A sting agonist-based lymph node targeting nanoparticle and preparation method and application thereof

Abstract

This invention belongs to the fields of biomedicine and molecular biology, specifically relating to a lymph node-targeting nanoparticle based on the STING agonist, its preparation method, and its applications. This invention utilizes a nano-drug delivery system—cationic polymer micelles pAA-pEPEMA—co-prepared with HBsAg and the STING agonist c-di-GMP to prepare a novel nanovaccine, which possesses the ability to target lymph nodes and increase antigen delivery; furthermore, this vaccine can effectively increase the phagocytic and antigen-presenting capacity of BMDCs. This invention demonstrates that this vaccine can safely and effectively clear HBV, induce the production of anti-HBs, and prevent HBV reinfection; simultaneously, this vaccine enhances DC activation and reverses CD4+ activation. + T, CD8 + By addressing the functional exhaustion of T cells, enhancing their immune response, this approach has significant practical value.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to a lymph node-targeting nanoparticle based on the STING agonist, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Hepatitis B is a global health problem caused by infection with the hepatitis B virus (HBV). HBV attacks the liver, leading to both acute and chronic diseases. It is estimated that 257 million people worldwide currently have chronic HBV and are at high risk of developing liver disease, cirrhosis, and hepatocellular carcinoma. While routine hepatitis B surface antigen (HBsAg) vaccination can induce protective antibodies in most healthy vaccinated individuals and effectively reduce the incidence of new HBV infections, HBsAg fails to induce an effective antibody response in animal models or clinical patients with chronic HBV (CHB) infection. Therefore, an effective treatment strategy to eliminate and eradicate CHB is urgently needed.

[0004] The reason why CHB is difficult to cure lies in the series of immune escape phenomena that occur after HBV invades the human body. These phenomena include abnormal function of innate immune cells such as dendritic cells, macrophages, and NK cells, increased expression of inhibitory cells or molecules such as Tregs and IL-10, and increased expression of CD4 and CD8. + T cells highly express immune checkpoints such as PD-1, LAG-3, and TIM-3, exhibiting a state of functional exhaustion. Currently, first-line drugs for chronic hepatitis B (CHB) are mainly based on third-generation nucleoside analogs (NUCs) and PEG-IFN-α. Although these drugs have some therapeutic effect, clinical trial follow-up results have shown that for CHB patients treated with NUCs, only 10% observed HBsAg clearance after five years of treatment; and for CHB patients treated with IFN-α, only 10%-20% achieved HBV cure, often accompanied by severe side effects. In conclusion, currently used drugs for CHB cannot completely cure CHB, and long-term use may lead to drug resistance. Therefore, overcoming these limitations and developing novel drugs for CHB is crucial.

[0005] Therapeutic vaccines are currently a hot topic in research on the treatment of chronic hepatitis B (CHB). Unlike preventative vaccines, therapeutic vaccines place greater emphasis on activating the CD8 receptors in the patient's body. +T-cell immune responses induce antiviral immune responses, thereby clearing HBV from the patient's body. Generally, therapeutic vaccines can be constructed by combining antigens with adjuvants. Adjuvants are non-specific immune enhancers that, when injected into the body along with or beforehand, can enhance the immunogenicity of the antigen, strengthen the immune response induced by the antigen, and upregulate the body's immune response level. Current research has identified several small molecules that can be used as adjuvants to construct novel human vaccines, including agonists such as TLR-like receptors (TLRs) and NOD-like receptors. Studies have shown that these small molecules, once ingested with antigens, can activate the function of immune cells such as dendritic cells (DCs), T cells, and B cells to some extent through the same or different pathways, enhancing the body's immune response and improving immunosuppression in patients.

[0006] The cCAS-STING pathway, a crucial component of the innate immune system, is widely present in dendritic cells (DCs) and regulates their activity. First, cGAS recognizes and binds to pathogen DNA, synthesizing GMP-AMP (cGAMP), which then binds to STING on the endoplasmic reticulum (ER) to form a polymer. The polymerized STING translocates from the ER to the Golgi apparatus. In the Golgi apparatus, STING recruits TBK1 and IRF3, activating TBK1, which in turn phosphorylates and activates IRF3. IRF3 can then translocate to the nucleus and induce the production of ISG and IFN-I. Furthermore, STING can activate signaling pathways such as NF-κB and STAT6, co-activating the body's antiviral function with IFN. Therefore, STING agonists have been used as adjuvants in the development of novel therapeutic vaccines. However, due to their small molecule nature, STING agonists rapidly distribute throughout the body after entering the system, which not only hinders their absorption by lymph node DCs but may also cause systemic toxicity. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a lymph node-targeting nanoparticle based on the STING agonist, its preparation method, and its applications. This invention utilizes a nano-drug delivery system—cationic polymer micelles pAA-pEPEMA—to co-prepare a novel nanovaccine (hereinafter referred to as PP-SG vaccine) with HBsAg and the STING agonist c-di-GMP. This nanovaccine possesses the ability to target lymph nodes and enhance antigen delivery; furthermore, the PP-SG vaccine can effectively increase the phagocytic and antigen-presenting capacity of BMDCs. Using an HBV-carrier mouse model and a "prime-boost" immunization strategy, the present invention demonstrates that the PP-SG vaccine can safely and effectively clear HBV, induce the production of anti-HBs, and prevent HBV reinfection; simultaneously, the PP-SG vaccine can effectively enhance the activation of DCs and reverse CD4+. + T, CD8 + By addressing the functional exhaustion of T cells and enhancing their immune response, this invention has significant practical application value. Based on the above research findings, this invention was completed.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a lymph node-targeting nanoparticle based on a STING agonist, the nanoparticle comprising cationic polymer micelles, and hepatitis B surface antigen HBsAg and a STING agonist loaded in the cationic polymer micelles.

[0010] The cationic polymer micelles are polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)methacrylate pAA-pEPEMA.

[0011] The STING agonist can be any known STING agonist. In one specific embodiment of the present invention, the STING agonist is c-di-GMP.

[0012] The nanoparticles have a hydrated particle size of 130-150 nm and a neutral surface zeta potential. Furthermore, transmission electron microscopy (TEM) images show that the nanoparticles are uniformly spherical with an actual particle size of approximately 30-40 nm.

[0013] A second aspect of the present invention provides a method for preparing the above-mentioned lymph node-targeting nanoparticles based on the STING agonist, the method comprising: adding an aqueous solution containing polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)methacrylate pAA-pEPEMA to a mixed aqueous solution containing HBsAg and the STING agonist to obtain a suspension; adjusting the pH of this suspension to neutral; continuing to stir and adding the above-mentioned aqueous solution containing polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)methacrylate pAA-pEPEMA; adjusting the pH to neutral again; and continuing to stir to obtain the final product.

[0014] A third aspect of the present invention provides the use of the above-described nanoparticles in the preparation of medicaments for the prevention and / or treatment of diseases related to hepatitis B virus (HBV) infection.

[0015] The hepatitis B virus infection-related diseases include, but are not limited to, acute hepatitis B, chronic hepatitis B, cirrhosis and liver cancer, as well as diseases such as glomerulonephritis, acute pancreatitis, cholangitis, cholecystitis, cardiomyopathy and agranulocytosis that may be caused when the hepatitis B virus invades organs such as the kidneys, pancreas, gallbladder, and heart; preferably, chronic hepatitis B.

[0016] In a fourth aspect, the present invention provides a medicament for the prevention and / or treatment of diseases related to hepatitis B virus infection, wherein the active ingredient of the medicament comprises the aforementioned nanoparticles.

[0017] The drug can be any known dosage form. In one specific embodiment of the present invention, the drug is a therapeutic vaccine injection. Specifically, it can be a subcutaneous injection dosage form, such as administering it subcutaneously using a "primer-booster" immunization strategy.

[0018] A fifth aspect of the present invention provides a method for preventing and / or treating diseases related to hepatitis B virus infection, the method comprising administering a therapeutically effective dose of the nanoparticles or drug to a subject.

[0019] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0020] The above technical solution provides a lymph node-targeting nanoparticle based on the STING agonist, its preparation method, and its application. The nanoparticles of this invention can serve as a therapeutic HBV vaccine, promoting CD8 activation by activating dendritic cells and their subsets. + T, CD4 +PP-SG nanoparticles enhance T cell proliferation, activation, and expression of functional molecules, disrupting the immunosuppressive microenvironment and reversing antigen-specific T cell depletion. This induces an HBV-specific cellular immune response to clear HBV and fosters long-term immune memory, preventing HBV reinfection. These results demonstrate the strong potential of PP-SG nanoparticles as a therapeutic HBV vaccine, offering a novel strategy and approach for the clinical cure of chronic HBV patients, thus possessing significant practical application value. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 Physicochemical properties of PP-SG vaccine. (A) DLS hydrated particle size of PP-SG vaccine. (B) Surface zeta potential of PP-SG vaccine. (C) Transmission electron microscopy image of PP-SG vaccine.

[0023] Figure 2 PP-SG vaccine can enhance APC uptake of antigens. (A) Flow cytometry analysis of BSA levels (percentage) in macrophages and dendritic cells (DCs) in lymph nodes. C57 BL / 6J mice were subcutaneously injected with PBS, BSA, or PP-BSA (BSA: 1.25g). Lymph nodes were isolated and detected 6 hours after subcutaneous injection. n = 3 per group. (B) Flow cytometry was used to determine the percentage of BMDCs in mouse bone marrow cells after in vitro induction with 10 ng / mL rmGM-CSF and 5 ng / mL rmIL-4 for 7 days. (C) BMDCs were treated in vitro with PBS, BSA, or PP-BSA. After 2, 4, and 8 hours, the BSA levels (mean fluorescence intensity) in BMDCs were analyzed by flow cytometry. Data are expressed as mean ± SEM (n≥5). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0024] Figure 3PP-SG vaccine promoted the maturation and phagocytosis of BMDCs. BMDCs were co-treated with PBS, SG, PP-S, or PP-SG vaccine (containing HBsAg: 0.15 μg / mL; c-di-GMP: 1.5 μg / mL) and 5 μg / mL FITC-BSA for 36 hours. (A) Flow cytometry was used to determine the expression of CD86 and MHC-II, and the results were displayed as histograms (percentage and mean fluorescence intensity). (B) Flow cytometry was used to analyze the phagocytosis of BMDCs, and the percentage (%) and mean fluorescence intensity (MFI) of FITC-BSA were displayed as histograms. Data are expressed as mean ± SEM (n≥5). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0025] Figure 4 PP-SG vaccine effectively clears HBV from HBV-carrying mice. HBV-carrying mice were subcutaneously injected with PBS, SG, PP-S, or PP-SG vaccine (containing HBsAg: 1 μg; c-di-GMP: 10 μg) on ​​days 0, 7, and 14, and serum was collected one day before each immunization. (A) HBsAg levels in the serum of mice in different treatment groups were measured using CLIA and expressed as relative expression. (B) HBV-DNA copy number in the serum of different treatment groups was detected by RT-PCR 21 days after treatment. (C) HBV-DNA, HBV-cccDNA, HBV-total-RNA, and HBV-3.5kb-RNA in the liver of different treatment groups were detected by RT-PCR 21 days after treatment. (D) HBcAg expression in different treatment groups was analyzed by immunohistochemical staining 21 days after treatment. (E) HBsAg levels in the serum of different treatment groups after treatment were detected by ELISA. All data are expressed as mean ± SEM (n≥5).

[0026] Figure 5 PP-SG vaccine can safely eliminate HBV in HBV-carrying mice. (A) Serum ALT concentration in experimental mice on day 28 after PP-SG vaccination was detected using the Reitman-Frankel method. A serum ALT concentration below 40 mIU / mL indicated normal physiological function. Data are expressed as mean ± SEM (n=5). (B) Liver tissue H&E staining of experimental mice on day 28 after PP-SG vaccination was analyzed.

[0027] Figure 6PP-SG vaccine can prevent HBV reinfection. HBV-carrying mice treated with PBS or vaccine were challenged again with 8 μg pAAV / HBV 1.2 on day 59 post-treatment and sacrificed on day 7 post-rechallenge. (A) HBsAg levels in mouse serum were detected by CLIA on days 2 and 4 post-HBV rechallenge. (B) Anti-HBs levels in mouse serum were detected by ELISA on days 2 and 4 post-HBV rechallenge. (C) ALT concentration in mouse serum was detected by Reitman-Frankel assay on day 7 post-HBV rechallenge. All data are expressed as mean ± SEM (n = 5). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0028] Figure 7 PP-SG vaccine downregulated PD-L1 expression on hepatic dendritic cells (DCs) in HBV-carrying mice. HBV-carrying mice treated with PBS or vaccine were rechallenged with 8 μg pAAV / HBV 1.2 on day 59 post-treatment and sacrificed on day 7 post-rechallenging. (A) Strategy for selecting DCs, cDC1, and cDC2 from hepatic mononuclear cells. (B) Flow cytometry detection of PD-L1 (MFI) expression in hepatic dendritic cells. (C) Percentage of cDC1 and cDC2 in hepatic DCs. (D) Flow cytometry analysis of PD-L1 (MFI) expression on hepatic cDC1 and cDC2. All data are presented as mean ± SEM (n≥5). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0029] Figure 8 PP-SG vaccine can activate hepatic dendritic cells (DCs) in HBV-carrying mice. (A) Flow cytometry analysis of MHC-I, MHC-II, and CD86 (MFI) expression in hepatic dendritic cells. (B) Flow cytometry analysis of MHC-I, MHC-II, and CD86 (MFI) expression in hepatic cDC1. (C) Flow cytometry analysis of MHC-I, MHC-II, and CD86 (MFI) expression in hepatic cDC2. Data are presented as mean ± SEM (n≥5). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0030] Figure 9 PP-SG vaccine can upregulate HBV-specific CD8 in HBV-carrying mice. + The proportion of T cells was increased, reversing their depletion. (AB) Flow cytometry detection of HBV-specific CD8+ in liver tissue. +(C) Flow cytometry analysis of HBV-specific CD8+ in liver tissue. + The expression of PD-1, LAG-3, and TIM-3 on T cells, plotted as a histogram. (D) Single, double, or triple positive HBV-specific CD8+ cells expressing PD-1, LAG-3, and TIM-3. + T cell subsets are shown in the pie chart (n≥5). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0031] Figure 10 PP-SG vaccine can induce CD8 in HBV-carrying mice + T cell proliferation and activation. (A) Flow cytometry analysis of HBV-specific CD8 in the liver. + T cell proliferation was analyzed using a histogram to show the percentage of Ki-67 and MFI. (B) HBV-specific CD8+ from the liver was analyzed by flow cytometry. + T cell activation was shown as a histogram of ICOS percentages and MFI. Data are presented as mean ± SEM (n≥5). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0032] Figure 11 PP-SG vaccine can increase CD8 + Expression of T cell functional molecules. Liver and lymph node mononuclear cells were stimulated with PMA / ionomycin for 4 hours and treated with BFA (5 g / mL). (A and C) Flow cytometry analysis of CD8+ in liver and lymph nodes. + The levels of IFN-γ, TNF-α, perforin, and IL-2 secreted by T cells were plotted as a histogram. (B and D) are presented as pie charts (n≥5). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0033] Figure 12 PP-SG vaccine can downregulate CD11a in the liver of HBV-carrying mice + CD4 + Expression of T-cell immune checkpoints. (A) Flow cytometry analysis of liver CD11a + CD4 + The expression of PD-1, LAG-3, and TIM-3 on T cells, plotted as a histogram. (B) Single, double, or triple positive CD11a cells expressing PD-1, LAG-3, and TIM-3. + CD4 +T cell subsets are shown as a pie chart (n≥5). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0034] Figure 13 PP-SG vaccine can activate CD4 in HBV-carrying mice. + T cells induce Th1 cell immune responses. (A) Flow cytometry analysis of spleen CD4 + (B) Flow cytometry analysis of spleen CD4 expression, with percentages and MFI represented by histograms. + Expression of ICOS in T cells, with percentage and MFI represented by a histogram. (C) Flow cytometry analysis of CD4 in the liver. + The levels of IFN-γ, TNF-α, and IL-2 produced by T cells are plotted as a histogram. (D) The pie chart shows the levels of single, double, or triple positive CD4+ cells secreting IFN-γ, TNF-α, and IL-2. + T cell subsets. All data are expressed as mean ± SEM (n≥5). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Detailed Implementation

[0035] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, 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 pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0038] As mentioned earlier, due to their small molecule characteristics, STING agonists are rapidly distributed throughout the body after entering the body, which not only hinders their absorption by lymph node DC cells, but may even cause systemic toxicity.

[0039] Lymph nodes, as important immune organs, are the sites where lymphocytes initiate their immune response to foreign antigens, and contain a large number of mature dendritic cells (DCs). Whether antigens and adjuvants can be phagocytosed by lymph node DCs is a crucial factor determining the strength of the immune response. Therefore, targeting antigens and adjuvants to lymph node DCs and increasing their residence time in the lymph nodes has become an important approach to enhancing the efficacy of therapeutic vaccines. Nanoparticles with a diameter of 10-100 nm can passively target lymph nodes through lymphatic capillaries.

[0040] In view of this, the present invention aims to optimize HBV therapeutic vaccines based on STING agonists using nanoparticles.

[0041] In a typical embodiment of the present invention, a lymph node-targeting nanoparticle based on a STING agonist is provided, the nanoparticle comprising cationic polymer micelles, and hepatitis B surface antigen HBsAg and a STING agonist loaded on the cationic polymer micelles;

[0042] The cationic polymer micelles are polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)methacrylate pAA-pEPEMA.

[0043] The STING agonist can be any known STING agonist. In one specific embodiment of the present invention, the STING agonist is c-di-GMP.

[0044] The nanoparticles have a hydrated particle size of 130-150 nm and a surface zeta potential of almost 0, indicating electrical neutrality. Furthermore, transmission electron microscopy (TEM) images show that the nanoparticles are uniformly spherical with an actual particle size of 30-40 nm.

[0045] In another specific embodiment of the present invention, a method for preparing the above-mentioned lymph node-targeting nanoparticles based on the STING agonist is provided. The preparation method includes: adding an aqueous solution containing polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)methacrylate pAA-pEPEMA to a mixed aqueous solution of HBsAg and the STING agonist to obtain a suspension; then adjusting the pH of the suspension to neutral; continuing to stir and adding the above-mentioned aqueous solution containing polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)methacrylate (pAA-pEPEMA); then adjusting the pH to neutral again and continuing to stir to obtain the final product.

[0046] In the mixed aqueous solution of HBsAg and STING agonist, the mass ratio of HBsAg to STING agonist is 1:5-20, preferably 1:10; the STING agonist can be cyclic diguanosine monophosphate c-di-GMP.

[0047] The polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)ethyl methacrylate (pAA-pEPEMA) can be prepared by the following method:

[0048] Polyacrylamide acetone oxime (PAA), (N-ethyl-N-propylamino) ethyl methacrylate (EPEMA) and 2,2'-azobis(2-methylpropionitrile) (AIBN) were dissolved in 1,4-dioxane. After deoxygenation by freeze-thaw cycles, the reaction solution was placed under heating conditions to initiate the polymerization reaction and stirred. The solution was then lyophilized by dialyzing to obtain the final product.

[0049] The molar ratio of PAA, EPEMA and AIBN is 0.01-0.05:1-2:0.005-0.05, preferably 0.0376:1.505:0.0113;

[0050] The heating conditions can be achieved using an oil bath, with the oil bath temperature controlled at 60-80℃, preferably 70℃, and the stirring and heating time controlled at 24-48 hours, preferably 36 hours, to ensure the completion of the polymerization reaction.

[0051] The PAA can be synthesized using known methods or purchased commercially, and will not be elaborated further here. Currently, the commonly used synthesis method uses acryloylacetone oxime (AA) as a starting material, obtained through a polymerization reaction.

[0052] The EPEMA monomer is prepared by the following method: 2-(N-ethyl-N-propyl)ethanolamine and triethylamine (TEA) are dissolved in acetonitrile to obtain a mixture. The mixture is stirred at low temperature, then methacryloyl chloride is added and stirred at low temperature for a while, and then reacted at room temperature. The product is then purified to obtain the final product.

[0053] The molar ratio of 2-(N-ethyl-N-propyl)ethanolamine to TEA is 1:0.5-5, preferably 22.90:22.93, and the low temperature condition is 0°C.

[0054] The specific steps for purifying the product include: filtering the product, drying the filtrate, redissolving it with dichloromethane to obtain the crude product, extracting the organic phase with water, collecting the organic phase layer, and drying it to obtain the final product.

[0055] The 2-(N-ethyl-N-propyl)ethanolamine can be prepared by the following method: N-ethylethanolamine, bromopropane, and sodium carbonate are placed in ethanol and heated and stirred. After the reaction is completed, the product is purified to obtain the 2-(N-ethyl-N-propyl)ethanolamine.

[0056] The molar ratio of N-ethylethanolamine, bromopropane, and sodium carbonate is 0.1-0.5:0.1-0.5:0.2-0.8, preferably 0.3:0.36:0.45. The specific conditions for heating and stirring are: heating temperature controlled at 70-90℃, preferably 80℃, and stirring for 12-36 hours, preferably 24 hours.

[0057] The specific steps for purifying the product include: filtering the product, drying the filtrate, redissolving it with dichloromethane to obtain the crude product, extracting the organic phase with water, collecting the organic phase layer, and drying it to obtain the final product.

[0058] In another specific embodiment of the present invention, the use of the above-mentioned nanoparticles in the preparation of drugs for the prevention and / or treatment of diseases related to hepatitis B virus (HBV) infection is provided.

[0059] The hepatitis B virus infection-related diseases mentioned include, but are not limited to, acute hepatitis B, chronic hepatitis B, cirrhosis, and liver cancer, as well as diseases such as glomerulonephritis, acute pancreatitis, cholangitis, cholecystitis, cardiomyopathy, and agranulocytosis that may be caused by hepatitis B virus invading organs such as the kidneys, pancreas, gallbladder, and heart; preferably, chronic hepatitis B. This invention demonstrates through experiments that the above-mentioned nanoparticles can serve as an HBV therapeutic vaccine, targeting lymph nodes and increasing antigen delivery; furthermore, this vaccine can effectively increase the phagocytic and antigen-presenting capacity of BMDCs. Simultaneously, this therapeutic vaccine can safely and effectively clear HBV, induce the production of anti-HBs, and prevent HBV reinfection; furthermore, this vaccine enhances the activation of DCs and reverses CD4+. + T, CD8 + By addressing the T-cell depletion state, the immune response function is enhanced, thereby effectively clearing HBV from the patient's body and achieving the therapeutic goal.

[0060] In another specific embodiment of the present invention, a medicament for preventing and / or treating diseases related to hepatitis B virus infection is provided, wherein the active ingredient of the medicament comprises the above-mentioned nanoparticles.

[0061] The drug can be any known dosage form. In one specific embodiment of the present invention, the drug is a therapeutic vaccine injection. Specifically, it can be a subcutaneous injection dosage form, such as a "prime-boost" immunization strategy administered subcutaneously.

[0062] According to the present invention, the drug may further include at least one non-pharmaceutical active ingredient, which may be any known excipient that meets the requirements of the pharmaceutical field, particularly excipients for vaccine injections.

[0063] In another specific embodiment of the present invention, the drug of the present invention can be administered into the body by known methods. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Subcutaneous injection is preferred.

[0064] Those skilled in the art will understand that the actual dose to be administered in this invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue, the general condition of the subject to be treated, the route of administration, the method of administration, etc.

[0065] In another specific embodiment of the present invention, the drug can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc.

[0066] In another specific embodiment of the present invention, a method for preventing and / or treating diseases related to hepatitis B virus infection is provided, the method comprising administering a therapeutically effective dose of the above-mentioned nanoparticles or drugs to a subject.

[0067] The subjects may be animals already being treated, observed, or experimented on, preferably mammals, and most preferably humans. The "therapeutic effective amount" refers to an amount of the active compound or agent, including the compound of the present invention, that can elicit a biological or medical response in an organ system, animal, or human sought by the researcher, veterinarian, physician, or other medical professional. This includes the reduction or partial reduction of symptoms of hepatitis B virus infection-related diseases, syndromes, symptoms, or disorders in the treated individual.

[0068] The hepatitis B virus infection-related diseases include, but are not limited to, acute hepatitis B, chronic hepatitis B, cirrhosis and liver cancer, as well as diseases such as glomerulonephritis, acute pancreatitis, cholangitis, cholecystitis, cardiomyopathy and agranulocytosis that may be caused when the hepatitis B virus invades organs such as the kidneys, pancreas, gallbladder, and heart; preferably, chronic hepatitis B.

[0069] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The following examples illustrate test methods with specific conditions, which are generally performed under conventional conditions.

[0070] Example

[0071] I. Experimental Methods:

[0072] 1. Synthesis of acid-sensitive polymers:

[0073] 1.1 Synthesis of Acryloylacetone oxime (AA):

[0074] AA was synthesized using a two-phase method. First, acetone oxime (3.18 g, 0.044 mol) dissolved in MilQ (32.5 mL) was placed in a cold hydrazine solution at 0 °C and stirred until 0 °C. Then, acryloyl chloride (4.0 g, 0.044 mol) was slowly added to the solution. After the addition was complete, the reaction mixture was brought to room temperature and stirred for 1 hour. After 1 hour, stirring was stopped, and the reaction mixture was allowed to stand to separate the aqueous and dichloromethane layers. The mixture was extracted with dichloromethane (3 × 30 mL) and collected. The collected dichloromethane was then concentrated by rotary evaporation. The concentrated organic phase was washed successively with saturated sodium bicarbonate aqueous solution and water. After drying the organic phase with Na₂SO₄, dichloromethane was removed by rotary evaporation to obtain AA.

[0075] 1.2 Synthesis of polymer PAA:

[0076] AA (Acryloylacetone oxime, 740.7 mg, 5.826 mmol), DCT (4-Cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid, 39.2 mg, 0.097 mmol), and AIBN (2,2'-azobis(2-methylpropionitrile, 3.2 mg, 0.0194 mmol)) were dissolved in 1,4-dioxane and placed in a Schlenk tube. After four freeze-thaw cycles to remove oxygen, the reaction solution was placed in an oil bath at 80°C to initiate the polymerization reaction and stirred for 3 hours. The reaction solution was cooled to room temperature to stop the reaction, and then added dropwise to a hexane:diethyl ether mixture of 3:1 (V / V) for precipitation. After centrifugation, the supernatant was discarded, and the bottom precipitate was redissolved in dichloromethane. This precipitation process was repeated twice more, and the final precipitate was PAA.

[0077] Synthesis of 1,3-(N-ethyl-N-propylamino)ethyl methacrylate and 2-(N-ethyl-N-propylamino)ethylmethacrylate (EPEMA):

[0078] First, 2-(N-ethyl-N-propyl)ethanolamine needs to be synthesized. The specific procedure is as follows: N-ethylethanolamine (27.42 g, 0.30 mol), bromopropane (44.28 g, 0.36 mol), and Na₂CO₃ (47.70 g, 0.45 mol) are added to a round-bottom flask and dissolved in ethanol (100 mL). The mixture is then heated to 80 °C and stirred continuously for 24 hours. After the reaction is complete, the reaction solution is filtered, the filtrate is collected, and the solvent is evaporated to dryness. The crude product is then redissolved in dichloromethane (DCM) (50 mL). The DCM organic phase is extracted with ultrapure water (3 × 50 mL). All DCM organic phases are collected, and the solvent is evaporated to dryness using a rotary evaporator to obtain the product. 1 Its structure was characterized by HNMR. Next, the synthesized 2-(N-ethyl-N-propyl)ethanolamine (3.00 g, 22.90 mmol) and TEA (2.32 g, 22.93 mmol) were added to a round-bottom flask and dissolved in acetonitrile (20 mL). This mixture was transferred to a cryogenic stirred reaction bath and cooled to 0 °C. Subsequently, methacryloyl chloride (2.39 g, 22.86 mmol) was added dropwise. After stirring the mixture at 0 °C for 2 hours, the reaction was carried out at room temperature for 12 hours. The reaction was stopped, filtered, and the filtrate was evaporated to dryness. The crude product was then redissolved in dichloromethane (DCM) (50 mL), and the organic phase was extracted with ultrapure water (3 × 50 mL). The DCM layer was collected, and the solvent was evaporated to dryness using a rotary evaporator to obtain the monomer EPEMA. 1 Its structure was characterized by HNMR.

[0079] 1.4 Synthesis of polyacrylacetone oxime-poly(2-(N-ethyl-N-propylamino)ethyl methacrylate)(pAA-pEPEMA):

[0080] pAA (211.4 mg, 0.0376 mmol), EPEMA (300 mg, 1.505 mmol), and 2,2'-azobis(2-methylpropionitrile) (AIBN) (1.85 mg, 0.0113 mmol) were dissolved in 1,4-dioxane and placed in a Schlenk tube. After four freeze-thaw cycles to remove oxygen, the reaction solution was placed in an oil bath at 70 °C to initiate the polymerization reaction and stirred for 36 hours. The reaction solution was cooled to room temperature to stop the reaction, and then transferred to a dialysis bag for dialyzing overnight. After lyophilization, pAA-pEPEMA was obtained.

[0081] Synthesis of 1.5 poly(2-(N-ethyl-N-propylamino)methyl methacrylate)-polymannose, Poly(2-(N-ethyl-N-propylamino)ethyl methacrylate)-polymannose (Mannose-DCT-pEPEMA):

[0082] pAA 41 -pEPEMA 32 Mannose (120 mg, 0.01 mmol), mannosamine hydrochloride (265.2 mg, 1.23 mmol), and TEA (0.379 mg, 0.0374 mmol) were dissolved in 1,4-dioxane and stirred at 50 °C for 48 hours under N2 protection. After the reaction was completed, the reaction solution was cooled and dialyzed against pure water for 2 days. After lyophilization, a brown powder, Mannose-DCT-pEPEMA, was obtained.

[0083] 2. Preparation of nano-vaccines:

[0084] (1) HBsAg + c-di-GMP (SG): The HBsAg raw material specification is 20 μg (1 mL); the c-di-GMP raw material specification is 1 mg. First, 1 mg of c-di-GMP is prepared into a 1 mg / mL c-di-GMP solution using injectable physiological saline; then, 1 mL of the HBsAg solution is added to 200 μL of the c-di-GMP solution and 2.8 mL of physiological saline, and mixed well to obtain 4 mL of vaccine solution. Each milliliter of the prepared vaccine solution contains 5 μg of HBsAg and 50 μg of c-di-GMP. The vaccine is stored at 4℃.

[0085] (2) pAA-pEPEMA+HBsAg (PP-S): A concentrated stock solution of polymer pEPEMA-pAA with a concentration of 20 mg / mL was prepared in sterile water at pH 5.5. 54 μL of concentrated HBsAg stock solution (240 μg / mL) was dispersed in 1.5 mL of sterile water at pH 6.5. While stirring, 150 μL of polymer pEPEMA-pAA was added dropwise to the above mixture. After 10 minutes, the pH of the suspension was adjusted to 7 with 0.1 M NaOH solution. After stirring for another 2 hours, another 150 μL of polymer pEPEMA-pAA was added dropwise to the suspension. After 10 minutes, the pH of the suspension was adjusted to 7 with 0.1 M NaOH solution, and stirring was continued for 6 hours to obtain the nanoparticle system.

[0086] (3) pAA-pEPEMA+HBsAg+c-di-GMP (PP-SG): A concentrated stock solution of polymer pEPEMA-pAA with a concentration of 20 mg / mL was prepared in sterile water at pH 5.5. 54 μL of concentrated HBsAg stock solution (240 μg / mL) and 130 μL of concentrated c-di-GMP stock solution (1 mg / mL) were dispersed in 1.5 mL of sterile water at pH 6.5 (HBsAg to c-di-GMP mass ratio 1:10). While stirring, 150 μL of polymer pEPEMA-pAA was added dropwise to the above mixture. After 10 minutes, the pH of the suspension was adjusted to 7 with 0.1 M NaOH solution. After stirring for another 2 hours, another 150 μL of polymer pEPEMA-pAA was added dropwise to the suspension. After 10 minutes, the pH of the suspension was adjusted to 7 with 0.1 M NaOH solution, and stirring was continued for 6 hours to obtain the nanoparticle system.

[0087] 3. Characterization of the properties of nano-vaccines:

[0088] Dynamic light scattering (DLS) was used to characterize the nanovaccines, determining their hydration size and zeta potential. The morphology of the nanovaccines was observed using transmission electron microscopy.

[0089] 4. Isolation and extraction of mononuclear cells from liver, spleen, and lymph nodes

[0090] 4.1 Isolation and extraction of liver mononuclear cells: Mice were sacrificed, and liver, spleen and lymph nodes were removed separately. The cells were cut and ground on a 200-mesh sieve, and the sieve was rinsed with 1×PBS (pH 7.4). The filtrate was transferred to a 15mL centrifuge tube, centrifuged, resuspended in Percoll solution, and resuspended after erythrocyte lysis to obtain the corresponding mononuclear cells.

[0091] 4.2 In vitro induction of BMDCs: C57 BL / 6J mice were sacrificed under aseptic conditions, and both femurs and tibias were harvested and immersed in sterile 1×PBS (pH 7.4) solution. Small holes were cut at both ends of the femurs and tibias with scissors. Using a 1mL syringe, sterile 1×PBS (pH 7.4) solution was drawn and injected into the bone marrow cavity through the small holes, flushing out the bone marrow. This process was repeated three times, and the bone marrow cell suspension was collected. After centrifugation, erythrocyte lysis and resuspending (1640 complete medium supplemented with penicillin-streptomycin mixture and cytokines rmIL-4 and rmGM-CSF), the corresponding suspension cells were obtained and cultured at 37°C and 5% CO2 for 48 hours. The culture medium was gently aspirated to remove the suspension cells from the 6-well plates. 1640 complete medium containing penicillin-streptomycin and cytokines was added again, and the cells were cultured at 37°C and 5% CO2 for another five days. Replace half of the medium with 1640 complete medium containing penicillin and streptomycin, and continue culturing until the seventh day. Gently pipette and collect all suspended and adherent cells, which are the enriched BMDCs.

[0092] 5. Testing of vaccine lymph node targeting and antigen delivery capability

[0093] 5.1 FACS detection of vaccine antigen uptake by lymph node APCs

[0094] Five- to six-week-old C57 BL / 6J mice were randomly divided into three groups and administered 1.25 μg of free FITC-BSA (BSA group), a nano-formulation containing 1.25 μg of FITC-BSA (PP-BSA group), or the same volume of sterile 1×PBS (pH 7.4) solution (PBS group) subcutaneously. Six hours after administration, the mice were sacrificed, and lymph node mononuclear cells were isolated. The levels of FITC-BSA on dendritic cells (DCs) and macrophages in the lymph nodes of each group were detected by flow cytometry and FACS.

[0095] 5.2 FACS detection of BMDCs' uptake of vaccine antigens

[0096] The induced BMDCs were divided into three groups. The experimental groups were stimulated with either free FITC-BSA or "PP-BSA", while the control group was stimulated with the same volume of sterile 1×PBS (pH 7.4). BMDCs were collected at 2, 4, and 8 hours after stimulation, stained with the corresponding flow cytometry antibodies, and the FITC-BSA level on the BMDCs was detected by FACS.

[0097] 5.3 FACS detection of BMDCs' phagocytic and antigen-presenting capabilities

[0098] The induced BMDCs were divided into four groups. The experimental groups were stimulated with a physical mixture of free HBsAg and c-di-GMP (SG group), a nanovaccine containing HBsAg (PP-S group), or a nanovaccine containing HBsAg and c-di-GMP (PP-SG group), respectively (HBsAg: 0.15 μg / mL; c-di-GMP: 1.5 μg / mL). 5 μg / mL FITC-BSA was also added to each group. After 36 hours, the stimulated cells were collected, stained with the corresponding flow cytometry antibodies, and the levels of MHC-II molecules, CD86, and FITC-BSA on the BMDCs were detected by FACS.

[0099] 6. Construction of the HBV-carrier mouse model

[0100] Five- to six-week-old C57BL / 6J mice were injected with 8 μg of pAAV / HBV1.2 plasmid via high-pressure injection into the tail vein. Five to six weeks later, peripheral blood was collected to separate serum, and the HBsAg level in the peripheral blood serum of the mice was detected. Mice with a serum HBsAg concentration higher than 500 ng / mL were considered to be successfully modeled HBV-carrier mice.

[0101] 7. Immune Strategy

[0102] HBV-carrier mice were divided into four groups: the experimental groups were the SG group (a physical mixture of free HBsAg and c-di-GMP), the PP-S group (a nanovaccine containing HBsAg), and the PP-SG group (a nanovaccine containing HBsAg and c-di-GMP); the control group was the PBS group (sterile 1×PBS solution). Each mouse in the experimental groups was subcutaneously injected with 1 μg of HBsAg from the SG group, PP-S group, or PP-SG vaccine group, and immunized three times at one-week intervals. Peripheral blood was collected one day before each immunization to separate serum, which was stored at -20℃ for later use.

[0103] 8. HBV reinfection

[0104] On day 59 after the start of treatment in the above four groups of mice, 8 μg of pAAV / HBV1.2 plasmid was injected into the tail vein of the mice again under high pressure (challenge). On day 61 and day 63, peripheral blood was collected to separate serum, which was stored at -20℃ for later use.

[0105] Blood and serum separation from mouse tail amputation

[0106] (1) Fix the mouse in a restraint device under sterile conditions, exposing the mouse tail. Disinfect the mouse tail by gently wiping it with 70% alcohol.

[0107] (2) Cut the tail about 2 mm from the end of the mouse's tail, massage from the base of the tail to the tip of the tail, and collect the blood flowing out from the tip of the tail.

[0108] (3) Let stand at room temperature for 30 minutes, centrifuge at 3000 rpm for 15 minutes, and take the supernatant as mouse peripheral blood serum, and store at -80℃.

[0109] 9. Detection of serum HBsAg levels in mouse peripheral blood using CLIA method

[0110] (1) Dilute the serum sample according to the experimental requirements.

[0111] (2) Add 50 μL of sample or standard (0, 0.05, 0.8, 10, 85, 250 ng / mL) to the corresponding coated wells respectively.

[0112] (3) Add 50 μL of enzyme conjugate to each well, gently shake to mix, cover with film, and incubate at 37°C for 1 hour.

[0113] (4) After the incubation is complete, remove the sealing film, shake off the liquid in the well, and wash the coated plate with HBsAg washing solution. Repeat the washing 5 times, and pat it dry as much as possible on the last time.

[0114] (5) Mix luminescent substrate A and luminescent substrate B in a 1:1 ratio. Add 50 μL of the mixed luminescent substrate to the coating wells, gently shake, and let stand at room temperature in the dark for 10 minutes.

[0115] (6) Use the Synergy2 multi-functional microplate reader to detect the luminescence intensity, plot a standard curve, and calculate the HBsAg concentration in the sample.

[0116] 10. Detection of HBsAb levels in mouse peripheral blood serum using ELISA.

[0117] (1) Dilute the serum sample according to the experimental requirements.

[0118] (2) Add 50 μL of sample or standard (10, 20, 40, 80, 100, 160 mIU / mL) to the corresponding coating wells respectively.

[0119] (3) Add 50 μL of enzyme-labeled reagent to each well, gently shake to mix, seal with film, and incubate at 37°C for 1 hour.

[0120] (4) After the incubation is complete, remove the sealing film, shake off the liquid in the well, and wash the coated plate with HBsAb washing solution. Repeat the washing 5 times, and pat it dry as much as possible on the last time.

[0121] (5) Add 50 μL each of colorimetric solution A and colorimetric solution B to the coated wells in sequence, gently shake to mix, and let stand at 37°C in the dark for 15 minutes.

[0122] (6) Add 50 μL of stop solution to the coated wells, gently shake to mix, use Synergy 2 multi-microplate reader to detect the absorbance at 450 nm / 630 nm, plot a standard curve, and calculate the HBsAb concentration in the sample.

[0123] 11. Extraction of genomic DNA and RNA from liver tissue and reverse transcription.

[0124] Liver tissue DNA / RNA detection, peripheral blood serum DNA detection, and peripheral blood serum ALT detection (Reiss method) were performed separately. Serum samples were diluted as needed for the experiment, and the absorbance values ​​at a wavelength of 510 nm were detected using a Synergy2 multi-functional microplate reader. ALT / GPT activity units were calculated based on the standard curve.

[0125] 12. Liver tissue sections and staining

[0126] Mice were euthanized, and liver tissue of appropriate size was collected, fixed in 4% paraformaldehyde, and left overnight. The tissue was then washed, dehydrated, cleared, embedded in paraffin, sectioned, dewaxed, hydrated, stained, dehydrated, cleared, mounted, and examined under a microscope.

[0127] 13. Immunohistochemical detection of HBcAg expression

[0128] Dewax and hydrate paraffin sections, add HBcAg primary antibody working solution, incubate overnight at 4°C in a humidified chamber, and wash three times with PBST solution. Add biotin-conjugated secondary antibody working solution, incubate at 37°C for 20 minutes in a humidified chamber, and wash three times with PBST solution. Add HRP-conjugated streptavidin working solution, incubate at 37°C for 15 minutes in a humidified chamber, and wash three times with PBST solution. Add DAB for staining for 3-5 minutes, observe the staining under a microscope, and rinse with deionized water. Stain with hematoxylin for 5 minutes, differentiate with 0.1% hydrochloric acid ethanol for 10 seconds, and then immerse in lithium carbonate solution for blue reversion for 10 minutes. Dehydrate, clear, mount with neutral resin, and examine under a microscope.

[0129] 14. Flow cytometry

[0130] 14.1 Cell Surface Molecular Detection

[0131] (1) Add 10% volume of rat serum to the mononuclear cell suspension and block at 4°C for 30 minutes.

[0132] (2) Add the corresponding diluted flow cytometry antibody, stain at 4°C for 30 minutes, add 1 PBS, centrifuge at 400 rcf for 5 minutes, and discard the supernatant.

[0133] (3) Flow cytometry was used to detect the data, and FlowJo10 software was used to analyze the data.

[0134] 14.2 Detection of molecules such as IFN-γ, TNF-α, perforin, and IL-2

[0135] (1) Prepare the culture medium for in vitro stimulation of lymphocytes: 1640 complete medium containing PMA (30ng / mL), Ionomycin (1μg / mL), IL-2 (100U / mL) and BFA (5μg / mL).

[0136] (2) The isolated mononuclear cells were resuspended in the above culture medium, plated, and stimulated at 37°C with 5% CO2 for 4 hours.

[0137] (3) Collect cells, add 10% volume of rat serum, and block at 4°C for 30 minutes.

[0138] (4) Add the corresponding diluted external standard flow cytometry antibody, stain at 4°C for 30 minutes, add 1×PBS (pH 7.4), centrifuge at 400 rcf for 5 minutes, and discard the supernatant.

[0139] (5) Add 100 μL of 1% paraformaldehyde solution, fix at 4°C in the dark for 30 minutes, add 1×PBS (pH 7.4), centrifuge at 400 rcf for 5 minutes, and discard the supernatant.

[0140] (6) Add 100 μL of membrane penetration solution and 10% volume of rat serum and let stand at 4°C in the dark for 30 minutes.

[0141] (7) Add the corresponding diluted internal standard flow cytometry antibody, stain at 4°C for 30 minutes, add 1×PBS (pH 7.4), centrifuge at 400rcf for 5 minutes, and discard the supernatant.

[0142] (8) Flow cytometry was used to detect the data, and FlowJo10 software was used to analyze the data.

[0143] 14.3 Intranuclear molecular detection

[0144] (1) Add 10% volume of rat serum to the mononuclear cell suspension and block at 4°C for 30 minutes.

[0145] (2) Add the corresponding diluted external standard flow cytometry antibody, stain at 4°C for 30 minutes, add 1 PBS, centrifuge at 400 rcf for 5 minutes, and discard the supernatant.

[0146] (3) Preparation of nucleope-related solutions: Prepare 1× Buffer by mixing 10× Permeabilization Buffer and ddH2O in a 1:9 ratio. Prepare the nucleope fixation solution by mixing the fixation nucleope concentration solution and the fixation nucleope dilution solution in a 1:3 ratio.

[0147] (4) After the external standard is completed, add 1 mL of 1×Buffer to each tube, centrifuge at 400 rcf for 5 minutes, and discard the supernatant (no need to discard the whole).

[0148] (5) Add 300 μL of fixative nucleolysis solution to a flow cytometer containing residual 1×Buffer and let stand at room temperature for 30 minutes.

[0149] (6) Add 1 mL of 1×Buffer to each tube, centrifuge at 400 rcf for 5 minutes, and discard the supernatant (no need to discard the whole).

[0150] (7) Add diluted internal standard antibody to a flow cytometer tube containing residual 1×Buffer and stain at room temperature for 30 minutes. Add 1×PBS, centrifuge at 400 rcf for 5 minutes, and discard the supernatant.

[0151] (8) Flow cytometry was used to detect the data, and FlowJo10 software was used to analyze the data.

[0152] II. Results

[0153] 2.1 Physicochemical properties of PP-SG vaccine

[0154] First, we measured the particle size and zeta potential of the PP-SG vaccine. The results are as follows: Figure 1 As shown in A and B, the hydrated particle size of this nanovaccine is between 130-150 nm, and its surface zeta potential is almost zero, indicating electroneutrality. Meanwhile, transmission electron microscopy results show that the nanovaccine is uniformly spherical, with an actual particle size between 30-40 nm. Figure 1 C).

[0155] 2.2 PP-SG vaccine can target lymph nodes and increase antigen uptake by APCs.

[0156] After antigens are taken up by APCs in lymph nodes, they undergo processing before being recognized by T cells, thereby inducing subsequent immune responses. Therefore, it is crucial that novel nanoparticle formulations can promote antigen phagocytosis by lymph node APCs. We used flow cytometry to detect the levels of FITC-BSA in mouse lymph node dendritic cells and macrophages 6 hours after subcutaneous administration. The results are as follows... Figure 2 As shown in Figure A, compared with mice injected with free BSA, the PP-BSA group showed significantly increased levels of BSA phagocytosis by macrophages and dendritic cells (DCs) in the lymph nodes. Furthermore, to further verify that this nanoformulation can increase the efficiency of antigen delivery to DCs, we induced BMDCs in vitro and treated them with free BSA antigen and PP-BSA, respectively, detecting the BSA levels in BMDCs at 2, 4, and 8 hours after treatment. Figure 2As shown in B and C, at 4 and 8 hours of treatment, the BSA level in BMDCs of the PP-BSA group was significantly higher than that of the free BSA group, indicating that higher levels of BSA entered the BMDCs, meaning that the nano-formulation increased the delivery efficiency of BSA. These results suggest that the novel nano-vaccine can increase the phagocytosis of antigens by APCs in lymph nodes, which is beneficial for inducing cellular immune responses.

[0157] 2.3 PP-SG vaccine can promote the maturation and phagocytic function of BMDCs.

[0158] Dendritic cells (DCs) are important active biological cells (APCs) and act as a bridge between innate and adaptive immunity. Literature has shown that DCs play a crucial role in activating CD8+. + T cell-mediated cellular immune responses are crucial. STING agonists can activate the cGAS-STING pathway in dendritic cells (DCs), inducing the expression of IFN-I and ISG, thereby activating DCs and regulating the body's immune function to establish an antiviral and antitumor immune state. To observe the effect of a novel nanovaccine on the maturation and activation of BMDCs, we induced BMDCs in vitro and then stimulated them with the SG group (a physical mixture of free HBsAg and free c-di-GMP), the PP-S group (a nanovaccine containing HBsAg), or the PP-SG vaccine group (a novel nanovaccine containing HBsAg and c-di-GMP), respectively. During treatment, FITC-labeled BSA antigen was added externally to evaluate the vaccine's effect on promoting antigen phagocytosis by BMDCs. Flow cytometry was used to detect the expression of MHC-II molecules and the co-stimulatory molecule CD86, important markers of BMDC surface maturation and activation, after 36 hours of treatment. Figure 3 As shown in Figure A, compared with the PBS-treated group, although the SG and PP-S groups increased the expression of MHC-II and CD86 to some extent, the PP-SG vaccine group showed a more significant upregulation of MHC-II and CD86 on the surface of BMDCs. Simultaneously, by detecting the level of BSA antigen in BMDCs, we found that the PP-SG vaccine group had a higher level of BSA antigen in BMDCs compared with other groups. Figure 3 B) indicates that PP-SG vaccine treatment effectively increases the phagocytosis of FITC-labeled BSA by BMDCs. These results suggest that the PP-SG vaccine promotes DC maturation and activation, and enhances the phagocytic and antigen-presenting functions of DCs.

[0159] 2.4 The PP-SG vaccine group can safely and effectively clear HBV and prevent HBV reinfection.

[0160] 2.4.1 The PP-SG vaccine group can effectively clear HBV.

[0161] To evaluate the therapeutic effect of the PP-SG vaccine group on CHB, we established an HBV-carrier mouse model by injecting 8 μg of pAAV / HBV1.2 plasmid into the tail vein of C57BL / 6J mice under high pressure. Previous studies have demonstrated that the pAAV / HBV1.2 plasmid injected using this method can persist and be continuously expressed in the liver of mice. HBV-carrier mice constructed using this method show high expression of HBsAg in their serum and contain various HBV DNAs, including the HBV replication intermediate HBV-cccDNA. Subsequently, we immunized HBV-carrier mice with the SG group, PP-S group, or PP-SG vaccine group, respectively. We used a "prime-boost" immunization strategy, administering the vaccine subcutaneously on day 0, followed by booster immunizations on days 7 and 14, and sacrificing the mice on day 28 for a series of immunological evaluations. First, we used the CLIA method to detect the relative expression level of HBsAg in the peripheral blood serum of HBV-carrier mice in each group during treatment. Figure 5 As shown in Figure A, the expression of HBsAg in the peripheral blood serum of mice treated with the SG group, PP-S group, or PP-SG vaccine group was significantly reduced, and it was almost undetectable after three immunizations, with the effect lasting for at least one week after treatment. Furthermore, qPCR results showed that after vaccine treatment, the levels of HBV-DNA in mouse serum, HBV-DNA in liver, HBV-cccDNA, HBV-total-RNA, and HBV-3.5kb-RNA were all significantly reduced. Figure 4 (B and C). Meanwhile, compared to the PBS group, the vaccine treatment group showed lower levels of HBcAg. + The number of hepatocytes was also significantly reduced. Figure 4 D). Although the SG group, PP-S group, or PP-SG vaccine group could effectively clear HBV in HBV-carrier mice, the PP-SG vaccine group induced the highest level of protective anti-HBs compared to other groups. Figure 4 E). The above results indicate that the PP-SG vaccine group can effectively clear HBV from HBV-carrier mice and induce protective anti-HBs during treatment.

[0162] 2.4.2 The PP-SG vaccine group can safely clear HBV.

[0163] After evaluating the efficacy of the vaccine, we further evaluated the safety of the PP-SG vaccine group. We measured the level of serum ALT in the peripheral blood of mice after treatment and observed the morphology of liver tissue in the mice after treatment using H&E staining to evaluate whether the vaccine would cause liver damage in the mice. Serum ALT results showed that the vaccine treatment did not affect liver function in the mice. Figure 5 A). Furthermore, no significant liver damage was observed in the H&E staining results of the liver sections. Figure 5 B). These results indicate that the PP-SG vaccine group can safely clear HBV.

[0164] 2.4.3 The PP-SG vaccine group can prevent HBV reinfection.

[0165] Next, we evaluated whether the PP-SG vaccine group could induce long-term immune memory and prevent HBV reinfection. We employed a "primary-boost" immunization strategy, immunizing HBV-carrier mice with the SG, PP-S, or PP-SG vaccine groups. On day 59 post-treatment, mice were re-challenged via high-pressure injection of 8 μg of pAAV / HBV1.2 plasmid into the tail vein. We measured HBsAg levels in peripheral blood serum at 2 and 4 days post-re-challenge. The results showed that, compared to other groups, the PP-SG vaccine group had the fewest mice with high HBsAg expression in peripheral blood. Figure 6 A). Meanwhile, the results of anti-HBs testing showed that the PP-SG vaccine group induced the highest levels of protective antibodies, which is beneficial for the body to neutralize invading HBV (A). Figure 6 B). Similarly, we also examined the serum ALT levels in mice after the re-challenge, such as... Figure 6 As shown in Figure C, serum ALT levels were normal. These results indicate that the PP-SG vaccine group can induce long-term immune memory against HBV infection, preventing HBV reinfection.

[0166] 2.5 The PP-SG vaccine group enhanced the function of DC cells in HBV-carrier mice.

[0167] 2.5.1 The PP-SG vaccine group downregulated the expression of PD-L1 on the surface of DC cells in HBV-carrier mice.

[0168] One of the key mechanisms of HBV immune escape is through affecting the function of host dendritic cells (DCs). This involves upregulating the expression of the inhibitory molecule PD-L1 on the DC surface while downregulating the expression of MHC-II, MHC-I molecules, and co-stimulatory molecules such as CD80, CD86, and CD40. This inhibits DC maturation and activation, leading to damage to the body's immune function and ultimately resulting in chronic infection and progressive liver disease due to the inability to clear the virus. Based on this, we first examined the effect of the PP-SG vaccine group on PD-L1 expression on the surface of DCs in HBV-carrier mice. The results showed that compared to the PBS group, PD-L1 expression in liver DCs of HBV-carrier mice decreased after treatment with the SG group, PP-S group, or PP-SG vaccine group. The downregulation of PD-L1 expression in liver DCs was most significant in the PP-SG vaccine group. Figure 7 (A and B).

[0169] Because DC cells are divided into different subsets, among which cDC1 mainly transforms into CD8 + T cells present antigens, while cDC2 primarily targets CD4 cells. + T cells present antigens, and the function of these subsets directly affects the activation of the body's adaptive immune response. Therefore, we further examined the proportions of the DC cell subsets cDC1 and cDC2 and their surface PD-L1 expression levels after vaccine treatment. The results showed that although PP-SG nanovaccine treatment did not increase the proportions of cDC1 and cDC2, it significantly reduced the expression levels of PD-L1 on the surfaces of cDC1 and cDC2. Figure 7 (A, C, and D). These results indicate that the PP-SG vaccine group can downregulate the expression of the inhibitory molecule PD-L1 on the surface of DC cells in HBV-carrier mice, which is beneficial in reversing the immunosuppressive microenvironment caused by CHB and helping the body generate an anti-HBV immune response.

[0170] 2.5.2 The PP-SG vaccine group can promote the maturation and activation of DC cells in HBV-carrier mice.

[0171] Next, we evaluated whether the PP-SG vaccine group could promote the maturation and activation of dendritic cells (DCs) in HBV-carrier mice. Similarly, we examined the expression of MHC-II, MHC-I molecules, and the co-stimulatory molecule CD86 on the surface of mouse liver DCs after vaccine treatment. The results showed that, compared with the PBS group, the expression of MHC-II and MHC-I molecules on the surface of mouse liver DCs was significantly increased after treatment in the SG group, PP-S group, or PP-SG vaccine group, with the most significant increase observed in the PP-SG vaccine group. However, when examining the level of the co-stimulatory molecule CD86 on the surface of DCs, we found no significant difference between the PP-SG vaccine group and the other vaccine treatment groups. Figure 8 A). Furthermore, the detection results of DC cell subsets cDC1 and cDC2 showed that, compared with other groups, the PP-SG vaccine group not only increased the levels of MHC-II and MHC-I molecules, but also upregulated the expression of the co-stimulatory molecule CD86. Figure 8 (B and C). The above results indicate that the PP-SG vaccine group can promote the maturation and activation of DC cells in HBV-carrier mice.

[0172] 2.6 The PP-SG vaccine group can increase antigen-specific CD8. + The proportion of T cells and reversing their exhaustion state.

[0173] During the body's clearance of HBV, CD8 + T cells, especially CD11a hi CD8α lo T cells play a crucial role; this group of CD8 cells + T cells, also known as antigen-specific CD8 cells + T cells. Therefore, we evaluated whether the PP-SG vaccine group could increase antigen-specific CD8 in HBV-carrier mice. + The proportion of T cells. For example... Figure 9 As shown in A and B, compared with the PBS group, the liver antigen-specific CD8 in mice in the SG group, PP-S group, or PP-SG vaccine group was significantly higher. + The proportion of T cells increased significantly in all groups, but the PP-SG vaccine group did not show any advantage over the SG or PP-S groups. Some literature indicates that antigen-specific CD8+ cells in CHB patients... + T cells are often in a state of functional exhaustion, with significantly upregulated expression of immune checkpoints such as PD-1, 2B4, TIM-3, LAG-3, and CTLA-4 on their surface. These immune checkpoints are closely related to T cell function, and T cells with high expression of immune checkpoints cannot properly clear HBV-infected cells. An ideal therapeutic HBV vaccine should be able to reverse antigen-specific CD8+ expression in CHB patients.+ To restore the normal function of T cells from their depleted state, we further examined the antigen-specific CD8+ of HBV-carrier mice after vaccine treatment. + The expression of T cell surface immune checkpoints was examined. Results showed that, compared to the PBS group, the SG and PP-S groups showed increased expression of liver antigen-specific CD8+ in HBV-carrier mice after vaccine treatment. + The expression of PD-1, LAG-3, and TIM-3 on T cells was significantly decreased, and the downregulation of these immune checkpoints was more pronounced in the PP-SG vaccine group. Simultaneously, we analyzed the antigen-specific CD8+ expression of PD-1, LAG-3, and TIM-3 in the livers of four groups of mice (single-positive, double-positive, and triple-positive). + The proportion of T cells showed that the PP-SG vaccine group had depleted antigen-specific CD8+ cells. + The proportion of T cells was the lowest. In summary, the PP-SG vaccine group showed an increase in antigen-specific CD8+. + Increasing the proportion of T cells and reversing their depletion state is beneficial for breaking the body's immune tolerance microenvironment.

[0174] 2.7 The PP-SG vaccine group can promote CD8. + T cell proliferation and activation, and upregulation of their antiviral function.

[0175] 2.7.1 The PP-SG vaccine group can promote CD8 + T cell proliferation and activation

[0176] The key to the effectiveness of HBV therapeutic vaccines is promoting the production of CD8 in the body. + T cell-mediated cellular immune responses; therefore, we investigated whether the PP-SG vaccine group could promote CD8+. + T cell proliferation and activation were evaluated. We examined CD8+ in the spleen of mice after vaccine treatment. + The expression levels of Ki-67 in the T cell nucleus and the surface co-stimulatory molecule ICOS, these two molecules can reflect the expression levels of CD8+ and CD6+, respectively. + The level of T cell proliferation and activation. For example... Figure 10 As shown in A and B, compared with the PBS group, the spleen CD8 of mice in the SG group and PP-S group had higher levels of CD8+. + The positivity rates of Ki-67 and ICOS and the MFI level in T cells were significantly increased, while the PP-SG vaccine group further enhanced CD8. + The expression of Ki-67 and ICOS molecules on T cells indicates that the PP-SG vaccine group can promote CD8 expression. + T cell proliferation and activation.

[0177] 2.7.2 The PP-SG vaccine group upregulated CD8. + Expression of T cell functional molecules

[0178] During the body's process of clearing viral infection, CD8 + T cells can release cytotoxic molecules such as perforin and granzyme B, which form pores in the cell membranes of virus-infected cells, altering the osmotic pressure of target cells and promoting the entry of killing molecules, thereby killing virus-infected cells. In addition, CD8... + T cells can also secrete cytokines such as IFN-γ, TNF-α, and IL-2, which directly or indirectly exert antiviral effects. These functional molecules also reflect CD8+. + The ability of T cells to kill target cells. Therefore, to demonstrate that novel nanovaccines based on SITNG agonists can enhance CD8... + To assess T cell function, we collected liver lymphocytes, stimulated them with PMA / Ionomycin, and then analyzed CD8 counts by flow cytometry. + The levels of IFN-γ, TNF-α, perforin, and IL-2 secreted by T cells. For example... Figure 11 As shown in Figure A, compared with the PBS group, treatment with the SG group, PP-S group, or PP-SG vaccine group significantly increased CD8+ in the liver of mice. + The expression levels of IFN-γ, TNF-α, perforin, and IL-2 on T cells were statistically analyzed, with the PP-SG vaccine group showing a significant advantage over the other two vaccine treatment groups. Furthermore, we also analyzed the expression levels of multifunctional CD8+ cells expressing various functional molecules after vaccine treatment. + The proportion of T cells. For example... Figure 11 As shown in Figure B, compared to the PBS group, the livers of mice in the three vaccine-treated groups showed multifunctional CD8 cells. + The proportion of T cells increased in all groups, with the PP-SG vaccine group showing the best results. Furthermore, to further demonstrate this phenomenon, we used the same method to detect CD8+ in lymph nodes. + Expression of functional molecules on T cells. Similar to the liver, the PP-SG vaccine group showed significantly upregulated CD8+ expression in lymph nodes compared to other groups. + The expression levels of IFN-γ, TNF-α, perforin, and IL-2 on T cells were increased, along with increased expression of multifunctional CD8. + The proportion of T cells ( Figure 11 (C and D). The above results indicate that the PP-SG vaccine group can upregulate CD8. + The expression of T cell functional molecules enhances CD8. + The killing and antiviral functions of T.

[0179] 2.8 The PP-SG vaccine group can regulate CD4. + T cell function

[0180] 2.8.1 The PP-SG vaccine group can reduce CD4. + Expression of inhibitory molecules on the surface of T cells

[0181] During HBV infection, CD4 + T cells can participate in the body's anti-HBV immune response by regulating the activity and function of other immune cells. Literature indicates that CD4... + T cells can signal CD8 + T cells provide activating factors to assist CD8 + T cells transform into effector T cells. On the other hand, CD4... + T cells can also help activate B cells and promote the production of neutralizing antibodies (anti-HBs). However, with CD8... + Similar to T cells, CD4 in CHB patients + T cells also exhibit high expression of immune checkpoints, leading to CD4+ expression. + T cell function was impaired. Therefore, we wanted to investigate whether the PP-SG vaccine group could also downregulate CD4 in HBV-carrier mice. + The expression level of T cell immune checkpoints improves CD4 expression. + Impaired T cell function was observed. To investigate this, we examined liver antigen-specific CD4 in mice after vaccine treatment. + The expression levels of immune checkpoints such as PD-1, LAG-3, and TIM-3 on the surface of T cells were observed. The results showed that, compared with the PBS group, the SG group, PP-S group, or PP-SG vaccine group all effectively reduced CD4+ expression in mouse liver after treatment. + The levels of PD-1, LAG-3, and TIM-3 molecules on the surface of T cells were measured, and the PP-SG vaccine group showed the lowest expression levels compared to other groups. Figure 12 A). Similarly, we also analyzed the antigen-specific CD4+ of PD-1, LAG-3, and TIM-3 single-positive, double-positive, and triple-positive cells in mouse liver. + T cell ratio, such as Figure 12 As shown in B, compared with all other groups, the PP-SG vaccine group expressed antigen-specific CD4+ of PD-1, LAG-3, or TIM-3. + The proportion of T cells was the lowest. These results indicate that the PP-SG vaccine group can reduce CD4+. + The expression of inhibitory molecules on the surface of T cells reverses the CD4+ induced by HBV. + Impaired T cell function.

[0182] 2.8.2 The PP-SG vaccine group can activate CD4. + T cells and induce Th1-type cell immune responses.

[0183] Furthermore, we evaluated the effect of the PP-SG vaccine group on CD4 activation. + The ability of T cells to proliferate and activate. We examined the CD4+ of the spleen in mice after vaccine treatment. + The expression levels of Ki-67 and ICOS molecules on T cells were measured. Results showed that treatment with the SG group, PP-S group, or PP-SG vaccine significantly upregulated spleen CD4 expression. + The percentages of T cell Ki-67 and ICOS molecules were correlated with MFI, and the PP-SG vaccine group showed the highest upregulation levels compared to other vaccine treatment groups. Figure 13 (A and B). This indicates that the PP-SG vaccine group can promote CD4. + T cell proliferation and activation. CD4 + After activation, T cells differentiate into functional subsets, such as Th1, Th2, and Th17 cells. Among them, Th1 cells can help CD8 cells... + T cells differentiate into CTLs, assisting CD8... + T cells clear viral infection. Th1 cells mainly regulate cellular immune responses by secreting cytokines such as IFN-γ, TNF-α, and IL-2. Therefore, we measured CD4 counts in mice after vaccine treatment. + The levels of IFN-γ, TNF-α, and IL-2 in T cells. For example... Figure 13 C shows that, compared with the PBS group, the liver CD4 levels in mice of the three vaccine-treated groups were significantly lower. + The expression levels of IFN-γ, TNF-α, and IL-2 on T cells were all increased, and the PP-SG vaccine group showed superior therapeutic efficacy compared to the other two vaccine treatment groups. Similarly, we also analyzed the levels of multifunctional CD4 in the livers of mice in each group. + The proportion of T cells, as shown in the pie chart, indicates the multifunctional CD4+ T cell group in the PP-SG vaccine group. + The highest proportion of T cells was observed (Figure 13D). Based on these results, it can be concluded that the PP-SG vaccine group can promote CD4 cell proliferation. + T cell proliferation and activation, and the induction of Th1-type cellular immune responses, are beneficial for assisting CD8 cell proliferation. + T cells clear HBV.

[0184] The difficulty in curing chronic HBV stems primarily from its ability to evade the body's immune surveillance in various ways. These include suppressing the activity of APCs such as dendritic cells (DCs) and macrophages, upregulating the expression of immune checkpoints like PD-1 and LAG-3 on T cell surfaces, blocking co-stimulatory signals between DCs and T cells, enhancing co-inhibitory signals, and interfering with the body's normal antiviral immune response. The inability to promptly eliminate HBV leads to its long-term coexistence with T cells, further inducing T cells to develop immune tolerance and causing T cell depletion. Furthermore, HBV can upregulate the expression of cells or molecules that suppress immune system function, such as Tregs and IL-10. These factors collectively create an immunosuppressive microenvironment within the patient's body. Therefore, reversing this immunosuppressive microenvironment and activating the functional activity of immune cells is crucial for curing chronic HBV with therapeutic vaccines.

[0185] Existing research indicates that activation of dendritic cells (DCs) in lymph nodes is crucial for activating CD8+ in the body. +T-cell-mediated cellular immune responses are crucial, suggesting that targeting dendritic cells (DCs) in lymph nodes with antigens and adjuvants could help vaccines better activate the immune system and clear HBV. cGAS-STING is one of the pathways that activates DCs and is a promising target for activating cellular immunity. Therefore, we chose the STING agonist c-di-GMP (as an adjuvant, used in combination with HBsAg to construct a novel therapeutic HBV vaccine). However, therapeutic vaccines constructed by physically mixing HBsAg and c-di-GMP do not have lymph node targeting, and due to its small molecular size, c-di-GMP rapidly distributes throughout the body after entering the body, making it difficult to remain in lymph nodes for a sufficient time. High doses may also induce systemic inflammation, hindering vaccine efficacy. Although intra-lymph node injection can alleviate this situation to some extent, this method still has limitations such as cumbersome operation and small dosage. Therefore, we chose nanomedicine technology to address this problem. Nanoparticle vaccines possess unique advantages: Firstly, due to their particle size, nanoparticles can enter lymph nodes through capillary lymphatic vessels, effectively enhancing the lymph node targeting of vaccines. Secondly, nanoparticle drug delivery systems can protect the carried antigens from the influence of the in vivo environment, safely delivering antigens and adjuvants to APCs. Furthermore, because the size and composition of nanoparticles are similar to cells, they can be rapidly taken up by cells through pinocytosis, exhibiting excellent antigen delivery efficiency. Current research has demonstrated that nanoparticles carrying HBsAg have a stronger ability to induce antibody production compared to ordinary HBsAg. In addition, the slow biodegradation rate of nanoparticle vaccines after entering APCs also facilitates the persistence of antigens in cells, prolonging the time for APCs to process antigens and promoting the initiation of subsequent adaptive immune responses. Based on this information, we believe that constructing novel HBV therapeutic nanoparticle vaccines based on c-di-GMP using nanoparticle drug delivery technology may have unique advantages in clearing HBV and inducing strong and long-lasting immune effects. Therefore, we used pAA-pEPEMA material to prepare a nano-vaccine, namely PP-SG vaccine group, by combining HBsAg and c-di-GMP.

[0186] By using FITC-labeled BSA as the antigen, we demonstrated in vitro and in vivo that this nanovaccine can target lymph nodes, promote antigen delivery, and prolong the duration of antigen presence in lymph nodes. To further elucidate the advantages of the PP-SG vaccine, we used a physical mixture of HBsAg and c-di-GMP (SG) and an HBsAg-containing nanovaccine (PP-S) as controls to evaluate the HBV clearance effect of the PP-SG vaccine group and to investigate its impact on the immune response. These experimental results indicate that the HBV vaccine prepared using nano-drug delivery technology can overcome the weaknesses of c-di-GMP and effectively target and deliver the antigen and adjuvant to lymph node dendritic cells (DCs). After entering DCs, c-di-GMP, as an adjuvant, can activate the cGAS-STING signaling pathway, stimulate the expression of IFN-I and some interferon-stimulating genes, thereby activating DCs and enhancing their antigen-presenting function, which undoubtedly benefits the subsequent adaptive immune response.

[0187] The "primary-boost" immunization strategy helps vaccines better activate the immune system. Therefore, we also used this method to immunize HBV-carrier mice, i.e., immunizing every other week for a total of three immunizations. Peripheral blood serum was collected from the mice the day before each immunization to analyze the relative expression level of HBsAg in the serum. Our results show that at the serological level, the SG group, PP-S group, or PP-SG vaccine group can effectively reduce the relative expression of HBsAg. However, from the results of liver tissue RNA and DNA levels, both the physical mixture of HBsAg and c-di-GMP and the nano-formulation containing only HBsAg had a worse clearance effect than the nano-vaccine PP-SG, which is a combination of HBsAg and c-di-GMP. At the same time, compared with other vaccine treatment groups, the PP-SG vaccine also induced the highest level of serum anti-HBs. Furthermore, we examined the vaccine's ability to induce long-term immune memory by reinfecting vaccine-treated HBV-carrier mice. The results showed that, compared to other vaccine groups, the PP-SG vaccine-treated group exhibited the lowest serum HBsAg levels and the highest serum anti-HBs levels. Clearly, using a nanocarrier to combine c-di-GMP with HBsAg has certain advantages in clearing HBV from HBV-carrier mice.

[0188] Dendritic cells (DCs) are the most important antigen-presenting cells. In patients with chronic HBV, impaired DC function is a significant reason why T cells cannot be activated. DCs are divided into plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). mDCs further exist in two subsets: cDC1 and cDC2, each with different functions. cDC1 has cross-presentation capabilities, delivering exogenous antigens via MHC-I molecules and inducing CD8+ expression. + T cell immune responses are primarily antigen-presenting cells, crucial for establishing the body's antiviral state. cDC2 cells mainly convert MHC-II to CD4+ antigens. + T cells deliver antigens, playing a supporting role in the humoral immune response. We found that PP-SG vaccine treatment effectively downregulated PD-L1 expression on the surface of dendritic cells (DCs) and their subsets, showing an absolute advantage in overall DC cell count. This is because the cDC1 subset plays a crucial role in activating CD8+. + Given the crucial role of T-cell immune responses, we focused on the effects of vaccines on the cDC1 subset. While the PP-SG vaccine did not increase the proportion of cDC1, it effectively reduced the level of PD-L1 on the cDC1 surface. Furthermore, PP-SG vaccine treatment significantly activated liver-derived DC cells and their subsets in mice, exhibiting the highest activation level among all vaccine treatment groups. Similarly, the PP-SG vaccine also activated spleen-derived DC cells and their subsets, but it did not show a significant advantage over the PP-S vaccine in spleen-derived DC cells. Overall, the PP-SG vaccine remained the most effective at activating DC cells and their subsets among the three vaccine treatment groups. Similar results were obtained in in vitro experiments. These results are consistent with their ability to clear HBV, prevent relapse, and induce anti-HBs.

[0189] CD8 + T cells are among the cells that directly exert antiviral functions, including antigen-specific CD8 cells. + T cells are crucial for the body to clear HBV, and the cellular immune response mediated by these T cells acts directly on HBV. Therefore, reversing antigen-specific CD8+... + The depletion of T cells is one of the key targets for breaking the immune tolerance microenvironment in patients with chronic HBV. All three vaccines—SG group, PP-S group, and PP-SG vaccine group—can upregulate antigen-specific CD8+ in the liver of HBV-carrier mice. + The proportion of T cells did not differ significantly among the three vaccine treatment groups. However, further testing for antigen-specific CD8... +When targeting T cell surface immune checkpoints such as PD-1, LAG-3, and TIM-3, we found that the PP-SG vaccine significantly downregulated the expression of these checkpoints, exhibiting a clear advantage over other vaccines. This indicates that the PP-SG vaccine can better block the transmission of co-inhibitory signals and break the state of T cell exhaustion compared to other vaccine groups. Furthermore, PP-SG vaccine treatment can effectively induce CD8+ expression. + T cells secrete effector molecules such as IL-2, IFN-γ, TNF-α, and perforin, and their effects are also superior to those of other vaccine treatment groups.

[0190] CD4 + T cells play an auxiliary role in the body's clearance of HBV and inducing the production of neutralizing antibodies. In patients with chronic HBV, CD4... + Impaired T-cell function is also one of the reasons for the decreased intensity of the immune response. Compared with other vaccine treatment groups, PP-SG vaccine treatment also downregulated CD4. + The PP-SG vaccine increases the expression of immune checkpoints such as PD-1, LAG-3, and TIM-3 on the surface of T cells, and promotes their activation and proliferation. Notably, the PP-SG vaccine upregulates CD4+. + The expression of cytokines such as IL-2, IFN-γ, and TNF-α on T cells indicates that the PP-SG vaccine promotes Th1-type cellular immune responses, which is beneficial for the body to generate antiviral cellular immune responses. Therefore, we believe that the PP-SG vaccine can reverse the co-inhibitory signaling between DC cells and T cells, restore the function of exhausted T cells, thereby breaking the immunosuppressive microenvironment and promoting the body's clearance of HBV.

[0191] In summary, the PP-SG vaccine can promote CD8 activation by activating dendritic cells and their subsets. + T, CD4 + The PP-SG vaccine promotes T cell proliferation, activation, and expression of functional molecules, disrupts the immunosuppressive microenvironment, reverses the depletion of antigen-specific T cells, and induces an HBV-specific cellular immune response to clear HBV. It also induces long-term immune memory to prevent HBV reinfection. Furthermore, compared to the SG and PP-S groups, the PP-SG vaccine demonstrated a significant advantage and the best therapeutic effect. These results indicate that the PP-SG vaccine has strong potential as a therapeutic HBV vaccine and provides a new strategy and approach for the clinical cure of chronic HBV patients.

[0192] Matters not covered in this invention are common knowledge.

[0193] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A STING agonist-based lymph node targeting nanoparticle, characterized in that, The nanoparticles comprise cationic polymer micelles, and hepatitis B surface antigen HBsAg and STING agonist loaded onto the cationic polymer micelles. The cationic polymer micelles are polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)methacrylate pAA-pEPEMA; The polyacrylacephenone oxime-poly(2-(N-ethyl-N-propylamino)ethyl methacrylate pAA-pEPEMA was prepared by the following method: Polyacrylamide acetone oxime (PAA), ethyl (N-ethyl-N-propylamino) methacrylate (EPEMA), and 2,2'-azobis(2-methylpropionitrile) (AIBN) were dissolved in 1,4-dioxane. After deoxygenation by freeze-thaw cycles, the reaction solution was placed under heating conditions to initiate the polymerization reaction and stirred. The solution was then lyophilized by dialyzing to obtain the final product. The molar ratio of PAA, EPEMA, and AIBN is 0.01-0.05:1-2:0.005-0.05; The EPEMA was prepared by the following method: 2-(N-ethyl-N-propyl)ethanolamine and triethylamine (TEA) were dissolved in acetonitrile to obtain a mixture. The mixture was stirred at low temperature, then methacryloyl chloride was added and stirred at low temperature for a while, and then reacted at room temperature. The product was purified to obtain the final product. The molar ratio of 2-(N-ethyl-N-propyl)ethanolamine to TEA is 1:0.5-5, and the low temperature condition is 0°C; The STING agonist is c-di-GMP (cyclic diguanosine monophosphate). The nanoparticles have a hydrated particle size of 130-150 nm and are electrically neutral; the nanoparticles are uniformly spherical and have an actual particle size of 30-40 nm. The mass ratio of HBsAg to STING agonist is 1:5-20.

2. The lymph node-targeting nanoparticles based on the STING agonist as described in claim 1, characterized in that, The mass ratio of HBsAg to STING agonist is 1:

10.

3. The lymph node-targeting nanoparticles based on the STING agonist as described in claim 1, characterized in that, The molar ratio of PAA, EPEMA and AIBN is 0.0376:1.505:0.0113.

4. The lymph node-targeting nanoparticles based on the STING agonist as described in claim 1, characterized in that, The heating conditions are achieved using an oil bath, with the oil bath temperature controlled at 60-80℃ and the stirring and heating time controlled at 24-48 hours.

5. The lymph node-targeting nanoparticles based on the STING agonist as described in claim 4, characterized in that, The oil bath temperature is controlled at 70℃.

6. The lymph node-targeting nanoparticles based on the STING agonist as described in claim 4, characterized in that, The stirring and heating time is controlled to be 36 hours.

7. The lymph node-targeting nanoparticles based on the STING agonist as described in claim 1, characterized in that, The specific steps for purifying the product include: filtering the product, drying the filtrate, redissolving it with dichloromethane to obtain the crude product, extracting the organic phase with water, collecting the organic phase layer, and drying it to obtain the final product.

8. The lymph node-targeting nanoparticles based on the STING agonist as described in claim 1, characterized in that, The molar ratio of 2-(N-ethyl-N-propyl)ethanolamine to TEA is 22.90:22.

93.

9. The method for preparing lymph node-targeting nanoparticles based on the STING agonist according to any one of claims 1-8, characterized in that, The preparation method includes: adding an aqueous solution containing polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)methacrylate pAA-pEPEMA to a mixed aqueous solution containing HBsAg and STING agonist to obtain a suspension; then adjusting the pH of the suspension to neutral; continuing to stir and adding the above aqueous solution containing polyacrylamide oxime-poly(2-(N-ethyl-N-propylamino)methacrylate pAA-pEPEMA; then adjusting the pH to neutral again and continuing to stir to obtain the final product.

10. The use of the nanoparticles according to any one of claims 1-8 in the preparation of a medicament for treating diseases related to hepatitis B virus infection; The hepatitis B virus infection-related disease mentioned is chronic hepatitis B.

11. A medicament for treating diseases related to hepatitis B virus infection, wherein the active ingredient of the medicament comprises the lymph node-targeting nanoparticles based on the STING agonist as described in any one of claims 1-8.

12. The medicament for treating hepatitis B virus infection-related diseases as described in claim 11, characterized in that, The drug is a therapeutic vaccine injection.

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