CN107617105A - Suppress Treg cytoactives and promote application of the material of Tfh cell differentiations in HBV infection is treated - Google Patents

Abstract

The invention discloses suppress Treg cytoactives and promote application of the material of Tfh cell differentiations in HBV infection is treated.Application disclosed by the invention includes：The material that suppresses regulatory T cells activity, the material for removing regulatory T cells and the two any of application in treatment and/or prevention and/or diagnosis HBV infection product is prepared with the reagent set and folliculus helper T lymphocyte and Germinal center B cell of HBV vaccines composition.It is demonstrated experimentally that removing Treg cells can repair the humoral immune response imbalance in Chronic HBV model mice in vivo, HBV is promoted to remove；CTLA4 blocking antibodies are injected intraperitoneally and suppress Treg cell functions, the humoral immune response imbalance in Chronic HBV model mice can be repaired, promote HBV to remove.Therefore, using regulatory T cells as target, suppress its function, strengthen HBV specificity T fh cell differentiations, HBV virus sweeps can be accelerated.

Description

Suppress Treg cytoactives and promote the material of Tfh cell differentiations in treatment HBV infection In application

Technical field

The present invention relates to suppress Treg cytoactives in biological technical field and promote the material of Tfh cell differentiations treating Application in HBV infection.

Background technology

Hepatitis type B virus (Hepatitis B Virus, HBV) is that one kind can be close by mother and baby, property contact and daily life Cut the DNA virus of contact transmission.According to statistics, there is nearly 3.5 hundred million HBV carrier in the current whole world, and wherein China just has about 100,000,000 people to take Band hepatitis B.HBV infection is likely to result in acute or chronic hepatitis, and develops into a series of serious chronic hepatic diseases. It is dead that the whole world there are about hepatic sclerosis, hepatic failure and hepatocellular carcinoma that 1,000,000 people induce by chronic HBV infection every year.

It is in the past few decades that chronic hepatitis virus infection, which is cured, so as to prevent the hepatopathys such as hepatic sclerosis or hepatocellular carcinoma The target that everybody pursues always.At present, only 7 kinds of granted treatments for chronic hepatitis B of medicine, including interferon-' alpha ', poly- second two Alcohol interferon-' alpha ' and nucleoside analog.Nucleoside analog selective depression varial polymerases, effectively suppress pgRNA to HBV-DNA's Reverse transcription, therefore HBV-DNA levels can be reduced well.But there is no effect to cccDNA, " curing the symptoms, not the disease ", therefore stop Recurred quickly after medicine, long-term treatment is easy to resistance again；Although ability of the interferon with certain degraded cccDNA, have anti- Virus and immunological regulation double action, but it is only effective to 20-40% patient, and side effect is excessive, also easily bounce-back after drug withdrawal. Generally, we are far away away from " healing hepatitis B " at present.

The research of hepatitis B virus infection is significantly limited by the shortage of toy HBV infection model.Lost without a kind of Pass and learn and all very clear and definite experimental animal models of immunological background, such as mouse and rat, can natural infection HBV, it is or similar Addicted to liver property DNA virus.The foundation of the small animal model of natural HBV infection is simulated to promoting HBV researchs very crucial.

The content of the invention

The technical problems to be solved by the invention are how to treat HBV infection.

In order to solve the above technical problems, present invention firstly provides a1), a2), a3) or a4) preparing treatment and/or pre- Application in anti-and/or diagnosis HBV infection product；

A1 the material of regulatory T cells activity) is suppressed；

A2 the material of regulatory T cells) is removed；

A3 the material and HBV vaccines of regulatory T cells activity) are suppressed；

A4 the material and HBV vaccines of regulatory T cells) are removed.

To exist in order to solve the above technical problems, present invention also offers folliculus helper T lymphocyte and/or Germinal center B cell Prepare the application in treatment and/or prevention and/or diagnosis HBV infection product.

In order to solve the above technical problems, present invention also offers promote folliculus helper T lymphocyte (Follicular Helper CD4+T cells, Tfh cell) material of differentiation and/or Germinal center B cell differentiation preparing treatment and/or pre- Application in anti-and/or diagnosis HBV infection product.

In order to solve the above technical problems, present invention also offers following A 1), A2), A3) or A4) in following B1)-B9) in Application in any：

A1) suppress regulatory T cells activity or remove the material of regulatory T cells；

A2) suppress regulatory T cells activity or remove the material and HBV vaccines of regulatory T cells；

A3) folliculus helper T lymphocyte and/or Germinal center B cell；

A4 the material of the differentiation of folliculus helper T lymphocyte and/or Germinal center B cell differentiation) is promoted；

B1) the application in treating and/or preventing and/or diagnose HBV infection；

B1) application in removing HBV products is being prepared；

B3) the application in HBV is removed；

B4) application in removing HBsAg product is being prepared；

B5) the application in HBsAg is removed；

B6) application in promoting HBV antibody-secreting products is being prepared；

B7) the application in HBV antibody-secretings are promoted；

B8) application in promoting HBsAg antibody-secreting product is being prepared；

B9) the application in HBsAg antibody-secreting is promoted.

In order to solve the above technical problems, present invention also offers the method for treating and/or preventing HBV infection, methods described Including：Following C1 are applied to be treated and/or object of prevention) or C2) or C3) treated and/or prevented：

C1) suppress regulatory T cells activity or remove the material of regulatory T cells；

C2 the material for) suppressing regulatory T cells activity or removing regulatory T cells is combined with HBV vaccines Obtained material；

C3 the material of the differentiation of folliculus helper T lymphocyte and/or Germinal center B cell differentiation) is promoted.

In order to solve the above technical problems, present invention also offers following H1) or H2)：

H1) treat and/or prevent HBV infection medicine and/or vaccine, its active component is H11) or H12) or H13)：

H11) the material for promoting the differentiation of folliculus helper T lymphocyte and/or Germinal center B cell differentiation；

H12) the material for suppressing regulatory T cells activity or removing regulatory T cells；

H13) by H12) material that is obtained together with HBV vaccine combinations；

H2 it is H21) to be used to treating and/or preventing the material X, the material X of HBV infection) or H22) or H23)：

H21) the material for promoting the differentiation of folliculus helper T lymphocyte and/or Germinal center B cell differentiation；

H22) the material for suppressing regulatory T cells activity or removing regulatory T cells；

H23) by H23) material that is obtained together with HBV vaccine combinations.

Above, the folliculus helper T lymphocyte can be HBsAg specificity folliculus helper T lymphocyte；The suppression Regulatory T cells activity processed or the material for removing regulatory T cells can be the material or diphtheria toxin for suppressing CTLA4 activity.Institute The material for stating suppression CTLA4 activity can be CTLA4 antibody.The CTLA4 antibody specifics can be BioXcell products, the diphtheria Toxin concretely Sigma-Aldrich products.

Above, the HBV infection is concretely chronic or acute HBV infection.The removing HBV is concretely removed slowly The HBV of property or acute HBV infection patient's body.The removing HBsAg concretely removes chronic or acute HBV infection The HBsAg of patient's body.The promotion HBV antibody-secretings concretely promote chronic or acute HBV infection patient's body Interior HBV antibody-secretings.The promotion HBsAg antibody-secreting concretely promotes chronic or acute HBV infection patient Internal promotion HBsAg antibody-secreting.

Above, the product can be medicine or vaccine.

Above, HBV vaccines concretely HBsAg vaccine.The HBsAg vaccine specifically can be by mixing Liquid and Imject^TMAlum Adjuvant are formed；The mixing liquid is by hepatitis B surface antibody (HBsAg), bacterium fat Polysaccharide (LPS) forms with PBS.The concentration of hepatitis B surface antibody concretely 140 μ g/ml, bacterium in the mixing liquid The concentration of lipopolysaccharides concretely 20 μ g/ml.Mixing liquid and Imject described in the HBsAg vaccine^TM Alum Adjuvant volume ratio concretely 1:1.Wherein, Imject^TMThe article No. that Alum Adjuvant are Thermo is 77161 Product.HBsAg concretely Beijing Key-Biotechnology Co., Ltd article No. be B204N001 blood source purify HBsAg.LPS concretely Sigma article No. be L4391 product.

Another technical problem to be solved by this invention is how to prepare HBV mouse model.

In order to solve the above technical problems, present invention firstly provides following D1) or method D2)：

D1) the construction method of acute HBV mouse model, including following E1 are applied to C57BL/6N mouse), E2) or E3), Obtain acute HBV mouse model；

E1) the carrier containing HBV genomic DNA or its DNA fragmentation；

E2) HBV genomic DNA or its DNA fragmentation；

E3)HBV；

D2) the construction method of Chronic HBV mouse model, including the E1 is applied to C57BL/6J mouse), the E2) or The E3), obtain Chronic HBV mouse model.

In the above method, C57BL/6N mouse and C57BL/6J mouse can be 6~8 week old mouse.C57BL/6N mouse It can be male mice with C57BL/6J mouse.

In the above method, the carrier of the DNA fragmentation of the genomic DNA containing HBV concretely pAAV/HBV1.2 matter Grain.

In order to solve the above technical problems, present invention also offers the complete sets of products of structure HBV animal models, the complete production Product are by C57BL/6N mouse or C57BL/6J mouse and the E1), the E2) or the E3) form.

In order to solve the above technical problems, the answering in HBV animal models are prepared present invention also offers the complete sets of products With

Above, C57BL/6N mouse are Beijing Vital River Experimental Animals Technology Co., Ltd.'s product, and C57BL/6J is small Mouse is Nanjing University-Nanjing biological medicine research institute product.

The animal concretely mammal, such as mouse.The animal is concretely dynamic containing diptheria toxin receptor Thing, such as Foxp3-DTR mouse.

In the present invention, specific may be embodied in of the suppression regulatory T cells activity suppresses regulatory T cells function, reduced Regulatory T cell number or limitation regulatory T cells differentiation etc..

In the present invention, the phenotype of the acute HBV mouse model includes：Occur core antibody, surface antigen and e in serum Antigen is quickly turned out cloudy, and HBV DNA level rapid decreases, cAg positive cell is eliminated, and with surface antibody and e antibody Produce.

The phenotype of the Chronic HBV mouse model includes：Occur core antibody in serum, and the surface of most of mouse resists Former and e antigen lasting masculins, HBV DNA continual high levels, cAg positive cell are persistently present, not surface antibody and E antibody produces.

Acute and chronic HBV mouse model provided by the present invention is to be utilized respectively two subbreed of C57BL/6 mouse C57BL/6N and C57BL/6J mouse carry out what pAAV/HBV1.2 plasmid Hydrodynamic injections obtained；It is provided by the present invention The novel immune therapeutic scheme of chronic HBV infection is transfers the Tfh cell responses for HBV, suppression is directed to HBV Treg cells Response, the Treg cell functions inhibitor including application CTLA4 blocking antibodies are exempted from as new hepatitis B virus infection Epidemic disease medicine, and above-mentioned therapeutic scheme is combined with HBV vaccine immunities.

The present invention is experimentally confirmed：(1) C57BL/6N mouse carry out pAAV/HBV1.2 plasmid Hydrodynamic injections Afterwards, HBV viral antigens are quickly removed, the horizontal rapid decreases of HBV-DNA, protection antibody response are produced, so as to establish mould Intend the acute HBV mouse model of clinically acute HBV infection person；And C57BL/6J mouse progress pAAV/HBV1.2 plasmid tails are quiet After arteries and veins high-pressure injection, HBV viral antigens can not be removed quickly, HBV-DNA continual high levels, it is impossible to which producing protection antibody should Answer, so as to establish the Chronic HBV mouse model for simulating clinically Patients with Chronic HBV Infection.(2) Chronic HBV model mice is present The Germinal center B cell differentiation defect and Tfh cell differentiations missing of HBV inductions.(3) the acute of Tfh cell differentiations is lacked HBV model mices lose generation surface antibody and remove the ability of viral antigen；Supplemented to Chronic HBV model mice B cell There is provided surface antigen specific effector T fh cells can then aid in it to produce surface antibody.(4) go out in Chronic HBV model mice The Treg cells of existing HBV inductions, and these Treg cells by suppressing Tfh cell differentiations so as to mediating Chronic HBV model mice In humoral immune response imbalance.(5) humoral immunity that removing Treg cells in vivo can repair in Chronic HBV model mice is answered Imbalance is answered, promotes virus sweep.(6) CTLA4 blocking antibodies are injected intraperitoneally and suppress Treg cell functions, can repair chronic Humoral immune response imbalance in HBV model mices, promotes virus sweep.

Therefore, using regulatory T cells as target, suppress its function, strengthen HBV specificity T fh cell differentiations, can accelerate HBV virus sweeps.

Brief description of the drawings

Fig. 1 is to establish acute and chronic HBV mouse model.(a) it is plasmid pAAV/HBV1.2 Hydrodynamic injections After C57BL/6N and C57BL/6J mouse, the dynamic change of hepatitis B virus surface antigen in serum, (b) is plasmid pAAV/ After HBV1.2 Hydrodynamic injection C57BL/6N and C57BL/6J mouse, the dynamic change of HBeAg in serum, (c) it is Hepatitis B Surface in serum after plasmid pAAV/HBV1.2 Hydrodynamic injection C57BL/6N and C57BL/6J mouse The dynamic change of antibody, (d) are blood after plasmid pAAV/HBV1.2 Hydrodynamic injection C57BL/6N and C57BL/6J mouse The dynamic change of hepatitis B e antibody in clear, (e) be plasmid pAAV/HBV1.2 Hydrodynamic injections C57BL/6N and After C57BL/6J mouse, hepatitis B virus core antigen dynamic change in hepatic tissue, (f) is that plasmid pAAV/HBV1.2 tail veins are high After pressure injection penetrates C57BL/6N and C57BL/6J mouse, the dynamic change of hepatitis B virus core antibody in serum, (g) is plasmid After pAAV/HBV1.2 Hydrodynamic injection C57BL/6N and C57BL/6J mouse, the dynamic of serum HBV DNA level becomes Change.

Fig. 2 is the Germinal center B cell and Tfh cell differentiation testing results that Chronic HBV model mice lacks HBV inductions. (a) (plasmid pAAV/HBV1.2 Hydrodynamic injections, similarly hereinafter)/control is modeled for C57BL/6N and C57BL/6J mouse HBV When handling (pAAV empty plasmid Hydrodynamic injections, similarly hereinafter) two weeks, in liver, liver draining lymph node and splenic B cells Germinal center B cell ratio, when (b) is C57BL/6N and C57BL/6J mouse HBV modelings/control treatment two weeks, liver, liver Tfh cell proportions in dirty draining lymph node and spleen, (c) C57BL/6N and C57BL/6J mouse HBV modelings/control treatment two Zhou Shi, using GAPDH as internal reference, analyze spleen CD4⁺Bcl6 and IL-21 mRNA relative expression levels in T cell.

Fig. 3 is that Tfh cells aid in the generation to Anti-HBV activity humoral immune response very crucial and promote virus sweep.(a) it is right The CD4-Cre Bcl6 of C57BL/6N backgrounds^wt/wtMouse (Tfh cell differentiations normal mouse) and CD4-Cre Bcl6^fl/flMouse (Tfh cell differentiations deficient mice) carries out control treatment/HBV modelings, in two to three week after modeling, analyzes the Tfh cells in spleen With Germinal center B cell ratio, (b) is the CD4-Cre Bcl6 to C57BL/6N backgrounds^wt/wtMouse and CD4-Cre Bcl6^{fl /fl}Mouse carries out HBV modelings, and the dynamic change of hepatitis B virus surface antigen, surface antibody and core antibody in serum, (c) is table Face mice immunized with antigen and the Tfh cells (CXCR5 in not immune mouse spleen source⁺PD-1⁺CD4⁺T cell), with Chronic HBV mould Type mouse (HBV models the C57BL/6J mouse of two weeks) splenic B cells are with 1:1 ratio carries out co culture system in vitro.Culture five days Afterwards, cell is transferred on the ELISpot planks for being coated with surface antigen, LPS is stimulated two days, detects surface antibody secretory cell Ratio, (d) is that auxiliary Chronic HBV model mice B cell secretes surface antibody in surface antigen specific effector T fh cell bodies Experiment proves schematic diagram, and (e) is surface antigen specificity T fh cells/non-surface antigen specificity T fh cells, with Chronic HBV mould Type mouse (HBV models the C57BL/6J mouse of two weeks) splenic B cells adopt to transplant jointly gives acceptor Rag1^-/-Mouse, second day After HBV modelings being carried out to Recipient mice, hepatitis B virus surface antigen and the dynamic change of surface antibody in serum.Wherein, CD4- Cre Bcl6^fl/flMouse represents the CD4-Cre Bcl6 of C57BL/6N backgrounds^fl/flMouse (i.e. C57BL/6N-CD4-Cre Bcl6^fl/flMouse), Cre Bcl6^wt/wtMouse represents the Cre Bcl6 of C57BL/6N backgrounds^wt/wtMouse (i.e. C57BL/6N- CD4-Cre Bcl6^wt/wtMouse).

Fig. 4 is that Chronic HBV model mice has suppression Anti-HBV activity humoral immune response and hinders the Treg of virus sweep thin Born of the same parents.(a) when being modeled two weeks for C57BL/6N and C57BL/6J control mices processing/HBV, liver, liver draining lymph node and Foxp3 in spleen⁺Treg cells account for CD4⁺T cell ratio, (b) are that the Treg cells of matching (c) are adopted transplantation experiments schematic diagram, (c) it is the CD4 of separate sources⁺CD25⁺C57BL/6N Recipient mices are given in the transplanting of adopting of Treg enrichment of cell, second day to it is all by After body mouse carries out HBV plasmid modelings, hepatitis B virus surface antigen and the dynamic change of surface antibody in serum, (d) is matching (e) Treg adopts transplantation experiments schematic diagram, the CD4 of (e) separate sources⁺CD25⁺Treg enrichment of cell adopts transplanting to C57BL/ 6N Recipient mices, the modeling of HBV plasmids or control treatment were carried out to Recipient mice in second day, after three weeks, Recipient mice splenic T fh is thin Born of the same parents and Germinal center B cell ratio.

Fig. 5 is that the Treg cells for removing Chronic HBV model mice in vivo recover its Anti-HBV activity humoral immune response ability and clear Except virus.(a) to remove experiment schematic diagram in the Treg cell bodies of matching (b), (b) is the Foxp3- to C57BL/6J backgrounds DTR mouse carry out HBV modelings, model and are classified as two groups latter week, one group of every mouse peritoneal injection 0.5 μ g DT, another group The PBS of same volume is injected intraperitoneally, then two groups of mouse are carried out with eye socket blood samplings, hepatitis B virus surface antigen and surface resist in serum The dynamic change of body, (c) are that experiment schematic diagram is removed in the Treg cell bodies of matching (d), and (d) is to C57BL/6J backgrounds Foxp3-DTR mouse carry out control treatment/HBV modelings, model and are classified as two groups latter week, one group of every mouse peritoneal injection 0.5 μ g DT, the PBS of another group of intraperitoneal injection same volume, after two weeks, mouse spleen Tfh cells and Germinal center B cell ratio, (e) to remove the experiment schematic diagram of joint surface antigen immune in the Treg cell bodies of matching (f), (f) is designated treatment The dynamic change of Hepatitis B surface antibody in Foxp3-DTR mice serums.

Fig. 6 is to suppress Treg cell functions to Chronic HBV model mice internal injection CTLA4 blocking antibodies, so as to extensive Its multiple Anti-HBV activity humoral immune response ability simultaneously removes virus.(a) closed for external CTLA4 antibody to the cell inhibiting work(of Treg The influence of energy, (b) are that CTLA4 blocking antibodies carry out Experiment on therapy schematic diagram to Chronic HBV model mice, and (c) is to chronic After HBV model mices inject CTLA4 blocking antibodies or control IgG albumen, hepatitis B virus surface antigen, e antigens, table in serum The dynamic change of face antibody, e antibody, (d) are that CTLA4 blocking antibodies or control IgG albumen are injected to Chronic HBV model mice Afterwards, the dynamic change of serum HBV DNA level, (e) are to inject progress control treatment/HBV to Chronic HBV model mice to build Mould, two groups are classified as within the 6th day after modeling, one group carries out intraperitoneal injection IgG, and another group of intraperitoneal injection is sealed with the CTLA4 of dosage Closing property antibody, after three weeks, mouse spleen Tfh cells and Germinal center B cell ratio.

Wherein, * represents that difference reaches significance, and * * represent that difference reaches pole significance.

Embodiment

The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.

Experimental method in following embodiments, it is conventional method unless otherwise specified.

Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.

PAAV/HBV1.2 plasmids in following embodiments are documented in United States Patent (USP) US7452696 B2 and (refer to https:// www.google.com.hk/patents/US7452696Dq=HBV+pAAV/HBV1.2＆hl=zh-CN＆sa=X＆ved= 0ahUKEwjY8_qsosbNAhVBQJQKHal3CEYQ6AEITTAH) with document (Huang L R, Wu H L, Chen P J, et al.An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection[J].Proceedings of the National Academy of Sciences,2006,103(47):In 17862-17867.), the public can obtain at applicant, and the biomaterial is only repetition Used in the related experiment of the present invention, it can not be used as other purposes.

PAAV empty plasmids in following embodiments are Beijing FivePlus Molecular Medicine Institute's product.

Experimental animal in following embodiments has：(Beijing dimension tonneau China experimental animal technology has male C57BL/6N mouse Limit company), male C 57 BL/6 J mouse (Nanjing University-Nanjing biological medicine research institute), the CD4-Cre of C57BL/6 backgrounds Bcl6^fl/flMouse (is received and taught from Toshitada Takemori, it is more than generation to pass four to C57BL/6N backgrounds, referring specifically to Deliver document：Hollister K,Kusam S,Wu H,et al.Insights into the role of Bcl6 in follicular Th cells using a new conditional mutant mouse model[J].The Journal of Immunology,2013,191(7):3705-3711.), the CD4-Cre Bcl6 of C57BL/6 backgrounds^wt/wtMouse (specific ginseng See and delivered document：Hollister K,Kusam S,Wu H,et al.Insights into the role of Bcl6 in follicular Th cells using a new conditional mutant mouse model[J].The Journal of Immunology,2013,191(7):3705-3711.)、B6.Rag1^-/-Mouse (Nanjing University-Nanjing biological medicine research Institute), Foxp3-DTR mouse (receive from Alexander Y.Rudensky teach, C57BL/6J backgrounds, containing diphtheria toxin by Body, referring specifically to having delivered document：Kim J M,Rasmussen J P,Rudensky A Y.Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice[J].Nature immunology,2007,8(2):191-197.), CD45.1 mouse (are received and taught from biophysics institute of Chinese Academy of Sciences Zhu Mingzhao, had Body is referring to having delivered document：Zhu M,Yang Y,Wang Y,et al.LIGHT regulates inflamed draining lymph node hypertrophy[J].The Journal of Immunology,2011,186(12):7156-7163.)。 CD4-Cre Bcl6 used in present patent application^wt/wtMouse and CD4-Cre Bcl6^fl/flMouse is C57BL/6N backgrounds, this Foxp3-DTR mouse used in patent are C57BL/6J backgrounds.Above mouse is raised according to standard SPF ranks and solved in Chinese people Fang Jun Military Medical Science Institutes animal platform.All zooperies are tested by Academy of Military Medicine, PLA The care of animal and ratified using the committee.

The kit used in following embodiments has：(the prosperous promise in Beijing is beautiful for hbv nucleic acid immue quantitative detection reagent box Enlightening technique of gene detection Co., Ltd)

SC-I perfusion liquids in following embodiments are made up of solvent and solute, and wherein solvent is perfusion liquid mother liquor, solute and Its concentration is respectively 5mM D-Glucoses, 20,000 UT/200ml heparin and 0.5mM EGTA, pH 7.3.Wherein, perfusion liquid mother liquor It is made up of solvent and solute, solvent is water, and solute and its concentration are：NaCl 136mM, KCl 5.3mM, NaH₂PO₄·2H₂O 0.5mM, Na₂HPO₄·7H₂O 0.4mM, HEPES 9.1mM, NaHCO₃4.1mM, pH 7.3.Note：SC-I can carry the previous day and match somebody with somebody Put, 0.22 μm of filter is filled into autoclaved vial.General SC-I is configured according to 50ml/ mouse.Perfusion liquid Mother liquor can prepare in advance, long-term storage.

Anti-mouse CD16/CD32, FITC Conjugated Anti-Mouse CD19, PE in following embodiments Conjugated Anti-Mouse FAS and FITC Conjugated Anti-Human/Mouse GL-7, Biotin Conjugated Anti-Mouse CXCR5、FITC Conjugated Anti-Mouse PD-1、streptavidin PE、 PE-Cy7 Conjugated Anti-Human/Mouse B220、APC Conjugated Anti-Mouse CD4、PE Conjugated Anti-Mouse Foxp3、APC Conjugated Anti-Mouse CD19、、APC Conjugated Anti-Human/Mouse B220, PE Conjugated Anti-Mouse CD25 are eBioscience Products； Goat Anti-Mouse IgG-HRP, Goat Anti-Mouse IgM-HRP are SouthernBiotech Products.

In following embodiments, PBS (pH7.4) is prepared as follows：Graduated cylinder measures 10 × PBS of 100ml, uses 900ml Deionized water is diluted to 1L, and pH value is adjusted to 7.4.4% paraformaldehyde (PFA) is prepared as follows：Add into 1LPBS Enter 40g paraformaldehydes, be positioned over 55 DEG C of oven overnight dissolvings, pH value is adjusted to 7.4, and 4 DEG C are kept in dark place after aseptic filtration.

Streaming dye solution (FACS Buffer) in following embodiments adds FBS and EDTA into PBS and obtained Liquid, wherein, FBS concentration is 1g/100ml, and EDTA concentration is 2.5mM.

The magnetic bead sorting buffer solution (MACS Buffer) used in following embodiments is prepared：PBS+2%FBS+1mM EDTA。

The kit and reagent used in following embodiments have：Endotoxin-free plasmid big extraction reagent kit (Tiangeng biochemical technology Co., Ltd), competent cell (Tiangeng biochemical technology Co., Ltd), modified form RPMI-RPMI-1640 culture mediums (Hyclone), penicillin streptomycin antibiotic (Sigma), domestic hyclone (Chinese holly), import hyclone (Gibco), 10 × phosphate buffer (Shanghai Yuan Mu bio tech ltd), erythrocyte cracked liquid (BD Bioscience), poly first Aldehyde (Chemical Reagent Co., Ltd., Sinopharm Group), EDTA (Amresco), hepatitis b virus s antigen diagnostic kit (section Magnificent biology), anti-HBs diagnostic kit (China of section biology), antihepatitis b e antibody diagnostic kit (China of section biology), hepatitis b virus s antigen diagnostic kit (China of section biology), anti-HBc diagnosis Kit (China of section biology), RNeasy Plus Mini Kit (Qiagen), RevertAid^TM First Strand cDNA Synthesis Kit (Fermentas), SYBR GREEN (TOYOBO), Ready-to-use TMB substrates (Mabtech), Foxp3 permeable membranes fix kit (eBioscience), OptiprepTM (Axis-Shield ProC AS), heparin (Chemical Reagent Co., Ltd., Sinopharm Group), EGTA (Amresco), glucose (Chemical Reagent Co., Ltd., Sinopharm Group), EasySep^TMMouse B Cell Isolation Kit (STEMCELL Technologies), diphtheria toxin DT (Sigma- Aldrich), CFSE (eBioscience), BSA (Amresco).

The foundation of embodiment 1, acute and chronic HBV mouse model

1. model is established

PAAV/HBV1.2 plasmids are injected to 6~8 week old hero using Hydrodynamic injection method (with normal saline dilution) In property C57BL/6N Mice Bodies.Injection dosage is every μ g pAAV/HBV1.2 plasmid of mouse 10, and volume injected is mouse weight 8%-12%, all volume injecteds are at the uniform velocity pushed into 5~7s, 9 pAAV/HBV1.2 plasmid transfection mouse moulds are obtained Type (hereinafter referred to as acute HBV mouse model).In order to facilitate tail vein injection, the needle point of 2ml syringes can be replaced with to 1ml notes The needle point of emitter.

According to the method described above, pAAV/HBV1.2 plasmids are replaced with into pAAV empty plasmids, other steps are constant, obtain The control mice of acute HBV mouse model, it is named as control mice N.

According to the method described above, male C57BL/6N mouse being replaced with into male C 57 BL/6 J mouse, other steps are constant, 10 pAAV/HBV1.2 plasmid transfections mouse models (hereinafter referred to as Chronic HBV mouse model) are obtained.

According to the method described above, male C57BL/6N mouse are replaced with into male C 57 BL/6 J mouse, and by pAAV/HBV1.2 Plasmid replaces with pAAV empty plasmids, and other steps are constant, obtains the control mice of Chronic HBV mouse model, is named For control mice J.

By the injection of pAAV/HBV1.2 plasmids as Zhou Jiwei the 0th week, promoting circulation of blood was entered to each mouse weekly at the 0-14 weeks respectively Final proof sheet, the collection of hepatic tissue and the detection of index of correlation, are specifically shown in step 2-4.

Hepatitis B virus surface antigen, e antigens, surface antibody, e antibody and core antibody in 2.ELISA detection mice serums

Serum sample is diluted with PBS, examined using the five indexes of hepatitis b of Shanghai Kehua Bio-engineering Co., Ltd Test agent box, instruct to carry out coherent detection to specifications, or commission Beijing North Institute of Biological Technology uses, and using radiation exempts from Epidemic disease method carries out the detection of index of correlation.

3.Q-PCR detection serum HBVs DNA

Test serum is collected, using hbv nucleic acid immue quantitative detection reagent box, under instructions direct, utilizes reality When quantitative real time PCR Instrument carry out coherent detection.

4. Liver immunity groupization detects cAg

Using paraffin section, according to routine immunization group program, anti-HbcAg (Beijing Zhong Shan Golden Bridge biotechnologys are utilized Co., Ltd) and goat anti-mouse/rabbit igg secondary antibody (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) progress HbcAg Dyeing, the coloration result of optical microphotograph Microscopic observation section simultaneously photograph to record.

As a result Fig. 1 is seen, the results showed that, the C57BL/6 mouse (C57BL/6N mouse and C57BL/6J mouse) of different subbreed, After injecting pAAV/HBV1.2 plasmids, acute and chronic HBV mouse model has been respectively obtained.Wherein, acute HBV mouse model Is there is core antibody in serum after modeling in phenotype, and surface antigen and e antigens are quickly turned out cloudy, HBV DNA level rapid decreases, core Heart antigen-positive cell is eliminated, and is produced with surface antibody and e antibody, and the phenotype of Chronic HBV mouse model is after modeling Occur core antibody in serum, and the surface antigen of most of mouse and e antigen lasting masculins, HBV DNA continual high levels, core Heart antigen-positive cell is persistently present, and surface antibody and e antibody do not produce.And to control mice N and control mice J not With above-mentioned phenotype, i.e., it is not acute or chronic HBV mouse.

The Germinal center B cell and Tfh cell differentiation feelings that HBV is induced in embodiment 2, acute and chronic HBV mouse model Condition is analyzed

Plasmid inject the 3rd week, in accordance with the following steps 1-3 method separate acute HBV mouse model in embodiment 1 respectively It is thin with control mice N and Chronic HBV mouse model and control mice J liver lymphocyte, liver draining lymph node lymph Born of the same parents and spleen lymphocyte, in accordance with the following steps 4 and 5 method detect Germinal center B cell ratio and the complementary T of folliculus respectively Cell (Follicular helper CD4⁺T cells, Tfh cell) ratio, analyzed according to the method for step 6 using Q-PCR CD4⁺The mater transcription factors Bcl6 of Tfh cell differentiations and the main effects cell factor IL-21 mRNA express water in T cell Flat, in triplicate, experiment every time sets three repetitions for experiment.

1. liver lymphocyte separates

(1) mouse is dissected：Make mouse deep anaesthesia to mouse peritoneal injection yellow Jackets anesthetic.Sprayed with alcohol small Mouse body surface, abdominal cavity is opened, enteron aisle etc. is pushed aside, exposes inferior caval vein, liver and portal vein.

(2) irrigate：From portal vein contact pin, perfusion liver is irrigated with SC-I, when the blood in liver is all poured totally When, stop perfusion.

(3) hepatic tissue is taken：Excise one's gallbladder, remove liver, be positioned in 200 mesh cell sieves, and by equipped with liver organization Cell sieve is placed on the 50ml centrifugation mouths of pipe.

(4) hepatocyte suspension is obtained：Hepatic tissue is shredded with ophthalmology small scissors, adds RPMI-1640 trainings of the 2ml containing 3%FBS After supporting base, hepatic tissue fragment is ground with the push rod of 2ml syringes, trained in grinding with the RPMI-1640 containing 3%FBS Foster base is washed, until only the connective tissue of white, the process operate on ice in cell screen clothes.Grind obtained cell Selection is filtered in the 50ml centrifuge tubes below cell sieve.

(5) low-speed centrifugal removes hepatic parenchymal cells and fragment of tissue：By above-mentioned 50ml centrifuge tubes under conditions of 50g, 4 DEG C 4min is centrifuged, the parenchyma of precipitation is removed, then repeats once.

(6) nonparenchymal cell is precipitated：Supernatant is carefully toppled over and is transferred to new 50ml centrifuge tubes, 4 DEG C, 500g, centrifuges 6- 8min, supernatant is abandoned, precipitate nonparenchymal cell.

(7) density gradient centrifugation：Cell precipitation is resuspended to 2.25ml with the RPMI-1640 culture mediums without serum, with 2.75ml 40%OptiPrep fully blow and beat mixing (being the equal of 22% OptiPrep).On the cell suspension of the density Face carefully slowly adds last layer serum-free RPMI-1640 culture mediums (3ml), forms two density gradients.Followed by density Gradient centrifugation, it is as follows to centrifuge related setting：Room temperature, 1100g, centrifuge 25min, raising speed 0, deceleration 0.

(8) aim cell is precipitated：After density gradient centrifugation is completed, by 22% density gradient layer and serum-free RPMI- Cell among 1640 culture medium layers suctions out (about 2-3ml), is transferred to new 15ml centrifuge tubes, adds culture medium and mixes (one Surely to overturn mixing, ensure that cell can be smoothly centrifugation down, cumulative volume 15ml) 500g centrifugation 8min, precipitation is For liver mixed lymphocytes.

2. liver draining lymph node separation of lymphocytes

(1) mouse is dissected：Disconnected neck puts to death mouse, sprays mouse body surface with alcohol, opens abdominal cavity, enteron aisle etc. is pushed aside, cruelly Expose inferior caval vein, liver and portal vein.

(2) lymph node is won：The portal vein lymph node and lymphonodi coeliaci two near portal vein are won with tweezers Liver draining lymph node.Remove two lymph nodes are placed on filter paper, the careful adipose tissue and mucous membrane for removing attachment.

(3) lymphocyte suspension is obtained：It will handle between clean lymph node is placed on the hair side of two panels slide, add a little RPMI-1640 culture mediums containing 3%FBS, gently rub two panels slide, is rinsed with the RPMI-1640 culture mediums containing 3%FBS, And filtered through 200 mesh cell sieves, cell suspension is collected into 1.5ml EP pipes.

(4) aim cell is precipitated：4 DEG C, 500g centrifugation 5min, precipitation is liver draining lymph node mixed lymphocytes.

3. spleen lymphocyte separates

(1) mouse is dissected：Disconnected neck puts to death mouse, sprays mouse body surface with alcohol, opens abdominal cavity.

(2) spleen cell mixing is obtained：Spleen is won with tweezers to be positioned over added with RPMI-1640 trainings of the 1ml containing 3%FBS In 12 orifice plates for supporting base, spleen is pressed lightly on the tack of the push rod of 2ml syringes, discharges splenocyte.It is and thin by what is obtained Born of the same parents' suspension is filtered into 1.5ml EP pipes via 200 mesh cell screen clothes.4 DEG C, 500g centrifugation 5min, abandon supernatant.

(3) erythrocyte splitting：With 500 μ l erythrocyte cracked liquid lysis at room temperature red blood cells 3min.Then 3%FBS is contained with 1ml RPMI-1640 culture mediums terminate it is red split, 4 DEG C, 500g centrifugation 5min.

(4) refilter：Supernatant is abandoned, precipitation is resuspended with RPMI-1640 culture mediums of the 1ml containing 3%FBS, with 200 mesh cell sieves Net filtration is into new 1.5ml EP pipes.

(5) aim cell is precipitated：4 DEG C, 500g centrifugation 5min, obtained precipitation is spleen mixed lymphocytes.

4. Flow cytometry Germinal center B cell ratio

(1) cell to be marked is washed one time with FACS Buffer, depending on visual cell's amount, with 50 μ l or 100 μ l FACS Cell precipitation is resuspended in Buffer.

(2) the Anti-mouse CD16/CD32 (FcR expressed on closing cell) of specified amount on antibody specification are added, FITC Conjugated Anti-Mouse CD19, PE Conjugated Anti-Mouse Fas, FITC Conjugated Anti-Human/Mouse GL-7, after fully mixing, on ice or 4 DEG C of lucifuges are incubated 30min (incubation period will be thin every 10min Born of the same parents mix, prevent because cell precipitation causes antibody incubation ineffective, similarly hereinafter).

(3) it is incubated after terminating, with 4 DEG C of 1ml FACS Buffer, 500g, 5min are washed twice, finally with 300-500 μ l Fixed cell is resuspended in 2%PFA, and being positioned over 4 DEG C of lucifuges can preserve 3 days or so, and BD LSRFortessa instruments obtain data, FlowJo softwares are analyzed data.

5. Flow cytometry Tfh cell proportions

(1) cell to be marked is washed one time with FACS Buffer, depending on visual cell's amount, with 50 μ l or 100 μ l FACS Cell precipitation is resuspended in Buffer.

(2) for 50 μ l systems, then 0.5 μ l Anti-mouse CD16/CD32 and 1 μ l Biotin Conjugated are added Anti-Mouse CXCR5；For 100 μ l systems, then add 1 μ l Anti-mouse CD16/CD32 and 2 μ l Biotin Conjugated Anti-Mouse CXCR5.After mixing, room temperature lucifuge on gyroscope is incubated 1.5h.

(3) 4 DEG C of 1ml FACS Buffer are used, 500g, 5min are washed twice.Depending on visual cell's amount, with 50 μ l or 100 μ l Cell precipitation is resuspended in FACS Buffer.

(4) in accordance with specification, the FITC Conjugated Anti-Mouse PD-1 of prescribed dose are added, Streptavidin PE, PE-Cy7 Conjugated Anti-Human/Mouse B220, APC Conjugated Anti- Mouse CD4.After mixing, DEG C lucifuge is incubated 30min on ice/4.

(5) it is incubated after terminating, with 4 DEG C of 1ml FACS Buffer, 500g, 5min are washed twice, finally with 300-500 μ l Fixed cell is resuspended in 2%PFA, and being positioned over 4 DEG C of lucifuges can preserve 3 days or so, and BD LSRFortessa instruments obtain data, FlowJo softwares are analyzed data.

6.Q-PCR analyzes CD4⁺In T cell the mater transcription factors Bcl6 of Tfh cell differentiations and main effects cell because Sub- IL-21 mRNA expressions

Sort spleen CD4⁺T cell, cell is washed into one time (500g, 5min) with PBS, wash away FBS that may contain etc. into Point.Intracellular is carried out to the cell that sorting obtains using micro RNA extracts kits (RNeasy Plus Mini Kit, Qiagen) Total RNAs extraction.It should be noted to test consumptive material used, environment ensures without RNase as far as possible, it is desirable to ambient operation, shorten experiment as far as possible Time, centrifuging temperature are not below 20 DEG C.After getting total serum IgE with reference to product description, each sample takes 500ng-2 μ g's RNA, using kit (RevertAid^TMFirst Strand cDNA Synthesis Kit, Fermentas) according to explanation Book carries out reverse transcription, obtains cDNA.Using the real-time fluorescence quantitative PCR based on SYBR Green, using GAPDH as internal reference, analysis Wherein Bcl6 and IL-21 relative expression levels.Sequence information table 1 used.

Table 1, primer sequence

Modeled by comparative analysis HBV or the C57BL/6N mouse and C57BL/6J mouse of control treatment, liver, liver draw Flow Germinal center B cell ratio, Tfh cell proportions and spleen CD4 in lymph node and spleen⁺Bcl6 and IL-21 table in T cell Up to level, data are shown in Fig. 2, the results showed that, Chronic HBV model mice lacks the Tfh cell differentiations and centrum germinativum B of HBV inductions Cell differentiation, acute HBV model mices have Tfh cell differentiations and the Germinal center B cell differentiation of HBV inductions.

Embodiment 3, the acute HBV model mices Germinal center B cell differentiation for lacking Tfh cell differentiations and surface antibody should Answer disappearance

CD4-Cre Bcl6^fl/flThe CD4 of mouse⁺The transcription factor for being responsible for Tfh cell differentiations is specifically lacked in T cell Bcl6, thus the CD4 of the mouse⁺It can not be divided into Tfh cells after T cell activation, and the CD4 of other subgroups⁺T cell effect point Change unaffected.By the CD4-Cre Bcl6 of C57BL/6 backgrounds^fl/flMouse is mated with C57BL/6N mouse, obtains F1 generation CD4-Cre Bcl6^wt/flMouse, then the CD4-Cre Bcl6 by F1 generation^wt/flMouse is mated with C57BL/6N mouse, is obtained To the CD4-Cre Bcl6 in F2 generations^wt/flMouse, repeat the operation, until obtaining the CD4-Cre Bcl6 in F4 generations^wt/flIt is small Mouse, the mouse have possessed the acute backgrounds of HBV (C57BL/6N backgrounds).Again by the female CD4-Cre Bcl6 in F4 generations^wt/flMouse and Male CD4-Cre Bcl6^wt/flMouse is mated, and obtains the CD4-Cre Bcl6 of littermate control^wt/wtMouse and CD4-Cre Bcl6^fl/flThe CD4-Cre Bcl6 of mouse, i.e. C57BL/6N backgrounds^wt/wtMouse and CD4-Cre Bcl6^fl/flMouse.

According to the method for embodiment 1, pAAV/HBV1.2 plasmids are injected to by 6~8 week old using Hydrodynamic injection method Male C57BL/6N-CD4-Cre Bcl6^fl/flIn Mice Body, HBV C57BL/6N-CD4-Cre Bcl6 are obtained^fl/flMouse, will With pAAV/HBV1.2 plasmids injection C57BL/6N-CD4-Cre Bcl6^fl/flThe mouse that mouse obtains is named as control For control mice CD4-Cre Bcl6^fl/fl。

According to the method for embodiment 1, pAAV/HBV1.2 plasmids are injected to by 6~8 week old using Hydrodynamic injection method Male C57BL/6N-CD4-Cre Bcl6^wt/wtIn Mice Body, HBV C57BL/6N-CD4-Cre Bcl6 are obtained^wt/wtMouse, will With pAAV/HBV1.2 plasmids injection C57BL/6N-CD4-Cre Bcl6^wt/wtThe mouse that mouse obtains is named as control For control mice CD4-Cre Bcl6^wt/wt。

Method according to embodiment 1 detects hepatitis B virus surface antigen, Hepatitis B Surface in above-mentioned each mice serum and resisted The dynamic change of body and hepatitis B virus core antibody.

Injected the 3rd week in plasmid, HBV C57BL/6N-CD4-Cre are separated according to the method for step 3 in embodiment 2 Bcl6^fl/flMouse and control mice Cre Bcl6^fl/flSpleen lymphocyte, and according to step 4 in embodiment 2 and 5 method Detect Tfh cell proportions and Germinal center B cell ratio in spleen mixed lymphocytes.

As a result a and b in Fig. 3 is seen, the results showed that, Tfh cell differentiations are blocked and completely inhibit in acute HBV models in hair tonic The differentiation and surface antibody response of heart B cell, and cause surface antigen is removed to slow down.And the core independent of T cell resists The generation of body is simultaneously uninfluenced.Also just say Tfh cell responses for Anti-HBV activity humoral immune response it is normal effectively produce and HBV antigens are removed very crucial.

Embodiment 4, surface antigen specificity T fh cell repair Chronic HBV model mice B cell antibody response defects

1. specific/non-specific Tfh cells of surface antigen obtain

The HBsAg (Beijing Key-Biotechnology Co., Ltd, article No. B204N001) and LPS that blood source is purified (Sigma, article No. L4391) is diluted with the PBS being sterile filtered, and obtains mixing liquid, and blood source purifies the dense of HBsAg in mixing liquid Spend for 2mg/ml, LPS concentration is 20 μ g/ml；By mixing liquid and Imject^TMAlum Adjuvant (Thermo, article No. 77161) HBsAg vaccine is obtained after being sufficiently mixed in equal volume uniformly, HBsAg vaccine immunity is injected intraperitoneally C57BL/6J mouse, injection dosage are 100 μ l/.

Two-wheeled is carried out to mouse to be immunized, and carries out within the 9th day after being immunized for the first time secondary immunity, second is immune latter 9th day, Examined according to the method separating mouse spleen lymphocyte of step 3 in embodiment 2, and according to step 4 in embodiment 2 and 5 method Survey Tfh cell proportions in spleen mixed lymphocytes.Flow sorter sorting purifies the spleen of the immune mouse of above-mentioned surface antigen Dirty Tfh cells.The streaming of wherein Tfh cells is defined as CD4⁺B220^-CXCR5⁺PD-1⁺.Obtained Tfh cells are named as HBsAg i.m.C57BL/6J Tfh cells (i.e. HBsAg specificity Ts fh cells).

According to the method described above, the Tfh cells of non-immunized C57BL/6J mouse are sorted, obtained cell is named as WT C57BL/6J Tfh cells (i.e. non-HBsAg specificity Ts fh cells).

The acquisition of 2.B cells

According to product description, the splenic B cells of chronic HBV infection mouse model in magnetic bead sorting embodiment 1 are used.

3.Tfh cells in vitro Help B Cells secretory antibodies

(1) co culture system in vitro：With complete medium (serum containing 10%Gibco, penicillin streptomycin antibiotic RPMI- 1640 culture mediums) the HBsAg i.m.C57BL/6J Tfh cells and B cell of an above-mentioned acquisition are washed, counted with calculating instrument Number.Co-cultured in flat 96 orifice plate, wherein T cell and B cell number is 1.5 × 10⁵Individual/hole.Add 10 μ g/ml RHBsAg (recombinant HBsAg (Beijing Key-Biotechnology Co., Ltd)), co-cultured 5 days with complete medium, it is middle A supplement rHBsAg of addition and complete medium.To not add the holes of rHBsAg or WT C57BL/6J Tfh cells as pair According to.

(2) ELISpot plates (Millipore) are transferred to：After co-culturing 5 days, washed with RPMI-1640 culture mediums thin twice Born of the same parents, it is transferred to and is coated with 2 μ g/ml rHBsAg ELISpot planks, addition 200ng/ml LPS is stimulated 2 days again.

(3) ELISpot is detected：Two days later, cell culture fluid is abandoned, board-washing, is incubated detection antibody, develop the color simultaneously read plate, detection Secrete the spot of the cell of surface antibody.

Help B Cells secretory antibody in 4.Tfh cell bodies

(1) construction method of the HBV infection model of foundation embodiment 1 is to 6-8 week old, male of the body weight in 20g or so B6.Rag1^-/-Mouse is modeled, and successfully obtains Chronic HBV mouse, is named as Chronic HBV Rag1^-/-Mouse, modeling are worked as It is designated as D-3.

(2) the splenic T fh cells in specified source and the acquisition methods of B cell are with step 1 and 2, weight after PBS is washed one time It is outstanding, count, blow even.

(3) in Chronic HBV Rag1^-/-The 3rd day (D0) after the injection of mouse plasmid, by 1 × 10⁶Individual Tfh cells and 5 × 10⁶ Individual B cell is adopted by tail vein injection is transplanted to Chronic HBV Rag1^-/-Mouse, obtain adopting the mouse after transplanting (in Fig. 3 (d))。

(4) different time points are taken a blood sample, and it is horizontal to detect surface antigen and surface antibody in serum.

Obtained HBsAg specificity T fh cells are sorted by mouse spleen is immunized from surface antigen and from non-immunized WT C57BL/6J mouse spleens sort obtained non-HBsAg specificity Ts fh cells, respectively with Chronic HBV mouse model splenic B cells Co-culture, data are shown in Fig. 3 (c), the results showed that HBsAg specificity T fh cells can Help B Cells produce surface antibody, and The non-HBsAg Tfh cells in WT C57BL/6J mouse source, or co-culture system do not add rHBsAg then without this phenomenon.Remove Experiment in vitro, inventor also utilize B6.Rag1^-/-Mouse has carried out experiment in vivo.B6.Rag1 used^-/-Mouse belongs to C57BL/6J backgrounds.B6.Rag1^-/-Mouse breaks up without the T cell and B cell of maturation, there is provided the clean acceptor of a comparison Environment.Select 6~8 week old, B6.Rag1 of the body weight in 20g or so^-/-Male mouse carries out HBV plasmid modelings.And after modeling, to its tail It is injected intravenously donor Tfh cells and B cell.Wherein donor B cell is all from Chronic HBV mouse model spleen；And Tfh cells Source is identical with above-mentioned experiment in vitro, is HBsAg specificity T fh cells and non-HBsAg specificity Ts fh cells respectively, with laggard Row serology ELISA is detected.Data are shown in Fig. 3 (e), the results showed that, chronic HBV infection mouse model B cell is in surface antigen Surface antibody can be secreted under specificity T fh cells auxiliary, and successfully removes HBsAg.Therefore, surface antigen specificity Tfh cells can aid in Chronic HBV mouse model B cell to secrete surface antibody.

Surface antigen specific Treg in embodiment 5, Chronic HBV mouse model is by suppressing Tfh cell differentiations So as to hinder the generation of surface antibody response

1.Treg cells are adopted transplantation experiments

(1) Treg cell purifications：It is most classical due to regulatory T cells (Regulatory T cells, Treg cell) Marker Foxp3 mark is needed to cell-permeant, influences cytoactive, therefore use CD4⁺CD25⁺T cell represents enrichment Treg cell masses.The spleen mixed lymphocytes of the acute HBV mouse of embodiment 1 are separated, after antibody labeling, use flow sorter It is purified into Treg cells (CD4 therein⁺CD25⁺T cells)。

(2) Treg cells are adopted transplanting：The Treg cells that above-mentioned sorting purifies to obtain are resuspended in PBS, with cell counter pair It is counted.To every acceptor C57BL/6N mouse tail vein injection 1.0 × 10⁶Individual CD4⁺CD25⁺Treg cells, injection Volume is 300 μ l/, and the week old of Recipient mice 6~8, body weight is in 20g or so.

(3) Recipient mice HBV is modeled：Adopt second day of transplanting receiving Treg cells, according to the method pair of embodiment 1 It carries out plasmid modeling, every μ g pAAV/HBV1.2 plasmid of mouse tail vein high-pressure injection 10, obtains Treg-HBV infection C57BL/6N mouse.And it is used as and is compareed by the use of the mouse after the transplanting of adopting of injection pAAV empty plasmids.

According to the method for step (1)-(3), adopted with the Treg cells of the Chronic HBV mouse of embodiment 1 and transplant C57BL/ 6J mouse, the modeling of pAAV/HBV1.2 plasmids is then injected, it is small that obtained mouse is named as into J-Treg-HBV infection C57BL/6J Mouse, the mouse for transplanting and injecting pAAV empty plasmids that will adopt through C57BL/6J mouse Treg cells is as control.

According to the method for step (1)-(3), adopted with the acute HBV mouse Treg cells of embodiment 1 and transplant C57BL/6J Mouse, the modeling of pAAV/HBV1.2 plasmids is then injected, it is small that obtained mouse is named as into N-Treg-HBV infection C57BL/6J Mouse；Adopted with the control mice J Treg cells of embodiment 1 and transplant C57BL/6J mouse, then inject pAAV/HBV1.2 plasmids Modeling, control-HBV infection C57BL/6J mouse are named as by obtained mouse；Adopted with PBS and transplant C57BL/6J mouse, so The modeling of pAAV/HBV1.2 plasmids is injected afterwards, and obtained mouse is named as PBS-HBV infection C57BL/6J mouse.

(4) Serologic detection：Specified time point carries out eye socket to various mouse and takes blood, ELISA detection serum after modeling The removing situation of middle surface antigen (100 times of dilutions) and the production of surface antibody (5 times of dilutions).

(5) cytology detects：When modeling three weeks, mouse is put to death, takes the spleen of mouse, is obtained according to the method for embodiment 2 Spleen mixed lymphocytes, liver mixed lymphocytes and liver draining lymph node mixed lymphocytes.2 stream as steps described below Formula analysis wherein Treg cell proportions, and detect Tfh cells and Germinal center B cell ratio according to the method in embodiment 2.

2. Flow cytometry Treg cell proportions

(1) cell to be marked is washed one time with FACS Buffer, depending on visual cell's amount, with 50 μ l or 100 μ l FACS Cell precipitation is resuspended in Buffer.

(2) padding：Padding is carried out to APC Conjugated Anti-Mouse CD4 antibody.

(3) fixed permeable membrane：After padding terminates, 1ml FACS Buffer are washed twice.Diluted with 500 μ l Cell precipitation is resuspended in Fixation/Permeabilization Buffer, and room temperature lucifuge is incubated 1h, permeable membrane is fixed.

(4) nuclear factor dyes：Add that 700 μ l have diluted into the cell suspension of fixed permeable membrane 1 × Permeabilization Buffer (the H of 10 × Permeabilization Buffer+9 part volumes of 1 part of volume₂O), room Warm 500g centrifuges 5min, then is washed again one time with 1 × Permeabilization of 1ml Buffer.Finally with 50 μ l or 100 μ l Cell is resuspended in 1 × Permeabilization Buffer, adds the PE Conjugated Anti-Mouse of specification specified amount Foxp3 antibody.Dark place lucifuge incubation at room temperature 30min, carries out nuclear factor dyeing.

(5) plus 1ml 1 × Permeabilization Buffer room temperatures 500g centrifuges 5min, washes twice.

(6) cell finally is resuspended with FACS Buffer, 4 DEG C of lucifuges can preserve 3 days or so, BD LSRFortessa instruments Data are obtained, FlowJo softwares are analyzed data.

Modeled by comparative analysis HBV or the C57BL/6N mouse and C57BL/6J mouse of control treatment, liver, liver draw Treg cell proportions in lymph node and spleen are flowed, data are shown in Fig. 4 (a), the results showed that, there is HBV and lured in Chronic HBV model mice The Treg cell differentiations led.

The C57BL/6N mouse of acute background are adopted the Treg cells of transplanting separate sources, adopt transplanting second day to by Body mouse carries out HBV plasmid modelings, to analyze the Treg cells of separate sources to acute HBV model mices surface antibody response Influence, as a result see in Fig. 4 (b, c).As a result show, only the Treg cells from Chronic HBV model mice spleen can show Write the surface antibody response for suppressing acute HBV model mices and then delay surface antigen to remove, and acute HBV model mices and right According to the C57BL/6J mouse spleens of processing Treg cells have no effect on the acute HBV model mices of acceptor surface antibody response and Surface antigen is removed.Also, found by analyzing Recipient mice splenic T fh cells and Germinal center B cell ratio, it is only chronic Suppress the Tfh cell differentiations and Germinal center B cell differentiation (Fig. 4 of HBV inductions in the Treg cell bodies in HBV model mices source Middle e and f), wherein the PBS for being labeled with " control treatment " represents injection PBS but modeled without HBV that another is not marked " at control The PBS of reason " represents injection PBS and carries out HBV modelings).It is above-mentioned it is demonstrated experimentally that HBV occur in Chronic HBV model mice body special Property Treg cell differentiations, and this part HBV specific Tregs can be resisted by restricted T fh cell responses so as to suppress surface Body produces and slowed down surface antigen removing.

Embodiment 6, the Tfh cell differentiations for removing the recovery HBV inductions of Chronic HBV model mice Treg cells in vivo simultaneously promote Surface antibody response and virus sweep

1.Treg cell clearances are tested

(1) plasmid models：Choose 6~8 week old, male Foxp3-DTR mouse of the body weight in 20g or so.According to embodiment 1 Method carry out plasmid modeling to it, every μ g pAAV/HBV1.2 plasmid of mouse tail vein high-pressure injection 10 will the injection same day The 0th day (D0) is designated as, obtains HBV mouse.Control is used as by the use of the control mice that injection pAAV empty plasmids obtain.

(2) DT/PBS is injected：In every kind of mouse single intraperitoneal injection diphtheria that modeling the 7th day (D7) obtains to step (1) Toxin (DT) is to remove Foxp3 positive cells (Treg cells).DT is dissolved and diluted with PBS, and injection dosage is 0.5 μ g/ Only, volume injected is 100 μ l/.The mouse injected by the use of the PBS of equal volume is used as negative control.

(3) Serologic detection：Eye socket is carried out after modeling and takes blood, ELISA detects surface antigen in serum (100 times of dilutions) Removing situation and surface antibody (5 times dilution) production.

(4) cytology detects：Put to death mouse within three weeks after modeling, take the spleen of mouse, obtain spleen mixed lymphocytes. Flow cytometer showed wherein Tfh cells and Germinal center B cell ratio.

Foxp3-DTR mouse are fixed point transgenic mices, give the mouse peritoneal injection diphtheria toxin (DT), can be selective Remove Foxp3 in ground⁺Treg cells, removed so as to realize in the Treg cell-specific bodies of specific phase.And the Foxp3-DTR Mouse is C57BL/6J subbreed.HBV plasmid modelings are carried out to Foxp3-DTR mouse, after modeling one week, DT/PBS is injected intraperitoneally, Then detection Treg is removed on influence caused by the removing of serum surface antigen and surface antibody.(b) is shown in Fig. 5, Foxp3-DTR C57BL/6J backgrounds are really belonged to, Chronic HBV model mice is developed into after the modeling of HBV plasmids；And when injection DT removings Treg is thin After born of the same parents, mouse successfully produces surface antibody response, quickly removes surface antigen.(d) is shown in same Fig. 5, internal Treg After cell is eliminated, effect differentiation and the centrum germinativum B for having recovered the Tfh cells that HBV is induced in Chronic HBV model mice are thin The differentiation of born of the same parents.These results indicate that Treg cells are that humoral immunity imbalance is viral so as to hinder to remove in mediation chronic HBV infection Important cells type, the novel targets of Treg cell clearances or function inhibitio as chronic HBV infection immunization therapy.

According to above-mentioned steps (1) and the method for (2), 14 days (D-14) first injects surface antigen to mouse before plasmid modeling Vaccine, plasmid modeling and DT/PBS injections are then carried out, as a result as shown in (f) in Fig. 5, is as a result shown, Chronic HBV model mice To surface antigen immune tolerance, it is impossible to surface antibody response is produced, and by after Treg cell clearances, then recover to surface antigen Immune responsibility (wherein, Chronic HBV model mice+surface antigen vaccine and the curve co-insides of Chronic HBV model mice, In institute, 0) detection time point value is.This result prompts Treg cell clearances or function inhibitio joint HBV vaccines, can conduct The new strategy of chronic HBV infection immunization therapy.

Wherein, the method for injecting surface antigen vaccine is as follows：Blood source is purified HBsAg (Beijing section jump in pattern biology skill Art Co., Ltd, article No. B204N001) diluted with LPS (Sigma, article No. L4391) with the PBS being sterile filtered, obtain mixed liquor Body, the concentration that blood source purifying HBsAg concentration is 140 μ g/ml, LPS in mixing liquid is 20 μ g/ml；By mixing liquid with Imject^TMAlum Adjuvant (Thermo, article No. 77161) are mixed in equal volume, obtain surface antigen vaccine.By surface antigen Vaccine intraperitoneal injection of mice, injection dosage are 100 μ l/.

Embodiment 7, CTLA4 blocking antibodies promote Chronic HBV model mice surface to resist by suppressing Treg cell functions Body response and virus sweep

1.Treg cells in vitro suppresses T cell proliferation experiment

(1) CD3 antibody wrapper sheet：Flat 96 orifice plate, 4 DEG C of wrapper sheets are stayed overnight, or 37 DEG C of wrapper sheets 5 hours.The anti-of purifying Mouse CD3 (BD Bioscience companies) wrapper sheet concentration is 1 μ g/ml, PBS dilutions, and 100 μ l are coated with per hole.

(2) Treg cells obtain：Utilize flow sorter, the Treg cells (CD4 of sorting WT C57BL/6J mouse spleens⁺ CD25⁺T cells)。

(3) responder T cells obtain：Utilize flow sorter, sorting CD45.1 mouse spleens CD4⁺CD25^-Initial T Cell.

(4) Responder T cells prepare：The obtained CD4 from CD45.1 mouse spleens will be sorted⁺CD25^-Initial T 4 DEG C of 500g centrifugation 8min of cell, sedimentation cell, and washed twice with PBS.The responder T cells of acquisition are counted, and 2~3 × 10 are diluted to the PBS containing 0.1%BSA⁶/ ml cell concentration.Place on ice, it is standby.

(5) CFSE prepares：CFSE to 8~10 μM is diluted with the PBS containing 0.1%BSA, prevents that on ice, lucifuge is standby.Pay attention to CFSE is dissolved with DMSO, is configured to the storing liquid of high concentration, and -20 DEG C are kept in dark place.It is finished every time after the CFSE of pipe packing Discard, it is impossible to 2 uses.

(6) the CFSE marks of Responder T cells：By isometric CFSE solution and responder T cell suspensions Mixed, pay attention to sufficiently being mixed, but not exert oneself pressure-vaccum or vortex.After mixing, avoid light place, room temperature mark 10 minutes.

(7) CFSE marks terminate：CFSE marks are terminated with the FBS serum of precooling on ice.With three times volume, (such as CFSE is marked Volume is 6ml, then plus 18ml FBS terminate) FBS.Avoid light place 2min on ice.Subsequent 4 DEG C of 500g are centrifuged 8 minutes, are then used Complete medium is washed one time, and cell count after complete medium is resuspended, lucifuge is standby on ice afterwards.

(8) plating cells：3 × 10 obtained will be sorted⁵Individual responder T cells and 3 × 10⁵Individual Treg cells.Simultaneously Add 100 μ g/ml IgG (Biogen companies) or CTLA blocking antibodies.

(9) CD28 antibody stimulates：1 μ g/ml of solubility addition purifying anti-mouse CD28 antibody (BD Bioscience companies), stimulate 3 days.

(10) CFSE detects cell propagation：After 3 days, cell is received, PBS is washed one time, and FACS Buffer are resuspended, flow cytometer detection The CD45.1 of propagation negative CFSE⁺Responder T cell ratios.

Blocking experiment in 2.CTLA4 bodies

(1) plasmid models：Choose 6~8 week old, C57BL/6J hero mouse of the body weight in 20g or so.Plasmid is carried out to it to build Mould, every μ g pAAV/HBV1.2 plasmid of mouse tail vein high-pressure injection 10.

(2) blocked in CTLA4 bodies：With PBS dilution anti-mouse CTLA (clone 9D9, BioXcell), modeling The the 6th, 9,12 day afterwards, anti-mouse CTLA three times or control IgG intraperitoneal injections are carried out respectively, per injection dosage is 500 μ Only, volume injected is 100 μ l/ to g/.

(3) Serologic detection：Eye socket is carried out after modeling and takes blood, ELISA detects hepatitis B virus surface antigen, e in serum Antigen, surface antibody, e antibody and HBV DNA change curves.

(4) cytology detects：Put to death mouse within five weeks after modeling, take the spleen of mouse, obtain spleen mixed lymphocytes. Flow cytometer showed wherein Tfh cells and Germinal center B cell ratio.

CTLA4 is very crucial for Treg cell functions, after Treg cell-specifics missing CTLA4, its internal and body Outer suppression sexual function is damaged.Inventor realizes the closing of CTLA4 signals by anti-mouse CTLA4 blocking antibodies.It is first First, the suppression sexual function of Treg cells can be blocked using the cell inhibiting experimental analysis CTLA4 antibody closings of external Treg.Figure (a) is clearly displayed in 6, and after CTLA4 is closed, Treg cells in vitro suppresses initial CD4⁺T cell multiplication capacity is remarkably decreased. Then, anti-mouse CTLA4 injections are carried out to Chronic HBV model mice, (c, d, e) is shown in Fig. 6, anit-mouse CTLA4 injections serve the action effect consistent with Treg cell clearances：The Tfh for significantly promoting Chronic HBV model mice is thin Born of the same parents and the response of GC B cells, antibody produce, so as to promote virus sweep.Illustrate that CTLA4 blocking antibodies can be removed, Ke Yiyong To treat chronic HBV infection.

Claims (10)

1. following a1), a2), a3) or the a4) application in treatment and/or prevention and/or diagnosis HBV infection product is prepared；

A1 the material of regulatory T cells activity) is suppressed；

A2 the material of regulatory T cells) is removed；

A3 the material and HBV vaccines of regulatory T cells activity) are suppressed；

A4 the material and HBV vaccines of regulatory T cells) are removed.

2. folliculus helper T lymphocyte and/or Germinal center B cell are preparing treatment and/or prevention and/or diagnosis HBV infection production Application in product.

3. the material of the differentiation of folliculus helper T lymphocyte and/or Germinal center B cell differentiation is promoted to prepare treatment and/or prevention And/or the application in diagnosis HBV infection product.

4. following A 1), A2), A3) or A4) in following B1)-B9) in it is any in application：

A1) suppress regulatory T cells activity or remove the material of regulatory T cells；

A2) suppress regulatory T cells activity or remove the material and HBV vaccines of regulatory T cells；

A3) folliculus helper T lymphocyte and/or Germinal center B cell；

A4 the material of the differentiation of folliculus helper T lymphocyte and/or Germinal center B cell differentiation) is promoted；

B1) the application in treating and/or preventing and/or diagnose HBV infection；

B2) application in removing HBV products is being prepared；

B3) the application in HBV is removed；

B4) application in removing HBsAg product is being prepared；

B5) the application in HBsAg is removed；

B6) application in promoting HBV antibody-secreting products is being prepared；

B7) the application in HBV antibody-secretings are promoted；

B8) application in promoting HBsAg antibody-secreting product is being prepared；

B9) the application in HBsAg antibody-secreting is promoted.

5. treatment and/or the method for prevention HBV infection, including：Following C1 are applied to be treated and/or object of prevention) or C2) or C3) treated and/or prevented：

C1) suppress regulatory T cells activity or remove the material of regulatory T cells；

C2 the material for) suppressing regulatory T cells activity or removing regulatory T cells is combined to obtain with HBV vaccines Material；

C3 the material of the differentiation of folliculus helper T lymphocyte and/or Germinal center B cell differentiation) is promoted.

6. according to any described application in claim 1-5 or the method described in claim 7, it is characterised in that：The filter Bubble helper T lymphocyte is HBsAg specificity folliculus helper T lymphocyte；The material for suppressing regulatory T cells activity To suppress the material or diphtheria toxin of CTLA4 activity.

7. following H1) or H2)：

H1) treat and/or prevent HBV infection medicine and/or vaccine, its active component is H11) or H12) or H13)：

H11) any the promotion folliculus helper T lymphocyte differentiation and/or Germinal center B cell differentiation in claim 1-6 Material；

H12) any material for suppressing regulatory T cells activity or removing regulatory T cells in claim 1-6；

H13) by H12) material that is obtained together with HBV vaccine combinations；

H2 it is H21) to be used to treating and/or preventing the material X, the material X of HBV infection) or H22) or H23)：

H21) any the promotion folliculus helper T lymphocyte differentiation and/or Germinal center B cell differentiation in claim 1-6 Material；

H22) any material for suppressing regulatory T cells activity or removing regulatory T cells in claim 1-6；

H23) by H23) material that is obtained together with HBV vaccine combinations.

8. following D1) or method D2)：

D1) the construction method of acute HBV mouse model, including following E1 are applied to C57BL/6N mouse), E2) or E3), obtain Acute HBV mouse model；

E1) the carrier containing HBV genomic DNA or its DNA fragmentation；

E2) HBV genomic DNA or its DNA fragmentation；

E3)HBV；

D2) the construction method of Chronic HBV mouse model, including the E1 is applied to C57BL/6J mouse), the E2) it is or described E3), Chronic HBV mouse model is obtained.

9. the complete sets of products of HBV animal models is built, described in C57BL/6N mouse or C57BL/6J mouse and claim 8 E1), the E2) or the E3) composition.

10. application of the complete sets of products described in claim 9 in HBV animal models are prepared.