CN115282278A

CN115282278A - Application of cholesterol regulator as antigen presentation promoter in treatment of hepatitis B

Info

Publication number: CN115282278A
Application number: CN202210820342.2A
Authority: CN
Inventors: 张建; 赵华俊; 邱学良; 韩秋菊; 胡一飞; 杨瑷璐; 王宇灿
Original assignee: Shandong Lishan Cell Medicine Development Co ltd; Shandong University
Current assignee: Shandong Lishan Cell Medicine Development Co ltd; Shandong University
Priority date: 2022-07-13
Filing date: 2022-07-13
Publication date: 2022-11-04

Abstract

The invention relates to the use of cholesterol modulators as antigen presentation promoters in the treatment of hepatitis B. The present study shows that the combination of cholesterol modulators and rHBV vaccines, through Prime-boost (Prime-boost) vaccination strategies, can be used for the treatment of HBV, achieving long-term effective virology control. The cholesterol regulator and the rHBV vaccine are combined and applied to the clinical HBV treatment process, so that the formation obstacle of cholesterol and lipid rafts on dendritic cells caused by chronic HBV infection can be recovered, the antigen presentation function of the dendritic cells and the cellular response and the humoral response of an organism are enhanced, and the complete elimination of HBV is realized. More importantly, the combination of the cholesterol regulator and the rHBV vaccine can induce the organism to form long-term immunological memory, protect the organism from re-infection of HBV and provide a new effective strategy for treating clinical chronic HBV.

Description

Application of cholesterol regulator as antigen presentation promoter in treatment of hepatitis B

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of cholesterol regulators such as simvastatin and lovastatin serving as antigen presentation promoters, and a novel HBV vaccine and a HBV treatment drug are also designed.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Chronic Hepatitis B (CHB) infection is a serious public health problem worldwide. At present, the drugs for clinically treating the CHB comprise two categories of nucleoside/nucleotide analogues and interferon alpha, but the two categories of drugs have certain limitations for treating the CHB and cannot realize long-term virology control. Therefore, there is an urgent need to develop new strategies to eliminate HBV and achieve durable immune control. The development of CHB infection is the result of the interaction between viral replication and the host immune response. Both viral and host factors contribute to the persistence of HBV. Therefore, understanding the mechanism by which HBV persists plays an important role in designing therapeutic strategies to eliminate HBV.

CHB is difficult to cure, mainly because an immunosuppressive microenvironment is formed in the liver of CHB patients, with a high proportion of regulatory T cells (tregs), myeloid-derived suppressor cells (MDSCs), and dysfunctional Antigen Presenting Cells (APCs). Dendritic cells serve as professional antigen presenting cells and are the bridge between innate and adaptive immune responses. Previous study reports demonstrated that dendritic cells from CHB patients have impaired antigen presentation and migratory capacity compared to healthy volunteers. In addition, CHB infection can induce dendritic cell dysfunction, reduce the expression of its co-stimulatory molecules, promote the depletion of HBV-specific T cells, and is mainly manifested by a significant increase in the expression of co-inhibitory receptors such as PD-1, CTLA-4, CD244 (2B 4), tim-3, etc., reduced cytotoxicity, altered transcriptional profiles, and metabolic disorders, ultimately leading to persistent infection and progression of liver disease in CHB patients. However, the mechanism by which CHB infection causes dendritic cell dysfunction has not been fully elucidated.

Cholesterol is an indispensable lipid molecule that plays an important role in a variety of biological processes, including the formation of lipid rafts, major Histocompatibility Complex (MHC) molecules, T Cell Receptors (TCR), B cell receptors, and Toll-like receptors. Cholesterol levels in immune cells reflect a dynamic balance between biosynthesis, uptake, efflux and esterification. Key regulators of cholesterol biosynthesis include sterol regulatory element binding protein 2 (SREBP 2) and two rate-limiting enzymes [ 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE) ]]. The Low Density Lipoprotein Receptor (LDLR) is the dominant factor responsible for the uptake of cholesterol in the circulation by peripheral immune cells. LDLR captures circulating LDL by endocytosis, after which LDL-carried cholesterol esters gradually progress to free cholesterol through the intracellular lysosomal system. Cholesterol efflux is mediated by ATP-binding cassette (ABC) transporters, including ABC subfamily a member 1 (ABCA 1) and ABC subfamily G member 1/5/8, and helps to treat or store excess cholesterol in cells as cholesterol esters. Esterification of cholesterol can be carried out by acyl-coa: cholesterol acyltransferases (ACATs, including ACAT1 and ACAT 2) convert free cholesterol into neutral cholesterol esters for storage in lipid droplets or for use as lipoprotein components to ensure cholesterol homeostasis. Previous studies have shown that inhibition of the cholesterol esterification pathway can drive esterificationThe cholesterol in the plasma membrane is translocated to the plasma membrane to reduce the accumulation of neutral lipid droplets, while the free cholesterol in the plasma membrane is a key structural component in the formation of lipid rafts. Lipid rafts serve as cholesterol-rich microdomains, playing a crucial role in signal transduction and in the regulation of immune cell phenotype and function. For example, CD8 ⁺ Free cholesterol enriched in the T cell plasma membrane promotes TCR aggregation and formation of immune synapses, while enhancing T cell proliferation and functional effects. At the same time, cholesterol accumulation promotes the formation of lipid rafts in Natural Killer (NK) cells and the activation of immune signaling, which in turn promotes the killing of NK cells on hepatoma cells. In macrophages, cholesterol accumulation induces activation of protein 3 (NLRP 3) inflammasome containing NOD, LRR and pyrin domains and MyD 88-mediated signaling pathways, exacerbating atherosclerotic progression. In addition, cholesterol-rich lipid rafts promote the accumulation of antigenic peptide-MHC class II complexes on APCs, promoting the activation of T cells; the hypercholesterolemia-induced increase in cholesterol on dendritic cells enhances antigen presentation and T cell activation, and promotes B cell proliferation, thereby increasing the risk of autoimmune disease. These findings confirm the important role of cholesterol in regulating immune cell activation; however, it is currently unclear whether CHB infection affects the cholesterol levels of DCs and subsequent dysfunction of DC-mediated anti-HBV immune responses.

Disclosure of Invention

The present study investigated the relationship between cholesterol levels and the development of CHB infection. The results indicate that CHB infection reduces free cholesterol levels and lipid raft formation on DCs, contributing to the persistent presence of HBV. Strategies to increase cholesterol accumulation on DCs by using drugs may improve the efficacy of clinical CHB treatment.

The invention applies the combination of rHBV vaccine and cholesterol regulator such as simvastatin or lovastatin to CHB infection model, the result shows that the cholesterol regulator can increase the accumulation of cholesterol on DC in chronic HBV infection, enhance the antigen presentation function, increase the proportion of CD8+ T cells with antigen specificity, reverse the exhaustion state, safely and effectively eliminate HBV, induce organism to form long-term immunological memory, protect organism to prevent HBV reinfection. Thus, increasing cholesterol accumulation on DCs may enhance the efficacy of HBV therapeutic vaccines.

Based on the research results, the invention provides the following technical scheme:

in a first aspect of the invention, there is provided the use of a cholesterol modulating agent as an antigen presentation enhancer.

In the research results of the invention, chronic HBV infection can cause cholesterol metabolism disorder in the body and cause the impairment of lipid raft formation; in particular, it is shown that the expression of free cholesterol (Filipin), lipid raft (CTxB) and cholesterol transporter LDLR on antigen presenting cells is reduced. The invention intervenes the process by adopting cholesterol regulating medicaments, and finds that cholesterol regulating agents such as simvastatin (Sim) or lovastatin (Lov) can increase the level of cholesterol and lipid rafts on dendritic cells (BMDC) of bone marrow sources and promote the synthesis and absorption of cholesterol; meanwhile, the increase of the cholesterol level can promote the intake of the BMDC cells to foreign antigens, increase the expression of mature molecules MHC II and CD86 and enhance the antigen presentation function.

Preferably, the cholesterol regulating agent is a cholesterol absorption enhancer or A Cholesterol Acyltransferase (ACAT) inhibitor;

further, selected from simvastatin, lovastatin, rosuvastatin or acyl coenzyme A; in a validated embodiment of the invention, the cholesterol modulating agent is simvastatin or lovastatin.

Therefore, the invention preferably provides the application of cholesterol regulator such as simvastatin or lovastatin as antigen presentation promoter.

The antigen presentation promoter mainly comprises an active preparation for improving the antigen uptake, processing and presentation functions of antigen presenting cells, and the specific application mode of the antigen presentation promoter comprises but is not limited to any one of the following:

(1) The simvastatin or lovastatin is applied to the preparation of the immunoregulation medicine;

(2) The application of simvastatin or lovastatin in the preparation of medicines for treating hepatitis B virus;

(3) Simvastatin or lovastatin is used as an HBV virus vaccine adjuvant;

(4) Simvastatin or lovastatin is used for preparing the antigen presenting cell culture reagent.

In the application of the aspect (1), based on the promotion effect of simvastatin or lovastatin on antigen presenting cells, the active ingredients are expected to be applied to the enhancement of the immune response of the body, such as the improvement of autoimmune diseases.

In the applications of the aspects (2) and (3), simvastatin or lovastatin and hepatitis b virus coat protein antigen (HBsAg) are co-injected, and the results show that the active ingredients can rapidly reduce the level of HBsAg in peripheral blood and liver, enhance the response capability of the body to antigen, eliminate HBV virus in the body, enhance the therapeutic effect of vaccine, and the inhibitory effect can be maintained for a long time.

In the application of the above aspect (4), the present invention also proves that the cleaning effect of simvastatin or lovastatin on HBV depends on the participation of dendritic cells, therefore, the antigen presenting cell is preferably dendritic cells; simvastatin or lovastatin can enhance the antigen presenting activity of dendritic cells, and can also be applied to the in vitro culture of the dendritic cells.

In a second aspect of the present invention, there is provided an HBV vaccine comprising an active dose of a cholesterol modulating agent.

Preferably, the cholesterol regulator is simvastatin or lovastatin; further, the active dosage of simvastatin or lovastatin can be adjusted according to the dosage of antigen in the vaccine, the subject, the purpose of administration and the like, and the active dosage can be obtained by the conventional method in the field.

In one embodiment provided by the invention, the HBV vaccine comprises HBsAg and simvastatin or lovastatin, and the dose ratio of HBsAg to simvastatin or HBsAg to lovastatin is 2 to 90-110; further, the HBV vaccine may further comprise other immunological adjuvants, such as aluminum hydroxide adjuvant, aluminum phosphate, calcium phosphate, paraffin oil, lanolin, surfactant, calcium alginate, polynucleotide, muramyl peptide, etc.

In a specific example, the HBV vaccine comprises simvastatin or lovastatin 100 μ g, HBsAg 2 μ g, and aluminum hydroxide 100 μ g per dose.

Preferably, the number of immunizations of the HBV vaccine is 2 to 3.

In a third aspect of the present invention, there is provided an HBV therapeutic drug, wherein the HBV vaccine of the second aspect is included in the therapeutic drug.

The HBV vaccine can be co-administered with other immunomodulators for clinically treating chronic HBV, such as immune check point treatment such as PD-1 and TIGIT, cell treatment such as TCR-T, CAR-T, and cytokine treatment such as IL-2, IL-12 and IL-15; therefore, the HBV treatment medicine also comprises other immune regulation medicines, including but not limited to PD-1, TIGIT monoclonal antibody, or TCR-T, CAR-T cell, or cytokines such as IL-2, IL-12, IL-15, etc.

The beneficial effects of one or more of the above technical schemes are:

(1) Clinical CHB sample experiments prove that the expression of free cholesterol on DC can be reduced by chronic HBV infection, so that the formation of lipid rafts is influenced, and the cholesterol level and the expression of the lipid rafts show obvious positive correlation; further research shows that chronic HBV infection affects the relevant pathways of cholesterol metabolism such as synthesis, esterification and the like. HBV infection mice experiments also confirmed that chronic HBV infection decreased DC (including CD11 b) ⁺ DC and CD103 ⁺ DC) and the expression of free cholesterol and lipid rafts, and the level of the cholesterol and the expression of the lipid rafts show obvious positive correlation, thereby defining the relevant mechanism of HBV infection and cholesterol metabolism disorder.

And cholesterol regulating agent such as simvastatin or lovastatin can up-regulate cholesterol level on bone marrow-derived dendritic cells (BMDC) and promote formation of lipid rafts; meanwhile, simvastatin or lovastatin promotes the uptake of exogenous cholesterol; further studies found that cholesterol regulating agents such as simvastatin or lovastatin promoted the expression of cholesterol synthesis-related genes on BMDCs, inhibiting esterification. In addition, in vitro experiments also demonstrated that the ability of BMDCs to phagocytose antigens, the expression of the co-stimulatory molecule CD86, and MHC-II molecules, was increased upon stimulation with cholesterol modulators such as simvastatin or lovastatin.

(2) The HBV treatment medicine provided by the invention adopts the combination of a clinically used preventive rHBV vaccine and a cholesterol regulator such as simvastatin or lovastatin, and is safe and nontoxic. The obtained preparation is safe, economical and convenient to prepare, and is beneficial to the development of immunization.

Proved by verification, the HBV therapeutic drug (combination of rHBV vaccine and cholesterol regulator such as simvastatin or lovastatin) can recover the levels of cholesterol and lipid raft on DC of HBV infected mice, enhance the antigen presenting function of the mice, and increase the antigen-specific CD8 ⁺ The proportion of T cells, reversing their depleted state. The HBV therapeutic medicine can be used for immunizing a mouse, can reduce the levels of HBsAg and HBV DNA in serum and HBcAg, HBV DNA and RNA in liver, effectively eliminate HBV in the body of the mouse infected by HBV, induce the body to generate long-term immunological memory, and protect the body from re-infection of HBV.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate exemplary embodiments of the invention and not to limit the invention;

FIG. 1: the construction of HBV-carrier mouse model and the treatment strategy of novel HBV treatment medicine;

wherein (a) - (f) are blank control group (a), negative control group (b), experimental group Sim-alone (c), experimental group Lov-alone (d), experimental group rHBV + Sim (e), and experimental group rHBV + Lov (f) in sequence as described in example 1;

FIG. 2: expression of cholesterol and lipid rafts on DCs from healthy volunteers and clinical CHB patients;

wherein, fig. 2A shows the expression of free cholesterol, lipid rafts and the cholesterol transporter LDLR on the peripheral blood-derived DCs of healthy volunteers and clinical CHB patients;

figure 2B is a graph of the correlation of cholesterol, LDLR levels with lipid rafts;

FIG. 2C shows the expression of the cholesterol synthesis related genes HMGCR, HMGCS1, SQLE, esterification related protein LCAT1 on mononuclear cells;

FIG. 3: chronic HBV infection reduces the expression of cholesterol and lipid rafts on HBV-carrier mouse derived DCs;

wherein, FIG. 3A is free cholesterol expression on DCs in liver, spleen and draining lymph nodes of wild mice and CHB infected mice;

FIG. 3B is the expression of the DC upstream cholesterol transporter LDLR in the liver, spleen and draining lymph nodes of wild mice and CHB infected mice;

FIG. 3C is the expression of lipid rafts on DCs in the liver, spleen and draining lymph nodes of wild mice and CHB infected mice;

FIG. 3D is MHC-II in spleen of HBV-carrier mouse ^hi CD11c ⁺ The result of expression of lipid rafts on antigen presenting cells of (a);

FIG. 3E is MHC-I in spleen of HBV-carrier mouse ^hi CD11c ⁺ (iv) expression of lipid rafts on antigen presenting cells _;

FIG. 3F is MHC-II in spleen of HBV-carrier mouse ^hi CD11c ⁺ And MHC-I ^hi CD11c ⁺ The association of cholesterol levels on antigen presenting cells of (a) with lipid rafts;

FIG. 3G shows co-localization of lipid rafts on DCs with expression of free cholesterol;

FIG. 4: the promotion effect of the cholesterol regulator on cholesterol and lipid rafts on bone marrow-derived dendritic cells (BMDCs);

wherein, FIG. 4A is the regulation of free cholesterol on bone marrow derived dendritic cells (BMDCs) by simvastatin or lovastatin;

FIG. 4B shows the regulation of lipid rafts on bone marrow derived dendritic cells (BMDCs) by simvastatin or lovastatin;

FIG. 4C is a positive correlation of lipid rafts on BMDC with free cholesterol expression;

FIG. 4D shows the effect of simvastatin or lovastatin on genes involved in DC maturation and activation;

FIGS. 4E-4G show the effect of simvastatin or lovastatin on genes for cholesterol synthesis and efflux;

FIG. 4H shows the effect of simvastatin or lovastatin on the ability to modulate the presentation of BMDC antigens.

FIG. 5: the cholesterol regulator restores the expression results of cholesterol and lipid rafts on DC from CHB infected mice;

wherein, FIG. 5A is the regulation of the levels of free cholesterol in DC of CHB-infected mice by simvastatin or lovastatin;

FIG. 5B shows the results of the regulation of the expression of LDLR, a cholesterol uptake-related molecule, by simvastatin or lovastatin;

FIG. 5C is a graph of the regulation of lipid raft levels on DCs by simvastatin or lovastatin;

FIGS. 5D-5E show the effect of simvastatin or lovastatin on the regulation of PDL1, MHCI expression in DC cells;

FIG. 6: enhancement of the function of HBV-carrier mouse antigen-specific T cells by increasing cholesterol accumulation on DCs;

wherein FIG. 6A shows the HBV-specific CD11a in the liver of simvastatin or lovastatin treated mice ^hi CD8α ^lo The proportion of cells;

FIG. 6B is a CD11a specific to HBV in spleen of simvastatin or lovastatin treated mice ^hi CD8α ^lo Expression of the terminally differentiated molecule KLRG1 on the cell;

FIG. 6C shows CD11a ^hi CD8α ^lo Expression of the immunosuppressive molecules LAG-3, tim-3, PD-1 on the cells;

FIG. 6D shows a CD11a ^hi CD8α ^lo Expression of TNF-alpha, IFN-gamma, IL-2 on cells;

FIG. 7: clearance of HBV in HBV-carrier mice by cholesterol modulators;

wherein, FIG. 7A shows the inhibitory effect of simvastatin or lovastatin on HBsAg in mouse serum;

FIG. 7B is a graph of the regulation of HBV DNA levels in peripheral serum by simvastatin or lovastatin;

FIG. 7C shows the result of immunohistochemical staining of HBcAg + in liver;

FIG. 7D shows the inhibitory effect of simvastatin or lovastatin on HBV-3.5kb-RNA, HBV-total-RNA, HBV DNA and HBV cccDNA expression in the liver;

FIG. 7E shows the result of serological conversion of anti-HBs;

FIG. 7F is the correlation of the anti-HBV effect of simvastatin and lovastatin with DC;

FIG. 8: the cholesterol regulator has the induction effect on the long-term immunologic memory of the organism;

FIG. 8A shows the result of HBsAg expression in serum of mice after being challenged with HBV;

FIG. 8B shows the expression of anti-HBs in an immunotherapeutic mouse after challenge with HBV;

FIG. 8C shows the expression results of HBV-3.5kb-RNA, HBV-total-RNA, HBV DNA and HBV cccDNA in the liver of an immunotherapeutic mouse challenged with HBV.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

The preparation method of the HBV therapeutic drug in the following examples:

raw materials and sources:

the rHBV vaccine is a recombinant hepatitis B vaccine (Hansenula polymorpha) product produced by da Lian Hanxin, and the specification is 1ml:20 μ g.

Simvastatin, lovastatin, purchased from invitogen.

The content of each main component in each dose of the HBV treatment medicine is as follows, the rHBV vaccine contains 2 mug of HBsAg, and simvastatin or lovastatin contains 100 mug of each.

In the following examples, the preparation of the HBV therapeutic agents is as follows:

1ml of rHBV vaccine is taken out and put in a sterile Ep tube, 1mg of simvastatin or lovastatin and PBS or normal saline are added respectively, and the mixture is mixed evenly to obtain 2ml of vaccine solution. Each milliliter of the prepared HBV treatment medicine contains 10 mug of hepatitis B surface antigen and 500 mug of simvastatin or lovastatin.

1. Construction of HBV-carrier mouse model and treatment strategy of novel HBV treatment drug

1.1HBV-Carrier mouse model construction

Construction of pAAV/HBV1.2 mouse model: taking normal adult 5-6w C57BL/6J mice, dissolving pAAV/HBV1.2 plasmid in sterile physiological saline, and injecting 8 mu g of plasmid into each mouse within 5-8s by tail vein high pressure injection, wherein the injection volume is 10% of the mass of the mouse. After 6w, mouse serum was collected. Mice with serum HBsAg concentrations greater than 500ng/ml were default to HBV-carrier mice.

Constructing a rAAV-HBV 1.3 mouse model: taking a normal adult C57BL/6J mouse with 5-6w, dissolving the rAAV-HBV 1.3 virus in sterile physiological saline, and injecting the virus into the mouse through tail vein. After 6w, mouse serum was collected. Mice with serum HBsAg concentrations greater than 500ng/ml were default to HBV-carrier mice. 1.2 immunization strategy for "Prime-boost" (Prime-boost) of novel HBV therapeutics

The experimental group immunized rHBV vaccine (containing HBsAg 2 μ g) + simvastatin (100 μ g) (abbreviated as "rHBV + Sim"), rHBV vaccine (containing HBsAg 2 μ g) + lovastatin (100 μ g) (abbreviated as "rHBV + Lov"), negative control immunized rHBV vaccine (containing HBsAg 2 μ g) (abbreviated as "rHBV-alone"), simvastatin alone (100 μ g) (abbreviated as "Sim-alone"), lovastatin alone (100 μ g) (abbreviated as "Lov-alone"), and blank control injected with PBS of the same volume and immunized 3 times (Prime-boost) at one week intervals, as shown in FIG. 1.

2. Reduced expression of cholesterol and lipid rafts in clinical CHB patient-derived DCs

2.1 test methods:

peripheral Blood Mononuclear Cells (PBMCs) of healthy volunteers and clinical CHB patients were isolated, the expression of free cholesterol (Filipin), lipid rafts (CTxB) and the cholesterol transporter LDLR on Dendritic Cells (DC) was detected by flow cytometry, and the expression of genes associated with cholesterol metabolism on Peripheral Blood Mononuclear Cells (PBMC) was detected by qPCR to analyze the effect of chronic HBV infection on cholesterol metabolism on DC.

2.2 test results:

as shown in fig. 2A, the expression of free cholesterol (Filipin), lipid rafts (CTxB), and the cholesterol transporter LDLR on peripheral blood-derived DCs was reduced in clinical CHB patients compared to healthy volunteers (fig. 2A); at the same time, cholesterol and LDLR levels showed a clear positive correlation with lipid rafts (fig. 2B); further studies found that CHB infection resulted in decreased expression of the cholesterol synthesis associated genes HMGCR, HMGCS1, SQLE on mononuclear cells (FIG. 2C) and increased expression of the esterification associated protein LCAT1 (FIG. 2D). Taken together, it can be seen that chronic HBV infection can cause cholesterol metabolism disorders on DCs, resulting in impaired lipid raft formation.

3. Chronic HBV infection reduces the expression of cholesterol and lipid rafts in HBV-carrier mouse-derived DCs

3.1 test methods:

liver, spleen and lymph node mononuclear cells of WT wild mice and CHB infected mice, peripheral Blood Mononuclear Cells (PBMC) of clinical CHB patients were isolated, the expression of free cholesterol (Filipin), lipid rafts (CTxB) and the cholesterol transporter LDLR on Dendritic Cells (DC) was detected by flow cytometry, and the co-localization of cholesterol and lipid rafts was analyzed by confocal microscopy to analyze the effect of chronic HBV infection on cholesterol metabolism on DC.

3.2 test results:

free cholesterol on DCs in liver, spleen and draining lymph nodes of CHB-infected mice compared to WT wild mice (Filipin, panel)3A) Decreased expression of cholesterol transporter LDLR (fig. 3B) and lipid raft (CTxB, fig. 3C); at the same time, CHB reduced MHC-II in HBV-carrier mice ^hi CD11c ⁺ And MHC-I ^hi CD11c ⁺ Expression of lipid rafts on antigen presenting cells (FIG. 3D)&E) (ii) a Furthermore, MHC-II ^hi CD11c ⁺ And MHC-I ^hi CD11c ⁺ The cholesterol level on the antigen presenting cells showed a clear positive correlation with lipid rafts (fig. 3F). Further studies found that expression of lipid rafts on DC showed significant co-localization with free cholesterol (fig. 3G). Taken together, it can be seen that chronic HBV infection can cause cholesterol metabolism disorders on DCs, resulting in impaired lipid raft formation.

4. Treatment with cholesterol regulator such as simvastatin or lovastatin can up-regulate cholesterol levels on bone marrow-derived dendritic cells (BMDC), promote formation of lipid rafts, and enhance antigen presentation

4.1 test methods:

in vitro induced bone marrow derived dendritic cells (BMDCs) were treated with cholesterol modulating agents such as simvastatin or lovastatin, free cholesterol (Filipin) and lipid raft (CTxB) expression on BMDCs and cholesterol transporter LDLR expression were examined by flow cytometry, co-localization of cholesterol and lipid rafts was analyzed by confocal microscopy to analyze the effect of chronic HBV infection on cholesterol metabolism on DCs.

4.2 test results:

cholesterol modulators such as simvastatin (Sim) or lovastatin (Lov) increased the levels of free cholesterol (Filipin, fig. 4A) and lipid rafts (CTxB, fig. 4B) on bone marrow derived dendritic cells (BMDC), and the expression of lipid rafts and free cholesterol on BMDC showed a clear positive correlation (fig. 4C). Transcriptome sequencing found that simvastatin (Sim) or lovastatin (Sim) treatment affected mainly genes associated with DC maturation and activation (fig. 4D), and at the same time, affected cholesterol metabolism, promoted cholesterol synthesis and absorption, and inhibited cholesterol efflux (fig. 4E-G). Further research has found that the increase of cholesterol level can promote the intake of foreign antigen by BMDC, increase the expression of mature molecules MHCII and CD86, and enhance the antigen presentation function (FIG. 4H). Taken together, it can be seen that treatment with a cholesterol-regulating agent such as simvastatin or lovastatin can up-regulate cholesterol levels on bone marrow-derived dendritic cells (BMDCs), promote formation of lipid rafts, and enhance their antigen-presenting functions.

5. Treatment with cholesterol regulator such as simvastatin or lovastatin can restore the expression of cholesterol and lipid rafts in DC derived from CHB-infected mice, and promote the antigen presentation function

5.1 test methods:

experimental groups subcutaneous immune rHBV vaccine (containing HBsAg 2 mug) + simvastatin (100 mug) (abbreviated as 'rHBV + Sim'), rHBV vaccine (containing HBsAg 2 mug) + lovastatin (100 mug) (abbreviated as 'rHBV + Lov'), blank control group injected PBS with same volume, immunized 3 times (Prime-boost) at one week interval, separating mouse liver, spleen and lymph node mononuclear cells, detecting the expression of cholesterol on DC (Filipin), lipid raft (CTxB), cholesterol transport protein LDLR and antigen presenting molecules PDL1 and MHCII by flow cytometry.

5.2 test results:

simvastatin (Sim) or lovastatin (Lov) restored levels of free cholesterol in DC in CHB-infected mice (fig. 5A), promoting expression of the molecule LDLR associated with cholesterol uptake (fig. 5B). Furthermore, simvastatin (Sim) or lovastatin (Lov) increased the level of lipid rafts on DCs (fig. 5C), increased their antigen presenting function, highly expressed MHCI molecules, and lowly expressed immunosuppressive molecules PDL1 (fig. 5D-E). From the above results, it can be seen that treatment with cholesterol regulator such as simvastatin or lovastatin can restore the expression of cholesterol and lipid rafts on DC from CHB-infected mouse, and promote the antigen presentation function.

6. Increasing cholesterol accumulation on DCs enhances antigen-specific CD8 in HBV-carrier mice ⁺ Function of T cells to reverse their depleted state

6.1 test methods:

the experimental group subcutaneous immune rHBV vaccine (containing HBsAg 2 mug) + simvastatin (100 mug) (abbreviated as 'rHBV + Sim'), rHBV vaccine (containing HBsAg 2 mug) + lovastatin (100 mug) (abbreviated as 'rHBV + Lov'),the blank control group was injected with the same volume of PBS, immunized 3 times at one week intervals (Prime-boost), after three immunizations, mouse liver and spleen mononuclear cells were isolated, and HBV-specific CD11a was detected by flow cytometry ^hi CD8α ^lo The difference in the proportion of cells and their absolute numbers; detection of HBV-specific CD11a by flow cytometry ^hi CD8α ^lo Expression of the terminally differentiated molecule KLRG1 on the cell; detection of HBV-specific CD11a by flow cytometry ^hi CD8α ^lo Expression of the immunosuppressive molecules LAG-3, tim-3, PD-1 on the cells; detection of HBV-specific CD11a by flow cytometry ^hi CD8α ^lo The secretion level of cytokines TNF-alpha, IFN-gamma, IL-2 on the cells.

6.2 test results:

the research finds that the antigen is specific to the CD11a ^hi CD8α ^lo The proportion of cells and their absolute number increased significantly in the liver of the mice after treatment (fig. 6A). Meanwhile, simvastatin (Sim) or lovastatin (Lov) can promote HBV-specific CD11a ^hi CD8α ^lo Expression of the terminal differentiation molecule KLRG1 on the cells promoted terminal differentiation (FIG. 6B). Furthermore, simvastatin (Sim) or lovastatin (Lov) treatment can reduce HBV-specific CD11a ^hi CD8α ^lo Expression of immunosuppressive molecules such as LAG-3, tim-3, PD-1 on cells (FIG. 6C), restoration of the expression of cytokines such as TNF-alpha, IFN-gamma, IL-2 (FIG. 6D), reversal of the state of immune depletion due to chronic HBV infection. Taken together, it can be seen that increasing cholesterol accumulation on DCs can enhance the function of antigen-specific CD8+ T cells in HBV-carrier mice, reversing their depleted state.

7. Increasing the accumulation of cholesterol on DCs can safely and effectively eliminate HBV in CHB mice and enhance the effect of HBV therapeutic vaccines

7.1 test methods:

experimental groups immunized s hbv vaccine (containing HBsAg 2 μ g) + simvastatin (100 μ g) (abbreviated as "r hbv + Sim"), r hbv vaccine (containing HBsAg 2 μ g) + lovastatin (100 μ g) (abbreviated as "r hbv + Lov"), blank control groups injected with PBS of the same volume at weekly intervals and immunized 3 times (Prime-boost). Whether increasing cholesterol accumulation on DC could safely and effectively eliminate HBV in CHB mice was evaluated by measuring the levels of peripheral blood HBsAg, HBV DNA, liver HBcAg, liver HBV DNA and RNA, anti-HBs. Conditional deletion of DC using HBV-carrier-CD11c-DTR mice, and observation of whether "rHBV + Sim" and "rHBV + Lov" treatment could effectively eliminate HBV after DC deletion, were performed to evaluate the role of cholesterol recovery of DC in anti-HBV.

As shown in fig. 7A, increased cholesterol accumulation on DC ("rbvb + Sim" and "rbvb + Lov" groups) reduced HBsAg levels in peripheral blood serum of CHB mice compared to untreated groups, with substantially no detectable HBsAg expression; also, the therapeutic effect can be maintained for a long time. Meanwhile, as shown in fig. 7B, the HBV DNA level in peripheral serum of CHB mice could be reduced, and immunohistochemical staining showed that hepatocytes of liver HBcAg + were almost disappeared after treatment (fig. 7C); increasing cholesterol accumulation on DC ("rbhbv + Sim" and "rbhbv + Lov" groups) reduced HBV-3.5kb-RNA, HBV-total-RNA, HBV DNA and HBV cccDNA expression in liver (fig. 7D), partially effecting seroconversion of anti-HBs (fig. 7E). Furthermore, we used HBV-carrier-CD11c-DTR mice to conditionally delete DC, and found that treatment of DC with "rHBV + Sim" and "rHBV + Lov" after deletion of DC impaired its ability to eliminate HBV, indicating that the anti-HBV effects of simvastatin and lovastatin were dependent on the participation of DC (FIG. 7F). From the above results, it can be seen that increasing the accumulation of cholesterol on DCs can safely and effectively eliminate HBV in CHB mice, enhancing the efficacy of HBV therapeutic vaccines.

8. Increasing the accumulation of cholesterol in DC can induce the body to develop long-term immunological memory, and can protect the body from re-infection of HBV

8.1 test methods:

experimental groups immunized s hbv vaccine (containing HBsAg 2 μ g) + simvastatin (100 μ g) (abbreviated as "r hbv + Sim"), r hbv vaccine (containing HBsAg 2 μ g) + lovastatin (100 μ g) (abbreviated as "r hbv + Lov"), blank control groups injected with PBS of the same volume at weekly intervals and immunized 3 times (Prime-boost). After three times of immunization, 8 mu g of pAAV/HBV1.2 plasmid is injected into the tail vein of 53d again to carry out HBV challenge, and HBsAg, anti-HBs, liver HBV DNA and RNA in peripheral blood serum are detected to evaluate whether the vaccine can form long-term immune protection.

8.2 test results:

mice in the group with increased cholesterol accumulation on DC ("hbv + Sim" and "hbv + Lov" groups) showed substantially no detectable HBsAg expression in peripheral blood serum upon secondary challenge infection compared to the untreated group (fig. 8A); more than 75% of mice produced high levels of protective anti-HBs (FIG. 8B), and HBV-3.5kb-RNA, HBV-total-RNA, HBV DNA, and HBV cccDNA in liver were essentially undetectable (FIG. 8C). From the above results, it can be seen that increasing the accumulation of cholesterol on DC can induce the body to generate long-term immunological memory, and can protect the body from re-infection of HBV.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a cholesterol modulating agent as an antigen presentation enhancer.

2. Use of a cholesterol modulator as an antigen presentation enhancer according to claim 1 wherein the cholesterol modulator is a cholesterol absorption enhancer or a cholesterol acyltransferase inhibitor;

further, selected from simvastatin, lovastatin, rosuvastatin or acyl coenzyme A;

further, the cholesterol regulator is simvastatin or lovastatin.

3. The use of the cholesterol regulating agent as an antigen presentation promoter according to claim 1, wherein the specific application mode of the cholesterol regulating agent as an antigen presentation promoter includes but is not limited to any one of the following:

(1) The simvastatin or lovastatin is applied to the preparation of the immunoregulation medicament;

(3) Simvastatin or lovastatin is used as an adjuvant of HBV virus vaccine;

4. The use of a cholesterol modulating agent as an antigen presentation enhancer according to claim 3, wherein in the use of the aspect (1), the immunomodulatory drug is used to enhance an immune response in a body; further, it can be used for improving autoimmune diseases.

5. The use of the cholesterol regulator as an antigen presentation promoter according to claim 3, wherein the use of the cholesterol regulator according to the above (2) or (3) comprises a hepatitis B virus coat protein antigen and one of simvastatin and lovastatin in the therapeutic agent for hepatitis B or the HBV vaccine.

6. The use of a cholesterol regulating agent as an antigen presentation enhancer according to claim 3, wherein in the use according to the aspect (4), the antigen presenting cell is preferably a dendritic cell.

7. An HBV vaccine comprising an active dose of a cholesterol modulating agent.

8. The HBV vaccine of claim 8, wherein the cholesterol modulator is simvastatin or lovastatin;

the dosage ratio of the HBsAg to the simvastatin or the HBsAg to the lovastatin is 2;

further, other immune adjuvants including, but not limited to, aluminum hydroxide adjuvant, aluminum phosphate, calcium phosphate, paraffin oil, lanolin, surfactant, calcium alginate, polynucleotide or muramyl peptide;

specifically, each dose of the HBV vaccine comprises simvastatin or lovastatin 100 mug, HBsAg 2 mug and aluminum hydroxide 100 mug;

preferably, the number of immunizations of the HBV vaccine is 2 to 3.

9. An HBV therapeutic drug, characterized in that, in the therapeutic drug, the HBV vaccine of claim 7 or 8 is included.

10. The HBV therapeutic agent of claim 9 further comprising other immunomodulatory drugs including but not limited to PD-1, TIGIT, or TCR-T, CAR-T cells, or IL-2, IL-12, IL-15 cytokines.