A preparation method for covalently coupling adjuvant and antigen of P24 virus-like particles

Abstract

The present invention provides a preparation method of covalently coupling an adjuvant and an antigen of a P24 virus-like particle, comprising: constructing a pCDNA3.4-P24 vector by molecular biology and molecular cloning and other experimental techniques, transfecting 293T cells to express and collect P24 VLPs, and covalently coupling a model antigen OVAt and a tumor antigen TRP-2, respectively, to prepare P24 VLPs-OVAt and P24 VLPs-TRP-2 vaccines. The present invention can effectively enhance the recognition of antigens by the immune system by preparing a vaccine in which an adjuvant and an antigen are integrated, thereby enhancing the killing of tumors that are not easily detected by the immune system, significantly enhancing the clinical anti-tumor effect of cancer vaccines, and preparing a more efficient and stable antigen delivery system and a released adjuvant system.

Description

Technical Field

[0001] The present invention relates to the field of vaccines, and in particular to a preparation method for covalently coupling an adjuvant and an antigen of a P24 virus-like particle and an adjuvant-antigen conjugate of the P24 virus-like particle prepared by the method. Background Art

[0002] According to the Global Cancer Report, tumor recurrence and metastasis are the main causes of death. In recent years, tumor immunotherapy has made major breakthroughs in the field of cancer treatment and has become a new first-line treatment for precision cancer treatment. Studies have reported that immune checkpoint inhibitors can significantly prolong patient survival. However, after repeated use, the efficacy is reduced and even drug resistance occurs, which still leads to tumor recurrence and metastasis. Therefore, there is still a long way to go to find new precision tumor treatment strategies. The new antigen peptides produced by tumor driver gene mutations can distinguish normal cells from tumor cells, and are recognized by T cells as new antigens, which are the most ideal targets for precision cancer treatment. The new antigen vaccine is a strategy to prepare personalized vaccines from new antigen epitopes screened from cancer patients to stimulate and enhance the body's specific immune system's attack ability, thereby achieving the goal of inhibiting and eliminating tumors. Clinical studies have shown that the clinical response rate of NY-ESO-1-specific new antigen vaccine in the treatment of advanced ovarian cancer with distant metastasis is as high as 62%; the response rate of NEO-PV-01 new antigen vaccine in the treatment of advanced melanoma with distant metastasis is 59%. Therefore, cancer vaccines targeting new antigens are expected to prevent and control tumor metastasis and recurrence, and even eliminate tumors, improving patients' quality of life and tumor cure rates. It is of great significance to conduct research on cancer vaccines based on neoantigens.

[0003] In recent years, with the continuous deepening of immunology research and the rapid development of genetic engineering technology, cancer vaccine types are mainly divided into long peptide vaccines, DC (Dendritic Cells) vaccines, mRNA vaccines, viral vector vaccines, etc. Due to the development of high-throughput technology and the advancement of biotechnology informatics technology, the screening of new antigens has made great progress. The various new antigens screened are prepared into new antigen cancer vaccines, which have strong specificity, targeting, and high safety, but weak immunogenicity, and the cellular immune response induced by the body is not strong enough. Therefore, it is particularly important to use adjuvants to enhance the immunogenicity of the host or enhance the host's protective response to the antigen.

[0004] Adjuvants are an important strategy to enhance the immune response to vaccine antigens. As early as 90 years ago, people have recognized the importance of adjuvants to vaccines. Due to the high standards of safety and efficacy and the limited understanding of the downstream mechanisms of adjuvants, there are currently 8 adjuvants approved by the FDA (Food and Drug Administration), most of which are newly approved adjuvants in the past 10 years. According to the type, they can be divided into Alum adjuvant, which is currently the most widely used adjuvant and has been approved for use in a variety of human vaccines; water-in-oil emulsion adjuvants such as MF59 and AS03, which are used in malaria and influenza virus vaccines; liposome adjuvant Zirosome, which is used in hepatitis A virus and influenza virus vaccines; TLR receptor agonists such as AS04, RC-529, Alhydroxiquim-Ⅱ and CpG ODN, which are used in human papillomavirus, hepatitis B virus and COVID-19 vaccines. Existing adjuvants are mainly used in the prevention and treatment of infectious diseases, mainly to induce B cell immune responses to produce neutralizing antibodies. Most adjuvants have the limitation of not being able to induce strong CTL responses and are difficult to be used in cancer vaccines. At the same time, existing protein adjuvants and traditional adjuvants such as aluminum salts still rely mainly on physical adsorption of isoelectric points to bind antigens to achieve the mixing of adjuvants and vaccines. Due to the diversity of antigens and the differences in isoelectric points, particle sizes and molecular weights between adjuvants and antigens, the adsorption and release of adjuvants on antigens are difficult and uncertain. Therefore, establishing a more efficient adjuvant-antigen "integrated" strategy that breaks through physical mixing may be more conducive to the role of adjuvants.

[0005] The effect principle of vaccines is based on the working principle of the immune system. It induces the body to produce an immune response by simulating infectious pathogens or specific antigens, thereby achieving the purpose of preventing and controlling infectious diseases. Its main principles include: simulating infection; vaccines usually contain part or all of microorganisms, attenuated, dead or recombinant proteins, nucleic acids, etc., simulating infectious pathogens entering the body, but not causing actual infection. Activating the immune system: The antigen components in the vaccine can be recognized by the body's immune system, activating and guiding the activity of immune cells. Immune memory: Immunity is achieved by activating memory immune cells

[0006] The production of memory B cells and memory T cells enables the body to produce a targeted immune response more quickly when it first comes into contact with a pathogen, thereby quickly eliminating the pathogen and avoiding the onset of disease. Formation of antibodies: Vaccines activate B cells, prompting them to differentiate into plasma cells and produce specific antibodies. These antibodies can bind to pathogens and neutralize their toxicity, or mark them for removal by other immune cells. Activation of cellular immune response: Vaccines can activate CD4 + Helper T cells and CD8 +T cell-mediated cytotoxic T cells (CTL), thereby inducing the body to produce a targeted cellular immune response, directly killing infected cells or abnormal cells. Establishing an immune barrier: The vaccine activates the body's immune system to form an "immune barrier" to prevent pathogens from entering the body, infecting cells or multiplying and spreading, thereby preventing the occurrence of the disease. In general, the principle of the vaccine effect is to improve the body's resistance to specific pathogens by simulating infection, activating the immune system, inducing immune memory and antibody production, and activating cellular immune responses, thereby achieving the purpose of preventing infectious diseases.

[0007] Humoral immunity and cellular immunity are two different but coordinated immune response types, which together constitute the body's defense system against pathogenic microorganisms and abnormal cells. Humoral immunity: Humoral immunity is mainly mediated by B cells, and its characteristic is to fight against foreign pathogenic microorganisms, such as bacteria, viruses, etc., by secreting antibodies. These antibodies can bind to specific antigens of pathogenic microorganisms to form antigen-antibody complexes, which cause the pathogenic microorganisms to be neutralized. Agglutination, precipitation and phagocytosis by macrophages, thereby eliminating pathogenic microorganisms. Humoral immunity also includes various cellular and molecular mechanisms that induce and maintain antibody responses, such as antibody class switching, antibody affinity maturation and the formation of memory B cells. Cellular immunity: Cellular immunity is mainly mediated by T cells, and its characteristic is to eliminate abnormal cells by identifying and killing infected cells, tumor cells and damaged cells. T cells recognize major histocompatibility complex (MHC) molecules presented by peptide fragments (antigens) through T cell receptors (TCRs) and mediate cell killing through cytokines, including the generation of signals that promote cell apoptosis, the release of cytotoxins, and the activation of macrophage killing.

[0008] Adjuvants play an important role in vaccines, such as enhancing immunogenicity: adjuvants can enhance the immunogenicity of antigens in vaccines, making them more effective in stimulating the immune system to produce immune responses. This enhancement can be manifested in improving the stability of antigens, prolonging their existence in the body, and enhancing the absorption and processing capacity of antigens; regulating immune responses: adjuvants can regulate the type and extent of immune responses, helping to achieve the desired immune effects. For example, some adjuvants can promote humoral immune responses, while others can enhance cellular immune responses, allowing vaccines to more comprehensively activate the body's immune system; improving vaccine stability: adjuvants can enhance the stability of vaccines, extend their shelf life, and reduce the loss of immunogenicity caused by vaccine denaturation or degradation; improving immune memory: some adjuvants can enhance the formation of immune memory, allowing the body to produce immune responses more quickly and more strongly when it comes into contact with the same pathogen again, thereby enhancing pathogen-specific immune responses. In general, adjuvants play a role similar to that of a medium in vaccines, which can effectively enhance the immunogenicity and protective effects of vaccines, and are an indispensable and important part of vaccine design.

[0009] At present, adjuvants play a key role in the field of vaccine research, and researchers are constantly working to develop new adjuvants to improve the immunogenicity and safety of vaccines. These adjuvants may include nanoparticles, biocolloids, oil emulsions, etc., which are aimed at improving the immune effect and protection durability of vaccines; Adjuvant-antigen interaction mechanism: Researchers conduct in-depth research on the mechanism of adjuvant-antigen interaction, and explore how to optimize the formulation and use conditions of adjuvants to achieve more effective antigen presentation and immune activation; Adjuvant regulation of immune response: Researchers focus on the regulatory effect of adjuvants on the type of immune response, and explore the differences between different adjuvants in inducing humoral immunity and cellular immunity, so as to make reasonable choices according to specific disease needs; Adjuvants and immunotoxicology: With the expansion of the scope of vaccination, immunotoxicology research on adjuvants has become particularly important. Researchers are committed to evaluating the potential effects of adjuvants on the immune system and organs to ensure the safety and reliability of vaccination. However, adjuvant research still faces some challenges and problems: Complexity of immune regulation mechanism: The immune system has a complex response mechanism for different types of adjuvants, and researchers need to have a deep understanding of these mechanisms to better guide the design and use of adjuvants. Balance between safety and immunogenicity: Some adjuvants may increase the risk of adverse reactions to vaccination while improving immunogenicity. Therefore, researchers need to find a balance between safety and immunogenicity to ensure the safety and effectiveness of vaccination. Individual differences and uncertainty in immune response: There may be differences between individuals in their immune response to adjuvants, which makes the design and application of adjuvants face certain uncertainties. Therefore, personalized vaccination strategies may be one of the future development directions. In summary, although adjuvants play a role in vaccine research, their research still needs to be further deepened to overcome existing challenges and continuously improve vaccine effectiveness and safety.

[0010] Virus-like particles are inactive particles that are similar in structure to real virus particles but do not contain the viral genome. They can mimic the appearance and structure of virus particles by expressing viral surface proteins or structural proteins, but are not infectious and therefore do not cause disease.

[0011] Prior art related to the present invention

[0012] Aluminum salt adjuvants are low-cost and easy to use. They are the most widely used adjuvant in the production of biological products and the first adjuvant approved by the US Food and Drug Administration (FDA) for use in human vaccines. Currently, the commonly used aluminum adjuvants are mainly aluminum hydroxide adjuvant (Alum) and aluminum phosphate adjuvant (Aluminum phosphate). Aluminum salts have many advantages as vaccine adjuvants. In addition to enhancing immune response, they also have very solid safety data. Although it can effectively induce humoral immune response, it has no obvious effect on cellular immunity and is difficult to induce cellular immune response.

[0013] In the current relevant scientific literature, the combination of antigen and adjuvant is mainly achieved by combining the effects and physical properties of different adjuvants to adsorb antigens, thereby achieving humoral immunity and cellular immune response at the same time. Vaccine preparations containing adjuvants are usually adjuvant-adsorbed antigenic vaccines, that is, the antigen in the vaccine is adsorbed on the adjuvant. Aluminum hydroxide adjuvant exists in the form of aluminum hydroxyl, in the form of fibrous particles, and its isoelectric point is usually around 10 to 11. Under neutral pH conditions, these micron-sized particles are positively charged, which is conducive to the adsorption of negatively charged CpG and negatively charged antigens. In contrast, aluminum phosphate adjuvant is a complex of hydroxyaluminum phosphate, which appears as an aggregate of spherical particles. Commercial aluminum phosphate adjuvants usually have a lower isoelectric point, about 4 to 5. Under neutral pH conditions, these micron-sized particles exist in a negatively charged form, which is not conducive to the adsorption of negatively charged CpG. In order to solve the problem of low adsorption rate of aluminum phosphate adjuvants, the prior art has proposed some solutions. For example, in China's patent CN106729702, the existing aluminum phosphate adjuvant formula and post-treatment process are improved to obtain an aluminum phosphate adjuvant with good adsorption performance and appearance. The particle size of the prepared aluminum phosphate can be effectively controlled between 7.5 and 15 nanometers, and the isotropic point is controlled between 4.0 and 6.0. Such improvements can stabilize the surface charge and particle size of the aluminum phosphate adjuvant, and the pH value is closer to the pH value of human body fluids.

[0014] However, due to the presence of adjuvants, the detection of some vaccine quality inspection items may be disturbed or even impossible. Therefore, in actual operation, it is necessary to provide an adjuvant system with good desorption effect, and to use appropriate methods to release the antigen adsorbed on the adjuvant for detection and analysis. For example, in China's patent 103471902A, a method for desorption of vaccines containing aluminum phosphate adjuvants is proposed. The method suggests using an alkaline solution to dissolve the vaccine containing adjuvant and using an acidic chemical reagent containing a metal chelator for neutralization. However, this method often leads to antigen degradation and lacks feasibility. On the contrary, in patent CN1572324A, an improvement is proposed on the adsorption method of the adjuvant system. The patent points out that for vaccines containing aluminum adjuvants and CpG adjuvants. This approach is more conducive to meeting the requirements of quality control. It should be noted that the size of the antigen is hundreds of times that of CpG, and theoretically it is impossible for the antigen to be adsorbed on CpG. Therefore, it adds difficulty and uncertainty to the adsorption and release of the adjuvant to the antigen.

[0015] Disadvantages of the prior art 1

[0016] 1. It is difficult for a single traditional adjuvant to induce both humoral immunity and cellular immunity at the same time, and usually requires physical mixing of two or more adjuvants;

[0017] 2. In the current relevant scientific literature, the combination of antigen and adjuvant is mainly achieved by adsorbing antigen through the physical properties of the combined adjuvant. Due to the diversity of antigens and the differences in isoelectric point, particle size and molecular weight between adjuvant and antigen, the adsorption and release of antigen by adjuvant increases difficulty and uncertainty. Summary of the invention

[0018] The purpose of the present invention is to solve the defects of the above-mentioned prior art and provide a method for preparing P24 virus-like particles by covalently coupling adjuvants and antigens. By covalently coupling P24VLPs and vaccine antigens, the adjuvant and vaccine antigen are co-expressed, presenting an adjuvant system for preparing stable antigen delivery and antigen release, which has good commercial value.

[0019] The present invention promotes the co-expression of adjuvant and vaccine antigen by covalently coupling P24 VLPs and vaccine antigen, presents an adjuvant system for preparing stable antigen delivery and antigen release, and has good commercial value.

[0020] The present invention adopts the following technical solution:

[0021] A method for preparing P24 virus-like particles by covalently coupling an adjuvant with an antigen, comprising the steps of:

[0022] Step (1). Prepare P24 VLPs: Prepare P24 VLPs in vitro. Obtain the cDNA sequence of P24 (NC_00 01802.1) from the website https: / / www.ncbi.nlm.nih.gov / gene / 155030, clone it into the mammalian expression vector pcDNA3.4, and construct the pcDNA3.4-P24 expression vector; transfect it into HEK293 cells for transient expression, then collect the cell supernatant and perform ultracentrifugation to collect P24 VBPs.

[0023] Step (2). Activate the surface of P24 VLPs: Use N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to activate the functional groups on the surface of P24 VLPs.

[0024] Step (3). Antigen preparation: synthesize a peptide segment of 9 amino acids that can be spliced, ensuring a purity of 90%.

[0025] Step (4). Activate the antigen surface: Similar to the activation of the P24 VLPs surface, activate the antigen to expose the reactive groups.

[0026] Step (5). Covalent linkage: Add P24 VLPs to a round-bottom flask and add 1×PBS to make the final protein concentration 15 mg / mL; add the prepared glutaraldehyde solution to the VLPs protein system and stir at room temperature for 1 hour; dialyze with 1×PBS at 4°C for 6 hours to remove free glutaraldehyde; dissolve the antigen peptide in 1×PBS solution; use Ellman's reagent to detect the thiol group in the antigen peptide in step (4): add 100 μL of antigen peptide solution to a 96-well plate and measure its ultraviolet absorbance at λ=412 nm using a Nano spectrophotometer. If the OD value is greater than 0.15, proceed to the next step; add the antigen peptide solution to VLPs-glutaraldehyde and react at room temperature with vertical mixing for 4 hours; use Ellman's reagent to detect the amino group in the polypeptide: add 100 μL of Ellman's reagent stock solution to a 96-well plate and then add 10 μL of the cross-linked antigen peptide solution and use a Nano spectrophotometer at λ=412 The UV absorbance was measured at nm.

[0027] Step (6) Purification: The reactants are purified by dialysis, column chromatography or gel electrophoresis to remove unreacted cross-linking reagents and other impurities.

[0028] Step (7) Identification and verification: The purified product was identified and verified by SDS-PAGE gel electrophoresis, transmission electron microscopy, WB and immunofluorescence to confirm that P24 VLPs and antigens were successfully covalently linked.

[0029] Step (8) Stability test: Detection of particle size and zeta potential of P24 VLPs-OVAt and P24 VLPs-TRP-2: Take about 0.2 mL of sample, place it in a 5 mL centrifuge tube, add 1 mL of water, and ultrasonically disperse for 5 min. Take 1 mL of the upper solution and slowly add it to the sample pool to avoid bubbles in the sample pool; use Malvern laser particle size analyzer ZS90 for testing, and preheat the instrument for 30 min; start the test software, select the Zeta (or particle size) test program, select water as the solvent, preheat at 25°C for 30 s and then measure, and each sample is measured in parallel 3 times.

[0030] In another aspect, the present invention also relates to the adjuvant-antigen covalent conjugate of P24 virus-like particles prepared by the above method.

[0031] On the other hand, the present invention also relates to the use of the adjuvant-antigen covalent conjugate of P24 virus-like particles in the preparation of vaccines.

[0032] On the other hand, the present invention also relates to a vaccine comprising the above-mentioned adjuvant-antigen covalent conjugate of P24 virus-like particles.

[0033] Beneficial effects of the present invention:

[0034] 1. High safety: VLP is self-assembled from viral surface proteins and does not contain viral genome, so it cannot replicate and infect. This makes VLP a safe vaccine candidate, avoiding the potential risks of the virus or inactivated virus used in traditional vaccines.

[0035] 2. Strong antigen immune response: The structure of VLP is very similar to natural virus particles, which can effectively simulate the real virus infection process, thereby causing the immune system to fully respond to the vaccine antigen. This makes VLP vaccines highly efficient and effective in inducing immune responses.

[0036] 3. Induce humoral immunity and cellular immunity at the same time, where VLP usually contains viral surface proteins, which can be recognized by the body's humoral immune system and trigger a humoral immune response corresponding to the vaccine antigen. In addition to inducing humoral immune responses, VLP can also enhance immune responses by activating the body's cellular immune system. After VLP enters the host cell, it can be taken up by antigen presenting cells such as dendritic cells and presented to T cells through MHC molecules, thereby activating the immune response of T cells specific to the vaccine antigen.

[0037] 4. Stable adjuvant delivery antigen system and antigen release system: By covalently coupling P24 VLPs and vaccine antigens, co-delivery of adjuvants and antigens is achieved. P24 VLPs viral vector vaccines enter the body and are taken up by host cells. These vaccine particles or cells are broken down by lysosomes or proteasomes in the cells, releasing their components. These components may be processed into antigen fragments and then presented to immune cells to trigger an immune response.

[0038] 5. High stability: VLP is relatively stable during preparation and storage. In PBS and 10% FBS in a refrigerator at 4 degrees for one week, its particle size and zeta potential do not change significantly, and it is not easily affected by external environmental factors such as temperature and pH value. This makes VLP more convenient to prepare, transport and store. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a diagram of the preparation model of P24 VLPs vaccine.

[0040] Figure 2 Transmission electron microscopy images, where (a) is P24VLPs, (b) is P24VLPs-OVAb, and (c) is P24VLPs-OVAt.

[0041] Figure 3 This is the DLS particle size detection diagram, where (a) represents the particle size and (b) is the particle size statistical result.

[0042] Figure 4 This is the DLS dispersion detection diagram.

[0043] Figure 5 For DLS Zeta potential detection.

[0044] Figure 6 P24 protein was detected by WB.

[0045] Figure 7 Immunofluorescence results show the coupling of peptides and P24 VLPs, where (a) is P24 VLPs, (b) is FITC-OVAt, (c) is P24 VLPs-OVAt, (d) is P24 VLPs, (e) is Cy5.5-OVAb, and (f) is P24 VLPs-OVAb.

[0046] Figure 8 Stability test of P24 VLPs. A and C: zeta potential; B and D: particle size.

[0047] Fig. 9 P24VLPs enhanced OVAt-specific CD8 +T cell response. A: HE staining shows the morphology of each group of heart, liver, spleen, lung and kidney; B: Specific T cell flow cytometry results; C: Statistical graph of flow cytometry results Figure B; D: Statistical graph of the proportion of specific T cells induced by the two groups of vaccines; E: Cytokine flow cytometry results; F: Statistical graph of flow cytometry results Figure E.

[0048] Fig.10 P24VLPs-OVAt significantly inhibited tumor growth. A: Vaccine immunization strategy; B: Tumor growth curves of each group; C: Statistical graph of tumor growth curves of each group.

[0049] Fig.11 P24VLPs can enhance DC antigen presentation. A: BMDCs in vitro induction strategy; B: BMDCs cell viability; C: CD86 flow cytometry results; D: Statistical graph of flow cytometry results in Figure C; E: CD80 flow cytometry results; F: Statistical graph of flow cytometry results in Figure E; G: flow cytometry results; H: Statistical graph of flow cytometry results in Figure G;

[0050] Fig.12 P24VLPs enhance tumor antigen-specific immune responses and specific immune memory. A: Flow cytometry results of specific T cell ratio; B: Statistical graph of flow cytometry results of Figure A; C: Flow cytometry results of T cell populations; D: Statistical graph of flow cytometry results of Figure C. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] 1. The present invention can induce humoral immunity and cellular immunity at the same time: It is difficult for a single traditional adjuvant to induce humoral immunity and cellular immunity at the same time, and usually two or more adjuvants need to be mixed by adsorption. However, the present invention can induce humoral immunity and cellular immunity at the same time.

[0053] 2. In the current relevant scientific literature, the combination of antigen and adjuvant is mainly achieved by adsorbing antigen through the physical properties of the combined adjuvant. Due to the diversity of antigens and the differences in isoelectric point, particle size and molecular weight between adjuvant and antigen, the adsorption and release of antigen by adjuvant increases difficulty and uncertainty. The focus of the present invention is to prepare an adjuvant system for stable delivery of antigens and antigen release.

[0054] Example

[0055] 1. Preparation of P24 VLPs

[0056] 1.1 Choose an appropriate expression system: First, you need to choose a suitable expression system. Commonly used expression systems include Escherichia coli, Saccharomyces cerevisiae, mammalian cells, etc. The HEK293 mammalian expression system is selected based on the properties of P24VLPs, production requirements, expression conditions and other factors.

[0057] 1.2 Construction of expression vector: Design a suitable expression vector according to the gene sequence of the target protein. The expression vector usually includes functional elements such as promoter, coding sequence (https: / / www.ncbi.nlm.nih.gov / gene / 155030 to obtain the cDNA sequence of P24 (NC_0001802.1)), signal sequence, tag, etc. to ensure that the target protein can be correctly expressed and localized in the host cell.

[0058] 1.3 Transfect host cells: Introduce the constructed expression vector into the HEK293 mammalian (such as mouse) expression system.

[0059] 1.4 Cultivate and express target protein: Culture the transfected or transformed cell lines in an incubator at 37°C and 5% carbon dioxide.

[0060] 1.5 Purification and assembly of P24 VLPs: The target protein is extracted from the culture by high-speed centrifugation and purified. Subsequently, the target protein is assembled into VLP particles under suitable conditions of pH = 6-7, appropriate amount of sodium ions, room temperature and moderate oxygen.

[0061] 1.6 Characterization and identification of P24 VLPs: Characterization and identification of the obtained VLP particles, including morphological observation (electron microscopy), biological property analysis (safety), physicochemical property analysis (such as particle size, charge, etc.). At the same time, quality control of P24 VLPs is also required to ensure that they meet the expected standards and safety requirements of biological preparations.

[0062] 1.7 Application and further research: After the preparation of P24 VLPs is completed, further application research can be carried out, such as in the fields of vaccine development, drug delivery systems, etc. At the same time, the preparation process can also be optimized and improved to improve the yield, stability and immunogenicity of P24 VLPs.

[0063] 2. Covalently linking proteins and antigens

[0064] 2.1 Prepare proteins and antigens: First, prepare the protein P24 VLPs and vaccine antigens to be connected.

[0065] Note: The antigens involved in the present invention are B cell and T cell antigen epitopes from tumors, pathogenic microorganisms such as bacteria and viruses. The present invention is applicable to the coupling of various antigen epitopes and has a universal type. Mix according to the mass ratio of 1:1.

[0066] 2.2 Activation of protein and antigen surface functional groups: Before connection, it is usually necessary to activate the surface functional groups of proteins and antigens to make them reactive. The activation reagents are N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).

[0067] 2.3 Preparation of cross-linking reagent: The cross-linking reagent is usually a compound containing a reactive functional group, such as an amino group, a carboxyl group, a hydroxyl group, etc. The cross-linking reagent used in the present invention is glutaraldehyde.

[0068] 2.4 Covalent ligation reaction: P24 VLPs were injected into a round-bottom flask and sufficient 1×PBS was added to make the final concentration of the protein reach 15 mg / mL. Subsequently, the pre-prepared glutaraldehyde solution was added dropwise to the P24 VLPs protein system and stirred for reaction at room temperature for 1 hour. Next, the system was dialyzed at 4°C for 6 hours to remove free glutaraldehyde. The peptide was dissolved in 1×PBS solution and then the thiol group in the peptide was detected using Ellman's reagent. If the peptide absorption value (OD value) detected in the 96-well plate is greater than 0.15, proceed to the next step. The peptide solution was added dropwise to the VLPs-glutaraldehyde mixture and mixed using a vertical mixer for reaction at room temperature for 4 hours. Finally, the amino group in the peptide was detected again using Ellman's reagent.

[0069] 2.5 Purification: The reaction product is purified by dialysis, column chromatography or gel electrophoresis to remove unreacted cross-linking reagents and other impurities.

[0070] 2.6 Purification of ligation product: After the ligation reaction is completed, the product needs to be purified and refined to remove unreacted reagents, impurities and by-products. Common purification methods include chromatography, electrophoresis and filtration.

[0071] 2.7 Verify the ligation product: Verify the ligation product to confirm the efficiency and stability of the ligation. Mass spectrometry, immunoblotting, functional experiments, etc. can be used for verification.

[0072] 2.8 Vaccine preparation: The purified ligation product is used as a vaccine component and mixed with appropriate auxiliary materials (such as adjuvants, stabilizers, etc.) to prepare a vaccine preparation.

[0073] 3. P24 VLPs vaccine-induced immune response

[0074] 3.1. Establishment of C57BL / 6J mouse immune model

[0075] Balb / c mice (8 weeks old, female) and C57BL / 6J mice (8 weeks old, male) were purchased from the Experimental Animal Center of our unit (Chongqing Medical University). Based on the previous research and literature review, the immunization dose of the antigen in this experiment was 25 μg / mouse, and the storage concentration was 1 mg / mL. 100 μL of antigen was mixed with different adjuvants according to the reagent instructions. After the water-in-oil adjuvant was emulsified, the state of oil-in-water was detected. During the immunization process, the ears and tail of the mouse were pinched with the left hand to expose the abdomen of the mouse; the armpits and abdomen were disinfected with alcohol cotton balls; the needle tip was tilted at 30 degrees, and the skin was lifted before injection to ensure that it was subcutaneous. 4-5 injections were performed according to the amount of 25 μg of antigen, and the total volume was 100 μL to avoid leakage during the injection process.

[0076] 3.2HE evaluation of the safety of P24VLPs: HE experiments detected the morphological structure of the heart, liver, spleen, lungs, and kidneys of C57BL / 6J mice after immunization (Wu J et al. Journal of Experimental&Clinical Cancer Research. 38:321).

[0077] 3.3. ELISA detection of cytokines: ELISA was used to detect the secretion of IFN-γ (Gao FX et al. iScience 22;25(12)) in serum or in the supernatant after in vitro co-incubation.

[0078] 3.4. Flow cytometry detection of DC or T cell activation molecules: Flow cytometry was used to detect the expression level and intensity of cell membrane molecules (Gao FX et al. Frontiers in immunology. 12(12):789905). BV510-CD45 (Clone: ​​30-F11), APC-CD11b (Clone: ​​ICRF44), Brilliant Violet 421™-CD80 (Clone: ​​16-10A1), PE / Cyanine7-CD86 (Clone: ​​BU63), PE-H-2Kb bound to SIINFEKL (Clone: ​​25-D1.16), Biotin H-2K(b)TRP-2 Monomer (SVYDFFVWL) (Cat#: 280061), APC Streptavidin (Cat#: 405207), PE / Cyanine7-CD86 (Clone: ​​GL-1), PerCP / Cyanine 5.5-CD4 (Clone: ​​GK1.5), FITC- CD8 (Clone: ​​53-6.7), PE / Cyanine7-CD69 (Clone: ​​H1.2F3), PE-CD137 (17B5Clone: ​​17B5), Brilliant Violet 421™-CD62L (Clone: ​​MEL-14), Alexa Fluor®700-CD44 (Clone: ​​IM7), FITC-TLR2 (Clone: ​​W15145C); centrifugation (300g, 5min). Flow cytometry was used to detect intracellular IFN-γ (Gao FX et al. Frontiers in immunology. 12(12): 789905). FACSCelesta flow cytometer was used to analyze and collect data.

[0079] 3.5. Tetramer detection of OVAt and TRP-2 specific T cells: The spleen cells of immunized mice were used as test samples. The necessary amount of cells was collected and the red blood cells were lysed. 20μL MTG-001 Clear Back was added and incubated for 5min (Blocking). 10μL OVA Tetramer and 10μL negative Tetramer were added to the control group, and the cells were incubated for 30min at 4°C. 10μL anti-mouse CD8-FITC was added and the cells were incubated for 30min at 4°C. Washed once with 2% FCS / 0.05% NaN3 / PBS. Detected on a BD FACSAria III flow cytometer. CD8-FITC (Clone:53-6.7), PE-SIINFEKLTetramer (MBL, TS-5001-1C), PE-SVYDFFVWL Tetramer (MBL, TB-5004-1).

[0080] Experimental results:

[0081] like Figure 1 As shown, the pCDNA3.4-P24 vector was constructed by molecular biology and molecular cloning techniques, 239T cells were transfected to express and collect P24 VLPs, which were covalently coupled to the model antigen OVAt and the tumor antigen TRP-2 to prepare P24VLPs-OVAt and P24 VLPs-TRP-2 vaccines.

[0082] OVAt antigen source:

[0083] https: / / rupress.org / jem / article / 215 / 5 / 1493 / 42587 / A-flexible-MHC-class-I-multimer-loading-system-for public website.

[0084] Antigen TRP-2 Source:

[0085] https: / / rupress.org / jem / article / 185 / 3 / 453 / 7069 / Identification-of-Tyrosinase-related-Protein-2-asA public website.

[0086] Characterization of P24 VLPs-OVAt and P24 VLPs-TRP-2: Using P24 VLPs to display CD8 +T cell antigen peptides OVAt and OVAb were used to prepare P24 VLPs-OVAt and P24 VLPs-OVAb vaccines, respectively. The successful coupling was verified by immunofluorescence and WB. The physical properties of the vaccine, such as morphology, particle size, potential, and stability, were characterized, and it was found that the physical properties of the vaccine were stable. Figure 2-Figure 8 :Transmission electron microscopy shows the morphology of P24 VLPs-OVAb and P24 VLPs-OVAt, confirming that proteins with P24 as the core can assemble into hollow structures similar to viruses. The scale bar of the transmission electron microscopy image indicates 100-200nM. Figure 3-Figure 5 The particle size and zeta potential of P24 VLPs-OVAb and P24 VLPs-OVAt confirmed that P24 VLPs are structurally stable and have stable physical properties. Figure 6 :WB detected the expression of P24 protein in P24 VLPs-OVAb and P24 VLPs-OVAt. Figure 7 : Immunofluorescence microscopy detected the coupling of P24 VLP with OVAb or OVAt. The scale bar of the immunofluorescence microscopy image indicates 20 μM, which proves that the peptide was successfully coupled and displayed on the outside of P24 VLPs.

[0087] The morphology of P24 VLPs-OVAt and P24 VLPs-TRP-2 in PBS solution is stable: Figure 8 A and B are in PBS solution; C and D are in 10% FBS solution; A and C: zeta potential. B and D: particle size. It is confirmed that P24 VLPs are structurally stable and physically stable in PBS and FBS solutions.

[0088] P24 VLPs-OVAt showed the potential to enhance cellular immunity and induced more OVAt-specific CD8 + The P24 VLPs-OVAt vaccine strategy after covalent coupling can induce stronger specific CD8 T cells than the injection of P24 VLPs and OVAt mixture. + T cell response. Fig. 9 Middle A: Morphology of heart, liver, spleen, and kidney after P24 VLPs vaccination. The scale bar of H&E staining image indicates 50 μM. Fig. 9 B and C: C57 mice immunized with OVAt in combination with different adjuvants. OVAt-specific CD8 + T cells were stained for tetramers. + T cells are gated. Fig. 9D: P24 VLPs-OVAt and the mixture of P24 VLPs and OVAt were used to immunize C57 mice for 8 weeks. The immunizations were repeated 3 times with an interval of 1 week between each. Fig. 9 E and F: Functional CD8 + T cells were stained for intracellular influx. *P<0.05, **P<0.01, ****P<0.0001.

[0089] The use of P24 VLPs-OVAt vaccine for cancer treatment significantly inhibited the growth of B16-OVA and had a more significant tumor treatment effect than Ploy (I:C) adjuvant combined with OVAt antigen. Fig.10 Middle A: Scheme showing the design of the animal experiment. Male BALB / c mice (n=5 mice per group) were inoculated subcutaneously (sc) with murine B16-OVA tumor cells (2*105 per mouse). The mice were then immunized with the indicated vaccine formulations on days 5, 10, and 17. Fig.10 Middle B: Detection of tumor growth curves of mice in each group. Fig.10 Middle C: Analysis of tumor growth curves of four groups. Tumor volume data were analyzed by two-way ANOVA. *P<0.05, **P<0.01. All error bars represent SEM.

[0090] Preliminary in vitro mechanism studies have found that P24 VLPs may promote DC to enhance the cross-presentation of OVAt, thereby promoting antigen peptide-specific CD8 + T cell immune response. Fig.11 Middle A: BMDC induction in vitro; Fig.11 Middle B: P24 VLPs-OVAt was co-cultured with BMDC. OVAt-specific CD8 + T cells were stained for tetramers. Fig.11 Middle C-F: flow staining of the maturation markers CD80 and CD86 on BMDC. Fig.11 Middle G) Specific presentation of SIINFEKL peptide in flow-stained BMDCs. **<0.001.****<0.00001.

[0091] P24 VLPs were used to display the tumor antigen TRP-2 to prepare the P24 VLPs-TRP-2 neoantigen vaccine for immunization of C57 mice. It was found that compared with the control adjuvant, P24 VLPs-TRP-2 induced higher levels of TRP-2 tumor antigen-specific CD8 in C57 mice. + T cell response and promoted the formation of T cell immune memory. Fig.12 Middle AB: Eight-week-old mice were used for immunization. Immunization was performed 3 times with 1 week interval. TRP-2 tetramer was used to detect TRP-2-specific CD8+ T cells. Pan refers to the number of each mouse. CD: Flow cytometry detection of memory CD8 induced by P24 VLPs-TRP-2 + T cell formation. *<0.05. **<0.001. ***<0.0001. Absence indicates ns.

[0092] After entering the body, the virus will cause the most intense immune response and has strong immunogenicity. The virus can induce a strong attack by the immune system: first, because its antigenic epitope is highly immunogenic, it can induce the body to produce neutralizing antibodies and CD8 + First, the virus can kill T cells; second, the size and shape of the virus can also be more easily discovered by the immune system. Therefore, loading antigens through polymers of virus-like particles with strong immunogenicity not only has stronger immunogenicity, but also can realize the integration of adjuvant and antigen, which has stronger delivery function than physical mixing; third, activating TLR receptors is an important way to activate antigen presenting cells. By looking for the mechanism of action of P24 VLPs, it was found that P24VLPs can enhance the antigen presentation of DC by activating the TLR2 pathway, and is a natural TLR2 receptor agonist.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing P24 virus-like particles by covalently coupling an adjuvant with an antigen, characterized in that: The steps include: Step (1). Prepare P24 VLPs: obtain the cDNA sequence of NC_0001802.1, clone it into the mammalian expression vector pcDNA3.4, construct the pcDNA3.4-P24 expression vector; transfect it into HEK293 cells for transient expression, then collect the cell supernatant, and collect P24 VLPs by ultracentrifugation; Step (2). Activating the surface of P24 VLPs: Activating the functional groups on the surface of P24 VLPs using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Step (3). Preparing antigens: preparing antigens OVAt and TRP-2; Step (4). Activating the antigen surface: Activating the antigen to expose the reactive groups, using activation reagents such as N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; Step (5). Covalent connection: Add the P24 VLPs in step (2) to a round-bottom flask, add 1×PBS to make the final protein concentration 15 mg / mL; add the prepared glutaraldehyde solution dropwise to the P24 VLPs protein system, stir and react at room temperature for 1 hour; dialyze with 1×PBS at 4°C for 6 hours to remove free glutaraldehyde; dissolve the antigen peptide in step (4) with 1×PBS solution; Detect the thiol group in the antigen peptide with Ellman reagent: add 100 μL of antigen peptide solution to a 96-well plate, measure its ultraviolet absorption value at λ=412nm with a Nano spectrophotometer, and proceed to the next step if the OD value is greater than 0.15; add the polypeptide solution dropwise to VLPs-glutaraldehyde, and react for 4 hours at room temperature with vertical mixing; detect the amino group in the antigen peptide with Ellman reagent: add 100 μL of Ellman reagent stock solution to a 96-well plate, then add 10 μL of the cross-linked antigen peptide solution, and measure the ultraviolet absorption value at λ=412nm with a Nano spectrophotometer; Step (6) purification: purifying the reactants by dialysis, column chromatography or gel electrophoresis to remove unreacted cross-linking reagents and other impurities; Step (7) identification and verification: The purified product was identified and verified by SDS-PAGE gel electrophoresis, transmission electron microscopy, WB and immunofluorescence to confirm that the P24 VLPs and the antigen were successfully covalently linked; Step (8) Stability test: Detection of particle size and zeta potential of P24 VLPs-OVAt and P24 VLPs-TRP-2: Take about 0.2 mL of sample, place it in a 5 mL centrifuge tube, add 1 mL of water, ultrasonically disperse for 5 min, take 1 mL of the upper solution, and slowly add it to the sample pool to avoid bubbles in the sample pool; use Malvern laser particle size analyzer ZS90 for testing, and preheat the instrument for 30 min; start the test software, select the Zeta test program, select water as the solvent, preheat at 25°C for 30 s and then measure, and each sample is measured in parallel 3 times.

2. The method according to claim 1, characterized in that Step (1) includes: obtaining the cDNA sequence, signal sequence, and tag of NC_0001802.1 through the website https: / / www.ncbi.nlm.nih.gov / gene / 155030.

3. The method according to claim 1, characterized in that Step (1) further comprises: culturing the transfected or transformed cell line in an incubator at 37° C. and 5% carbon dioxide; The target protein is extracted from the culture by high-speed centrifugation and purified, and then assembled into VLP particles under pH = 6-7, appropriate amount of sodium ions, room temperature and moderate oxygen conditions; The obtained VLP particles are characterized and identified, including morphological observation, biological property analysis, and physicochemical property analysis; at the same time, quality control of P24 VLPs is also required to ensure that they meet the expected biological preparation standards and safety requirements.

4. The method according to claim 1, characterized in that: Step (6) also includes: purifying the ligation product, and purifying and refining the product using chromatography, electrophoresis and filtration methods.

5. An adjuvant-antigen covalent conjugate of P24 virus-like particles prepared according to the method of any one of claims 1 to 4.

6. Use of the adjuvant-antigen covalent conjugate of the P24 virus-like particles according to claim 5 in the preparation of vaccines.

7. A vaccine, characterized in that An adjuvant-antigen covalent conjugate comprising the P24 virus-like particles according to claim 5.

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