to HBV's
Remove.
(6) the adjuvant using CpG M362 as HBV therapeutic vaccines, antigen in liver, spleen can be promoted specific
CD8+The terminal differentiation of T cell；Meanwhile reduce the specific CD8 of antigen in liver, spleen+T cell Eomes expression, promote
Differentiation of the MPECs to SLECs.
(7) the adjuvant using CpG M362 as HBV therapeutic vaccines, body can be induced to produce prolonged immunological memory,
Body can be protected to prevent HBV subinfection again.
In summary, as the adjuvant of HBV preventative vaccines body can be induced to produce protectiveness CpG M362
Anti-HBs, promote the CD8 of antigentic specificity+T cell response, protection body prevent HBV infection；Can be with by CpG M362
As the adjuvant of HBV therapeutic vaccines, HBV can be safely and effectively removed, is improved immunosuppressive caused by chronic HBV infection
Microenvironment, induction body form long-term immunological memory, and protection body prevents HBV subinfection again.Therefore, CpG M362 can be with
Simultaneously as HBV preventative vaccines and the adjuvant of therapeutic vaccine.
Brief description of the drawings
The Figure of description for forming the part of the present invention is used for providing a further understanding of the present invention, and of the invention shows
Meaning property embodiment and its illustrate be used for explain the present invention, do not form inappropriate limitation of the present invention.
Applications of the 1.C types CpG as adjuvant in HBV preventative vaccines
Fig. 1：Adjuvant using CpG M362 as HBV preventative vaccines, induction body produce the assessment of anti-HBs abilities
As a result；
Fig. 2：Adjuvant using CpG M362 as HBV preventative vaccines, promote the assessment result of the activation of T cell；
Fig. 3：Adjuvant using CpG M362 as HBV preventative vaccines, induction body produce the CD8 of antigentic specificity+T is thin
Born of the same parents, promote CD8+The assessment result of the end last Memorability differentiation of T cell；
Fig. 4：Adjuvant using CpG M362 as HBV preventative vaccines, the assessment result of inducing mouse permanent immunity protection；
Fig. 5：Alhydroge 2% and CpG M362 is combined the adjuvant as HBV preventative vaccines, and induction body produces
Anti-HBs abilities and the assessment result of permanent immunity protection；
Applications of the 2.C types CpG as adjuvant in HBV therapeutic vaccines
Fig. 6：Adjuvant using CpG M362 as HBV therapeutic vaccines, remove the assessment result of HBV abilities；
Fig. 7：Adjuvant using CpG M362 as HBV therapeutic vaccines, induce the assessment result of DC activation capacities；
Fig. 8：Adjuvant using CpG M362 as HBV therapeutic vaccines, improve the assessment knot that immunity of organism suppresses microenvironment
Fruit；
Fig. 9：Adjuvant using CpG M362 as HBV therapeutic vaccines, strengthen CD8+The assessment result of T cell function；
Figure 10：Adjuvant using CpG M362 as HBV therapeutic vaccines, promote CD8+The assessment knot of T cell terminal differentiation
Fruit；
Figure 11：Adjuvant using CpG M362 as HBV therapeutic vaccines, induce and the permanent of HBV-carrier mouse is exempted from
The assessment result of epidemic disease protection.
Embodiment
It is noted that described further below is all exemplary, it is intended to provides further instruction to the present invention.It is unless another
Indicate, all technologies used herein and scientific terminology are with usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation and/or combinations thereof.
As background technology is introduced, need a kind of effective Anti-HBV activity vaccine of new type of safe of exploitation badly in the prior art and use
In HBV prevention and treatment, in consideration of it, the present invention proposes a kind of c-type CpG M362 is preparing the preventative epidemic diseases of HBV as adjuvant
Application in seedling.
In a preferred embodiment of the present invention, the HBV preventative vaccines, its active component include hepatitis type B virus
Surface antigen (HBsAg), aluminium hydroxide and CpG M362.
Wherein, HBsAg is the hepatitis B of the restructuring expressed by Hansenula yeast of Dalian Hanxin Biology Pharmacy Co., Ltd's production
Viral surface antigen (HBsAg).RHBV vaccines prepared by this strain HBsAg are applied to clinic for many years, body can be stimulated to produce
The immunity of anti-hepatitis B virus, it can effectively prevent hepatitis B.Aluminium hydroxide, for the production of invivogen companiesAdjuvant 2%, the preventative rHBV vaccines of clinic are widely used to, it is safe, effectively.CpG M362,
For the c-type CpG of invivogen companies production, body can be induced to produce certain Th1/Th2 type immune responses, its nucleotides
Sequence is：5’-tcgtcgtcgttc:gaacgacgttgat-3’(25mer).
In a preferred embodiment of the present invention, in every dose of HBV preventative vaccines, the content of each principal component is as follows：HBsAg
2 2%100 μ g, CpG M36210 μ g of μ g, Alhydrogel.
In a preferred embodiment of the present invention, the special of the immunization strategy of " just exempt from-strengthen " (Prime-boost) is used
Property；Embodiment is as follows：HBV preventative vaccines were with the time interval of two weeks, and continuous immunity is three times.
It can not only induce body to produce high-caliber anti-HBs (humoral response) after being immunized three times, while can induce
Body produces high-caliber cell response, the shortcomings that overcoming traditional vaccine cell response ability weaker.
In a preferred embodiment of the present invention, a kind of c-type CpG M362 are also provided and are preparing induction body as adjuvant
Produce anti-HBs while produce the application in the medicine of cell response.
In a preferred embodiment of the present invention, a kind of c-type CpG M362 are also provided and are preparing promotion T cell as adjuvant
Activation, enhancing CD4+Application in the horizontal medicine of T cell secrete cytokines IFN-γ.
In a preferred embodiment of the present invention, a kind of c-type CpG M362 are also provided and are preparing induction body as adjuvant
Produce the CD8 of antigentic specificity+T cell, promote CD8+Application in the medicine of the end last Memorability differentiation of T cell.
In a preferred embodiment of the present invention, the HBV preventative vaccines and other clinical prevention Chronic HBVs is immune
Conditioning agent is used in conjunction with.
In a preferred embodiment of the present invention, the preparation method of above-mentioned HBV preventative vaccines, this method include：By hydrogen
Aluminum oxide is added dropwise in HBsAg solution, mixing of turning upside down, then adds CpG M362 solution, you can it is preventative to obtain HBV
Vaccine, this vaccine liquid can be made into the bacterin preparation of the injection of unit dose by packing.
In a preferred embodiment of the present invention, the present invention also provides a kind of c-type CpG M362 and treated as adjuvant in HBV
Application in property vaccine.
In a preferred embodiment of the present invention, the HBV therapeutic vaccines, its active component include rHBV vaccines and
CpG M362。
Wherein, rHBV vaccines are the recombinant hepatitis B vaccine (Hansenula yeast) of Dalian Chinese letter production.This strain vaccine should
For clinic for many years, body can be stimulated to produce the immunity of anti-hepatitis B virus, can effectively prevent hepatitis B.CpG
M362, for the c-type CpG of invivogen companies production, body can be induced to produce certain Th1/Th2 type immune responses.
In a preferred embodiment of the present invention, in every dose of HBV therapeutic vaccines, the content of each principal component is as follows：rHBV
Vaccine μ g, CpG M36210 μ g containing HBsAg 2.
Foregoing every dose refers to each bacterin preparation, such as each injection, or every injection, or uses every time
The injection medicament of amount.
In a preferred embodiment of the present invention, the special of the immunization strategy of " just exempt from-strengthen " (Prime-boost) is used
Property；Embodiment is as follows：HBV therapeutic vaccines were with the time interval of one week, and continuous immunity is three times.
The virological controls of long-term HBV can be realized after being immunized three times.
In a preferred embodiment of the present invention, a kind of c-type CpG M362 are also provided and are preparing promotion periphery as adjuvant
Blood, spleen, lymph node position DC recruitment, maturation and differentiation, raising BMDC swallow the ability of antigen, cause costimulatory molecules
Application in the increased medicine of expression of CD80, CD86 and MHC-II molecule.
In a preferred embodiment of the present invention, a kind of c-type CpG M362 are also provided to prepare and can improve as adjuvant
The microenvironment that immunity of organism suppresses, reverse liver, the CD8 of spleen antigentic specificity+The exhaustion of T cell, reduce liver, spleen table
Face immunosuppression molecule LAG-3, TIGIT, Tim-3, CTLA-4 expression, reduce and negative tune cell T is immunized in liverregS ratio
Example, the level of TGF-β in serum is reduced, increase the CD8 of antigentic specificity+In the medicine of the expression of T cell propagation nuclear antigen
Using.
In a preferred embodiment of the present invention, a kind of c-type CpG M362 are also provided to prepare and can strengthen as adjuvant
CD8+The activation of T cell and CD107a expression, induction body produce IFN-γ, increase the CD8 of antigentic specificity+T cell
Application in the increased medicine of ratio.
In a preferred embodiment of the present invention, a kind of c-type CpG M362 are also provided to prepare and can promote as adjuvant
The specific CD8 of antigen in liver, spleen+The terminal differentiation of T cell, reduce the specific CD8 of antigen in liver, spleen+T is thin
Born of the same parents Eomes expression, promote applications of the MPECs into the medicine of SLECs differentiation.
In a preferred embodiment of the present invention, the HBV therapeutic vaccines and other clinical treatment Chronic HBVs is immune
Conditioning agent is used in conjunction with.
In a preferred embodiment of the present invention, the preparation method of above-mentioned HBV therapeutic vaccines, this method include：Take
RHBV vaccines, CpG M362 solution is added, mixed, you can obtain HBV therapeutic vaccine liquid, this vaccine liquid can be made by packing
Into the bacterin preparation of the injection of unit dose.
Bacterin preparation in the present invention using CpG M362 as adjuvant can induce body to form immunological memory after being immunized three times,
Individual after immune can 100% prevention HBV infection.
The bacterin preparation using CpG M362 as adjuvant in the present invention can be used for clinical HBV immunization campaign and chronic
HBV, HBV related HCC clinical treatment.
In order that technical scheme can clearly be understood by obtaining those skilled in the art, below with reference to tool
The embodiment of body describes technical scheme in detail.
Test material used is the conventional test material in this area in the embodiment of the present invention, can pass through commercial channel
It is commercially available.
Applications of the 1.C types CpG as adjuvant in HBV preventative vaccines
Embodiment 1
The preparation method of the preventative hepatitis B vaccine of the present invention：
Raw material and source：
HBsAg is the product in the Hansenula yeast source of Dalian Han Xin biological products Co., Ltd production, and concentration is 200 μ g/
ml。
Alhydrogel Invivogen companies, specification：250ml, Al (OH)3Concentration：9-10mg/ml
CpG M362, invivogen companies, specification：1mg.1mg/ml is configured to injection physiological saline, with 200 μm
Sterilised membrane filter filtration sterilization, freeze in -20 DEG C.
The preparation method of vaccine：
The μ l of HBsAg 100 are taken in sterile Ep pipes, add the μ l of physiological saline 700, are mixed；Then 100 μ l are taken
Alhydrogel2%, it is added dropwise in HBsAg solution, turns upside down and mix 5min；Finally add CpG M362 solution
100μl.Every milliliter of the vaccine liquid prepared contains μ g of hepatitis B surface antigen 20, Al (OH)3The μ g of 100 μ g, CpG M362 100.
The immunization strategy of preventative hepatitis B vaccine：
Experimental group subcutaneous inoculation HBsAg (2 μ g)+Alhydrogel 2% (100 μ g)+CpG M362 (10 μ g), HBsAg (2
μ g)+CpG M362 (10 μ g), HBsAg (2 μ g)+Alhydrogel 2% (100 μ g), negative control group is immunized in positive controls
Immune HBsAg (2 μ g), with the time interval of two weeks, it is carried out continuously and (Prime-boost) is immunized three times.
Test example 1：Adjuvant using CpG M362 as HBV preventative vaccines, induction body produce anti-HBs abilities
Assessment result
1.1 test method：
Experimental group subcutaneous inoculation HBsAg (2 μ g)+CpG M362 (10 μ g), positive controls be immunized HBsAg (2 μ g)+
Alhydrogel 2% (100 μ g), negative control group are immunized HBsAg (2 μ g), with the time interval of two weeks, are carried out continuously three times
Immune (Prime-boost), 2w after 2w, secondary immunity after ELISA detection primary immune responses, be immunized three times after in 2w peripheral blood serum
anti-HBs。
1.2 result of the test：
As shown in figure 1, compared with negative control " HBsAg ", CpG can produce one separately as adjuvant after being immunized three times
Fixed horizontal anti-HBs.
With positive control, " compared with HBsAg+Alhydrogel 2% ", CpG produces anti-HBs water separately as adjuvant
It is flat relatively low.
Test example 2：Adjuvant using CpG M362 as HBV preventative vaccines, promote the assessment result of the activation of T cell
2.1 test method：
Experimental group subcutaneous inoculation HBsAg (2 μ g)+CpG M362 (10 μ g), positive controls be immunized HBsAg (2 μ g)+
Alhydrogel 2% (100 μ g), negative control group are immunized HBsAg (2 μ g), with the time interval of two weeks, are carried out continuously three times
Immune (Prime-boost), 1w peripheral bloods after FACS detections are immunized three times, liver, CD44, CD69 in the T cell of lymph node position,
NKG2D expression and CD4+The level of T cell secretion of gamma-IFN.
2.2 result of the test：
As shown in Fig. 2 with " compared with the positive controls of HBsAg+Alhydrogel 2% ", individually using CpG M362 as
The adjuvant of HBV preventative vaccines, CD44, CD69 and NKG2D of T cell expression can be promoted, strengthen CD4+T cell is secreted
The level of cell factor IFN-γ.
Test example 3：Adjuvant using CpG M362 as HBV preventative vaccines, induction body produce the CD8 of antigentic specificity+T cell, promote CD8+The assessment result of the end last Memorability differentiation of T cell
3.1 test method：
Experimental group subcutaneous inoculation HBsAg (2 μ g)+CpG M362 (10 μ g), positive controls be immunized HBsAg (2 μ g)+
Alhydrogel 2% (100 μ g), negative control group are immunized HBsAg (2 μ g), with the time interval of two weeks, are carried out continuously three times
Immune (Prime-boost), the total CD8 in 1w peripheral bloods position after FACS detections are immunized three times+T cell, antigentic specificity CD8+T
The ratio of cell, antigentic specificity CD8+KLRG1 expression and antigentic specificity CD8 in T cell+MPEC, SLEC in T cell
Ratio.
3.2 result of the test：
As shown in figure 3, with " compared with the positive controls of HBsAg+Alhydrogel 2% ", individually using CpG M362 as
The adjuvant of HBV preventative vaccines, total CD8 in human peripheral blood+The ratio of T cell does not influence significantly, but increases antigen
Specific C D8+The ratio of T cell；Meanwhile promote antigentic specificity CD8+KLRG1 expression in T cell, and antigentic specificity
CD8+The terminal differentiation of T cell.
Test example 4：Adjuvant using CpG M362 as HBV preventative vaccines, the assessment of inducing mouse permanent immunity protection
As a result
4.1 test method：
Experimental group subcutaneous inoculation HBsAg (2 μ g)+CpG M362 (10 μ g), positive controls be immunized HBsAg (2 μ g)+
Alhydrogel 2% (100 μ g), negative control group are immunized HBsAg (2 μ g), with the time interval of two weeks, are carried out continuously three times
Immune (Prime-boost), 30d after being immunized three times, the μ g pAAV/HBV 1.2 of Hydrodynamic injection 8 plasmid, simulate HBV
Infection, ELISA detection serum in anti-HBs, HBsAg level.
4.2 result of the test：
As shown in figure 4, compared with " HBsAg " cloudy control group, the independent assistant using CpG M362 as HBV preventative vaccines
Agent, body can be protected to prevent HBV infection, meanwhile, body produces the anti-HBs of protectiveness；With " HBsAg+
Alhydrogel2% " positive controls are compared, individually the adjuvant using CpG M362 as HBV preventative vaccines, although can protect
Body prevents HBV infection, and still, body produces the horizontal lower slightly of the anti-HBs of protectiveness.
Test example 5：Alhydroge 2% and CpG M362 is combined the adjuvant as HBV preventative vaccines, induction body production
Raw anti-HBs abilities and the assessment result of permanent immunity protection
5.1 test method：
Experimental group subcutaneous inoculation HBsAg (2 μ g)+Alhydrogel 2% (100 μ g)+CpG M362 (10 μ g) are positive right
According to immune HBsAg (2 μ the g)+Alhydrogel 2% (100 μ g) of group, HBsAg (2 μ g) is immunized in negative control group, with two weeks when
Between be spaced, be carried out continuously three times be immunized (Prime-boost), three times be immunized after 30d, the μ g pAAV/HBV of Hydrodynamic injection 8
1.2 plasmid, simulates HBV infection, and ELISA detects anti-HBs in serum, HBsAg level.
5.2 result of the test：
As shown in figure 5, with positive control " compared with HBsAg+Alhydrogel 2% ", Alhydroge 2% and CpG
M362 is combined the adjuvant as HBV preventative vaccines, and comparable levels of anti-HBs can be produced in immunologic process；Together
When, body can be induced to produce the CD8 of high-caliber antigentic specificity+T response, more efficiently body is protected to prevent HBV
Infection.
Applications of the 2.C types CpG as adjuvant in the therapeutic property vaccines of HBV
Embodiment 1
The preparation method of the hepatitis B vaccines of the present invention：
Raw material and source：
RHBV vaccines are that the Dalian Chinese believes recombinant hepatitis B vaccine (Hansenula yeast) product produced, specification 1ml：20μg.
Purity is not less than 98%, and aluminium hydroxide must not cross 1mg per 1ml, and thimerosal must not cross 0.01% per 1ml, and formaldehyde must not mistake per 1ml
0.01%.
CpG M362, purchased from invivogen companies, specification：1mg.1mg/ml is configured to injection physiological saline, with
200 μm of sterilised membrane filter filtration sterilization, freezes in -20 DEG C.
The following rHBV vaccines of content μ g, CpG containing HBsAg 2 of each principal component in every dose of the HBV therapeutic vaccines of the present invention
M362 10μg。
The preparation method of vaccine：
Recombinant hepatitis B vaccine 1ml is taken in sterile Ep pipes, adds the μ l of CpG M362 solution 100 and physiological saline
900 μ l, it is 2ml vaccine solution after mixing.Every milliliter of the vaccine liquid prepared contains the μ g of hepatitis B surface antigen 10, CpG M362
50μg。
Test example 1：The immunization strategy of the structure of HBV-carrier mouse models and new HBV therapeutic vaccines
1.1HBV-carrier mouse models are built
The 5-6w of normal adult C57BL/6J mouse are taken, pAAV/HBV1.2 plasmids are dissolved in sterile physiological saline,
By way of Hydrodynamic injection, 8 μ g plasmid is injected to every mouse in 5-8s, volume injected is mouse quality
10%.After 6w, mice serum is collected.Mouse of the serum HBsAg concentration more than 500ng/ml is defaulted as HBV-carrier mouse.
The immunization strategy of " just exempt from-strengthen " (Prime-boost) of 1.2 new HBV therapeutic vaccines
Experimental group subcutaneous inoculation rHBV vaccine (μ g containing HBsAg2)+CpG M362 (10 μ g), rHBV is immunized in negative control group
Vaccine (μ g containing HBsAg2), blank control group inject the physiological saline of same volume, with the time interval of one week, are immunized 3 times
(Prime-boost)。
Test example 2：Adjuvant using CpG M362 as HBV therapeutic vaccines, remove the assessment result of HBV abilities
1.1 test method：
Experimental group subcutaneous inoculation rHBV vaccine (μ g containing HBsAg2)+CpG M362 (10 μ g), rHBV is immunized in negative control group
Vaccine (μ g containing HBsAg2), blank control group inject the physiological saline of same volume, with the time interval of one week, are immunized 3 times
(Prime-boost).By detecting peripheral blood HBsAg, HBV DNA, liver HBcAg, HE and ALT level, assess new
HBV therapeutic vaccines remove HBV ability.Untr groups are physiological saline group in caption, and cHBV-vaccine groups are new HBV
Therapeutic vaccine (CpG M362 are used in combination with rHBV vaccines) group.
1.2 result of the test：
As shown in Figure 6A, compared with non-treatment group, new HBV therapeutic vaccines mouse peripheral blood blood after immunization therapy three times
HBsAg level significantly decreases in clear, is substantially not detectable HBsAg expression；Also, therapeutic effect can remain longer
Time.Meanwhile as shown in Figure 6B, HBV DNA levels are reduced, and immunohistochemical staining shows that liver HBcAg+ liver cell exists
Almost (Fig. 6 C) is disappeared after treatment；In addition, liver HE section statinings and serology ALT level monitoring are shown in treatment cycle
In, the liver of mouse has no obvious lymphocytic infiltration (Fig. 6 D) and hepatocellular injury (Fig. 6 E).Based on the above results can be with
Find out, the adjuvant using CpG M362 as HBV therapeutic vaccines, can safely and effectively remove HBV.
Test example 3：Adjuvant using CpG M362 as HBV therapeutic vaccines, induce the assessment result of DC activation capacities
3.1 test method：
3.1.1 experimental group subcutaneous inoculation rHBV vaccines (μ g containing HBsAg2)+CpG M362 (10 μ g), blank control group injection
The physiological saline of same volume, with the time interval of one week, is immunized 3 times (Prime-boost), and FACS is detected after 7d is immunized three times
Macrophage, DC ratio and its expression of costimulatory molecules, new HBV therapeutic vaccines are assessed to DC activation capacities
Influence.Untr groups are physiological saline group in caption, cHBV-vaccine groups for new HBV therapeutic vaccines (CpG M362 with
RHBV vaccines are used in combination) group.
3.1.2 external evoked BMDC, CpG M362 stimulation then is carried out to it, observes and BMDC phagocytosiss and its submission are resisted
The influence of proper energy power.
3.2 result of the test：
It is significantly raised that the expression of DC cell surfaces CD80, CD86 in mouse peripheral blood after treatment are can be seen that such as Fig. 7 A＆B,
Meanwhile antigen presenting cell Macrophage, DC ratio and absolute number increase.Further analysis, it has been found that in liver
The high expression MHC-II molecules of Macrophage, meanwhile, the high expression costimulatory molecules CD80, CD86 (figures of liver, the DC at spleen position
7C&D).For further influences of the checking CpG M362 to DC functions, we are studied using external evoked BMDC.Can be with
It was observed that with not plus compared with CpG M362 treatment groups, when stimulating BMDC with the CpG M362 of various concentrations, can substantially increase DC
Phagocytosis (Fig. 7 D) to OVA antigens；Meanwhile the water of DC surfaces MHC-II and costimulatory molecules CD86 percentage and MFI
Put down has obvious up-regulation after CpG M362 stimulations, and in the increase (Fig. 7 E) of concentration dependent.Based on the above results as can be seen that
Adjuvant using CpG M362 as HBV preventative vaccines, DC activation capacities are induced, recover chronic HBV infection patient
Middle DC function.
Test example 4：Adjuvant using CpG M362 as HBV therapeutic vaccines, improve immunity of organism and suppress commenting for microenvironment
Estimate result
4.1 test method：
Experimental group subcutaneous inoculation rHBV vaccines (μ g containing HBsAg 2)+CpG M362 (10 μ g), blank control group injection are identical
The physiological saline of volume, with the time interval of one week, is immunized 3 times (Prime-boost), and FACS detection livers are thin after 7d is immunized three times
The upper PD-L1 of born of the same parents, liver TregS, the CD8 of the antigentic specificity of serum TGF-β, liver and spleen position+LAG-3 in T cell,
CTLA-4, TIGIT, PD-1, Tim-3 expression；Meanwhile the CD8 of FACS detection antigentic specificities+Breed nuclear antigen in T cell
Ki-67 expression.Untr groups are physiological saline group in caption, and cHBV-vaccine groups are new HBV therapeutic vaccines (CpG
M362 is used in combination with rHBV vaccines) group.
4.2 result of the test：
Not only HBV- can be reduced with the expression (Fig. 8 A) of PD-L1 on liver cell after new HBV therapeutic vaccines treatment
Carrier mouse livers Treg ratio (Fig. 8 B), and the level (figure of immunosuppression molecule TGF-β in serum can be reduced
8C)；Meanwhile the specific CD8 of HBV-carrier murine antigens after vaccine therapy+The multiple immunologic test point LAG-3 of T cell,
CTLA-4, TIGIT, PD-1, Tim-3 expression reduces (Fig. 8 D), and the splenocyte nuclear antigen Ki67 related to propagation expression
Substantially up-regulation (Fig. 8 E) after the treatment, it can preferably improve the immunosupress microenvironment of body.It can see based on the above results
Go out, the adjuvant using CpG M362 as HBV therapeutic vaccines, can significantly improve the immunosupress microenvironment of body, reverse
Immune tolerance caused by chronic HBV infection.
Test example 5：Adjuvant using CpG M362 as HBV therapeutic vaccines, strengthen CD8+The assessment result of T cell function
5.1 test method：
Experimental group subcutaneous inoculation rHBV vaccines (μ g containing HBsAg 2)+CpG M362 (10 μ g), blank control group injection are identical
The physiological saline of volume, with the time interval of one week, it is immunized 3 times (Prime-boost), after 7d is immunized three times, FACS detection livers
Dirty, lymph node position CD8+T cell CD69, CD107a, CD62L expression, ELISA detect the level of IFN-γ in serum,
FACS detections peripheral blood, liver, the spleen position specific CD11a of HBVhi CD8αloThe ratio of cell and its difference of absolute number
It is different；After being immunized three times, separating mouse CD11ahi CD8αloAnd CD11alo CD8αhiCell, transferred to new untreated
In HBV-carrier mouse, the expression for detecting HBcAg in HBsAg, HBV DNA and liver in HBV-carrier mouse is poor
It is different.Untr groups are physiological saline group in caption, cHBV-vaccine groups for new HBV therapeutic vaccines (CpG M362 with
RHBV vaccines are used in combination) group.
5.2 result of the test：
It was found that mouse liver, lymph node position CD8 after treatment+T cell CD69, CD107a, CD62L expression increase
(Fig. 9 A＆B), meanwhile, there is high-caliber IFN-γ to produce (Fig. 9 C) in peripheral blood serum.Further analysis finds, antigen-specific
The CD11a of propertyhi CD8αloThe spleen of mouse, liver and peripheral blood have substantially the ratio and its absolute number of cell after the treatment
Increase (Fig. 9 D＆E).In order to further determine that antigentic specificity CD11ahi CD8αloWork of the cell in cHBV vaccine therapies CHB
With we separate CD11a after cHBV treatmentshi CD8αloAnd CD11alo CD8αhiCell.With CD11 αhi CD8αloTransfer a group phase
Than transferring CD11ahiCD8αloGroups of cells can significantly reduce HBsAg and serum HBV in HBV-carrier mouse models
DNA level (Fig. 9 F＆G).Meanwhile Liver immunity histochemical staining shows HBcAg+Liver cell almost disappear (Fig. 9 H).It is comprehensive
Result above can be seen that the adjuvant using CpG M362 as HBV therapeutic vaccines, body can be induced to produce Anti-HBV activity special
Property CD11 αhi CD8αloCell, removing of the body to virus can be mediated.
Test example 6：Adjuvant using CpG M362 as HBV therapeutic vaccines, promote CD8+The assessment of T cell terminal differentiation
As a result
6.1 test method：
Experimental group subcutaneous inoculation rHBV vaccines (μ g containing HBsAg 2)+CpG M362 (10 μ g), blank control group injection are identical
The physiological saline of volume, with the time interval of one week, it is immunized 3 times (Prime-boost), after 7d is immunized three times, FACS detections are outer
CD8 in all blood+The upper KLRG1 of T dynamics change, and peripheral blood, liver, the CD8 of spleen position antigentic specificity+In T cell
MPEC and SLEC ratio
6.2 result of the test：
It was found that CD8 during cHBV vaccine therapies+The high expressing K LRG1 of T cell (Figure 10 A), meanwhile, it can induce
Antigentic specificity CD8+T cell produces the memory cells (CD127 of the effect sample of differentiation-its terminal differentiation of Memorabilitylo
KLRG1hi) ratio and its absolute number increase (Figure 10 B-D).Based on the above results as can be seen that being treated CpG M362 as HBV
The adjuvant of property vaccine, the CD8 of antigentic specificity can be promoted+The formation of the terminal differentiation and Memorability of T cell.
Test example 7：Adjuvant using CpG M362 as HBV therapeutic vaccines, is induced to the permanent of HBV-carrier mouse
The assessment result of immunoprotection
7.1 test method：
Experimental group subcutaneous inoculation rHBV vaccines (μ g containing HBsAg 2)+CpG M362 (10 μ g), blank control group injection are identical
The physiological saline of volume, with the time interval of one week, it is immunized 3 times (Prime-boost).After being immunized three times, in 53d tail veins
Inject 8 μ g pAAV/HBV1.2 plasmids again, by detecting peripheral blood HBsAg in serum, anti-HBs, in liver HBcAg with
And can HBV DNA assessment of levels vaccine form long-term immunoprotection.Meanwhile detected using FACS in immunological memory process
The level of middle cell response, include the CD8 of antigentic specificity+The absolute number of T cell and the immune negative expression for adjusting molecule, MPEC
And SLEC ratio and the level etc. of absolute number.Untr groups are physiological saline group in caption, and cHBV-vaccine groups are new
HBV therapeutic vaccines (CpG M362C are used in combination with rHBV vaccines) group of type.
7.2 result of the test：
Compared with non-treatment group, the adjuvant using CpG M362 as HBV preventative vaccines, outside superinfection early stage mouse
HBsAg expression (Figure 11 A) is substantially not detectable in all blood serum；More than 100% mouse produces high-caliber protectiveness
Anti-HBs (Figure 11 B), serum HBV DNA are substantially not detectable (Figure 11 C)；Meanwhile humoral response in treatment group's Mice Body
Related Tfh ratios substantially increase (Figure 11 D).It was found that the specific CD8 of antigen in liver, spleen+T cell it is absolute
Number increases (Figure 11 E) during superinfection, antigentic specificity CD8+Immune negative adjust such as T cell low expression LAG-3, PD-1 is divided
Sub (Figure 11 F).Simultaneously, it has been found that the mouse after cHBV vaccine therapies, high expression IL-12 (Figure 11 G) in peripheral blood serum.Enter
The change of one step analysis liver, spleen position MPEC and SLEC, it has been found that SLEC ratio and absolute number substantially increases
Add.Based on the above results as can be seen that using CpG M362 as the adjuvant of HBV preventative vaccines, can induce to HBV-
The permanent immunity protection of carrer mouse.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of c-type CpG M362 as adjuvant prepare that HBV is preventative or therapeutic vaccine in application.
2. a kind of HBV preventative vaccines, it is characterized in that：Its active component is hepatitis b virus s antigen (HBsAg), hydrogen-oxygen
Change aluminium and CpG M362.
3. HBV preventative vaccines as claimed in claim 2, it is characterized in that：In every dose of HBV preventative vaccines, each principal component
Content is as follows：2%100 μ g, the CpG M36210 μ g of μ g, Alhydrogel of HBsAg 2.
4. HBV preventative vaccines as claimed in claim 2, it is characterized in that：
The preventative vaccine can induce body to produce anti-HBs while produce cell response；
The preventative vaccine can promote the activation of T cell, enhancing CD4+The level of T cell secrete cytokines IFN-γ；
The preventative vaccine can induce the CD8 of body generation antigentic specificity+T cell, promote CD8+The end memory eventually of T cell
Property differentiation.
5. the preparation method of the HBV preventative vaccines any one of claim 2~4, it is characterized in that, this method includes：
Aluminium hydroxide is added dropwise in HBsAg solution, mixing of turning upside down, then adds CpG M362 solution, you can it is pre- to obtain HBV
Anti- property vaccine.
6. a kind of HBV therapeutic vaccines, it is characterized in that：Its active component is rHBV vaccines and CpG M362.
7. HBV therapeutic vaccines as claimed in claim 6, it is characterized in that：In every dose of HBV therapeutic vaccines, each principal component
Content is as follows：RHBV vaccines μ g, CpG M36210 μ g containing HBsAg 2.
8. HBV therapeutic vaccines as claimed in claim 6, it is characterized in that：
The therapeutic vaccine can promote peripheral blood, spleen, lymph node position DC recruitment, maturation and differentiation, raising BMDC
The ability for swallowing antigen, the expression increase for causing costimulatory molecules CD80, CD86 and MHC-II molecule；
The therapeutic vaccine can improve the microenvironment of immunity of organism suppression, reverse liver, the CD8 of spleen antigentic specificity+T is thin
The exhaustion of born of the same parents, the expression of liver, spleen surface immunosuppression molecule LAG-3, TIGIT, Tim-3, CTLA-4 is reduced, reduce liver
In be immunized and negative adjust cell TregS ratio, the level of TGF-β in serum is reduced, increase the CD8 of antigentic specificity+T cell is bred
The expression of nuclear antigen；
The therapeutic vaccine can strengthen CD8+The activation of T cell and CD107a expression, induction body produce IFN-γ, increased
Add the CD8 of antigentic specificity+The ratio increase of T cell；
The therapeutic vaccine can promote the specific CD8 of antigen in liver, spleen+The terminal differentiation of T cell；Meanwhile reduce liver
The specific CD8 of antigen in dirty, spleen+T cell Eomes expression, promote differentiation of the MPECs to SLECs.
9. HBV therapeutic vaccines as claimed in claim 6, it is characterized in that：Using CpG M362 as the bacterin preparation of adjuvant and its
He is used in conjunction with the immunomodulator of clinical treatment Chronic HBV.
10. the preparation method of the HBV therapeutic vaccines any one of claim 6~9, it is characterized in that, this method includes：
RHBV vaccines are taken, add CpG M362 solution, are mixed, you can obtain HBV therapeutic vaccine liquid.
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