CN117547519A - Lymph node targeting nanoparticle based on STING agonist and preparation method and application thereof - Google Patents
Lymph node targeting nanoparticle based on STING agonist and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to a lymph node targeting nanoparticle based on STING agonist, and a preparation method and application thereof. The invention prepares a novel nano vaccine by utilizing a nano drug carrying system-cationic polymer micelle pAA-pEPEMA, HBsAg and STING agonist c-di-GMP together, and has the capability of targeting lymph nodes and increasing antigen delivery; and the vaccine can effectively increase phagocytosis and antigen presentation capacity of BMDCs. The vaccine can safely and effectively remove HBV, induce the generation of anti-HBs and prevent the reinfection of HBV; at the same time, the vaccine enhances the activation of DCs cells, reversing CD4 + T、CD8 + The T cell has the function of eliminating the state and enhancing the immune response function, so that the T cell has good practical application value.
Description
Technical Field
The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to a lymph node targeting nanoparticle based on STING agonist, and a preparation method and application thereof.
Background
The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Hepatitis b is a global health problem caused by Hepatitis B Virus (HBV) infection, which attacks the liver, resulting in acute and chronic diseases. It is estimated that currently 2.57 million people worldwide are chronic HBV patients and have a high risk of developing liver disease, cirrhosis and hepatocellular carcinoma. Although conventional hepatitis b surface antigen (HBsAg) vaccination can induce protective antibodies in most healthy vaccinated populations and effectively reduce the incidence of new HBV infection, HBsAg fails to induce an effective antibody response in animal models or in patients with clinical Chronic HBV (CHB) infection. Thus, an effective therapeutic strategy to eliminate and eradicate CHB is highly desirable.
CHB is difficult to cure, and is characterized by a series of immune escape phenomena after HBV invades human body, including abnormal functions of natural immune cells such as DC cells, megaphaga cells and NK cells, and increased expression of inhibitory cells or molecules such as Tregs and IL-10, and CD4 and CD8 + T cells highly express immune checkpoints such as PD-1, LAG-3, TIM-3 and the like, and show a functional exhaustion state and the like. The current line of drugs used clinically against CHB is mainly based on the drugs developed by the third generation nucleotide analogs (NUCs) and PEG-IFN- α. Although these drugs have therapeutic effects to some extent, clinical trial follow-up results have found that HBsAg elimination is observed in only 10% of patients after five years of treatment for CHB patients treated with NUC; whereas, patients with CHB using IFN- α, only 10% -20% of them can achieve HBV cure, and are often accompanied by serious side effects. In summary, currently clinically used medicines for treating CHB cannot thoroughly cure CHB, and may cause drug resistance in case of long-term use. Therefore, overcoming the above limitations, it is important to develop a novel drug for CHB.
Therapeutic vaccines are one of the hot spots of current research for treating CHB. Unlike prophylactic vaccines, therapeutic vaccines emphasize how CD8 activates the patient's body more + T cell immune responses to induce an antiviral immune response, thereby clearing HBV in the patient. In general, we can construct therapeutic vaccines by combining an antigen with an adjuvant. The adjuvant is a nonspecific immunopotentiator, and can enhance immunogenicity of antigen, enhance intensity of immune response caused by antigen, and up regulate immune response level of organism by injecting together with antigen or into organism in advance. Recent studies have found that a variety of small molecules can be used as adjuvants to construct novel human vaccines, including agonists of TLR-like receptors (TLRs), NOD-like receptors, and the like. Research shows that after the small molecules enter the body together with the antigen, the functions of immune cells such as DC cells, T, B cells and the like can be activated to a certain extent through the same or different paths, so that the immune response of an organism is enhanced, and the immune suppression phenomenon of a patient is improved.
The cas-STING pathway is an important component of the innate immune system, widely present in DC cells, and regulates its activity. First, cGAS recognizes binding to pathogenic DNA, synthesizes GMP-AMP (cGAMP), and then binds to STING on the endoplasmic reticulum to form multimers. Polymerized STING is transferred from the endoplasmic reticulum to the golgi apparatus. In the golgi, STING recruits TBK1 and IRF3 and activates TBK1, which in turn phosphorylates IRF3.IRF3 can then migrate to the nucleus and induce the production of ISG and IFN-I. In addition, STING can activate signals such as NF- κB and STAT6, and together with IFN, activate antiviral function of organism. Thus, research has been conducted on using STING agonists as adjuvants to construct novel therapeutic vaccines. However, STING agonists, due to their small molecular nature, rapidly distribute throughout the body after entry, not only being unfavorable for uptake by lymph node DC cells, but may even cause systemic toxicity.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a lymph node targeting nanoparticle based on STING agonist, and a preparation method and application thereof. The invention utilizes a nano drug-carrying systemThe cationic polymer micelle pAA-pEPEMA and HBsAg and STING agonist c-di-GMP together prepare a novel nano vaccine (also called PP-SG vaccine for short in the invention) which has the ability of targeting lymph nodes and increasing antigen delivery; and the PP-SG vaccine can effectively increase phagocytosis and antigen presentation capacity of BMDCs. The invention uses HBV-carrier mouse model and uses prime-boost (prime-boost) immunization strategy for subcutaneous administration, which proves that the PP-SG vaccine can safely and effectively remove HBV, induce anti-HBs and prevent HBV from re-infection; meanwhile, the PP-SG vaccine can effectively enhance the activation of DCs cells and reverse CD4 + T、CD8 + The T cell has the function of eliminating the state and enhancing the immune response function, so that the T cell has good practical application value. Based on the above results, the present invention has been completed.
In order to achieve the technical purpose, the technical scheme provided by the invention is as follows:
in a first aspect of the present invention, there is provided a STING agonist-based lymph node targeting nanoparticle comprising a cationic polymer micelle, and hepatitis b surface antigens HBsAg and STING agonist loaded on the cationic polymer micelle;
wherein the cationic polymer micelle is polyacrylacetonoxime-poly (2- (N-ethyl-N-propylamine) ethyl methacrylate pAA-pEPEMA.
The STING agonist may be any currently known STING agonist, and in one embodiment of the present invention, the STING agonist is cyclodiguanylate c-di-GMP.
The hydration particle size of the nano particles is 130-150nm, and the surface zeta potential is electrically neutral. Meanwhile, the shooting result of a transmission electron microscope shows that the nano particles are uniformly spherical, and the actual particle size is about 30-40nm.
In a second aspect of the present invention, there is provided a preparation method of the above-mentioned STING agonist-based lymph node targeting nanoparticle, the preparation method comprising: adding an aqueous solution containing the poly (2- (N-ethyl-N-propylamino) ethyl methacrylate pAA-pEPEMA) into a mixed aqueous solution containing the HBsAg and the STING agonist to obtain a suspension, regulating the pH of the suspension to be neutral, continuously stirring, adding the aqueous solution containing the poly (2- (N-ethyl-N-propylamino) ethyl methacrylate pAA-pEPEMA), regulating the pH to be neutral, and continuously stirring.
In a third aspect of the present invention, there is provided the use of the above nanoparticle for the preparation of a medicament for the prevention and/or treatment of a disease associated with Hepatitis B Virus (HBV) infection.
Wherein, the hepatitis B virus infection related diseases include, but are not limited to, acute hepatitis B, chronic hepatitis B, liver cirrhosis, liver cancer, and diseases such as glomerulonephritis, acute pancreatitis, cholangitis, cholecystitis, cardiomyopathy, and granulocytopenia which are possibly caused when the hepatitis B virus invades organs such as kidney, pancreas, gall bladder, heart, etc.; preferably chronic hepatitis B.
In a fourth aspect of the present invention, there is provided a medicament for preventing and/or treating a disease associated with hepatitis b virus infection, the medicament comprising the above nanoparticles as an active ingredient.
The medicament can be any type of known medicament, and in one embodiment of the invention, the medicament is a therapeutic vaccine injection. In particular, it may be in the form of a subcutaneous injection, for example, which may be administered subcutaneously using a "prime-boost" immunization strategy.
In a fifth aspect of the present invention, there is provided a method for preventing and/or treating a disease associated with hepatitis b virus infection, the method comprising administering to a subject a therapeutically effective dose of the nanoparticle or drug described above.
The beneficial technical effects of one or more of the technical schemes are as follows:
the technical scheme provides a lymph node targeting nanoparticle based on STING agonist, and a preparation method and application thereof. The nanoparticle can be used as an HBV therapeutic vaccine for promoting CD8 by activating DC cells and subpopulations thereof + T、CD4 + T cell proliferation, activation and expression of functional molecules, breaks the immune suppression microenvironment of the organism, reverses the depletion state of antigen specific T cells, thereby inducing the organism to generate HBV specific cell immune response to clear HBV and inducingLong-term immunological memory is induced and HBV re-infection is prevented. The results show that the PP-SG nanoparticles have strong potential as HBV therapeutic vaccines, and provide a new strategy and thought for clinically curing chronic HBV patients, so the PP-SG nanoparticles have good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
Fig. 1: physicochemical properties of PP-SG vaccine. (A) DLS hydrated particle size of PP-SG vaccine. (B) surface zeta potential of PP-SG vaccine. And (C) transmission electron microscope images of the PP-SG vaccine.
Fig. 2: the PP-SG vaccine can enhance the uptake of APC into antigen. (A) Flow cytometry analyzed BSA levels (percent) of macrophages and DCs in lymph nodes. C57 BL/6J mice were subcutaneously injected with PBS, BSA or PP-BSA (BSA: 1.25 g), and lymph nodes were isolated and examined 6 hours after the subcutaneous injection. n=3/group. (B) The percentage of BMDC in mouse bone marrow cells after 7 days of in vitro induction of 10ng/mL rmGM-CSF and 5ng/mL rmIL-4 was determined by flow cytometry. (C) BMDCs were treated in vitro with PBS, BSA or PP-BSA, and after 2, 4, 8 hours, BSA levels (average fluorescence intensity) in BMDCs were analyzed by flow cytometry. Data are expressed as mean.+ -. SEM (n.gtoreq.5). P <0.05, < P <0.01, < P <0.001, < P < 0.0001);
fig. 3: the PP-SG vaccine can promote the maturation and phagocytosis of BMDC. BMDC was co-treated with PBS, SG, PP-S or PP-SG vaccine (containing HBsAg: 0.15. Mu.g/mL; c-di-GMP: 1.5. Mu.g/mL) and 5. Mu.g/mL FITC-BSA for 36 hours. (A) Flow cytometry determines the expression of CD86 and MHC-II and displays it as a histogram (percentage and average fluorescence intensity). (B) The phagocytosis of BMDC was analyzed by flow cytometry and the percent (%) and Mean Fluorescence Intensity (MFI) of FITC-BSA were shown as histograms. Data are expressed as mean.+ -. SEM (n.gtoreq.5). P <0.05, < P <0.01, < P <0.001, < P < 0.0001);
Fig. 4: the PP-SG vaccine can effectively remove HBV in HBV-carrying mice. HBV-bearing mice were subcutaneously injected with PBS, SG, PP-S or PP-SG vaccine (containing HBsAg: 1. Mu.g; c-di-GMP: 10. Mu.g) on days 0, 7, 14, and serum was collected one day prior to each immunization. (A) Serum HBsAg levels were determined in mice from different treatment groups by the CLIA method and expressed as relative expression. (B) RT-PCR method was used to detect HBV-DNA copy number in serum after 21 days of treatment in different treatment groups. (C) The HBV-DNA, HBV-cccDNA, HBV-total-RNA and HBV-3.5kb-RNA in the liver after 21 days of treatment in the different treatment groups were examined by RT-PCR. (D) The expression of HBcAg was analyzed by immunohistochemical staining after 21 days of treatment in the different treatment groups. (E) ELISA method detects HBsAg level in serum after different treatment groups. All data are expressed as mean.+ -. SEM (n.gtoreq.5).
Fig. 5: the PP-SG vaccine can safely eliminate HBV in HBV-carrying mice. (A) ALT concentration in serum at day 28 after PP-SG vaccine vaccination was measured in experimental mice using the Reitman-Frankel method. ALT concentration in serum was less than 40mIU/mL, indicating physiological normation. Data are expressed as mean ± SEM (n=5) (B) experimental mice were analyzed for H & E staining of liver tissue on day 28 after PP-SG vaccination.
Fig. 6: the PP-SG vaccine can prevent HBV re-infection. HBV-carrying mice treated with PBS or vaccine were again challenged with 8 μg pAAV/HBV 1.2 on day 59 post-treatment and sacrificed on day 7 post-re-challenge. (A) CLIA detects HBsAg in mice serum at day 2 and day 4 after HBV re-challenge. (B) ELISA method for detecting anti-HBs level in serum of experimental mice at day 2 and day 4 after HBV re-excitation. (C) ALT concentration in serum of experimental mice at day 7 after HBV re-challenge was measured using the Reitman-Frankel method. All data are expressed as mean ± SEM (n=5). P <0.05, < P <0.01, < P <0.001, < P < 0.0001);
fig. 7: the PP-SG vaccine can down regulate the expression of PD-L1 on HBV-carrying mouse liver DC. HBV-carrying mice treated with PBS or vaccine were re-challenged with 8 μg pAAV/HBV 1.2 on day 59 post-treatment and sacrificed on day 7 post-re-challenged. (A) Strategy for gating DC, cDC1, cDC2 from liver mononuclear cells. (B) Flow cytometry detects the expression of hepatic dendritic cells PD-L1 (MFI). (C) percentage of cDC1 and cDC2 in liver DC. (D) Flow cytometry was used to analyze PD-L1 (MFI) expression on livers cDC1 and cDC 2. All data are expressed as mean.+ -. SEM (n.gtoreq.5). P <0.05, < P <0.01, < P <0.001, < P < 0.0001);
Fig. 8: the PP-SG vaccine can activate liver DC of HBV-carrying mice. (A) Flow cytometry was used to analyze hepatic dendritic cell MHC-I, MHC-II and CD86 (MFI) expression. (B) Flow cytometry was used to analyze the expression of MHC-I, MHC-II and CD86 (MFI) on liver cDC 1. (C) Flow cytometry was used to analyze the expression of MHC-I, MHC-II and CD86 (MFI) on liver cDC 2. Data are expressed as mean.+ -. SEM (n.gtoreq.5). P <0.05, < P <0.01, < P <0.001, < P < 0.0001);
fig. 9: PP-SG vaccine can up-regulate HBV-specific CD8 of HBV-carrying mice + T cell ratio, reverse its failure. (A-B) detection of HBV-specific CD8 in liver tissue by flow cytometry + T cell ratio. (C) Analysis of HBV-specific CD8 in liver tissue by flow cytometry + Expression of T cells PD-1, LAG-3 and TIM-3 was used as histograms. (D) Single, double or triple positive HBV specific CD8 expressing PD-1, LAG-3 and TIM-3 + T cell subsets are shown in the sector (n.gtoreq.5). (xP)<0.05，**P<0.01，***P<0.001，****P<0.0001)；
Fig. 10: PP-SG vaccine can induce HBV to carry mouse CD8 + T cell proliferation and activation. (A) Flow cytometry analysis of intrahepatic HBV-specific CD8 + Proliferation of T cells and percent Ki-67 and MFI are shown as histograms. (B) Analysis of HBV-specific CD8 from liver by flow cytometry + T cell activation and ICOS percentages and MFI are shown as histograms. Data are expressed as mean.+ -. SEM (n.gtoreq.5). (xP)<0.05， **P<0.01，***P<0.001，****P<0.0001)；
Fig. 11: the PP-SG vaccine can increase CD8 + Expression of T cell functional molecules. Mononuclear cells of liver and lymph nodes 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 + IFN-gamma, TNF-alpha, perforin and IL-2 levels secreted by T cells were used as histograms. (B and D) are shown in a sector (n.gtoreq.5). (xP)<0.05，**P<0.01， ***P<0.001，****P<0.0001)；
Fig. 12: PP-SG vaccine can down regulate CD11a in liver of HBV-carrying mice + CD4 + Expression of T cell immune checkpoints. (A) Flow cytometry analysis of liver CD11a + CD4 + Expression of PD-1, LAG-3 and TIM-3 on T cells was used as histograms. (B) Single, double or triple positive CD11a expressing PD-1, LAG-3 and TIM-3 + CD4 + T cell subsets are shown as sector patterns (n.gtoreq.5). (xP)<0.05，**P<0.01，***P<0.001， ****P<0.0001)；
Fig. 13: PP-SG vaccine can activate CD4 in HBV-carrying mice + T cells, induce Th1 cellular immune responses. (A) Flow cytometry analysis of spleen viscera CD4 + Expression of Ki-67 in T cells and percentage and MFI are shown as histograms. (B) Flow cytometry analysis of spleen CD4 + ICOS expression in T cells and percentage and MFI are shown as histograms. (C) Flow cytometry analysis of CD4 in liver + IFN-gamma, TNF-alpha and IL-2 levels produced by T cells were used as histograms. (D) The pie charts show single, double or triple positive CD4 secretion of IFN-gamma, TNF-alpha and IL-2 + A subpopulation of T cells. All data are expressed as mean.+ -. SEM (n.gtoreq.5). (xP)<0.05，**P<0.01，***P<0.001，****P<0.0001)。
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. 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 present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
As mentioned above, STING agonists, due to their small molecular nature, rapidly distribute throughout the body after entry, not only being poorly absorbed by lymph node DC cells, but may even cause systemic toxicity.
The lymph node is an important immune organ of the organism, is a part of the lymphocyte which starts the immune response to the external antigen, and a large number of mature DC cells exist in the lymph node. Whether antigen and adjuvant can be engulfed by lymph node DC cells is one of the important links in determining the intensity of an immune response. Thus, targeting antigens and adjuvants to lymph node DC cells and increasing their residence time in the lymph nodes become important pathways for enhancing therapeutic vaccine effects. Whereas nanoparticles with particle sizes of 10-100nm can be passively targeted to lymph nodes through lymphatic capillaries.
In view of this, the present invention decides to optimize HBV therapeutic vaccines based on STING agonists by means of nanoparticles.
In one exemplary embodiment of the present invention, there is provided a STING agonist-based lymph node targeting nanoparticle comprising a cationic polymer micelle, and hepatitis b surface antigen HBsAg and STING agonist supported on the cationic polymer micelle;
wherein the cationic polymer micelle is polyacrylacetonoxime-poly (2- (N-ethyl-N-propylamine) ethyl methacrylate pAA-pEPEMA.
The STING agonist may be any currently known STING agonist, and in one embodiment of the present invention, the STING agonist is cyclodiguanylate c-di-GMP.
The hydration particle diameter of the nano particle is 130-150nm, the surface zeta potential is almost 0, and the nano particle is electrically neutral. Meanwhile, the shooting result of a transmission electron microscope shows that the nano particles are uniformly spherical, and the actual particle size is 30-40nm.
In still another embodiment of the present invention, there is provided a method for preparing the above-mentioned STING agonist-based lymph node targeting nanoparticle, the method comprising: adding an aqueous solution containing the poly (2- (N-ethyl-N-propylamine) ethyl methacrylate pAA-pEPEMA) into a mixed aqueous solution of the HBsAg and the STING agonist to obtain a suspension, regulating the pH of the suspension to be neutral, continuously stirring, adding the aqueous solution containing the poly (2- (N-ethyl-N-propylamine) ethyl methacrylate) pAA-pEPEMA, regulating the pH to be neutral, and continuously stirring to obtain the anti-aging agent.
Wherein, in the mixed aqueous solution of the HBsAg and the STING agonist, the mass ratio of the HBsAg to the STING agonist is 1:5-20, preferably 1:10; the STING agonist may be cyclodiguanylate c-di-GMP.
The polyacrylacetonoxime-poly (2- (N-ethyl-N-propylamino) ethyl methacrylate (pAA-pEPEMA) can be prepared by the following method:
and (3) dissolving the Polyacrylacetonoxime (PAA), (N-ethyl-N-propylamine) ethyl methacrylate (EPEMA) and 2,2' -azobis (2-methylpropanenitrile) (AIBN) in the 1, 4-dioxane, carrying out freeze thawing circulation to remove oxygen, placing the reaction solution under heating condition to initiate polymerization reaction, stirring, and then carrying out dialysis and freeze drying to obtain the product.
Wherein the molar ratio of the PAA to the EPEMA to the AIBN is 0.01-0.05:1-2:0.005-0.05, preferably 0.0376:1.505:0.0113;
the heating condition can be realized by adopting an oil bath mode, the temperature of the oil bath is controlled to be 60-80 ℃, preferably 70 ℃, and the stirring and heating time is controlled to be 24-48 hours, preferably 36 hours, so that the completion of the polymerization reaction is ensured.
The PAA may be synthesized by a known method or purchased in a commercially available manner, and will not be described herein. The synthesis method commonly used at present takes acryloylacetonoxime (AA) as a raw material and is obtained through polymerization reaction.
The EPEMA monomer is prepared by the following method: dissolving 2- (N-ethyl-N-propyl) ethanolamine and Triethylamine (TEA) in acetonitrile to obtain a mixture, stirring the mixture at a low temperature, adding methacryloyl chloride, continuously stirring at a low temperature, reacting at room temperature, and purifying the product.
Wherein the molar ratio of the 2- (N-ethyl-N-propyl) ethanolamine to the TEA is 1:0.5-5, preferably 22.90:22.93, and the low temperature condition is 0 ℃;
the specific steps of product purification comprise: filtering the product, drying the filtrate, re-dissolving the filtrate with dichloromethane to obtain a crude product, extracting an organic phase with water, collecting an organic phase layer, and drying to obtain the product.
The 2- (N-ethyl-N-propyl) ethanolamine can be prepared by the following method: and (3) placing the N-ethylethanolamine, bromopropane and sodium carbonate into ethanol, heating and stirring the mixture, and purifying the product after the reaction is finished.
Wherein the molar ratio of the N-ethylethanolamine to the bromopropane to the 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 of the heating and stirring treatment are as follows: the heating temperature is controlled to 70-90 ℃, preferably 80 ℃, and stirring is carried out for 12-36 hours, preferably 24 hours.
The specific steps of product purification comprise: filtering the product, drying the filtrate, re-dissolving the filtrate with dichloromethane to obtain a crude product, extracting an organic phase with water, collecting an organic phase layer, and drying to obtain the product.
In yet another embodiment of the present invention, there is provided the use of the above nanoparticle for the preparation of a medicament for preventing and/or treating a disease associated with Hepatitis B Virus (HBV) infection.
Wherein, the hepatitis B virus infection related diseases include, but are not limited to, acute hepatitis B, chronic hepatitis B, liver cirrhosis, liver cancer, and diseases such as glomerulonephritis, acute pancreatitis, cholangitis, cholecystitis, cardiomyopathy, and granulocytopenia which are possibly caused when the hepatitis B virus invades organs such as kidney, pancreas, gall bladder, heart, etc.; preferably chronic hepatitis B. Experiments prove that the nanoparticle can be used as an HBV therapeutic vaccine, can target lymph nodes and can increase antigen delivery; furthermore, the vaccine can effectively increase phagocytosis of BMDCsAnd antigen presenting ability. Meanwhile, the therapeutic vaccine can safely and effectively remove HBV, induce the generation of anti-HBs and prevent the reinfection of HBV; at the same time, the vaccine enhances the activation of DCs cells and reverses CD4 + T、CD8 + The function of T cells is exhausted, and the immune response function is enhanced, so that HBV in a patient is effectively cleared, and the treatment purpose is realized.
In still another embodiment of the present invention, there is provided a medicament for preventing and/or treating a disease associated with hepatitis b virus infection, the medicament comprising the above-mentioned nanoparticle as an active ingredient.
The medicament can be any type of known medicament, and in one embodiment of the invention, the medicament is a therapeutic vaccine injection. In particular, it may be in the form of a subcutaneous injection, for example, administered subcutaneously using a prime-boost immunization strategy.
According to the invention, the medicament may further comprise at least one non-pharmaceutically active ingredient, which may be any known adjuvant which meets the requirements of the pharmaceutical field, in particular for vaccine injections.
In yet another embodiment of the invention, the medicament of the invention may be administered to the body in a known manner. For example, by intravenous systemic delivery or local injection into the tissue of interest. Administration by subcutaneous injection is preferred.
It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.
In yet another embodiment of the present invention, the subject to be administered can be human or non-human mammal, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.
In yet another embodiment of the present invention, there is provided a method for preventing and/or treating a disease associated with hepatitis b virus infection, the method comprising administering to a subject a therapeutically effective dose of the nanoparticle or drug described above.
The subject may be an animal, preferably a mammal, most preferably a human, who is already the subject of treatment, observation or experiment. By "therapeutically effective amount" is meant that amount of active compound or pharmaceutical agent, including a compound of the present invention, which causes a biological or medical response in a tissue system, animal or human that is sought by a researcher, veterinarian, medical doctor or other medical personnel, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder associated with the hepatitis B virus infection being treated.
Wherein, the hepatitis B virus infection related diseases include, but are not limited to, acute hepatitis B, chronic hepatitis B, liver cirrhosis, liver cancer, and diseases such as glomerulonephritis, acute pancreatitis, cholangitis, cholecystitis, cardiomyopathy, and granulocytopenia which are possibly caused when the hepatitis B virus invades organs such as kidney, pancreas, gall bladder, heart, etc.; preferably chronic hepatitis B.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are noted, and are generally conducted under conventional conditions.
Examples
1. The test method comprises the following steps:
1. synthesis of acid sensitive polymers:
1.1 Synthesis of Acylacetonoxime, acryloylacetone oxime (AA):
AA was synthesized using a two-phase method. Acetone oxime (3.18 g,0.044 mol) dissolved in MiliQ (32.5 mL) was first placed in cold hydrazine at 0deg.C and stirred to 0deg.C. Subsequently, acryloyl chloride (4.0 g,0.044 mol) was slowly added to the above solution. After the addition, the reaction solution was allowed to warm to room temperature and stirred for 1 hour. After 1 hour, stirring was stopped and the reaction solution was allowed to stand to separate an aqueous layer and a dichloromethane layer. Extracted with dichloromethane (3X 30 mL) and collected, followed by rotary evaporation of the collected dichloromethane. The concentrated organic phase is sequentially treated with saturated sodium bicarbonate waterThe solution and water were washed. Na for organic phase 2 SO 4 And (3) after drying, removing dichloromethane by rotary evaporation to obtain AA.
1.2 Synthesis of Polymer PAA:
AA (Acryloylacetone oxime,740.7mg,5.826 mmol) and DCT (4-Cyano-4- (dodelsholtzianyiocarboyl) sulfanyl pentanoic acid,39.2mg,0.097 mmol) were dissolved in 1, 4-dioxane and placed in a Schlenk tube, after 4 freeze-thawing cycles to remove oxygen, the reaction solution was placed in an oil bath at 80℃to initiate polymerization and stirred for 3 hours, the reaction was cooled to room temperature and was stopped by dropping in a mixed solution of n-hexane: diethyl ether=3:1 (V/V) to precipitate, after centrifugation, the supernatant was discarded, the bottom precipitate was re-dissolved with methylene chloride, and the above precipitation operation was repeated two more times to obtain a final precipitate, namely PAA.
1.3 Synthesis of ethyl (N-ethyl-N-propylamino) methacrylate, 2- (N-ethyl-N-propelamino) ethyl methacrylate (EPEMA):
firstly, 2- (N-ethyl-N-propyl) ethanolamine is synthesized by the following steps of N-ethyl ethanolamine (27.42 g,0.30 mol), bromopropane (44.28 g,0.36 mol), na 2 CO 3 (47.70 g,0.45 mol) was added to the round bottom flask and dissolved with ethanol (100 mL) and heated to 80℃with continued stirring for 24 hours. After completion of the reaction, the reaction mixture was filtered, the filtrate was taken and the solvent was spin-dried, the crude product obtained was then reconstituted with Dichloromethane (DCM) (50 mL), the DCM organic phase (3X 50 mL) was extracted with ultrapure water, all the DCM organic phase was collected, the solvent was spin-dried using a rotary evaporator to obtain the product, and the solvent was spin-dried by 1 HNMR characterizes its structure. Next, the synthesized 2- (N-ethyl-N-propyl) ethanolamine (3.00 g,22.90 mmol), TEA (2.32 g,22.93 mmol) was added to a round bottom flask and dissolved with acetonitrile (20 mL), and the mixture was transferred to a low temperature constant temperature stirring reaction bath, stirred and cooled to 0 ℃. Subsequently, methacryloyl chloride (2.39 g,22.86 mmol) was added dropwise. After stirring the mixture at 0℃for 2 hours, it was reacted at room temperature for 12 hours. The reaction was stopped, filtered, the filtrate was dried by spinning, and the resulting crude product was subsequently redissolved with dichloromethane DCM (50 mL) and the organic phase was extracted with ultra pure water (3X) 50 mL), collecting DCM layer, spin drying the solvent with a rotary evaporator to obtain monomer EPEMA, and passing 1 HNMR characterizes its structure.
1.4 Synthesis of Polyacrylacetoxime-poly (ethyl 2- (N-ethyl-N-propylamino) methacrylate), poly acryloylacetoneoxime-poly (2- (N-methyl-N-proplamino) ethyl methacrylate) (pAA-pEPEMA):
pAA (211.4 mg,0.0376 mmol), EPEMA (300 mg,1.505 mmol), 2' -azobis (2-methylpropanenitrile) (AIBN) (1.85 mg,0.0113 mmol) was dissolved in 1, 4-dioxane and placed in a Schlenk tube. After removal of oxygen by 4 freeze-thaw cycles, the reaction solution was placed in an oil bath at 70 ℃ to initiate polymerization and stirred for 36 hours. The reaction solution was cooled to room temperature to stop the reaction, and then put into a dialysis bag for dialysis overnight, and pAA-pEPEMA was obtained after lyophilization.
1.5 Synthesis of Poly (2- (N-ethyl-N-propylamino) ethyl methacrylate) -polymannose, poly (2- (N-ethyl-N-proplamino) ethyl methacrylate) -Poly Mannose (Mannose-DCT-pEPEMA):
pAA is prepared 41 -pEPEMA 32 (120 mg,0.01 mmol), mannosamine hydrochloride (265.2 mg,1.23 mmol), TEA (0.379 mg,0.0374mmol) dissolved in 1, 4-dioxane, N 2 Stirring is carried out for 48 hours at 50 ℃ under protection. After the reaction is finished, cooling the reaction solution, dialyzing with purified water for 2 days, and freeze-drying to obtain brown powder Mannose-DCT-pEPEMA.
2. Preparation of the nanovaccine:
(1) hbsag+c-di-GMP (SG): HBsAg stock gauge was 20 μg (1 mL); the specification of the c-di-GMP raw material is 1 mg. First, 1mg of c-di-GMP is prepared into 1mg/mL of c-di-GMP solution using physiological saline for injection; then, 1mL of HBsAg solution was taken, 200. Mu.L of c-di-GMP solution and 2.8mL of physiological saline were added thereto, and the mixture was homogenized to obtain 4mL of vaccine solution. The prepared vaccine liquid contains HBsAg 5 mug and c-di-GMP 50 mug per milliliter. The vaccine was stored at 4 ℃.
(2) pAA-pEPEMA+HBsAg (PP-S): a concentrated stock of the polymer pEPEMA-pAA was prepared in sterile water at pH5.5 at a concentration of 20mg/mL. 54. Mu.L of HBsAg concentrate (240. Mu.g/mL) was dispersed in 1.5mL of sterile water at pH6.5. 150. Mu.L of the polymer pEPEMA-pAA was added dropwise to the above mixture with stirring, and after 10 minutes the pH of the suspension was adjusted to 7 with 0.1M NaOH solution. After stirring for 2 hours, 150. Mu.L of the polymer pEPEMA-pAA was added dropwise to the suspension, and after 10 minutes, the pH of the suspension was adjusted to 7 with 0.1M NaOH solution, and stirring was continued for 6 hours to obtain a nanoparticle system.
(3) pAA-pEPEMA+HBsAg+c-di-GMP (PP-SG): a concentrated stock of the polymer pEPEMA-pAA was prepared in sterile water at pH5.5 at a concentration of 20mg/mL. 54. Mu.L of HBsAg concentrate (240. Mu.g/mL) and 130. Mu.L of c-di-GMP concentrate (1 mg/mL) were each dispersed in 1.5mL of sterile water at pH6.5 (mass ratio of HBsAg to c-di-GMP 1:10). 150. Mu.L of the polymer pEPEMA-pAA was added dropwise to the above mixture with stirring, and after 10 minutes the pH of the suspension was adjusted to 7 with 0.1M NaOH solution. After stirring for 2 hours, 150. Mu.L of the polymer pEPEMA-pAA was added dropwise to the suspension, and after 10 minutes, the pH of the suspension was adjusted to 7 with 0.1M NaOH solution, and stirring was continued for 6 hours to obtain a nanoparticle system.
3. Characterization of nanovaccine:
the nanovaccines were characterized by Dynamic Light Scattering (DLS) to determine their hydrated particle size and zeta potential. The morphology of the nanovaccine was observed by transmission electron microscopy.
4. Separation and extraction of mononuclear cells of liver, spleen and lymph node
4.1 isolation and extraction of liver mononuclear cells: mice are sacrificed, livers, spleens and lymph nodes are respectively picked, crushed and ground on a 200-mesh screen, a filter screen is washed by 1 XPBS (pH 7.4), filtrate is transferred into a 15mL centrifuge tube, and the corresponding mononuclear cells are obtained through operations such as adding percoll solution for resuspension, red blood cell lysis, resuspension and the like.
4.2 BMDCs induction in vitro: c57 BL/6J mice were sacrificed under aseptic conditions, and the femur and tibia on both sides were immersed in sterile 1 XPBS (pH 7.4) solution. Cutting small holes on both ends of femur and tibia with scissors, sucking sterile 1 XPBS (pH 7.4) solution with 1mL syringe, penetrating into bone marrow cavity via small holes, flushing bone marrow, repeating for 3 times, collecting bone marrow cell suspension, centrifuging, and re-suspending after erythrocyte lysis (adding penicillin and cytogene into 1640 complete culture mediumSub rmIL-4 and rmGM-CSF) to obtain corresponding suspension cells, 37 ℃ and 5% CO 2 Cells were cultured for 48 hours. The culture broth was gently aspirated and the suspension cells were removed from the 6-well plate. To this was again added 1640 complete medium containing penicillin and cytokines, and 37℃was continued with 5% CO 2 Cells were cultured until day five. Half-volume liquid exchange is carried out on the complete culture medium 1640 containing the streptomycin and the cytokines, the culture is continued until the seventh day, and all suspended and adherent cells are collected by gentle blowing, thus obtaining the enriched BMDCs.
5. Vaccine lymph node targeting and antigen delivery capability detection
5.1 FACS detection of uptake of vaccine antigens by lymph node APCs
C57 BL/6J mice, 5-6 weeks old, were divided into 3 groups, and 1.25. Mu.g of free FITC-BSA (BSA group), a nano-preparation containing 1.25. Mu.g of FITC-BSA (PP-BSA group) or a sterile 1 XPBS (pH 7.4) solution (PBS group) of the same volume was injected subcutaneously. Mice were sacrificed 6 hours after dosing, mouse lymph node mononuclear cells were isolated, stained with the corresponding flow antibody, and levels of FITC-BSA on individual groups of mouse lymph node DC cells and macrophages were detected by FACS.
5.2 FACS detection of uptake of vaccine antigens by BMDCs
Induced BMDCs were divided into 3 groups, each stimulated with free FITC-BSA or "PP-BSA" and the control group was added with the same volume of sterile 1 XPBS (pH 7.4) solution. At 2, 4, 8 hours of stimulation, BMDCs were collected after stimulation, stained with the corresponding flow antibody, and FACS detected FITC-BSA levels on BMDCs.
5.3 FACS detection of phagocytosis and antigen presentation Capacity of BMDCs
Induced BMDCs were divided into 4 groups, stimulated with a physical mixture of free HBsAg and c-di-GMP (SG group), a nanovaccine with HBsAg (PP-S group) or a nanovaccine with HBsAg and c-di-GMP (PP-SG group) respectively (concentration of HBsAg: 0.15. Mu.g/mL; c-di-GMP: 1.5. Mu.g/mL) and 5. Mu.g/mL FITC-BSA was added to each group. After 36 hours, stimulated cells were collected, stained with the corresponding flow antibody and FACS detected MHC-II molecules, CD86 and FITC-BSA levels on BMDCs.
6. Construction of HBV-carrier mouse model
Taking C57BL/6J mice of 5-6 weeks old, injecting 8 mug of pAAV/HBV1.2 plasmid into tail vein at high pressure, taking peripheral blood after 5-6 weeks to separate serum, and detecting the HBsAg level in the peripheral blood serum of the mice, wherein the serum HBsAg concentration is higher than 500ng/mL, which is HBV-carrier mice successfully molded.
7. Immunization strategies
HBV-carrier mice were divided into 4 groups, and the experimental groups were SG group (physical mixture of free HBsAg and c-di-GMP), PP-S group (nano vaccine containing HBsAg) and PP-SG group (nano vaccine containing HBsAg and c-di-GMP), respectively; the control group was PBS group (sterile 1 XPBS solution). Each mouse of the experimental group was subcutaneously injected with 1 μg of SG, PP-S or PP-SG vaccine group containing HBsAg, immunized three times at a time interval of one week, and peripheral blood was taken and serum was isolated on the day before each immunization, and stored at-20 ℃ for later use.
8. HBV reinfection
On day 59 after the start of the treatment of the above 4 groups of mice, 8. Mu.g of pAAV/HBV 1.2 plasmid (challenge) was injected again into the tail vein of the mice at high pressure, and peripheral blood was taken on days 61 and 63 to isolate serum, which was stored at-20℃for use.
Mouse tail-breaking blood-taking and serum-separating
(1) The mice were fixed in a holder under sterile conditions, exposing the tails of the mice. The mice were gently rubbed with 70% alcohol to disinfect their tails.
(2) The tail end of the mouse was cut at about 2mm, and the tail was massaged from the tail root to the tail tip, and the blood flowing out of the tail tip was collected.
(3) Standing at room temperature for 30 min, centrifuging at 3000rpm for 15 min, collecting supernatant, and storing at-80deg.C.
9. CLIA method for detecting serum HBsAg level of mouse peripheral blood
(1) Serum samples were diluted as required for the experiment.
(2) mu.L of sample or standard (0, 0.05, 0.8, 10, 85, 250 ng/mL) was added to the corresponding coated wells, respectively.
(3) 50 μl of enzyme conjugate was added to each well, mixed with gentle shaking, sealed with plate membrane, and incubated at 37deg.C for 1 hr.
(4) After the incubation, the sealing plate membrane is removed, the liquid in the wells is forced to be thrown away, the coated plate is washed by HBsAg washing liquid, the washing is repeated for 5 times, and finally the coated plate is patted dry as much as possible.
(5) Luminescent substrate a and luminescent substrate B were mixed according to 1: mixing in proportion of 1. 50 mu L of the mixed luminescent substrate is added into the coating hole, the mixture is gently shaken, and the mixture is kept away from light at room temperature for 10 minutes.
(6) And detecting the luminous intensity by using a Synergy2 multifunctional enzyme-labeled instrument, making a standard curve, and calculating the concentration of HBsAg in the sample.
10. ELISA (enzyme-Linked immunosorbent assay) for detecting serum HBsAb level of mouse peripheral blood
(1) Serum samples were diluted as required for the experiment.
(2) 50. Mu.L of sample or standard (10, 20, 40, 80, 100, 160 mIU/mL) was added to the corresponding coated wells, respectively.
(3) 50 mu L of enzyme-labeled reagent is added into each hole, the mixture is gently shaken and mixed, a sealing plate film is attached, and the mixture is incubated for 1 hour at 37 ℃.
(4) After the incubation, the sealing plate membrane is removed, the liquid in the wells is forced to be thrown away, the coated plate is washed by HBsAb washing liquid, the washing is repeated for 5 times, and finally the coated plate is patted dry as much as possible.
(5) 50 mu L of each of the color development liquid A and the color development liquid B is sequentially added into the coating hole, and the mixture is gently shaken and mixed uniformly, and the mixture is kept away from light for 15 minutes at 37 ℃.
(6) Adding 50 mu L of stop solution into the coating hole, gently shaking and uniformly mixing, detecting the absorbance at 450nm/630 nm by using a Synergy2 multifunctional enzyme-labeled instrument, taking a standard curve, and calculating the concentration of HBsAb in the sample.
11. Extraction of genomic DNA, RNA and reverse transcription of liver tissue
Respectively performing liver tissue DNA/RNA detection, peripheral blood serum DNA detection and peripheral blood serum ALT detection (a rice method), diluting a serum sample according to experimental requirements, detecting a 510nm wavelength absorbance value by a Synergy2 multifunctional enzyme-labeled instrument, and calculating ALT/GPT activity units according to a standard curve.
12. Liver tissue section and staining
Mice were sacrificed and liver tissue of appropriate size was fixed in 4% paraformaldehyde overnight. Washing, dehydrating, transparentizing, wax dipping, embedding, slicing, dewaxing, hydrating, dyeing, dehydrating, transparentizing, and performing lens sealing and microscopic examination.
13. Immunohistochemical detection of HBcAg expression
Paraffin sections were dewaxed, hydrated, HBcAg primary antibody working solution was added dropwise, incubated overnight in a wet box at 4 ℃ and washed 3 times with PBST solution. The secondary antibody working solution coupled with biotin is dripped, incubated for 20 minutes at 37 ℃ in a wet box, and washed 3 times with PBST solution. HRP-conjugated streptavidin working solution was added dropwise, incubated in a wet box for 15 min at 37 ℃ and washed 3 times with PBST solution. DAB is dripped for 3-5 minutes, the dyeing is observed under a mirror, and the solution is washed by deionized water. Hematoxylin is stained for 5 minutes, 0.1% ethanol hydrochloride is differentiated for 10 seconds, and the mixture is placed into a lithium carbonate solution to return to blue for 10 minutes. Dehydrating, transparent, and performing microscopic examination on the neutral resin sealing piece.
14. Flow cytometry
14.1 cell surface molecule detection
(1) To the mononuclear cell suspension 10% by volume of rat serum was added and blocked for 30 minutes at 4 ℃.
(2) The corresponding diluted flow antibody was added, stained at 4℃for 30 minutes, centrifuged at 1PBS,400rcf for 5 minutes, and the supernatant was discarded.
(3) Flow cytometer detection, flowJo10 software analysis data.
14.2 Molecular detection of IFN-gamma, TNF-alpha, performin and IL-2
(1) Preparing a culture medium for lymphocyte in vitro stimulation: 1640 complete medium containing PMA (30 ng/mL), ionomycin (1. Mu.g/mL), IL-2 (100U/mL) and BFA (5. Mu.g/mL).
(2) The isolated mononuclear cells were resuspended in the above medium, plated at 37℃with 5% CO 2 Stimulation was performed for 4 hours.
(3) Cells were collected, 10% by volume of rat serum was added, and the mixture was blocked at 4℃for 30 minutes.
(4) The corresponding diluted external standard flow antibody was added, stained at 4℃for 30 minutes, 1 XPBS (pH 7.4) was added, centrifuged at 400rcf for 5 minutes, and the supernatant was discarded.
(5) 100. Mu.L of 1% paraformaldehyde solution was added, the mixture was fixed at 4℃for 30 minutes in the absence of light, 1 XPBS (pH 7.4) was added, and the mixture was centrifuged at 400rcf for 5 minutes, and the supernatant was discarded.
(6) 100. Mu.L of the membrane-penetrating fluid and 10% by volume of rat serum were added thereto and allowed to stand at 4℃for 30 minutes in the dark.
(7) The corresponding diluted internal standard flow antibody was added, stained at 4℃for 30 minutes, 1 XPBS (pH 7.4) was added, centrifuged at 400rcf for 5 minutes, and the supernatant was discarded.
(8) Flow cytometer detection, flowJo10 software analysis data.
14.3 detection of Nuclear molecules
(1) To the mononuclear cell suspension 10% by volume of rat serum was added and blocked for 30 minutes at 4 ℃.
(2) The corresponding diluted external standard flow antibody was added, stained at 4℃for 30 minutes, centrifuged at 1PBS,400rcf for 5 minutes, and the supernatant was discarded.
(3) Preparing a nucleus penetrating related solution: with 10X Permeabilization Buffer and ddH 2 O was formulated as 1 XBuffer in a 1:9 ratio. Preparing the fixed nucleus penetrating liquid by using the fixed nucleus penetrating concentrated liquid and the fixed nucleus penetrating diluent according to the proportion of 1:3.
(4) After completion of the external standard, 1mL of 1 XBuffer was added to each tube, and the supernatant was discarded (without discarding the supernatant) by centrifugation at 400rcf for 5 minutes.
(5) To the flow tube with 1 XBuffer remaining, 300. Mu.L of the immobilization through-core solution was added, and the mixture was allowed to stand at room temperature for 30 minutes.
(6) 1mL of 1 XBuffer was added to each tube, and the supernatant was discarded (without discarding) by centrifugation at 400rcf for 5 minutes.
(7) The diluted internal standard antibody was added to a flow tube with 1 XBuffer remaining, and stained at room temperature for 30 minutes. Add 1×pbs, centrifuge at 400rcf for 5 min, discard supernatant.
(8) Flow cytometer detection, flowJo10 software analysis data.
2. Results
2.1 Physicochemical Properties of PP-SG vaccine
First, we measured the particle size and zeta potential of the PP-SG vaccine. As shown in FIGS. 1A and B, the hydration particle size of the nano vaccine is 130-150nm, the surface zeta potential is almost 0, and the nano vaccine is electrically neutral. Meanwhile, the transmission electron microscope shooting result shows that the nano vaccine is in a uniform sphere shape, and the actual particle size is between 30 and 40nm (figure 1C).
2.2 PP-SG vaccine can target lymph node and increase uptake of APCs into antigen
After the antigen is ingested by APCs at the lymph nodes, the antigen can be recognized by T cells after processing, and further subsequent immune response is induced. It is therefore important whether the novel nanoformulation promotes phagocytosis of antigens by lymph node APCs. We examined levels of FITC-BSA in lymph node DCs and macrophages in mice 6 hours after subcutaneous administration using flow cytometry. The results are shown in FIG. 2A, where the level of phagocytic BSA by macrophages and DC cells in the lymph nodes of the PP-BSA group mice was significantly increased compared to the mice injected with the free BSA group. In addition, to further demonstrate that the nanoformulations can increase the efficiency of antigen delivery into DC cells, we induced BMDCs in vitro and treated BMDCs with free BSA antigen and PP-BSA, respectively, and tested the levels of BSA in BMDCs at 2, 4, 8 hours of treatment. As shown in fig. 2B and C, BSA levels in PP-BSA group BMDCs were significantly higher than free BSA group at 4, 8 hours of treatment, indicating that higher levels of BSA entered the BMDCs, i.e., the nanoformulation increased the BSA delivery efficiency. The results show that the novel nano vaccine can increase the phagocytosis of APCs in lymph nodes to antigens, and is beneficial to inducing the cellular immune response of organisms.
2.3 The PP-SG vaccine can promote the maturation and phagocytic function of BMDCs
DC cells are one of the important APCs, being the bridge between innate immunity and adaptive immunity. The literature has shown that DC cells are useful for activating CD8 + T cell mediated cellular immune responses are critical. The STING agonist can activate DC cell by activating cGAS-STING channel in DC cell to induce IFN-I and ISG expression, so as to regulate immunity of organism and establish antiviral and antitumor immune state. To observe the effect of the novel nanovaccine on maturation and activation of BMDCs, we induced BMDCs in vitro, and subsequently stimulated BMDCs with SG group (physical mixture of free HBsAg and free c-di-GMP), PP-S group (nanovaccine with HBsAg) or PP-SG vaccine group (novel nanovaccine with HBsAg and c-di-GMP), respectively, simultaneously with FIT during the treatmentC-labeled BSA antigen to assess the effect of the vaccine in promoting phagocytosis of antigens by BMDCs. By flow cytometry we examined the expression of the important marker MHC-II molecules and the costimulatory molecule CD86 for surface maturation and activation of BMDCs treated for 36 hours. As shown in FIG. 3A, although the SG and PP-S groups increased the expression of MHC-II and CD86 to some extent compared to the PBS-treated groups, the PP-SG vaccine groups treated BMDCs had more significant upregulation of MHC-II and CD86 than the other groups. Meanwhile, by detecting the level of BSA antigen in BMDCs, we found that the level of BSA antigen in BMDCs treated with PP-SG vaccine group was higher than that in other groups (FIG. 3B), indicating that PP-SG vaccine group treatment was effective in increasing the phagocytosis of FITC-labeled BSA by BMDCs. These results indicate that the PP-SG vaccine group can promote maturation and activation of DC cells, and enhance phagocytosis and antigen presentation functions of DC cells.
2.4 The PP-SG vaccine group can safely and effectively clear HBV and prevent HBV from reinfection
2.4.1 The PP-SG vaccine group can effectively remove HBV
To evaluate the therapeutic effect of the PP-SG vaccine group on CHB, we injected C57BL/6J mice with 8 μg of pAAV/HBV1.2 plasmid at high pressure in the tail vein, and established HBV-carrier mouse models. Previous studies demonstrated that the pAAV/HBV1.2 plasmid injected by this method can be long-term and continuously expressed in mouse liver, HBV-carrier mouse serum constructed by this method highly expressed HBsAg, and that there are a variety of HBV DNA including HBV replication intermediate HBV-cccDNA. Afterwards, we immunized HBV-carrier mice with SG group, PP-S group or PP-SG vaccine group, respectively. We used a "Prime-boost" (Prime-boost) immunization strategy to immunize mice, subcutaneously injected mice on day 0, then re-immunized on days 7 and 14, and mice sacrificed on day 28 for a series of immunological evaluations. First, we used the CLIA method to measure the relative expression levels of HBsAg in the serum of peripheral blood of each group of HBV-carrier mice during treatment. As shown in fig. 5A, the HBsAg expression in the peripheral blood of the mice treated with SG, PP-S or PP-SG vaccine was significantly reduced, and the effect was hardly detectable after three immunizations and maintained at least until one week after the treatment. In addition, q The PCR results showed a significant decrease in serum HBV-DNA, intrahepatic HBV-DNA, HBV-cccDNA, HBV-total-RNA and HBV-3.5kb-RNA levels in mice after vaccine treatment (FIGS. 4B and C). Meanwhile, compared with PBS group, vaccine treatment group HBcAg + The number of hepatocytes was also significantly reduced (fig. 4D). Although the SG, PP-S or PP-SG vaccine groups were effective in clearing HBV from HBV-carrier mice, the PP-SG vaccine group treatment induced the highest level of protective anti-HBs compared to the other groups (fig. 4E). 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.
2.4.2 The PP-SG vaccine group can safely remove HBV
After completion of the evaluation of vaccine effectiveness, we again evaluated the safety of the PP-SG vaccine group. We examined the levels of serum ALT in the peripheral blood of mice after treatment and observed the morphology of liver tissue of mice after treatment by H & E staining to evaluate whether the vaccine would cause liver damage to mice. Serologic ALT results indicated that mouse liver function was not affected during vaccine treatment (fig. 5A). At the same time, no significant liver damage was observed in the staining results of liver H & E sections (fig. 5B). These results indicate that the PP-SG vaccine group can safely clear HBV.
2.4.3 The PP-SG vaccine group can prevent HBV from re-infection
Afterwards, we evaluated whether the PP-SG vaccine group could induce the body to produce long-term immunological memory, preventing HBV reinfection. We used a "prime-boost" immunization strategy to immunize HBV-carrier mice with SG, PP-S or PP-SG vaccine groups, and re-injected mice with 8 μg of pAAV/HBV1.2 plasmid (re-challenge) at high pressure in the tail vein on day 59 post-treatment. We examined HBsAg levels in mouse peripheral blood serum 2 days and 4 days after re-challenge. The results showed that the number of mice highly expressing HBsAg in peripheral blood was minimal in the PP-SG vaccine group mice compared to the other groups (FIG. 6A). Meanwhile, the results of detection of anti-HBs showed that the PP-SG vaccine group induced the highest level of protective antibodies, which was beneficial for the neutralization of invasive HBV in the body (FIG. 6B). Similarly, we also examined serum ALT levels in mice after re-challenge, as shown in FIG. 6C, with serum ALT levels normal. The results show that the PP-SG vaccine group can induce the organism to generate long-term immune memory for resisting HBV infection and prevent HBV from re-infection.
2.5 The PP-SG vaccine group can enhance the function of DC cells in HBV-carrier mice
2.5.1 PP-SG vaccine group can down regulate expression of PD-L1 on DC cell surface in HBV-carrier mouse
One of the important mechanisms of HBV immune escape is to up-regulate the expression of the DC cell surface inhibitory molecule PD-L1 by affecting the function of the host DC cell, and down-regulate the expression of the surface MHC-II, MHC-I molecules, and co-stimulatory molecules CD80, CD86, CD40 and the like, so as to inhibit the maturation and activation of the DC cell, thereby damaging the immune function of the organism and further causing chronic infection and developing liver diseases caused by the fact that viruses cannot be cleared in patients. Based on this, we first examined the effect of the PP-SG vaccine group on DC cell surface PD-L1 expression in HBV-carrier mice. The results showed that HBV-carrier mice had decreased liver DC cell PD-L1 after treatment with SG, PP-S or PP-SG vaccine groups, which showed the most significant decrease in liver DC cell PD-L1 expression compared to PBS groups (FIGS. 7A and B).
Since DC cells are divided into different subsets, wherein cDC1 is directed primarily to CD8 + T cells present antigen, cDC2 is directed primarily to CD4 + T cells present antigens and the function of these sub-populations directly influences the activation of the adaptive immune response of the body. We therefore further examined the proportion of DC cell subsets cDC1 and cDC2 and their surface PD-L1 expression levels after vaccine treatment. The results indicate that, although PP-SG nanovaccine treatment did not increase the ratio of dcs 1 and dcs 2, the expression level of PD-L1 on the dcs 1, dcs 2 surfaces could be significantly reduced (fig. 7A, C and D). These results indicate that the PP-SG vaccine group can down-regulate the expression of the DC cell surface inhibitor molecule PD-L1 in HBV-carrier mice, is favorable for reversing the immunosuppression microenvironment caused by CHB and helps the organism generate an anti-HBV immune response.
2.5.2 The PP-SG vaccine group can promote the maturation and activation of DC cells in HBV-carrier mice
Afterwards, we evaluated whether the PP-SG vaccine group could promote maturation and activation of DC cells 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 DC cells after vaccine treatment. The results show that the expression of MHC-II and MHC-I molecules on the surfaces of mouse liver DC cells is obviously increased after the treatment of SG group, PP-S group or PP-SG vaccine group compared with PBS group, and the increase of PP-SG vaccine group is most obvious. However, i found that there appeared to be no significant difference between the PP-SG vaccine group and the other vaccine treatment groups when the DC cell surface co-stimulatory molecule CD86 level was detected (fig. 8A). Further, the results of the detection of DC cell subsets cDC1, cDC2 showed that the PP-SG vaccine group not only increased the levels of MHC-II, MHC-I molecules, but also up-regulated the expression of the co-stimulatory molecule CD86 compared to the other groups (FIGS. 8B and C). The results show that the PP-SG vaccine group can promote the maturation and activation of DC cells in HBV-carrier mice.
2.6 The PP-SG vaccine group can increase antigen-specific CD8 + Proportion of T cells and reversal of their depleted state
CD8 during HBV elimination process + T cells, in particular CD11a hi CD8α lo T cells have a critical role, this group of CD8 + T cells are also known as antigen-specific CD8 + T cells. Thus, we evaluated whether the PP-SG vaccine group could increase antigen-specific CD8 in HBV-carrier mice + T cell ratio. As shown in fig. 9A and B, the SG, PP-S or PP-SG vaccine groups mice liver antigen specific CD8 compared to the PBS group + There was a significant increase in the proportion of T cells, but the PP-SG vaccine group showed no advantage over the SG group or the PP-S group. There is literature showing that antigen-specific CD8 in patients with CHB + T cells are often in a state of functional exhaustion with significantly up-regulated immune checkpoint expression at their surfaces, PD-1, 2B4, TIM-3, LAG-3, CTLA-4, and the like. These immune checkpoints are closely related to the function of T cells, and T cells that highly express immune checkpoints cannot normally clear HBV infected cells. An ideal therapeutic HBV vaccine should be able to reverse antigen-specific CD8 in patients with CHB + T cellTo restore its normal function, we further examined HBV-carrier mouse antigen-specific CD8 after vaccine treatment + Expression of T cell surface immune checkpoints. The results showed that HBV-carrier mouse liver antigen specific CD8 after vaccine treatment of SG group and PP-S group compared with PBS group + T cells PD-1, LAG-3 and TIM-3 expression were significantly reduced, while the effect of the PP-SG vaccine group on downregulating these immune checkpoints was more pronounced. At the same time, we counted the antigen-specific CD8 of PD-1, LAG-3, TIM-3 single positive, double positive and triple positive in the livers of four groups of mice + T cell ratio, results show that the PP-SG vaccine group depleted antigen-specific CD8 + T cell ratios were lowest. From the above results, it can be seen that the PP-SG vaccine group can increase antigen-specific CD8 + The proportion of T cells and reverse the exhaustion state of the T cells are favorable for breaking the immune tolerance microenvironment of the organism.
2.7 The PP-SG vaccine group can promote CD8 + T cell proliferation and activation and up-regulation of its antiviral function
2.7.1 The PP-SG vaccine group can promote CD8 + T cell proliferation and activation
The key to the action of HBV therapeutic vaccine is to promote the production of CD8 by human body + T cell mediated cellular immune response, therefore, whether we could promote CD8 for the PP-SG vaccine group + T cell proliferation and activation were evaluated. We examined mice spleen CD8 after vaccine treatment + Expression levels of Ki-67 and the surface Co-stimulatory molecule ICOS in the T cell nucleus, which reflect CD8, respectively + Proliferation level and activation degree of T cells. As shown in FIGS. 10A and B, the SG group, PP-S group mice spleen CD8 compared to the PBS group + Positive rate and MFI levels of Ki-67 and ICOS in T cells were significantly increased, whereas the PP-SG vaccine group could even further enhance CD8 + Expression of T cell Ki-67 and ICOS molecules. The results show that the PP-SG vaccine group can promote CD8 + T cell proliferation and activation.
2.7.2 The PP-SG vaccine group can up-regulate CD8 + Expression of T cell functional molecules
CD8 during the process of eliminating virus infection in human body + T cells can release cytotoxicity related molecules perforin, granzymeB and the like, form holes on virus-infected cell membranes, change the osmotic pressure of target cells, and promote the entry of killer molecules, thereby killing virus-infected cells. In addition, CD8 + T cells can also secrete cytokines such as IFN-gamma, TNF-alpha, IL-2 and the like, and directly or indirectly play an antiviral role. These functional molecules also reflect CD8 + T cells kill the target cells. Thus, to demonstrate that novel nanovaccines based on SITNG agonists can enhance CD8 + Function of T cells we collected liver lymphocytes and after stimulation with PMA/Ionomycin, CD8 was detected by flow cytometry + T cells secrete IFN-gamma, TNF-alpha, performin and IL-2 levels. As shown in FIG. 11A, the liver CD8 of mice was significantly increased after treatment with SG, PP-S or PP-SG vaccine groups compared to PBS groups + The expression levels of T cells IFN-gamma, TNF-alpha, performin and IL-2, and the PP-SG vaccine group showed significant advantages over the other two vaccine treatment groups. Meanwhile, we also counted the multifunctional CD8 expressing multiple functional molecules after vaccine treatment + T cell ratio. As shown in fig. 11B, three vaccine-treated mice had liver multifunctional CD8 relative to PBS group + The T cell ratio was increased, and the PP-SG vaccine group showed the best effect. Furthermore, to further demonstrate this phenomenon, we have again used the same method to detect lymph node CD8 + Expression of functional molecules on T cells. The detection result is similar to that of liver, and compared with other groups, the PP-SG vaccine group significantly up-regulates lymph node CD8 + IFN-gamma, TNF-alpha, performin and IL-2 expression levels on T cells and increased multifunctional CD8 + T cell ratio (fig. 11C and D). The results show that the PP-SG vaccine group can up-regulate CD8 + Expression of T cell functional molecules to enhance CD8 + Killing and antiviral functions of T.
2.8 The PP-SG vaccine group can regulate CD4 + Function of T cells
2.8.1 The PP-SG vaccine group can reduce CD4 + Expression of T cell surface inhibitory molecules
During the course of the HBV infection,CD4 + t cells can participate in the anti-HBV immune response of the body by modulating the activity and function of other immune cells. The literature shows that CD4 + T cells can direct to CD8 + T cells provide activating factors to assistCD8 + T cells transition to effector T cells. CD4, on the other hand + T cells can also assist in activating B cells, promoting the production of neutralizing antibodies anti-HBs. However, with CD8 + T cells are similar, CD4 in CHB patients + T cells also have the phenomenon of high expression immune checkpoints, resulting in CD4 + T cell function is impaired. I therefore want to investigate whether the PP-SG vaccine group could also down-regulate HBV-carrier mouse CD4 + Expression level of immune checkpoint of T cells, improving CD4 + Phenomenon of impaired T cell function. For this, we examined mouse liver antigen-specific CD4 after vaccine treatment + Expression levels of immune checkpoints such as PD-1, LAG-3 and TIM-3 on the surface of T cells. The results show that the SG group, the PP-S group or the PP-SG vaccine group effectively reduce the CD4 of the liver of mice after treatment compared with the PBS group + Levels of the T cell surface PD-1, LAG-3 and TIM-3 molecules, and the PP-SG vaccine group showed the lowest expression levels compared to the other groups (FIG. 12A). Similarly, we also counted the antigen-specific CD4 for PD-1, LAG-3, TIM-3 single positive, double positive and triple positive in mouse livers + T cell ratios, as shown in FIG. 12B, PP-SG vaccine groups expressed antigen-specific CD4 of PD-1, LAG-3 or TIM-3 compared to all other groups + T cell fraction was minimal. These results demonstrate that the PP-SG vaccine group can reduce CD4 + Expression of T cell surface inhibitory molecules, reversing HBV-caused CD4 + T cell function is impaired.
2.8.2 The PP-SG vaccine group can activate CD4 + T cells and induction of Th1 type cellular immune response
Further, we evaluated the PP-SG vaccine group to promote CD4 + T cell proliferation and activation capacity. We examined mice spleen CD4 after vaccine treatment + T cell Ki-67 and ICExpression level of OS molecules. The results showed that treatment with SG, PP-S or PP-SG vaccine group significantly up-regulated spleen CD4 + The percentage of T cells Ki-67 and ICOS molecules was the highest compared to MFI and PP-SG vaccine groups up-regulated compared to other vaccine treated groups (FIGS. 13A and B). This suggests that the PP-SG vaccine group promotes CD4 + Proliferation and activation of T cells. CD4 + T cells differentiate into functionally distinct subsets, such as Th1, th2, th17, etc., after activation. Wherein Th1 type cells are capable of assisting in CD8 + T cell differentiation to CTL, helping CD8 + T cells clear viral infection. Th1 type cells play a role in regulating cellular immune response mainly by secreting cytokines such as IFN-gamma, TNF-alpha and IL-2. Thus, we examined mouse CD4 after vaccine treatment + Levels of T cells IFN-gamma, TNF-alpha and IL-2. As shown in fig. 13C, three vaccine-treated groups mice liver CD4 compared to PBS group + The levels of IFN-gamma, TNF-alpha and IL-2 expression were elevated in T cells and the PP-SG vaccine group also showed superior therapeutic efficacy compared to the other two vaccine treatment groups. Similarly, we also counted the multifunctional CD4 in the livers of mice in each group + T cell ratio, as shown in the sector, of PP-SG vaccine group multifunctional CD4 + The T cell fraction was highest (FIG. 13D). From the above results, the PP-SG vaccine group can promote CD4 + Proliferation and activation of T cells and induction of Th1 type cellular immune response, facilitating helper CD8 + T cells clear HBV.
Chronic HBV is difficult to cure mainly because it can escape from immune surveillance of the body in a variety of ways in the patient, including inhibiting the activity of APCs such as DC cells, macrophages, up-regulating the expression of immune checkpoints such as PD-1, LAG-3 on the surface of T cells, blocking co-stimulatory signals between DC cells and T cells, enhancing co-inhibitory signals, interfering with the normal antiviral immune response of the body, etc. The long-term coexistence of HBV and T cells caused by the inability to timely clear HBV further induces the development of T cells to immune tolerance, resulting in the depletion of T cells. In addition, HBV can up-regulate expression of cells or molecules inhibiting immune system functions such as Treg and IL-10, and the like, and these factors together create an immunosuppressive microenvironment in the patient. Thus, reversing the immunosuppressive microenvironment, activating the functional activity of immune cells is critical for the treatment of chronic HBV with therapeutic vaccines.
Existing studies indicate that activation of DC cells in lymph nodes is responsible for activating CD8 in the body + The T cell mediated cellular immune response is very important suggesting that targeting antigens and adjuvants to DC cells in lymph nodes will help the vaccine to better activate the immune system, exerting an effect of scavenging HBV. cGAS-STING is one of the pathways of activating cells in DC cells, and is a very potential target for activating cellular immunity. Thus, we selected the STING agonist c-di-GMP (as an adjuvant, in combination with HBsAg to construct a new HBV therapeutic vaccine). However, therapeutic vaccines constructed by physical mixing of HBsAg with c-di-GMP do not possess lymph node targeting, and c-di-GMP, due to its small molecule nature, rapidly distributes throughout the body after entry into the body, and is difficult to stay in the lymph nodes for a sufficient period of time, while high doses may cause systemic inflammatory responses that are detrimental to vaccine performance. Although intra-lymph node injection can alleviate the situation to a certain extent, the method still has the limitations of complex operation, smaller administration dosage and the like. Based on this we have chosen nano-drug delivery technology to address this problem. The nanovaccine has the unique advantages that: on one hand, due to the particle size of the nano particles, the nano particles can enter lymph nodes through capillary lymphatic vessels, so that the lymph node targeting of the vaccine is effectively enhanced; on the other hand, nano-drug delivery systems can protect the carried antigen from the in vivo environment, safely deliver antigen and adjuvant to APCs. Moreover, since the size and composition of the nanoparticle are similar to those of cells, the nanoparticle can be rapidly taken up by the cells through pinocytosis, and has good antigen delivery efficiency. There have been studies to date that nanoparticles carrying HBsAg have a greater capacity to induce antibody production than normal HBsAg. In addition, the slow biodegradation rate of the nano-vaccine after entering the APCs is beneficial to the continuous existence of the antigen in the cells, prolongs the time of the APCs for treating the antigen, and is beneficial to starting the subsequent adaptive immune response. Based on this information we recognize For the purpose of constructing a novel HBV therapeutic nano vaccine based on c-di-GMP by using nano administration technology, the novel HBV therapeutic nano vaccine has unique advantages in terms of HBV elimination, strong induction and long-acting immune effect. Thus, we prepared the nano-vaccine, PP-SG vaccine group, from HBsAg and c-di-GMP using pAA-pema material.
By using fluorescent molecule FITC-labeled BSA as antigen, we demonstrate that the nanovaccine can target lymph nodes in vitro and in vivo, and promote antigen delivery, and prolong the duration of antigen 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), a HBsAg-containing nanovaccine (PP-S) as a control, evaluated the effect of the PP-SG vaccine group on HBV clearance, and studied the effect of the PP-SG vaccine on immune response. These experimental results show that HBV vaccines prepared by using nano-dosing technology can overcome the weakness of c-di-GMP, effectively targeted delivery of antigen and adjuvant into lymph node DC cells. After c-di-GMP as an adjuvant enters DC cells, the expression of IFN-I and some interferon stimulatory genes can be stimulated by activating the cGAS-STING signaling pathway, thereby activating DC cells and enhancing their antigen presenting function, which is clearly advantageous for the subsequent generation of adaptive immune responses.
The "prime-boost" immunization strategy is advantageous for the vaccine to better exert the effect of activating the immune system, so we also immunized HBV-carrier mice in this way, i.e. by three times a week, and taken the mouse peripheral blood serum the day before each immunization for analysis of HBsAg relative expression levels in the serum. The research results of the application show that the relative expression of the HBsAg can be effectively reduced in the serology level in the SG group, the PP-S group or the PP-SG vaccine group, but the clearance effect of the nano vaccine PP-SG prepared by combining the HBsAg and the c-di-GMP is poorer in the physical mixture of the HBsAg and the c-di-GMP or the nano preparation only containing the HBsAg from the results of the liver tissue RNA and DNA levels; meanwhile, the PP-SG vaccine also induced the highest level of serum anti-HBs relative to other vaccine treatment groups. Furthermore, we examined the vaccine-induced long-term immune memory function, and reinfected HBV-carrier mice after vaccine treatment, and the results showed that PP-SG vaccine-treated groups showed the lowest serum HBsAg and the highest serum anti-HBs levels relative to other vaccine groups. It is clear that the use of nanocarriers to combine c-di-GMP with HBsAg has certain advantages in scavenging HBV from HBV-carrier mice.
DC cells are the most important antigen presenting cells. For chronic HBV patients, impaired function of DC cells is a significant cause of T cell failure to be activated. DC cells are divided into plasma cell-like DCs (pDC) and myeloid DCs (mDC), which in turn exist in two subsets, cDC1 and cDC2, respectively, with different DC cell subsets having different functions. cDC1 has cross-presentation function, and can deliver exogenous antigen through MHC-I molecule, and induce CD8 + The primary antigen presenting cells of the T cell immune response are critical to the establishment of an antiviral state in the body. cDC2 is directed to CD4 primarily through MHC-II + T cells deliver antigen and play a corresponding auxiliary role in the humoral immune effect of the body. We found that PP-SG vaccine treatment can effectively down-regulate expression of PD-L1 on the surface of DC cells and sub-populations thereof, and shows absolute advantages in overall DC cells. Activation of CD8 due to a cDC1 subset + Important role of T cell immune response we also focused on the effect of the vaccine on the dcs 1 subpopulation. Although the PP-SG vaccine does not raise the proportion of cDC1, it is still effective in reducing the level of PD-L1 on the surface of cDC 1. In addition, PP-SG vaccine treatment significantly activated mouse liver-derived DC cells and sub-populations thereof, with the highest degree of activation among all vaccine treatment groups. Similarly, PP-SG vaccine also has an activating effect on spleen-derived DC cells and subpopulations thereof, but PP-SG vaccine does not show a significant advantage over PP-S vaccine in spleen-derived DC cells. Overall, the effect of PP-SG vaccine on activating DC cells and their subpopulations remained the most excellent of the three vaccine treatment groups. In addition, similar results were obtained from in vitro experiments. These results are consistent with their ability to clear HBV, prevent relapse and induce anti-HBs.
CD8 + T cells are one of the cells that directly exert antiviral function, in which antigen-specific CD8 + T is thinCells are important for the body to clear HBV, and the cell immune response mediated by T cells directly acts on HBV. Thus, reversing antigen-specific CD8 + The depletion state of T is one of the important targets for breaking the immune tolerance microenvironment of chronic HBV patients. The three vaccines of SG group, PP-S group or PP-SG vaccine group can up-regulate antigen specific CD8 in liver of HBV-carrier mouse + There was no significant difference in T cell ratio between the three vaccine treatment groups. However, further detection of antigen-specific CD8 + When T surface PD-1, LAG-3, TIM-3 and other immune checkpoints are detected, the PP-SG vaccine can obviously lower the expression of the immune checkpoints and has obvious advantages compared with other vaccines. This indicates that PP-SG vaccine can better block the transmission of co-suppression signals than other vaccine groups, breaking the state of T cell depletion. Furthermore, the PP-SG vaccine treatment can also effectively induce CD8 + T cells secrete IL-2, IFN-gamma, TNF-alpha, and performin effector molecules, which are also better than other vaccine treatment groups.
CD4 + T cells have auxiliary effects on eliminating HBV and inducing neutralizing antibody production, and CD4 of chronic HBV patient + Impaired T cell function is also one of the reasons for reduced intensity of immune response. PP-SG vaccine treatment also down-regulates CD4 compared to other vaccine treatment groups + Expression of immune checkpoints such as PD-1, LAG-3 and TIM-3 on the surface of T cells and promotion of activation and proliferation thereof. Notably, the PP-SG vaccine upregulated CD4 + The expression of cytokines such as T cell IL-2, IFN-gamma, TNF-alpha and the like shows that the PP-SG vaccine promotes Th1 type cellular immune response and is beneficial to the organism to produce antiviral cellular immune response. Therefore, we consider that the PP-SG vaccine can restore the function of the exhausted T cells by reversing the co-suppression signal between DC cells and T cells, thereby breaking the microenvironment of the body immunosuppression and promoting the body to clear HBV.
Taken together, the PP-SG vaccine can promote CD8 by activating DC cells and subpopulations thereof + T、CD4 + T cell proliferation, activation and expression of functional molecules, breaking the body's immunosuppressive microenvironment and reversing antigen specificityThe depletion state of T cells, thereby inducing the organism to generate HBV specific cellular immune response to clear HBV, and inducing long-term immunological memory to prevent HBV from reinfecting. Meanwhile, compared with vaccines of the SG group and the PP-S group, the PP-SG vaccine has absolute advantages and has optimal treatment effect. The results show that the PP-SG vaccine has strong potential as HBV therapeutic vaccine, and provides a new strategy and thought for clinically curing chronic HBV patients.
The invention is not a matter of the known technology.
The above embodiments are provided to illustrate 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 the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (10)
1. A STING agonist-based lymph node targeting nanoparticle, wherein the nanoparticle comprises a cationic polymer micelle, and hepatitis b surface antigens HBsAg and STING agonist loaded on the cationic polymer micelle;
wherein the cationic polymer micelle is polyacrylacetonoxime-poly (2- (N-ethyl-N-propylamine) ethyl methacrylate pAA-pEPEMA.
2. The nanoparticle of claim 1, wherein the STING agonist is cyclic diguanylate c-di-GMP.
3. The nanoparticle of claim 1, wherein the nanoparticle has a hydrated particle size of 130-150nm and is electrically neutral; the nano particles are in uniform spherical shape, and the actual particle size is 30-40nm.
4. A method of preparing STING agonist-based lymph node targeting nanoparticles according to any of claims 1 to 3, comprising: adding an aqueous solution containing the poly (2- (N-ethyl-N-propylamino) ethyl methacrylate pAA-pEPEMA) into a mixed aqueous solution containing the HBsAg and the STING agonist to obtain a suspension, regulating the pH of the suspension to be neutral, continuously stirring, adding the aqueous solution containing the poly (2- (N-ethyl-N-propylamino) ethyl methacrylate pAA-pEPEMA), regulating the pH to be neutral, and continuously stirring.
5. The method of claim 4, wherein the mass ratio of HBsAg to STING agonist in the aqueous mixture of HBsAg and STING agonist is 1:5-20, preferably 1:10; the STING agonist is cyclodiguanylate c-di-GMP.
6. The method of claim 4, wherein the polyacrylacetone oxime-poly (ethyl 2- (N-ethyl-N-propylamino) methacrylate pAA-pEPEMA is prepared by:
dissolving Polyacrylacetonoxime (PAA), (N-ethyl-N-propylamine) ethyl methacrylate EPEMA and 2,2' -azo-bis (2-methylpropanenitrile) (AIBN) in 1, 4-dioxane, after freeze thawing and cyclic deoxidization, placing the reaction liquid under heating condition to initiate polymerization reaction and stirring, and then dialyzing and freeze-drying to obtain the product;
preferably, the molar ratio of PAA, EPEMA and AIBN is 0.01-0.05:1-2:0.005-0.05, more preferably 0.0376:1.505:0.0113;
preferably, the heating condition is realized by adopting an oil bath mode, the temperature of the oil bath is controlled to be 60-80 ℃, more preferably 70 ℃, and the stirring heating time is controlled to be 24-48 hours, more preferably 36 hours.
7. The preparation method of claim 4, wherein the EPEMA monomer is prepared by the following method: dissolving 2- (N-ethyl-N-propyl) ethanolamine and Triethylamine (TEA) in acetonitrile to obtain a mixture, stirring the mixture at a low temperature, adding methacryloyl chloride, continuously stirring at a low temperature, reacting at room temperature, and purifying the product to obtain the catalyst;
Preferably, the molar ratio of the 2- (N-ethyl-N-propyl) ethanolamine to TEA is 1:0.5-5, more preferably 22.90:22.93, and the low temperature condition is 0 ℃;
the specific steps of product purification comprise: filtering the product, drying the filtrate, re-dissolving the filtrate with dichloromethane to obtain a crude product, extracting an organic phase with water, collecting an organic phase layer, and drying to obtain the product.
8. Use of the nanoparticle according to any one of claims 1 to 3 for the preparation of a medicament for the prophylaxis and/or treatment of diseases associated with hepatitis b virus infection.
9. The use according to claim 8, wherein the hepatitis b virus infection-related diseases include acute hepatitis b, chronic hepatitis b, cirrhosis and liver cancer, and glomerulonephritis, acute pancreatitis, cholangitis, cholecystitis, cardiomyopathy and granulocytopenia caused when hepatitis b virus invades organs such as kidney, pancreas, gall bladder, heart; preferably chronic hepatitis B.
10. A medicament for preventing and/or treating a disease associated with hepatitis b virus infection, the active ingredient of which comprises the above nanoparticle;
preferably, the medicament is a therapeutic vaccine injection.
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