DNA vaccine for preventing and treating echinococcosis multilocularis and preparation method thereof

Abstract

The invention discloses a preparation method of a DNA vaccine for preventing and treating echinococcosis multilocularis. The preparation method comprises the following steps: (1) preparing a nucleic acid vaccine plasmid (pVAX1T / B); (2) synthesizing chitosan selenium nano particles (CS-SeNPs); (3) obtaining a stable transfection cell strain for expressing a DEC 205 antibody on the surface of a cell membrane through a cell membrane display technology, crushing the cell through an ultrasonic crusher, and then separating through a density gradient centrifugation method to obtain cell membrane nano-vesicles; (4) mixing the pVAX1T / B obtained in the step (1) with the CS-SeNPs obtained in the step (2) under the condition of room temperature, so as to obtain a chitosan selenium nanoparticle loaded nucleic acid vaccine compound (pVAX1T / B coated CS-SeNPs); and (5) carrying out ultrasonic mixing on the cell nano-vesicles extracted in the step (3) and the pVAX1T / B-coated CS-SeNPs obtained in the step (4), and carrying out membrane extrusion on the cell nano-membrane vesicles so that the pVAX1T / B-coated CS-SeNPs are entrapped by the cell nano-membrane vesicles, thereby obtaining the chitosan selenium nanoparticles (NVs pVAX1T / B-coated CS-SeNPs) entrapped by the cell membrane vesicles. The DNA vaccine can be used for preventing and treating alveolar echinococcosis.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA vaccines, and in particular to a DNA vaccine for preventing and treating multilocular echinococcosis and a preparation method thereof. Background Art

[0002] Echinococcosis, also known as echinococcosis, is a serious zoonosis caused by infection with the larvae of Echinococcus tapeworm. It is mainly divided into two types: cystic echinococcosis and alveolar echinococcosis. Among them, alveolar echinococcosis (AE) is a zoonosis parasitic helminth disease caused by infection with the tapeworm larvae of Echinococcus multilocularis (Em), so it is also called multilocular echinococcosis. The disease is distributed all over the world, but China is one of the countries with the most serious epidemic of the disease, mainly in livestock and poultry areas such as Gansu, Ningxia, Qinghai, and Xinjiang.

[0003] Multilocular echinococcosis is a serious disease with complex clinical symptoms, which may be accompanied by symptoms such as liver pain, shortness of breath, and chest pain. Multilocular echinococcosis can grow infiltratively throughout the liver within 1 to 2 years, and can also metastasize to the lungs and brain through the blood circulation and lymphatic vessels. It is extremely harmful and is called "worm cancer" because of its tumor-like growth and metastasis ability. The liver is the organ with the highest incidence of echinococcosis. Patients with hepatic echinococcosis have no obvious clinical symptoms in the early stage. Most patients are diagnosed only in the late stage of echinococcosis survey, which seriously affects the health of people in pastoral areas. At present, the only reliable conventional treatment strategy for this disease is surgical removal of the parasitic mass, supplemented by drug therapy. Since most patients have multifocal lesions and are diffusely distributed in the liver, less than one-third of patients can undergo surgical resection. For patients who cannot undergo surgical resection, long-term use of albendazole is required to prevent further growth and spread of the lesions. In clinical applications, due to the poor water solubility and low bioavailability of albendazole, the drug can only achieve the purpose of "inhibiting insects" rather than "killing insects". Although many new technologies have been used to treat patients with multilocular echinococcosis and have achieved certain therapeutic effects, the overall treatment effect of multilocular echinococcosis patients is still unsatisfactory.

[0004] The transmission of Echinococcus multilocularis mainly occurs in the wild animal cycle between the definitive host such as foxes and the intermediate host such as rodents. Echinococcus multilocularis parasitizes in the small intestine of the definitive host, and the gravid segments and eggs are excreted in the feces. The intermediate host is infected by ingesting food or water contaminated with the eggs. The eggs hatch into hexacanthellae in the small intestine, which invade the liver through the bloodstream and develop into alveolar larvae. When mice or animal organs carrying alveolar larvae are ingested by definitive hosts such as foxes, dogs and wolves, the protoscolex can generally develop into adults and excrete gravid segments and eggs after 45 days. Humans become accidental hosts of Echinococcus multilocularis by mistakenly ingesting vegetables and raw water containing eggs. However, there are currently no effective measures for the prevention and control of wild definitive hosts such as foxes, wolves and jackals.

[0005] In summary, at present, there is neither an effective drug for the prevention and treatment of Echinococcus multilocularis disease nor an effective measure for the prevention and treatment of the wild terminal hosts of Echinococcus multilocularis in the existing technology. Therefore, vaccines are the most economical and effective way to control the disease.

[0006] However, currently, no effective vaccine for preventing and treating Echinococcosis has been reported in the prior art. Summary of the invention

[0007] The present invention provides a DNA vaccine for preventing and treating multilocular echinococcosis and a preparation method thereof, which can effectively solve the above problems.

[0008] The present invention is achieved in that:

[0009] A method for preparing a DNA vaccine for preventing and treating multilocular echinococcosis comprises the following steps:

[0010] (1) The dominant epitopes of Echinococcus multilocularis were concatenated and cloned into the pVAX1 vector to prepare a nucleic acid vaccine plasmid (pVAX1 T / B);

[0011] (2) Prepare chitosan solution with acetic acid, add ascorbic acid solution dropwise, stir and mix, and finally add Na2SeO3 solution, continue stirring, and when the color changes from colorless to dark red, the synthesis of chitosan selenium nanoparticles (CS-SeNPs) is completed, and then dialyze the CS-SeNPs with ultrapure water for 22-26 hours, filter with a 0.22 μm pore size filter membrane after dialysis, and store at 4°C;

[0012] (3) obtaining a stable transfected cell line expressing DEC 205 antibody on the cell membrane surface by cell membrane display technology, culturing the cells to a certain amount, collecting the cells and washing them twice with pre-cooled PBS, then scraping the cells, adding PBS containing protease inhibitors and sodium deoxycholate surfactant, resuspending the cells, breaking the cells by ultrasonic disruptor, and then separating the cell membrane nanovesicles by density gradient centrifugation;

[0013] (4) mixing the pVAX1 T / B obtained in step (1) with the CS-SeNPs obtained in step (2) at room temperature to obtain a chitosan selenium nanoparticle-loaded nucleic acid vaccine complex (pVAX1T / B@CS-SeNPs);

[0014] (5) Ultrasonic mixing the cell nanovesicles extracted in step (3) and the pVAX1T / B@CS-SeNPs obtained in step (4), and then encapsulating the cell nanomembrane vesicles with pVAX1T / B@CS-SeNPs by membrane extrusion to obtain cell membrane vesicle-encapsulated chitosan selenium nanoparticles (NVs pVAX1T / B@CS-SeNPs), i.e., the DNA vaccine for preventing and treating multilocular echinococcosis.

[0015] In some embodiments, in step (1), the dominant epitopes of Echinococcus multilocularis include LAP, 14-3-3, EMY162 and TSP3.

[0016] In some embodiments, in step (2), the mass fraction of the chitosan solution is 0.05-0.15%, the concentration of the ascorbic acid solution is 0.20-0.25M, and the concentration of the Na2SeO3 solution is 0.45-0.55M; the volume ratio of the chitosan solution, ascorbic acid solution, and Na2SeO3 solution is 70-80:35-40:1.

[0017] In some embodiments, in step (3), the operation of obtaining a stably transfected cell line expressing the DEC 205 antibody on the cell membrane surface by cell membrane display technology is: obtaining the DEC 205 antibody gene sequence, fusing the transmembrane sequence of PDGFR at the C-terminus of the heavy chain constant region, introducing a Human Fc Tag tag between the heavy chain constant region and the transmembrane sequence, and constructing the sequence into a plasmid vector; using the plasmid vector to prepare a lentivirus, and obtaining a stably transfected cell line expressing the DEC 205 antibody on the cell membrane surface by lentiviral transfection.

[0018] In some embodiments, in step (3), the density gradient centrifugation technique is as follows: ultrasonically disrupt the cells at 22.5 W for 15 s, place on ice for 3 min, repeat 4 times, and after ultrasonic disruption, centrifuge the suspension at 2000 rpm and 4°C for 10 min to remove the cytoplasm and nucleus; retain the supernatant and further centrifuge at 5000 rpm and 4°C for 10 min to remove the organelles; finally, centrifuge the supernatant at 15000 rpm and 4°C for 1 h to separate the precipitate, which is the cell membrane nanovesicle.

[0019] In some embodiments, in step (4), the mass ratio of CS-SeNPs to pVAX1 T / B is 0.8-1.2:1.

[0020] In some embodiments, in step (5), the mass ratio of the cell membrane nanovesicles to pVAX1T / B@CS-SeNPs is 0.8-1.2:1.

[0021] A DNA vaccine prepared by the above method for preventing and treating multilocular echinococcosis.

[0022] A use of the above DNA vaccine in the preparation of a DNA vaccine preparation for preventing and treating multilocular echinococcosis.

[0023] A cell membrane nanovesicle, the preparation method of which is as follows: a stable transfected cell line expressing a DEC 205 antibody on the cell membrane surface is obtained by cell membrane display technology, the cells are cultured to a certain amount, the cells are collected and washed twice with pre-cooled PBS, then the cells are scraped off, PBS containing a protease inhibitor and a sodium deoxycholate surfactant is added, the cells are resuspended, the cells are broken by an ultrasonic disruptor, and then the cell membrane nanovesicles are separated by density gradient centrifugation.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a DNA vaccine for preventing and treating multilocular echinococcosis, which is stabilized by selenium nanoparticles and targeted to dendritic cells (DCs) through cell membrane vesicles displaying DEC 205 antibodies as delivery vectors, so that exogenous DNA can activate the STING pathway in BMDCs and stimulate BMDCs maturation, DNA is transcribed and translated in DCs, and antigen-specific CD8+T cells are activated through endogenous presentation, inducing the body to produce a strong and lasting immune response, thereby achieving the purpose of preventing and treating alveolar echinococcosis.

[0026] The present invention utilizes electrostatic adsorption to combine CS-SeNPs with pVAX1 T / B, thereby increasing the DNA encapsulation rate and effectively improving the stability of the DNA vaccine and the lysosomal escape efficiency.

[0027] The chitosan selenium nanoparticles of the present invention can inhibit DCs from producing ROS and prolong the in vivo survival time of DCs.

[0028] The DNA vaccine for preventing and treating multilocular echinococcosis overcomes the current situation of lack of immunotherapy for alveolar echinococcosis, can be mass-produced, and is easy to replace antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The dominant epitope sequences are the codon-optimized LAP, 14-3-3, EMY162, and TSP3 of the Echinococcus multilocularis tapeworm in the embodiments of the present application.

[0031] Figure 2 TEM image, particle size and potential of NVs pVAX1 T / B@CS-SeNPs in the examples of this application.

[0032] Figure 3 This is the protective effect of CS-SeNPs on pVAX1 T / B in the examples of this application.

[0033] Figure 4 It is the encapsulation efficiency of pVAX1 T / B@CS-SeNPs in the examples of this application.

[0034] Figure 5 In the example of this application, DC2.4 cells take up NVs pVAX1T / B@CS-SeNPs through DEC 205 receptors.

[0035] Figure 6 In the examples of this application, NVs pVAX1 T / B@CS-SeNPs can induce BMDCs maturation.

[0036] Figure 7 In the examples of this application, NVs pVAX1 T / B@CS-SeNPs can activate the STING pathway of BMDCs.

[0037] Figure 8 In the examples of this application, NVs pVAX1 T / B@CS-SeNPs can be translated in BMDCs.

[0038] Fig. 9 In the examples of this application, NVs pVAX1 T / B@CS-SeNPs can inhibit the production of ROS in BMDCs.

[0039] Fig.10 NVs pVAX1 T / B@CS-SeNPs were enriched in mouse lymph nodes in the examples of this application.

[0040] Fig.11In the present application, flow cytometry was used to detect the maturation level of DCs in lymph nodes and CD8 + T, CD4 + The proportion of T cells and the level of related cytokine secretion and humoral immunity were detected by ELISA.

[0041] Fig.12 The flow cytometry method was used to detect the proportion of lymphocytes in the liver in the mouse alveolar echinococcosis treatment model in the examples of the present application.

[0042] Fig.13 The ultrastructural changes of the lesion site in the mouse alveolar echinococcosis treatment model in the examples of this application.

[0043] Fig.14 This is the in vivo safety evaluation of NVs pVAX1 T / B@CS-SeNPs in mice in the examples of this application. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0045] The present invention is further described in detail below in conjunction with embodiments and drawings, but the implementation method of the present invention is not limited thereto.

[0046] The present invention provides a method for preparing a DNA vaccine for preventing and treating multilocular echinococcosis, comprising the following steps:

[0047] (1) The dominant epitopes of Echinococcus multilocularis were concatenated and cloned into the pVAX1 vector to prepare a nucleic acid vaccine plasmid (pVAX1 T / B);

[0048] (2) Prepare chitosan solution with acetic acid, add ascorbic acid solution dropwise, stir and mix, and finally add Na2SeO3 solution, continue stirring, and when the color changes from colorless to dark red, the synthesis of chitosan selenium nanoparticles (CS-SeNPs) is completed, and then dialyze the CS-SeNPs with ultrapure water for 22-26 hours, filter with a 0.22 μm pore size filter membrane after dialysis, and store at 4°C;

[0049] (3) obtaining a stable transfected cell line expressing DEC 205 antibody on the cell membrane surface by cell membrane display technology, culturing the cells to a certain amount, collecting the cells and washing them twice with pre-cooled PBS, then scraping the cells, adding PBS containing protease inhibitors and sodium deoxycholate surfactant, resuspending the cells, breaking the cells by ultrasonic disruptor, and then separating the cell membrane nanovesicles by density gradient centrifugation;

[0050] (4) mixing the pVAX1 T / B obtained in step (1) with the CS-SeNPs obtained in step (2) at room temperature to obtain a chitosan selenium nanoparticle-loaded nucleic acid vaccine complex (pVAX1T / B@CS-SeNPs);

[0051] (5) Ultrasonic mixing the cell nanovesicles extracted in step (3) and the pVAX1T / B@CS-SeNPs obtained in step (4), and then encapsulating the cell nanomembrane vesicles with pVAX1T / B@CS-SeNPs by membrane extrusion to obtain cell membrane vesicle-encapsulated chitosan selenium nanoparticles (NVs pVAX1T / B@CS-SeNPs), i.e., the DNA vaccine for preventing and treating multilocular echinococcosis.

[0052] The DNA vaccine is encapsulated by chitosan selenium nanoparticles, which can significantly improve its stability and lysosomal escape efficiency. It is then delivered to DCs via cell membrane vesicles displaying the DEC 205 antibody, inducing a potent and lasting immune response in the body, thereby achieving the purpose of preventing and treating alveolar echinococcosis.

[0053] In certain embodiments, step (1) involves identifying and selecting dominant epitopes with significant immunogenicity in Echinococcus multilocularis, which play a key role in immune response. Specifically, these dominant epitopes include LAP (cysteine ​​protease-like antigen protein), 14-3-3 (a multifunctional protein involved in multiple signal transduction pathways), EMY162 (a protein closely related to the growth and development of parasites), and TSP3 (a protein that may be involved in regulating host immune response).

[0054] In certain embodiments, with regard to the operational details of step (2), the mass fraction of the chitosan solution is set within the range of 0.05% to 0.15%. Such a concentration helps to ensure that the chitosan can be evenly dispersed in the solution, while not causing the solution to be too viscous due to excessive concentration, thereby affecting the subsequent reaction process. As for the ascorbic acid solution, its concentration is precisely controlled within the range of 0.20 to 0.25 mol / l (M). Such a concentration range can effectively serve as a reducing agent to promote the reduction reaction of the selenium element while avoiding the side reactions that may be produced due to excessive concentration. For the Na2SeO3 solution, its concentration is set within the range of 0.45 to 0.55 mol / l (M). This concentration range can ensure that there are enough selenium sources to participate in the reaction while avoiding the unstable effects that may be caused to the reaction system due to excessive selenium concentration.

[0055] When preparing these solutions, the volume ratio of chitosan solution, ascorbic acid solution and Na2SeO3 solution is precisely controlled within the range of 70-80:35-40:1. Such a volume ratio helps to ensure that the proportions of the components in the reaction system are appropriate, so that chitosan can fully react with selenium to form stable nanoparticles. If the proportion of chitosan solution is too high, the chitosan concentration in the reaction system may be too high, affecting the formation and stability of nanoparticles; and if the proportion of ascorbic acid solution is too high, the reduction reaction may be too intense, affecting the size and morphological distribution of the nanoparticles. Therefore, by precisely controlling the volume ratios of these solutions, the synthesis process of nanoparticles can be effectively controlled to obtain nanoparticles with desired properties.

[0056] In certain embodiments, the specific step (3) involves the process of obtaining a transfected cell line that stably expresses the DEC 205 antibody on the cell membrane surface by cell membrane display technology, which can be described in detail as follows: First, the gene sequence of the DEC 205 antibody is obtained, and then the transmembrane sequence of the platelet-derived growth factor receptor (PDGFR) is fused to the C-terminus of the heavy chain constant region of the gene sequence. A Human Fc Tag tag is introduced between the heavy chain constant region and the transmembrane sequence to enhance the stability and expression efficiency of the antibody. Next, the modified gene sequence is constructed into a plasmid vector to form a recombinant plasmid containing the target gene. Then, the recombinant plasmid is used to prepare lentiviral particles, and the lentiviral particles containing the DEC 205 antibody gene are introduced into the host cell by a lentiviral-mediated transfection method. Finally, through screening and cultivation, a transfected cell line that can stably express the DEC 205 antibody on the cell membrane surface is obtained. This process not only ensures the correct expression of the antibody gene, but also improves the transfection efficiency and stability of the cell line through lentiviral transfection technology, providing a reliable cell model for subsequent cell membrane display and antibody function research.

[0057] In some embodiments, in step (3), the density gradient centrifugation technique is as follows: ultrasonically disrupt the cells at 22.5 W for 15 s, place on ice for 3 min, repeat 4 times, and after ultrasonic disruption, centrifuge the suspension at 2000 rpm and 4°C for 10 min to remove the cytoplasm and nucleus; retain the supernatant and further centrifuge at 5000 rpm and 4°C for 10 min to remove the organelles; finally, centrifuge the supernatant at 15000 rpm and 4°C for 1 h to separate the precipitate, which is the cell membrane nanovesicle.

[0058] In certain specific embodiments, in step (4), the weight ratio of the prepared chitosan-selenium nanoparticles (CS-SeNPs) to the carrier pVAX1 T / B is set between 0.8 and 1.2. Such a ratio is conducive to the formation of a stable complex between the nanoparticles and the carrier, thereby ensuring the best effect in subsequent biological applications.

[0059] In some embodiments, in step (5), the weight ratio of cell membrane nanovesicles to CS-SeNPs loaded with pVAX1 T / B is also set between 0.8 and 1.2. The selection of this ratio is based on the results of experimental optimization, aiming to ensure that the nanovesicles can effectively encapsulate the pVAX1 T / B@CS-SeNPs complex while maintaining its stability and biological activity in the body, so as to play its intended role in the treatment or prevention of the disease. A DNA vaccine for the prevention and treatment of multilocular echinococcosis prepared by the above method.

[0060] A use of the above DNA vaccine in the preparation of a DNA vaccine preparation for preventing and treating multilocular echinococcosis.

[0061] The present invention relates to a cell membrane nanovesicle, and its preparation method utilizes cell membrane display technology, and first obtains a stable transfected cell line expressing DEC 205 antibody on the cell membrane surface. The specific steps include: first, the cells are cultured to a certain number, then the cells are collected, and they are washed with pre-cooled phosphate buffered saline (PBS), and repeated twice to ensure the removal of impurities. Next, the cells are scraped from the culture medium and transferred to PBS containing protease inhibitors and sodium deoxycholate surfactant to prevent cell protein from being degraded and to help separate the cell membrane. Afterwards, the cell suspension is resuspended, and the cells are crushed using an ultrasonic crusher to release the cell contents while retaining the cell membrane structure. Finally, the crushed cell suspension is separated by density gradient centrifugation to obtain pure cell membrane nanovesicles.

[0062] This cell membrane nanovesicle has similar biocompatibility and bioactivity to the original cell membrane, and can effectively encapsulate and protect DNA vaccines to prevent them from being degraded by enzymes in the body, thereby improving the stability and immune effect of the vaccine. In addition, since DEC 205 antibodies are expressed on the surface of cell membrane nanovesicles, they can specifically bind to the DEC205 receptors on the surface of dendritic cells to enhance the targeted delivery and immunogenicity of the vaccine. Therefore, this cell membrane nanovesicle has broad application prospects in vaccine delivery systems, especially in improving the delivery efficiency and immune response of DNA vaccines.

[0063] Example 1

[0064] A preparation method for a DNA vaccine for preventing and treating multilocular echinococcosis comprises the following steps:

[0065] (1) Preparation of nucleic acid vaccine plasmid. The T and B dominant epitopes of LAP, 14-3-3, EMY162, and TSP3 of Echinococcus multilocularis were concatenated and codon optimized ( Figure 1 a is the T epitope, Figure 1 b is B epitope), the sequence was synthesized and cloned into pVAX1 vector (pVAX1 T / B), as Figure 1 c.

[0066] (2) Preparation of chitosan selenium nanoparticle (CS-SeNPs) solution. Prepare chitosan solution with acetic acid, then add ascorbic acid solution dropwise, stir and mix, finally add Na2SeO3, continue stirring, when the color changes from colorless to dark red, it indicates that CS-SeNPs are synthesized, then use dialysis bag to dialyze CS-SeNPs with ultrapure water for 24h, filter with a 0.22μm pore size filter membrane after dialysis, and store at 4℃. The mass fraction of chitosan solution is 0.1%, 15mL, and it needs to be prepared and used immediately. The ascorbic acid concentration is 0.23M, add 7.5mL, stir for 30min, add Na2SeO3 dropwise to 0.51M, add 200μL.

[0067] (3) Preparation of cell membrane vesicles. The DEC 205 antibody sequence (GenBank: AGH72060.1) was constructed into the vector. At the C-terminus of the heavy chain constant region (CH), the transmembrane domain (TM) of PDGFR was fused to ensure that the antibody could be anchored on the cell membrane. VH was added to the N-terminus of CH, which is the antibody heavy chain variable region gene, and VL is the antibody light chain variable region gene. In order to detect the correct assembly of the light chain and heavy chain on the cell membrane surface, we introduced the HumanFc Tag tag between CH and TM. After preparing lentivirus with the plasmid, a stably transfected cell line expressing DEC 205 antibody on the cell membrane surface was obtained by lentiviral transfection, and the cells were cultured in a 100 mm cell culture dish. When the cell confluence reached about 90%, the cells were collected and washed twice with cold PBS, the cells were scraped off with a cell scraper, 1 mL of PBS containing 1% PMSF protease inhibitor and 5% sodium deoxycholate surfactant was added, and the cells were resuspended in a sterile centrifuge tube, the cells were broken by an ultrasonic disruptor, and then the cell membrane nanovesicles were separated by density gradient centrifugation. Density gradient centrifugation technology is to use an ultrasonic disruptor to sonicate the cells at 22.5W for 15s, let them stand on ice for 3min, and repeat 4 times. After ultrasonic disruption, the suspension is centrifuged at 2000rpm and 4℃ for 10min to remove the cytoplasm and nucleus; the supernatant is retained and further centrifuged at 5000rpm and 4℃ for 10min to remove the organelles; finally, the supernatant is centrifuged at 15000rpm and 4℃ for 1h to separate the precipitate, which is the cell membrane nanovesicles.

[0068] (4) Preparation of chitosan selenium nanoparticle-loaded nucleic acid vaccine complex (pVAX1T / B@CS-SeNPs). The pVAX1 T / B obtained in step (1) and the CS-SeNPs obtained in step (2) were mixed at room temperature to obtain pVAX1 T / B@CS-SeNPs. The CS-SeNPs were mixed with the nucleic acid vaccine at a mass ratio of 1:1.

[0069] (5) Preparation of cell membrane vesicles loaded with chitosan selenium nanoparticles (NVs pVAX1 T / B@CS-SeNPs nanoparticles) displaying DEC 205 antibody. The cell vesicles extracted in step (3) were ultrasonically mixed with the pVAX1 T / B@CS-SeNPs obtained in step (4), and then the cell membrane vesicles were loaded with pVAX1 T / B@CS-SeNPs by membrane extrusion to obtain NVs pVAX1 T / B@CS-SeNPs. The cell membrane vesicles and pVAX1 T / B@CS-SeNPs were mixed at a mass ratio of 1:1.

[0070] The morphology of NVs pVAX1 T / B@CS-SeNPs was observed by transmission electron microscopy, and the hydrated particle size and Zeta potential of NVs pVAX1 T / B@CS-SeNPs were measured by Malvern laser particle size analyzer at room temperature. The experimental results are shown in Figure 2 As shown. Figure 2 It can be seen that the synthesized CS-SeNPs are round, with a hydrated particle size of about 200nm, have a high specific surface area and are easily phagocytosed by antigen-presenting cells; they are negatively charged, with an average potential of about -15mV, and can deliver antigens to lymph nodes more efficiently.

[0071] Verify that CS-SeNPs can increase the stability of pVAX1 T / B. The same concentration of pVAX1T / B@CS-SeNPs, pVAX1 T / B (100 μg / mL) and 1 active unit of DNase I were incubated at 37°C and gel electrophoresis was used to verify the protective effect of CS-SeNPs on pVAX1 T / B. The experimental results are shown in the figure. Figure 3 As shown. Figure 3 It can be seen that CS-SeNPs can improve the stability of DNA vaccines.

[0072] The vesicles expressing the DEC 205 antibody (anti-DEC205 NVs) were labeled with DiD and pVAX1 was labeled with FITC. The encapsulation efficiency was verified by nanoflow cytometry. The experimental results are shown in 4. Figure 4 It can be seen that CS-SeNPs can improve the stability of DNA vaccines and increase the encapsulation rate.

[0073] Example 2 DC2.4 uptake experiment on NVs pVAX1 T / B@CS-SeNPs

[0074] First, DC2.4 cells (mouse bone marrow-derived dendritic cells) were cultured at 2×10 4 After the cells were inoculated in a confocal culture dish, DEC 205 antibody (1:200 dilution) was added to the antibody blocking group after the cells adhered to the wall and cultured overnight. Subsequently, NVs VAX1 T / B@CS-SeNPs (where the final concentration of pVAX1 T / B was 50 ng / mL) and pVAX1 T / B (final concentration of 50 ng / mL) were added to the unblocked DC2.4 cells, and NVs VAX1T / B@CS-SeNPs (where the final concentration of pVAX1 T / B was 50 ng / mL) were added to the blocked DC2.4 cells and incubated for 24 hours, which verified that NVs pVAX1 T / B@CS-SeNPs entered the cells through the DEC 205 receptor-mediated endocytosis pathway on the surface of the DC2.4 cell membrane ( Figure 5 ).

[0075] Example 3NVs pVAX1 T / B@CS-SeNPs stimulate BMDCs maturation experiment

[0076] The mature bone marrow-derived dendritic cells (BMDCs) will show increased expression of antigen differentiation clusters CD80 and CD86 and high expression of MHC-II on the cell membrane surface. Therefore, the maturity level of BMDCs can be indirectly inferred by detecting the expression levels of antigen differentiation clusters and MHC-II on the BMDC cell surface.

[0077] BMDCs were extracted from the bone marrow of C57BL / 6J mice and 3-6×10 6 / mL BMDCs cells were seeded in 6-well plates, and then NVs pVAX1 T / B@CS-SeNPs (where the final concentration of pVAX1 T / B was 1 μg / mL) were added and cultured for 24 h. Figure 6 As shown in the figure, flow cytometry analysis revealed that the expression levels of BMDC surface differentiation antigen clusters CD80 and CD86 in the NVs pVAX1 T / B@CS-SeNPs group were significantly increased compared with the PBS control group, indicating that NVs pVAX1 T / B@CS-SeNPs can effectively promote the maturation of BMDCs. At the same time, the expression of MHC-II class molecules also increased significantly, indicating that NVs pVAX1T / B@CS-SeNPs can also promote the antigen presentation ability of BMDCs. 293T NVs pVAX1T / B@CS-SeNPs are pVAX1T / B@CS-SeNPs encapsulated in cell membrane vesicles that do not express DEC 205 antibodies.

[0078] Example 4 Experiment on NVs pVAX1 T / B@CS-SeNPs activating the STING pathway of BMDCs

[0079] 3-6×10 6 / mL BMDCs were seeded in a 6-well plate, and NVs pVAX1T / B@CS-SeNPs (where the final concentration of pVAX1 T / B was 1μg / mL) were added for incubation for 24h, and the activation of the STING pathway of BMDCs was detected. First, the secretion of cytokine IFN-β in the culture medium was detected by ELISA; secondly, the mRNA transcription level of CXCL-10 was detected by qPCR; finally, WB was used to further verify the expression of related proteins in the STING pathway in BMDCs. Figure 7As shown in the figure, compared with the PBS control group, the secretion of IFN-β and the expression level of CXCL-10 mRNA in the NVs pVAX1 T / B@CS-SeNPs group were significantly increased, and the related proteins in the STING pathway were expressed, indicating that NVs pVAX1T / B@CS-SeNPs can effectively activate the STING pathway of BMDCs.

[0080] Example 5 NVs pVAX1 T / B@CS-SeNPs translation and transcription experiment in BMDCs

[0081] After NVs pVAX1 T / B@CS-SeNPs are taken up by antigen-presenting cells, the antigen protein is translated and transcribed in the cell. The antigen is processed by the proteasome and delivered to the surface of the antigen-presenting cell by MHC-I. In this process, the antigen-presenting cells mature and activate the host immune-related cells. Therefore, the successful expression of Echinococcus multilocularis antigens by NVs pVAX1T / B@CS-SeNPs in DC cells is crucial for triggering an effective immune response. Therefore, we replaced the T / B cell antigen epitope of Echinococcus multilocularis with the luciferase reporter gene Luc and co-incubated it with BMDCs. The firefly luciferase reporter gene detection kit (Biyuntian, RG005) was used to quantitatively verify whether NVs pVAX1 Luc@CS-SeNPs can deliver pVAX1 Luc to BMDCs and express it in cells. 3-6×10 6 / mL BMDCs were seeded in 6-well plates, and then NVs pVAX1 Luc@CS-SeNPs (where the final concentration of pVAX1 Luc was 1 μg / mL) were added and incubated for 24h-48h. Figure 8 As shown in the figure, the Luc expression in the NVs pVAX1Luc@CS-SeNPs group was significantly upregulated, indicating that NVs pVAX1 Luc@CS-SeNPs can be successfully expressed in BMDCs cells, while there was no significant difference in the Luc expression between the free pVAX1 Luc group and the PBS group, indicating that free pVAX1 Luc cannot be expressed in BMDCs. Therefore, this experiment proves that NVs pVAX1Luc@CS-SeNPs can deliver pVAX1 Luc to antigen-presenting cells and achieve antigen expression.

[0082] Example 6 Experiment on the inhibition of ROS production in BMDCs by NVs pVAX1 T / B@CS-SeNPs

[0083] Se is an essential trace element for the body, which has antioxidant activity and regulates cellular redox reactions. 6 / mL BMDCs were seeded in 6-well plates and stimulated with LPS (1 μg / mL) to mature BMDCs. At the same time, NVspVAX1 T / B@CS-SeNPs (where the final concentration of pVAX1 T / B was 1 μg / mL) were added to verify its antioxidant activity. Fig. 9 As shown in the figure, the ROS content in BMDCs was significantly reduced after adding NVs pVAX1 T / B@CS-SeNPs, which proved that it could inhibit the production of ROS in mature BMDCs.

[0084] Example 7 Distribution of NVs pVAX1 T / B@CS-SeNPs in vivo

[0085] In the lymph nodes, DCs will further differentiate and mature, and then activate the body's immune response. Therefore, lymph node enrichment is crucial for activating the body's immune response. First, NVs pVAX1T / B@CS-SeNPs were labeled with DiD, and 100 μg / mouse was injected subcutaneously at the base of the tail. The fluorescence signal in the mouse was observed by a small animal fluorescent in vivo imager 12h and 24h after injection to verify the biological distribution of the nanovaccine. Fig.10 As shown in the figure, at 12 hours after immunization, obvious fluorescence signals can be detected in the inguinal lymph nodes of mice. The fluorescence signal increased at 24 hours, indicating that NVs pVAX1 T / B@CS-SeNPs continued to be enriched in the inguinal lymph nodes. Finally, the mice were dissected, and the main organs and inguinal lymph nodes were taken out to further detect the enrichment of the nanovaccine in the main organs of the mice. Compared with other organs, there was a strong fluorescence signal in the lymph nodes. Statistical analysis of the fluorescence values ​​showed that the fluorescence intensity of the lymph nodes was significantly higher than that of other organs. This shows that after entering the body, NVs pVAX1 T / B@CS-SeNPs can be taken up by antigen-presenting cells and quickly enriched in the lymphatic system.

[0086] Example 8 NVs pVAX1 T / B@CS-SeNPs at the level of immune cells in mouse spleen and lymph nodes

[0087] C57BL / 6 female mice were selected and immunized by subcutaneous injection of nanovaccines (containing 100 μg of pVAX1T / B) at the base of the tail once a week. After three immunizations, the immune effect of the mice was evaluated one week after the third immunization. The mice were humanely killed and peripheral blood, spleen and lymph nodes were obtained. The maturation of DCs in the mouse lymph nodes was detected by flow cytometry. The experimental results are shown in Figure 2. Fig.11As shown in the figure, compared with the PBS group, the maturity of lymph node DCs in the NVs pVAX1 T / B@CS-SeNPs group was significantly increased, and significantly higher than that in the 293T NVs pVAX1 T / B@CS-SeNPs group and the pVAX1 T / B@CS-SeNPs group. At the same time, the proportions of CD8+T and CD4+T cells in spleen lymphocytes were detected. Compared with 293T NVs pVAX1T / B@CS-SeNPs, pVAX1 T / B@CS-SeNPs and pVAX1 T / B, the proportions of CD8+T and CD4+T cells in the NVs pVAX1T / B@CS-SeNPs group were significantly increased. ELISA was used to detect spleen lymphocytes after restimulation with total protein of Echinococcus multilocularis. It can be observed that the secretion of TNF-α, IFN-γ and other cytokines by spleen lymphocytes in the NVs pVAX1T / B@CS-SeNPs group mice was the highest in all groups, and there was a statistical difference compared with other groups. Peripheral blood was collected to detect the IgG antibody level in the mice. The results showed that the IgG antibody level in the peripheral blood of the mice in the NVs pVAX1 T / B@CS-SeNPs experimental group was significantly increased compared with the control group. The above results show that the NVspVAX1 T / B@CS-SeNPs nanovaccine can efficiently induce the maturation of DCs in the body and produce antigen-specific cellular immunity and humoral immunity.

[0088] Example 9: Mouse alveolar echinococcosis treatment model

[0089] C57BL / 6 mice were selected and a multilocular echinococcosis model was established by intraperitoneal injection of 0.2 mL (about 2000 protoscoleci) of echinococcosis suspension. After the model was successfully established, the mice were immunotherapyed by subcutaneous injection once a week for three consecutive weeks. The pVAX1 T / B in the control nanovaccine group was 100 ug / mouse. After 3 weeks of treatment, the mice were humanely killed one week after the last treatment. Fig.12 As shown in the figure, in the treatment experiment, the proportion of liver-related CD3+CD8+T and CD3+CD4+T cells in the NVs pVAX1 T / B@CS-SeNPs group was significantly higher than that in the control group, and the proportion of CTL / Tregs was also increased, indicating that NVspVAX1 T / B@CS-SeNPs can not only improve the host's immune killing ability but also regulate the immune microenvironment to inhibit the growth and reproduction of worms. The lesion sites of the PBS group and the NVs pVAX1 T / B@CS-SeNPs group were taken and the changes in their ultrastructure were observed by TEM ( Fig.13). The germinal layer (GL) contains a large number of germinal cells, which are considered to be the only proliferative cells of Echinococcus multilocularis and are related to driving the growth and development of the parasite in the host. Compared with the control group, the structure of the germinal layer in the NVs pVAX1 T / B@CS-SeNPs group became looser; microtubule structure (microtriches) is related to the nutrient absorption of alveolar larvae. In the NVs pVAX1 T / B@CS-SeNPs group, it can be seen that this structure is significantly destroyed. At the same time, the laminated layer (LL) secreted by Echinococcus multilocularis also becomes uneven in thickness in the nucleic acid vaccine group. Therefore, it is shown that the nucleic acid vaccine can effectively destroy the ultrastructure of Echinococcus multilocularis and inhibit the growth and reproduction of hydatids in the body.

[0090] Example 10 In vivo safety verification

[0091] The in vivo safety of NVs pVAX1 T / B@CS-SeNPs was evaluated. C57BL / 6 female mice, 7 to 8 weeks old, were selected and injected subcutaneously at the base of the tail with nanovaccines (containing 100 μg of pVAX1 T / B). After 24 hours, peripheral blood was collected from the mice through the canthus to test the liver, kidney, and heart function, including alanine aminotransferase, aspartate aminotransferase, serum creatinine, and creatine kinase isoenzymes to evaluate the biosafety of the nanovaccine. At the same time, the heart, liver, lung, and kidney were taken for H&E staining to analyze whether it caused damage to important organs in the body. In addition, the weight of the mice was monitored throughout the immunization cycle to evaluate whether the nanovaccine was likely to be toxic. Fig.14 As shown, the liver, kidney and heart functions of the mice were normal, there were no obvious lesions in the major organs, and there was no significant difference in body weight, indicating that the nucleic acid vaccine has high safety.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a DNA vaccine for preventing and treating multilocular echinococcosis, characterized in that: The following steps are involved: (1) The dominant epitopes of Echinococcus multilocularis were concatenated and cloned into the pVAX1 vector to prepare the nucleic acid vaccine plasmid pVAX1T / B; (2) Prepare chitosan solution with acetic acid, add ascorbic acid solution dropwise, stir and mix, and finally add Na2SeO3 solution, continue stirring, and when the color changes from colorless to dark red, the synthesis of chitosan selenium nanoparticles CS-SeNPs is completed, and then dialyze CS-SeNPs with ultrapure water for 22-26 hours, filter with a 0.22 μm pore size filter membrane after dialysis, and store at 4°C; (3) obtaining a stable transfected cell line expressing DEC 205 antibody on the cell membrane surface by cell membrane display technology, culturing the cells to a certain amount, collecting the cells and washing them twice with pre-cooled PBS, then scraping the cells, adding PBS containing protease inhibitors and sodium deoxycholate surfactant, resuspending the cells, breaking the cells by ultrasonic disruptor, and then separating the cell membrane nanovesicles by density gradient centrifugation; (4) mixing the pVAX1 T / B obtained in step (1) with the CS-SeNPs obtained in step (2) at room temperature to obtain a chitosan selenium nanoparticle-loaded nucleic acid vaccine complex pVAX1T / B@CS-SeNPs; (5) Ultrasonic mixing the cell nanovesicles extracted in step (3) and the pVAX1T / B@CS-SeNPs obtained in step (4), and then encapsulating the cell nanomembrane vesicles with pVAX1T / B@CS-SeNPs by membrane extrusion to obtain cell membrane vesicle-encapsulated chitosan selenium nanoparticles NVs pVAX1T / B@CS-SeNPs, which is the DNA vaccine for preventing and treating multilocular echinococcosis.

2. The preparation method according to claim 1, characterized in that: In step (1), the dominant epitopes of Echinococcus multilocularis include LAP, 14-3-3, EMY162 and TSP3.

3. The preparation method according to claim 1, characterized in that: In step (2), the mass fraction of the chitosan solution is 0.05-0.15%, the concentration of the ascorbic acid solution is 0.20-0.25M, and the concentration of the Na2SeO3 solution is 0.45-0.55M; the volume ratio of the chitosan solution, ascorbic acid solution, and Na2SeO3 solution is 70-80:35-40:

1.

4. The preparation method according to claim 1, characterized in that: In step (3), the operation of obtaining a stably transfected cell line expressing the DEC 205 antibody on the cell membrane surface by cell membrane display technology is as follows: obtaining the DEC 205 antibody gene sequence, fusing the transmembrane sequence of PDGFR at the C-terminus of the heavy chain constant region, introducing a HumanFc Tag tag between the heavy chain constant region and the transmembrane sequence, and constructing the sequence into a plasmid vector; using the plasmid vector to prepare a lentivirus, and obtaining a stably transfected cell line expressing the DEC 205 antibody on the cell membrane surface by lentiviral transfection.

5. The preparation method according to claim 2, characterized in that: In step (3), the operation of the density gradient centrifugation technique is as follows: ultrasonically disrupt the cells at 22.5 W for 15 s, place on ice for 3 min, repeat 4 times, and after ultrasonic disruption, centrifuge the suspension at 2000 rpm and 4°C for 10 min to remove the cytoplasm and nucleus; retain the supernatant and further centrifuge at 5000 rpm and 4°C for 10 min to remove the organelles; finally, centrifuge the supernatant at 15000 rpm and 4°C for 1 h to separate the precipitate, which is the cell membrane nanovesicle.

6. The preparation method according to claim 2, characterized in that: In step (4), the mass ratio of CS-SeNPs to pVAX1 T / B is 0.8-1.2:

1.

7. The preparation method according to claim 2, characterized in that: In step (5), the mass ratio of the cell membrane nanovesicles to the pVAX1 T / B@CS-SeNPs is 0.8-1.2:

1.

8. A DNA vaccine for preventing and treating Echinococcosis prepared by the method according to any one of claims 1 to 7.

9. Use of the DNA vaccine according to claim 8 in the preparation of a DNA vaccine preparation for preventing and treating multilocular echinococcosis.

10. A cell membrane nanovesicle, characterized in that: The preparation method is as follows: a stable transfected cell line expressing DEC 205 antibody on the cell membrane surface is obtained by cell membrane display technology, the cells are cultured to a certain amount, the cells are collected and washed twice with pre-cooled PBS, the cells are then scraped off, PBS containing protease inhibitors and sodium deoxycholate surfactant is added, the cells are resuspended, the cells are broken by an ultrasonic disruptor, and then the cell membrane nanovesicles are separated by density gradient centrifugation.

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