A monkeypox virus nanoparticle vaccine based on hbv-vlp carrier, and a preparation method and application thereof

Abstract

This invention discloses a monkeypox virus nanoparticle vaccine based on an HBc-VLP vector, its preparation method, and its application, belonging to the field of biomedical technology. The vaccine uses HBc-VLP as a backbone with SpyCatcher sites on its surface. Through a SpyTag / SpyCatcher coupling system, antigenic polypeptides derived from the monkeypox virus M1R protein (containing overlapping epitopes for B cells and T cells) are loaded onto the VLP surface in a high-density, directed, and stable manner. This design mimics the structure of natural viral particles and can efficiently activate humoral and cellular immunity. The epitopes, screened by immunoinformatics, exhibit high antigenicity, broad-spectrum HLA affinity, and are non-toxic and non-sensitizing. This invention also provides an immunomodulatory composition containing this vaccine and its application in the preparation of drugs for the prevention or treatment of monkeypox virus infection. The vaccine preparation process is simple, has strong antigen compatibility, and has good industrialization prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a monkeypox virus nanoparticle vaccine based on the HBc-VLP vector, its preparation method, and its application. Background Technology

[0002] Monkeypox virus (MPXV) belongs to the Poxviridae family ( Poxviridae ) genus of poxvirus ( Orthopoxvirus Monkeypox virus (SPOV) is an enveloped double-stranded DNA virus. Its viral particles are rectangular, approximately 200-300 nm in diameter, with a genome length of about 190-200 kb, encoding approximately 200 proteins. SPOV is highly homologous to other members of the Orthopox genus, such as smallpox virus, vaccinia virus, and vaccinia virus, in terms of antigenicity, genomic structure, and biological characteristics. Therefore, those previously vaccinated against smallpox have approximately 85% cross-protection against SPOV. However, with the cessation of routine smallpox vaccination worldwide, population immunity to Orthopox virus has gradually declined, creating conditions for the spread and prevalence of SPOV. Based on its genetic evolutionary characteristics, SPOV can be divided into two branches: the Central African branch (Clade I) and the West African branch (Clade II). The Central African branch is mainly prevalent in the Congo Basin and surrounding areas, exhibiting high pathogenicity in humans, with a mortality rate as high as 10% in unvaccinated populations. The West African branch is mainly distributed in West African countries, exhibiting lower pathogenicity, with a mortality rate of approximately 3.6%.

[0003] Monkeypox, caused by monkeypox virus infection, is a self-limiting disease, with most patients recovering spontaneously within 2-4 weeks. However, newborns, children, and immunocompromised individuals are prone to serious complications such as secondary bacterial infections, bronchopneumonia, and encephalitis, resulting in a high mortality rate. The pathogenesis of monkeypox virus is complex, primarily related to the virus's ability to evade the immune system, damage to host cells, and abnormal host immune responses. After infecting the host, the virus suppresses both innate and adaptive immune responses through various immunomodulatory proteins, creating conditions for viral replication and spread. Simultaneously, the virus replicates extensively within host cells, leading to cell damage and apoptosis, resulting in clinical symptoms such as skin rashes and lymphadenopathy. Furthermore, an excessive immune response in the host can cause a cytokine storm, exacerbating tissue damage and triggering serious complications. Currently, there are no specific treatments for monkeypox virus; clinical treatment focuses on symptomatic support, and the development of vaccines and antiviral drugs is ongoing.

[0004] The MIR protein (encoded by the M1R gene) is a core structural protein on the surface of mature intracellular monkeypox virus (IMV). It belongs to the conserved protein genus *Orthopoxvirus* and shares high sequence similarity (over 80% amino acid identity) with its homologous proteins of vaccinia virus (L1R protein) and smallpox virus. The MIR protein is a transmembrane glycoprotein with a molecular weight of approximately 35 kDa. Its extracellular domain contains multiple antigenic epitopes, playing a crucial role in viral adsorption and invasion of host cells. The virus initiates the infection process by binding the MIR protein to sulfated glycosaminoglycans (GAGs) on the host cell surface. Studies have found that the MIR protein not only participates in viral invasion but also mediates immune escape through multiple mechanisms. The MIR protein mediates the initial adsorption of viral particles by binding to sulfated glycosaminoglycans (GAGs) on the host cell surface, thereby promoting viral invasion. Furthermore, the MIR protein also participates in immune escape processes, such as masking viral surface immunodominant epitopes, inhibiting T cell activation, and interfering with complement system activation, thus enhancing the virus's persistent infectivity. Therefore, MIR protein is one of the important targets for the development of monkeypox virus vaccines and antibody drugs.

[0005] However, traditional vaccines based on attenuated or inactivated viruses pose biosafety risks and have complex preparation processes. Although studies have confirmed that MIR proteins are important protective antigens, the immunogenicity of single recombinant proteins is generally weak, making it difficult to induce strong cellular immunity and durable protective effects. In recent years, nanoparticle technology has shown great potential in vaccine development. Virus-like particles (VLPs), due to their advantages such as morphological mimicry of natural viruses, lack of genetic material, and high-density antigen display, have become an important platform for next-generation vaccines. The hepatitis B virus core protein (HBc) is a structural protein of hepatitis B virus (HBV) and can autonomously assemble into virus-like particles (VLPs). Due to its unique advantages such as strong immunogenicity, high plasticity, high expression efficiency, good safety, and outstanding targeting, HBc has become a research hotspot in the field of vaccine vectors and is widely used in the development of HBV self-vaccines, vaccines against other pathogens, tumor vaccines, and therapeutic vaccines. This platform has been successfully applied to the development of vaccines against viruses such as SARS-CoV-2, demonstrating excellent immunogenicity and protective efficacy.

[0006] In summary, based on the key role of MIR protein in monkeypox virus invasion and immune escape, and combined with the advantages of the HBc-VLP nanoparticle platform, developing a multi-epitope nanoparticle vaccine that can simultaneously activate humoral and cellular immunity has significant scientific and clinical application value. Summary of the Invention

[0007] In view of the current situation where existing monkeypox vaccines are mostly based on attenuated live viruses or recombinant proteins, which have problems such as insufficient immunogenicity, difficulty in simultaneously activating humoral and cellular immunity, low antigen display efficiency, and complex production processes, and pose safety risks to immunocompromised individuals, and are difficult to respond quickly to viral mutations and global pandemic needs, this invention aims to provide a monkeypox virus nanoparticle vaccine based on HBc-VLP vector, its preparation method and application.

[0008] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a monkeypox virus nanoparticle vaccine based on an HBc-VLP vector, comprising: A hepatitis B virus core protein virus-like particle vector, wherein the hepatitis B virus core protein virus-like particle vector is fused with SpyCatcher protein; And monkeypox virus antigen peptide, wherein the monkeypox virus antigen peptide is fused with a SpyTag tag; The amino acid sequence of the monkeypox virus antigen polypeptide is shown in SEQ ID NO.1.

[0009] The monkeypox virus antigen peptide is covalently coupled to the surface of the hepatitis B virus core protein virus-like particle carrier via heteropeptide bonds formed between its fused SpyTag tag and the SpyCatcher protein, forming nanoparticles displaying the monkeypox virus antigen peptide.

[0010] The amino acid sequence of the monkeypox virus antigen polypeptide fused with the SpyTag tag is shown in SEQ ID NO.2; the amino acid sequence of the hepatitis B virus core protein virus-like particle vector fused with SpyCatcher protein is shown in SEQ ID NO.3.

[0011] Preferably, the particle size of the monkeypox virus nanoparticle vaccine is 30-50 nm; more preferably, the particle size is 35-40 nm.

[0012] This invention provides a polynucleotide, the polynucleotide comprising: The first nucleotide sequence encodes the hepatitis B virus core protein virus-like particle vector fused with SpyCatcher protein; and / or The second nucleotide sequence encodes the monkeypox virus antigenic epitope polypeptide fused with the SpyTag tag.

[0013] The present invention provides an expression vector comprising the aforementioned polynucleotide.

[0014] The present invention provides a host cell comprising the expression vector, wherein the host cell is used to express the hepatitis B virus core protein virus-like particle backbone or the monkeypox virus antigen polypeptide.

[0015] This invention provides a method for preparing a monkeypox virus nanoparticle vaccine based on an HBc-VLP vector, comprising the following steps: (1) Construct a virus-like particle vector for hepatitis B virus core protein fused with SpyCatcher protein; (2) Synthesize and purify an antigenic epitope polypeptide derived from monkeypox virus M1R protein fused with a SpyTag tag; (3) The hepatitis B virus core protein virus-like particle vector obtained in step (1) is mixed with the antigenic epitope polypeptide obtained in step (2), and covalently reacted through the SpyTag / SpyCatcher system to obtain a monkeypox virus nanoparticle vaccine displaying the antigenic polypeptide.

[0016] In step (1), the hepatitis B virus core protein virus-like particle vector fused with SpyCatcher protein is prepared by a prokaryotic expression system; in step (3), the molar ratio of the hepatitis B virus core protein virus-like particle vector to the antigen peptide is 1:1 to 1:5.

[0017] Preferably, the prokaryotic expression system is an Escherichia coli expression system, and the induction conditions are: adding IPTG to a final concentration of 0.1-1.0 mM and inducing at 16-25℃ for 10-16 hours.

[0018] Preferably, the purification includes affinity chromatography and gel filtration chromatography to obtain a protein with a purity >95%.

[0019] The antigenic polypeptide fused with the SpyTag tag described in step (2) was synthesized using a solid-phase synthesis method and purified by HPLC with a purity >95%. Preferably, in step (3), the mixing incubation temperature is 4-25℃ and the incubation time is 8-24 hours.

[0020] The present invention provides an immune composition comprising the aforementioned monkeypox virus nanoparticle vaccine, and a pharmaceutically acceptable adjuvant or carrier.

[0021] Preferably, the adjuvant is QS-21.

[0022] The application of the monkeypox virus nanoparticle vaccine based on the HBc-VLP vector, or the polynucleotide, or the expression vector, or the host cell in the preparation of drugs for the prevention or treatment of monkeypox virus infection.

[0023] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a monkeypox virus nanoparticle vaccine based on an HBc-VLP vector. This monkeypox virus nanoparticle vaccine comprises an "HBc-VLP backbone + SpyCatcher / SpyTag conjugation system + M1R antigen peptide." Irreversible heteropeptide bonds are formed between the SpyTag and SpyCatcher, ensuring the antigen is displayed on the VLP surface in the correct orientation and stable conformation, avoiding antigen inactivation or detachment caused by physical adsorption or non-directional conjugation. The antigen peptide, derived from the monkeypox virus M1R protein, ensures that the selected epitopes are overlapping epitopes of B cells and T cells. Combining the nanoscale advantage of VLP particles, this vaccine can not only be recognized by B cells but also efficiently phagocytosed by antigen-presenting cells and presented to T cells, thereby simultaneously activating humoral and cellular immunity, solving the problem of traditional vaccines' inability to simultaneously activate two types of immune responses.

[0024] Furthermore, the overlapping B-cell and T-cell epitopes obtained through immunoinformatics screening were predicted and verified to be antigenic (VaxiJen score 0.8381), capable of simultaneously activating humoral and cellular immunity. These epitopes exhibit high affinity for various HLA class I and II molecules, covering over 91% of the global population, making them suitable for recipients with diverse genetic backgrounds. Bioinformatics predictions indicate that the sequence is non-toxic and non-sensitizing, making it suitable as a vaccine antigen. By introducing a flexible linker peptide (GGGGS repeat) and a SpyTag sequence at the C-terminus of the antigenic peptide, the flexible linker peptide reduces steric hindrance between the SpyTag and the antigenic peptide, ensuring proper exposure of the antigenic epitope and facilitating immune recognition. The SpyTag sequence can rapidly form covalent bonds with the SpyCatcher on the VLP surface, exhibiting high coupling efficiency without the need for additional chemical cross-linking agents, thus avoiding non-specific reactions. The fusion expression design simplifies the preparation and purification of the antigenic peptide, eliminating the need for complex chemical modifications. The HBc core protein retains complete self-assembly domains, enabling efficient expression in prokaryotic expression systems and spontaneous assembly into uniform VLP particles with a diameter of approximately 30-35 nm.

[0025] The nucleotides provided by this invention can be used to obtain recombinant expression vectors through genetic engineering, enabling efficient and stable expression of VLP backbone and antigenic peptides, laying the foundation for large-scale vaccine production; two polynucleotides can be selected to be used alone or in combination as needed, adapting to different expression systems and production strategies.

[0026] The preparation method provided by this invention simplifies the vaccine development process. The VLP backbone and antigenic peptides are prepared separately and then assembled via coupling, avoiding the complex integrated expression and folding process, simplifying the process flow, facilitating rapid screening and replacement of different antigens, and possessing a "plug-and-play" platform advantage, accelerating vaccine development. Using the SpyTag / SpyCatcher system, antigens can be covalently and site-specifically linked to the VLP, ensuring consistent orientation and structural stability. SpyTag / SpyCatcher coupling can be performed at room temperature and neutral pH, without the need for harsh reaction conditions, which is beneficial for maintaining antigen activity. Combining the Tag / Catcher HBc platform with the monkeypox multi-epitope vaccine design concept has broad antigen compatibility. Antigen compatibility is independent of gene fusion, capable of displaying complex protein antigens of varying sizes and structures (from small peptides to trimers exceeding 300 kDa) while maintaining their native conformation. This ensures that each monkeypox epitope antigen is displayed in the correct orientation and conformation, which is particularly important for inducing protective antibodies against conformation-dependent epitopes. This process is easy to scale up from laboratory scale to industrial production and has good prospects for industrialization.

[0027] The immune composition provided by this invention contains an adjuvant or a carrier. The adjuvant (such as QS-21) can synergistically work with VLP nanoparticles to further enhance the level of immune response. The pharmaceutically acceptable carrier can improve the storage stability and in vivo delivery efficiency of the vaccine. Appropriate adjuvants and formulations can be flexibly selected according to different vaccination populations and routes of administration.

[0028] The application provided by this invention is a vaccine used to prevent or treat monkeypox virus infection, providing legal protection for subsequent clinical research and application. The M1R protein is a conserved protein of the orthopoxvirus genus, and vaccines targeting this protein may have cross-protective effects against non-branched, West African branch, and other orthopoxviruses of monkeypox. This invention provides a novel, safe, and highly effective vaccine candidate for responding to monkeypox outbreaks, and has significant social value and market prospects. Attached Figure Description

[0029] Figure 1 This is an SDS-PAGE electrophoresis image of the tandem multiepitope protein coupled with HBc-Spy according to the present invention (M: protein molecular weight standard marker; 1: HBc VLP vector (control group); 2: HBc VLP coupled with tandem multiepitope protein at a molar ratio of 1:1; 3: HBc VLP coupled with tandem multiepitope protein at a molar ratio of 1:2). Figure 2 Electron microscopy examination of HBc-Spy before and after conjugation with tandem epitope protein. In the image, a is the TEM morphology of HBc-Spy VLP without antigen conjugation, and b is the morphology of P1-HBc vaccine sample after conjugation of HBc-Spy with epitope protein. Figure 3 This is a bar chart showing the antibody titers of each group in Example 5 of the present invention; Figure 4 The values ​​represent the IFN-γ factor levels produced by T cell responses in each group in Example 6 of this invention, where a represents the statistical results of spot data for each group, and b represents the immunospot scan images for each group. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0031] This patent discloses a method for preparing a novel monkeypox virus nanoparticle vaccine based on the AP205-VLP vector, comprising the following steps: 1. Antigen sequence screening The M1R protein sequence from the 2022 monkeypox epidemic strain MPXV_USA_2022_MA001 (GenBank accession no. ON563414.2) was downloaded from the NCBI database, and dominant antigenic epitopes were screened using computer simulation and immunoinformatics tools. HLA class I (HLA-A) 01:01, -A 02:01, -A 03:01, -A 11:01, -A 24:02, -B 07:02, -B 08:01, -B 15:01, -B 40:01 and -C 07:02) It can cover 91.7% of the global population. HLA-DR (HLA-DRB1) 01:01, -DRB1 03:01, -DRB1 04:01, -DRB1 07:01, -DRB1 11:01 and -DRB1 (15:01) This covers 70.6% of the global population. Therefore, to obtain T-cell epitopes with high population coverage, T-cell epitope prediction was mainly conducted within the above HLA types. Epitope sequences with strong binding affinity to HLA class I (10 types) and HLA class II (6 types) (covering over 91% of the global population) were screened, and overlapping epitope sequences of classes I and II were further screened. Simultaneously, B-cell epitope prediction was performed, and overlapping T and B-cell epitopes were screened to obtain candidate epitopes that could induce both cellular and humoral immunity. Subsequently, the immunogenicity and biochemical characteristics of each epitope were analyzed, and candidate epitopes with immunogenicity, non-toxicity, and non-sensitizing properties were selected, resulting in one polypeptide antigen epitope (Table 1). This epitope was then used for polypeptide synthesis and subsequent experimental validation.

[0032] 2. VLP Particle Preparation This patent describes the construction of the expression plasmid pET-28a(+)-HBc-Spy, a self-assembled HBc-VLP nanovaccine vector, using genetic engineering methods. After successful enzyme digestion and sequencing identification, the vector was transformed into host bacteria for engineering. HBc-VLP was expressed and purified in a prokaryotic expression system, yielding a recombinant self-assembled HBc-VLP protein vector with a purity greater than 95% (Table 2). Further immunoblotting identification and VLP morphology and particle size determination were performed. This HBc-VLP protein self-assembles a nano-core backbone, enabling the target antigen to attach to the backbone surface via heteropeptide coupling to form a vaccine. The selected peptide antigen P1 epitope sequence was tagged and then coupled using the SpyTag system to obtain the P1-HBc nanoparticle vaccine.

[0033] 3. Pharmacodynamic evaluation This patent conducts a preliminary pharmacodynamic evaluation of the P1-HBc nanoparticle vaccine by immunizing BALB / c mice. Simultaneously, the immunization effects were compared with a positive control group (M1R protein) and a P1-KFE8 (a self-assembled nanovaccine based on the KFE8 carrier) control group. The immunization method was as follows: three immunizations were administered via subcutaneous injection at multiple sites in the hind neck, 100 μL per mouse, with each immunization spaced two weeks apart. 100 μL of physiological saline per mouse served as a control. Ten days after the third immunization, blood was collected from the eyeballs to obtain mouse antiserum, and specific antibodies were detected by ELISA. Neutralizing antibody titers were further detected by competitive binding ELISA. PBMCs from each group of mice were collected, and antigen-specific T cell activation was detected by the Elispot assay. The peptide vaccine prepared in this patent exhibited certain antigenicity in both antibody titers and T cell responses in mice, demonstrating significant advantages in vaccine construction, preparation, and safety.

[0034] The technical solution of this patent will be described in detail below with reference to specific embodiments and accompanying drawings. These embodiments do not constitute a limitation on this patent.

[0035] In this invention, Escherichia coli BL21 (DE3) and E. coli DH5α (used for plasmid amplification) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; pET-28a (+), carrying a kanamycin resistance selection marker and an N-terminal His-Tag tag, was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0036] Example 1: Screening and Design of Monkeypox Virus M1R Protein Epitopes Download the amino acid sequence of the M1R protein of the 2022 monkeypox epidemic strain MPXV_USA_2022_MA001 (GenBank accession number: ON563414.2) from the NCBI database. The sequence is 250 amino acids long and is the core structural protein on the surface of monkeypox virus, which is highly conserved in the orthopoxvirus genus.

[0037] (1) T cell epitope prediction T-cell epitopes are recognized by two independent T-cell subsets, CD8 (MHC-I) and CD4 (MHC-II). Using the T-cell epitope prediction tool from the Immune Epitope Database (IEDB, http: / / www.iedb.org / ), MHC-I and MHC-II molecularly restricted T-cell epitopes were predicted from the M1R protein sequence. To obtain T-cell epitopes with high population coverage, the HLA allele combinations with the highest coverage in the global population were selected for prediction. MHC class I (HLA-A) 01:01, -A 02:01, -A 03:01, -A 11:01, -A 24:02, -B 07:02, -B 08:01, -B 15:01, -B 40:01 and -C (07:02) It can cover 91.7% of the global population.

[0038] MHC class II (HLA-DRB1) 01:01, -DRB1 03:01, -DRB1 04:01, -DRB1 07:01, -DRB1 11:01 and -DRB1 (15:01) It can cover 70.6% of the global population.

[0039] Prediction parameter settings: Epitope length set to 12-20 amino acids (MHC-II) and 8-10 amino acids (MHC-I), binding affinity threshold set to IC50. 50 ≤200nM. Screen for epitope sequences that strongly bind to the above HLA molecules, and further screen for overlapping epitopes that can bind to both MHC-I and MHC-II molecules simultaneously.

[0040] (2) B-cell epitope prediction The BPred server (http: / / crdd.osdd.net / raghava / bcpred / ) was used to predict linear B-cell epitopes of the M1R protein. Parameter settings were: epitope length set to 16 amino acids, threshold set to 0.8, and other parameters left as default. BPred identified linear B-cell epitopes and selected those with higher scores. This epitope is crucial for inducing humoral immune responses that stimulate B lymphocytes to produce antibodies.

[0041] (3) Screening of overlapping epitopes The predicted T-cell epitopes were compared with the predicted B-cell epitopes to screen for overlapping regions that possess the potential of both B-cell and T-cell epitopes. Screening criteria: The region appears in both B-cell and T-cell epitope predictions and can bind to multiple HLA molecules.

[0042] A candidate epitope sequence of 19 amino acids, named P1, was obtained through screening and located at amino acids 213-231 of the M1R protein. This sequence contains both B-cell and T-cell epitopes and can be predicted to bind to various MHC class I and MHC class II molecules.

[0043] (4) Population coverage analysis The population coverage of the selected MHC-I and MHC-II epitopes and related HLA binding alleles was calculated using the IEDB population coverage tool (http: / / tools.iedb.org / population / ). The results showed that the HLA allele combination corresponding to the epitope can cover more than 91% of the global population, demonstrating good broad applicability.

[0044] (6) Immunogenicity and safety analysis Immuninformatics analysis was performed on the screened P1 epitopes to assess their feasibility as vaccine antigens: a. Antigenicity prediction The VaxiJen v2.0 web software (http: / / www.ddg-pharmfac.net / vaxijen / ) was used to predict antigenicity, with a default threshold of 0.4. The results showed that the VaxiJen score for the P1 epitope was 0.8381, exceeding the threshold of 0.4, indicating antigenicity.

[0045] b. Toxicity prediction ToxinPred software (https: / / webs.iiitd.edu.in / raghava / toxinpred / ) was used to predict toxicity. The results showed that the P1 epitope was determined to be non-toxic (Non-Toxin).

[0046] c. Allergenicity prediction Allergenicity was predicted using the AllerTOP 2.0 web tool (https: / / www.ddg-pharmfac.net / AllerTOP / ). The results showed that the P1 epitope was not sensitizing.

[0047] d. Immunogenicity score The physicochemical properties, including molecular weight, theoretical isoelectric point, and hydrophilicity, were analyzed using the ProtParam tool in ExPASy software (https: / / web.expasy.org / protparam / ). The results showed that the epitope is physicochemically stable and suitable for peptide synthesis.

[0048] (7) Spatial localization analysis of epitopes The location of epitopes in antigen proteins was simulated using MOE (Molecular Operating Environment) software to observe whether they were exposed on the protein surface. The simulation results showed that the P1 epitope was located in the exposed surface region of the M1R protein, which is easily recognized by the immune system and meets the spatial conformation requirements of antigen epitopes.

[0049] (8) Candidate epitope determination The epitope sequences, immunogenicity, and biological characteristics obtained from the above screening are detailed in Table 1. Figure 1 As shown.

[0050] Table 1: P1 sequences of peptide epitopes screened from antigen M1R protein

[0051] Table 1 shows the specific amino acid sequence, length, source protein, MHC coverage, antigenicity, and immunogenicity data of the polypeptide epitopes from the M1R protein screened in Example 1. These polypeptides are overlapping epitope sequences of B cells and T cells, with a length not exceeding 50 amino acids, are easy to synthesize, and possess certain antigenicity.

[0052] Example 2: Expression and purification of the HBc-VLP backbone This embodiment details the preparation process of the HBc-VLP self-assembled nanocore backbone based on the SpyTag / SpyCatcher system. The recombinant expression plasmid pET-28a(+)-HBc-Spy was constructed using genetic engineering methods, transformed into *E. coli* BL21(DE3) for induced expression, and purified to obtain a high-purity (>95%), correctly self-assembled HBc-Spy fusion protein VLP core backbone, which will be used for subsequent covalent coupling of the target antigen via the SpyTag / SpyCatcher system.

[0053] (1) Synthesis and plasmid construction of HBc-Spy fusion gene Based on the HBcAg gene sequence (Gene ID: 944568) published in GenBank, a nucleotide sequence encoding a flexible peptide linker (GGGGSGGGGSGGGGS) is linked to the coding sequence of SpyCatcher at its C-terminus. The nucleotide sequence of the entire HBc-Spy fusion gene was optimized for E. coli preferred codons, and its amino acid sequence is shown in SEQ ID NO. 3. The gene was synthesized in its entirety by Sangon Biotech (Shanghai) Co., Ltd.

[0054] Table 2: HBc-Spy related sequences

[0055] The synthesized HBc-Spy gene fragment was double-digested with NdeI and XhoI, and the pET-28a(+) vector was linearized using the same enzymes. The digestion products were recovered and ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells, plated on LB agar plates containing kanamycin (50 μg / mL), and incubated overnight at 37°C. Single colonies were picked, plasmids were extracted, and double-digested with NdeI / XhoI. Analysis by 1% agarose gel electrophoresis showed the size was consistent with the expected size. Positive clones identified by enzyme digestion were sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing. BLAST alignment of the sequencing results showed that the inserted HBc-Spy gene sequence was completely correct, with no reading frame errors and no base mutations; the correctly identified recombinant plasmid was named pET-28a(+)-HBc-Spy.

[0056] (2) Induced expression of HBc-Spy fusion protein The correctly sequenced pET-28a(+)-HBc-Spy plasmid was transformed into the expression host bacterium *E. coli* BL21 (DE3) and plated on LB agar plates containing kanamycin, incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB medium containing kanamycin, and cultured overnight at 37°C with shaking at 220 rpm. The overnight culture was then transferred to fresh LB medium containing kanamycin at a 1:100 volume ratio and cultured at 37°C with shaking at 220 rpm until the bacterial culture reached OD500. 600 Approximately 0.6. Add IPTG to a final concentration of 0.5 mM, and continue induction culture at 20℃ and 180 rpm for 4 hours. Lyse using the FHPure® E. coli Lysis Clarification Kit (Xi'an Maiboteike, model 11-230016-10) and centrifuge. First, purification was performed using a cation exchange chromatography column. The lysed precipitate was dissolved in 20 mM Tris-HCl, 1 mM EDTA, and 10 mM MTT at pH 8.0 at a ratio of 1:20. After centrifugation, the supernatant was purified using a D6246 cation exchange column (CV = 400 mL) at a flow rate of 30 mL / min. The equilibration buffer was 20 mM Tris-HCl at pH 8.0. After equilibration for 5 CVs, the protein was loaded onto the column and eluted with a gradient of 20 mM Tris-HCl + 1 M sodium chloride at pH 8.0 to collect the protein peak. Further purification was then performed using molecular sieve chromatography. The target protein was further purified using a Chromdex-75 molecular sieve (CV = 1600 mL) at a flow rate of 10 mL / min using a buffer of 20 mM Tris-HCl + 150 mM sodium chloride at pH 8.0 to collect the target protein peak. Finally, the protein was purified and concentrated using a Source 30S column. Further purification and concentration were performed on a Source 30S cation exchange column (CV = 70 mL). The equilibration buffer was 20 mM Tris-HCl, pH = 8.0. After equilibration for 5 CVs, the protein was loaded and eluted with a gradient of 20 mM Tris-HCl + 1 M sodium chloride, pH = 8.0 to collect the protein peak. The purity of the target protein was determined by SDS-PAGE.

[0057] (3) Identification and Analysis SDS-PAGE: Samples from before induction, after induction, and after purification were subjected to 15% SDS-PAGE electrophoresis. Protein purity was analyzed after Coomassie Brilliant Blue R-250 staining. Analysis using a gel imaging system showed that the purity of the purified target protein was >95%.

[0058] Western Blot: Protein from SDS-PAGE gels was transferred to PVDF membranes using a semi-dry method. After blocking with 5% skim milk powder, the membranes were incubated overnight at 4°C with mouse anti-His-tagged monoclonal antibody (1:5000 dilution, Kangwei Century). After washing, the membranes were incubated at room temperature for 1 hour with the corresponding HRP-labeled secondary antibody (1:5000 dilution). ECL chemiluminescence staining showed a specific band at approximately 25 kDa, confirming that the purified protein was a His-tagged HBc fusion protein.

[0059] TEM and DLS: A suitable amount of purified protein sample (approximately 0.1 mg / mL) was dropped onto a copper grid covered with a carbon membrane, allowed to stand for 2 minutes, and excess liquid was absorbed with filter paper. The sample was negatively stained with 2% phosphotungstic acid (pH 6.5) at room temperature for 2 minutes, excess stain was absorbed with filter paper, and the sample was dried at room temperature before observation under a transmission electron microscope. At an accelerating voltage of 80 kV, numerous spherical particles with a diameter of approximately 30-35 nm, uniform in morphology, and spherical or quasi-spherical virus-like particles were observed, confirming the successful self-assembly of the purified HBc-Spy fusion protein.

[0060] The purified protein sample was diluted to approximately 0.5 mg / mL, filtered through a 0.22 μm filter membrane, and then added to a quartz cuvette. The particle size distribution was determined using a dynamic light scattering particle size analyzer at 25 °C. The results showed that the average hydrated particle size of the sample was 32.4 nm, and the polydispersity index (PDI) was <0.1, indicating that the VLPs had uniform morphology and good dispersibility.

[0061] This embodiment successfully established a complete process from gene construction, protein expression, purification to identification, obtaining a high-purity, highly uniform, and correctly self-assembling HBc-Spy fusion protein VLP core backbone. This HBc-VLP protein self-assembles a nano-core backbone, which can attach the target antigen to the backbone surface via heteropeptide coupling to form a vaccine. This provides a stable and reliable vector platform for subsequent covalent coupling of P1 peptide antigen with the SpyTag / SpyCatcher system to construct P1-HBc nanoparticle vaccines.

[0062] Example 3: Conjugation preparation and characterization of P1-HBc nanoparticle vaccine This embodiment details the entire process of obtaining a P1-HBc nanoparticle vaccine by covalently coupling the P1 antigen peptide derived from the monkeypox virus M1R protein obtained in Example 1 to the surface of the HBc-VLP nanocore framework prepared in Example 2 using the SpyTag / SpyCatcher system. The purity, coupling efficiency, and morphology of the conjugated product were also characterized.

[0063] HBc-VLP core framework: HBc-Spy fusion protein (carrying the SpyCatcher domain) with a purity >95% prepared in Example 2 was dissolved in PBS buffer (pH 7.4) at a concentration of 2 mg / mL.

[0064] (1) Synthesis of P1-tag antigen peptide Based on the P1 antigen polypeptide sequence (SEQ ID NO.1) obtained in Example 1, a SpyTag sequence (AHIVMVDAYKPTK) was linked to its C-terminus via a flexible linker peptide (GGGGSGGGGSGGGGS) to obtain the fusion polypeptide P1-tag, the amino acid sequence of which is shown in SEQ ID NO.2 (see Table 3). This fusion polypeptide was synthesized by Sanyou Biopharmaceutical (Shanghai) Biotechnology Co., Ltd. using a solid-phase synthesis method and purified by HPLC to a purity >95%. It was stored as a lyophilized powder and dissolved in sterile PBS to a concentration of 1 mg / mL before use.

[0065] Table 3: P1 epitope sequence after tagging

[0066] (2) Coupling of P1-HBc nanoparticles Both the HBc VLP vector and the fusion peptide were dialyzed into coupling buffer (20 mM Tris-HCl + 50 mM NaCl, pH 8.5) for 24 h, with two buffer changes. Then, HBc VLP and the fusion peptide P1-tag were mixed at molar ratios of 1:1 and 1:2 in 50 mL solutions and incubated overnight at 4°C for coupling. The next day, the reaction was terminated by adding 10 mM Tris-HCl (pH 8.0).

[0067] (3) Identification and characterization of coupling products A. SDS-PAGE identification of coupling: Electrophoresis: After determining protein concentration using the BCA method, samples were loaded for 15% SDS-PAGE electrophoresis. Coomassie Brilliant Blue staining was performed after electrophoresis. Result analysis: The positions of protein bands in each lane were observed and compared. The theoretical size of the HBc-Spy monomer is approximately 29 kDa (21 kDa HBc + 8 kDa SpyCatcher and linker). The size of the P1-tag peptide is approximately 4 kDa. After successful P1-HBc coupling, due to the formation of a covalent isopeptide bond between SpyTag and SpyCatcher, a new fusion band of approximately 30 kDa should appear, while the HBc-Spy monomer band should significantly weaken or disappear. The coupling efficiency can be calculated by analyzing the grayscale using a gel imaging system. SDS-PAGE results are shown (see [link]). Figure 1It can be seen that the coupling efficiency is highest when the molar ratio of HBc VLP to tandem multi-epitope protein is 1:2.

[0068] B. Morphological observation by transmission electron microscopy (TEM) Take an appropriate amount of purified P1-HBc vaccine sample (approximately 0.1 mg / mL) and perform negative staining and TEM observation as described in Example 2. Compare the morphology with that of the unconjugated HBc-Spy VLP in Example 2 to observe whether the VLP particles still maintain a complete spherical shape after antigen conjugation, and whether there is aggregation or deaggregation. See Appendix. Figure 2 .

[0069] A typical VLP should present a hollow spherical structure without nucleic acids, with clear boundaries and uniform morphology, consisting of attached... Figure 2 It can be seen that the HBc VLP vector, after being coupled with tandem multi-epitope proteins, exhibits a typical VLP morphology.

[0070] C. Dynamic Light Scattering (DLS) Particle Size Analysis Take an appropriate amount of purified P1-HBc vaccine sample (approximately 0.5 mg / mL) and perform DLS determination according to the method described in Example 2. Record the average hydrated particle size and polydispersity index, and compare them with the particle size of HBc-Spy VLP before conjugation to analyze the changes in particle size and uniformity after antigen conjugation.

[0071] Example 4: Animal immunization and sample collection This embodiment details the experimental protocol for immunizing BALB / c mice with the P1-HBc nanoparticle vaccine prepared in Example 3, including specific operational steps for animal grouping, immunization dosage, immunization procedure, serum sample collection, and spleen lymphocyte isolation, providing sample materials for subsequent antibody titer detection (Example 5) and T cell response detection (Example 6).

[0072] (1) Experimental groups: Female BALB / c mice (17-19g, purchased from the Animal Center of Air Force Medical University) were divided into 4 groups of 5 mice each for immunization, as follows: Negative control group: (physiological saline, 100 μL / animal), without adjuvant Positive control group: (M1R 15μg / animal) + 15 μg adjuvant (QS-21) / animal P1-HBc group: P1-HBc nanoparticle vaccine (100 μg / animal) + 15 μg adjuvant (QS-21) / animal; P1-KFE8 control group: P1-KFE8 self-assembled nanovaccine (200 nmol / animal) + 15 μg adjuvant (QS-21 / animal); (2) Vaccine preparation P1-HBc vaccine preparation: Take the P1-HBc nanoparticle vaccine (concentration 1 mg / mL) prepared in Example 3, and calculate the required volume based on 100 μg (i.e., 100 μL) per mouse. Mix with adjuvant QS-21 (15 μg / mouse), and add sterile PBS to make up to 100 μL of immunization volume per mouse. Prepare fresh before use.

[0073] M1R protein vaccine preparation: Take M1R recombinant protein (concentration 0.15 mg / mL) (Novazia, RM2152-02), and calculate the required volume based on 15 μg (i.e., 100 μL) per mouse. Mix with adjuvant QS-21 (15 μg / mouse) (MCE, HY-101092), and add sterile PBS to a final immunization volume of 100 μL per mouse. Prepare fresh before use.

[0074] P1-KFE8 vaccine preparation: Wherein, P1 is the polypeptide screened in Example 1 of this invention; the amino acid sequence of the PADRE helper T cell epitope is AKFVAAWTLKAAA, as shown in SEQ ID NO.7; the sequence of KFE8 is (acetylated) Ac-FKFEFKFE-NH2 (amidated), as shown in SEQ ID NO.8; the components and proportions of the P1-KFE8 vaccine are shown in Table 4. Table 4: Components and Proportions of P1-KFE8 Vaccine

[0075] Mix the components according to the above proportions, add adjuvant QS-21 (15 μg / mouse), and supplement with sterile PBS to make up to 100 μL of immunization volume per mouse. Prepare fresh before use.

[0076] (3) Immunization regimen The immunization method was as follows: 100 μL was injected subcutaneously at multiple points behind the neck into each mouse, for a total of three immunizations, at week 0 (primary immunization), week 2 (day 14, first booster immunization), and week 4 (day 28, second booster immunization), with each immunization spaced two weeks apart. Ten days after the third immunization, blood was collected from the eyeballs of the mice, and spleen cells were isolated.

[0077] Blood collection: Blood was collected from the eyeballs of mice. The obtained mouse blood was left at room temperature for 2 hours, centrifuged at 12000g for 20 minutes, and the supernatant was the mouse antiserum, which was stored at -20℃.

[0078] Splenic cell extraction: After blood was collected from the eyeballs of mice, the mice were euthanized by cervical dislocation. Spleen tissue from each group of immunized mice was collected and soaked in PBS. A 70µm filter was placed on a 50mL centrifuge tube, moistened with PBS, and the spleen was placed in the filter. The cells were ground with a pipette tip, and the filter was washed with PBS to obtain a single-cell suspension. The suspension was centrifuged at 400g for 5 min, and the supernatant was discarded. 1-2mL of cytosolic lysate was added, and the suspension was incubated on ice for 5 min. The cytosolic lysate was stopped by adding PBS, and the suspension was centrifuged at 400g for 5 min, and the supernatant was discarded. The cells were washed with PBS, centrifuged at 400g for 5 min, and the supernatant was discarded. The cells were resuspended in 1640 complete culture medium and counted for later use.

[0079] Throughout the immunization process, all mice in each group survived, maintained good mental condition, and had normal appetites. No adverse reactions such as redness, swelling, or ulceration were observed at the injection sites, indicating that the vaccines had good safety profiles. Serum and spleen lymphocyte samples were successfully collected from each group of mice. The serum samples were clear and transparent, with no hemolysis; the spleen lymphocyte yield met the requirements for subsequent testing.

[0080] Example 5: Detection of antibody titers induced by P1-AP205 nanoparticle vaccine This embodiment uses an enzyme-linked immunosorbent assay (ELISA) to detect the specific antibody titer in the serum of immunized mice in Example 4. Using monkeypox virus M1R protein as the coating antigen, the levels of specific antibodies against M1R protein in the serum of each group of mice (negative control group, positive control group, P1-HBc group, and P1-KFE8 group) were detected to evaluate the humoral immunization effect of the P1-HBc nanoparticle vaccine.

[0081] Serum samples: The serum samples were obtained from the mice after immunization in each group collected in Example 4, including the negative control group (physiological saline), the positive control group (M1R protein immunization), the P1-HBc group, and the P1-KFE8 group, with 5 mice in each group. The samples were stored at -20°C for later use.

[0082] Reagents: Coating buffer: 0.05 M carbonate buffer (pH 9.6), formulation: Na₂CO₃ 1.59 g, NaHCO₃ 2.93 g, diluted with distilled water to 1000 mL; Blocking buffer: PBST containing 5% skim milk powder. Secondary antibody: HRP-labeled goat anti-mouse IgG secondary antibody, purchased from Kangwei Century Company. Chromogenic solution: TMB single-component chromogenic solution, purchased from Xinsaimei Biotechnology Company. Stop solution: 2 M H₂SO₄.

[0083] Coating: Take a 96-well ELISA plate, dissolve the antigen protein M1R (Novizan, model BM2152-01, purity 95%) in coating buffer (0.5 μg / mL), add 100 μL / well to the plate wells, and coat overnight at 4°C.

[0084] Discard the coating solution, add 250 μL of PBST washing buffer to each well, let stand for 2 minutes and then discard. Add 200 μL of PBST washing buffer to each well and pat dry on absorbent paper. Repeat the washing process 3 times. Add 200 μL of blocking buffer to each well and block at room temperature for 2 h.

[0085] Discard the blocking solution and wash 6 times with PBST washing solution, 2 min each time; Primary antibody incubation: Mouse serum from each group was serially diluted with PBST at dilution ratios of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and 1:51200. 100 μL of diluted serum was added to each well, with three replicates per serum sample. PBST was added to the negative control wells instead of serum, and 100 μL of blocking buffer was added to the blank control wells. The samples were incubated at room temperature for 1 hour. After incubation, the serum was discarded, and the mice were washed six times with PBST for 2 minutes each time. Secondary antibody incubation: Add HRP-labeled goat anti-mouse IgG diluted 1:5000 with PBST, 100 μL / well, and incubate at room temperature for 1 h; discard the secondary antibody and wash 6 times with PBST washing buffer, 3 min each time; Color development and termination: Add 100 μL of TMB color development solution to each well and incubate at room temperature for 10-20 min. When the color in the well turns blue, add 50 μL of 2M H2SO4 stop solution to terminate the reaction. The color in the well will change from blue to yellow.

[0086] The absorbance (OD) of each well at 490 nm was measured using a microplate reader. 490 The results of the detection of specific antibody titers in the serum of mice in each group are as follows: Figure 3 As shown. From the appendix Figure 3 Data showed that the specific antibody titers against monkeypox virus M1R protein in the serum of immunized mice in each group were successfully detected by indirect ELISA. The P1-HBc nanoparticle vaccine induced mice to produce high-titer specific antibodies, with the highest antibody titer level (average 26,880), significantly better than the M1R protein positive control group and the P1-KFE8 control group. The antibody titers of all antigen-containing immunization groups (positive control group, P1-KFE8 group, and P1-HBc group) were above 10,000, indicating that all vaccines had good immunogenicity. The P1-HBc nanoparticle vaccine, through high-density display of the P1 antigen on the HBc-VLP vector, significantly enhanced the humoral immune response, confirming the immunomodulatory effect of the HBc-VLP vector.

[0087] The results of this embodiment validate the superior humoral immune effect of the P1-HBc nanoparticle vaccine. Compared with soluble M1R protein, the P1-HBc nanoparticle vaccine displays the P1 antigen at a high density on the surface of HBc-VLP, forming a repeating structure that effectively cross-links B cell receptors, significantly enhancing B cell activation efficiency and thus inducing higher titers of specific antibodies. Compared with the P1-KFE8 self-assembled nanovaccine, the HBc-VLP vector has a more regular icosahedral structure and superior immunogenicity, thus inducing a stronger antibody response.

[0088] Example 6: Detection of T-cell immune response induced by HBc-VLP nanoparticle vaccine This embodiment details the experimental method for detecting antigen-specific T cell responses in spleen lymphocytes of immunized mice in Example 4 using the IFN-γ ELISpot method. Using P1 polypeptide as a stimulus, the frequency of IFN-γ-secreting T cells in spleen cells of each group of mice (negative control group, positive control group, P1-HBc group, and P1-KFE8 group) was detected to evaluate the cellular immunogenicity of the P1-HBc nanoparticle vaccine.

[0089] Spleen cell samples: derived from mouse spleen lymphocytes isolated in Example 4, including negative control group (physiological saline), positive control group (M1R protein immunization), P1-HBc group, and P1-KFE8 group, with 5 mice in each group, freshly isolated or cryopreserved and thawed for use.

[0090] ELISpot pre-coated plate: ELISpot Plus: Mouse IFN-γ (HRP) pre-coated plate (MBT-3321-4HST-2, Mabtech). This plate is pre-coated with anti-mouse IFN-γ capture antibody.

[0091] Cell culture medium: RPMI 1640 complete medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamine, 1% sodium pyruvate, and 0.1% β-mercaptoethanol.

[0092] Stimulant: P1 polypeptide (SEQ ID NO.1), synthesized by Chengdu Shengnuo Biotechnology Co., Ltd., with a purity >95%, dissolved in sterile PBS to 1 mg / mL, aliquoted and stored at -80℃ for later use. Dilute to working concentration with complete culture medium before use.

[0093] Positive control stimulus: ConA (concanavalin A), diluted to 5 μg / mL with complete culture medium.

[0094] Negative control stimulus: Complete culture medium (no irritant).

[0095] Detection antibody: Biotin-labeled anti-mouse IFN-γ detection antibody (as per the kit).

[0096] Secondary antibody: HRP-labeled streptavidin (as per kit).

[0097] Developing solution: TMB developing solution (specifically for ELISpot, included with the kit).

[0098] Washing solution: PBS (PBST) containing 0.05% Tween-20.

[0099] 1) Activation of pre-coated plates: Take an ELISpot Plus: Mouse IFN-γ (HRP) pre-coated plate (Mabtech, catalog number: 3321-4HST-2), remove the Mouse IFN-γ ELISpot pre-coated plate, add 200 μL of sterile PBS to each well, let stand at room temperature for 2 minutes, discard the PBS, and repeat the washing 4 times; after the last PBS is discarded, add 200 μL of RPMI 1640 complete culture medium to each well and let stand at room temperature for 30 minutes.

[0100] 2) Co-incubation with cell stimulants: Discard the culture medium. Cell suspension preparation: The spleen cells isolated in Example 4 were adjusted to a concentration of 2 × 10⁻⁶ using RPMI 1640 complete medium. 6 cells / mL: Stimulant preparation: Experimental well stimulus: Dilute P1 peptide with complete culture medium to 20 μg / mL (final concentration 10 μg / mL); Positive control stimulus: Dilute PMA + Ionomycin with sterile PBS and add 10 μL of working solution to each well (final concentration PMA 500ug / mL + Ionomycin 10 μg / mL); Negative control stimulus: Complete culture medium (no stimulus). According to the experimental design, 10 μL of the stimulant was added to each well, and three replicates were set up for each sample.

[0101] Add 100 μL of cell suspension to each well, wrap the plate with aluminum foil or plastic wrap to prevent evaporation, and incubate in a 37°C, 5% CO2 cell incubator for 32 h.

[0102] (Previously, aseptic procedures were followed) 3) Antibody incubation: After incubation, discard the cell suspension, add 200 μL PBST to each well, incubate at 4°C for 3-5 min, then discard; repeat washing 5 times.

[0103] Biotin-labeled anti-mouse IFN-γ detection antibody (1 mg / mL) was diluted 1:1000 with PBS containing 0.5% FCS to a working concentration of 1 μg / mL. 100 μL of the diluted antibody was added to each well and incubated at 37°C for 2 h. The detection antibody was discarded and the well was washed 5 times with PBST (PBS containing 0.05% Tween-20), 200 μL / well each time.

[0104] 4) HRP secondary antibody incubation: Dilute streptavidin-HRP 1:1000 with PBS containing 0.5% FBS, add 100 μL to each well, and incubate at 37°C for 1 h. Discard the secondary antibody, wash 3 times with PBST, 200 μL / well each time.

[0105] 5) Color development: Wash the plate, add 100 μL TMB color development solution to each well, develop the color at room temperature in the dark for 10 min, rinse with deionized water to stop the color development, peel off the back film and wash the plate.

[0106] 6) Air dry at room temperature. Automated counting and analysis of the spots were performed using an ELISPOT Reader software. Results were expressed as per 2 × 10⁻⁶. 5 The number of spot-forming cells (SFC) per cell is represented by the number of spots forming cells (SFC). See the appendix for detailed results. Figure 4 As shown.

[0107] From the appendix Figure 4 Data showed that the positive control group, P1-KFE8 group, and P1-HBc group were all significantly higher than the negative control group (P<0.05); the P1-HBc group was significantly higher than the positive control group and P1-KFE8 group (P<0.01 or P<0.001), indicating that it induced the highest level of IFN-γ secretion and had the strongest immunogenicity. It is evident that all vaccine groups could induce a certain degree of T-cell immune response in mice, with the P1-HBc vaccine inducing the strongest T-cell immune response and significantly higher IFN-γ secretion levels than the other groups.

[0108] The results of this embodiment verify the excellent cellular immune response of the P1-HBc nanoparticle vaccine. IFN-γ is a key effector molecule for Th1-type cellular immunity, mainly composed of CD8+. + Cytotoxic T cells and CD4 + Th1 cell secretion plays a crucial role in antiviral immunity. The high levels of IFN-γ secretion induced by the P1-HBc nanoparticle vaccine indicate that the HBc-VLP carrier can efficiently promote antigen cross-presentation: VLP particles can be efficiently taken up, processed, and presented by antigen-presenting cells (such as dendritic cells), activating CD8 cells through the MHC-I molecular pathway. +T cells induce a strong cellular immune response. The P1 epitope contains CD8. + and CD4 + T cell epitopes: P1, screened in Example 1, is an overlapping epitope of B cells and T cells, capable of simultaneously activating CD4. + Helper T cells and CD8 + Cytotoxic T cells. High-density display enhances T cell activation: The P1 antigen displayed at high density on the surface of HBc-VLP can be effectively recognized by T cell receptors, enhancing T cell activation efficiency.

[0109] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A monkeypox virus nanoparticle vaccine based on an HBc-VLP vector, characterized in that, Include: A hepatitis B virus core protein virus-like particle vector, wherein the hepatitis B virus core protein virus-like particle vector is fused with SpyCatcher protein; And monkeypox virus antigenic epitope polypeptide, wherein the monkeypox virus antigenic polypeptide is fused with a SpyTag tag; The amino acid sequence of the monkeypox virus antigenic epitope polypeptide is shown in SEQ ID NO.

1.

2. The monkeypox virus nanoparticle vaccine based on the HBc-VLP vector according to claim 1, characterized in that, The monkeypox virus antigenic epitope polypeptide is covalently coupled to the surface of the hepatitis B virus core protein virus-like particle carrier via an isopeptide bond formed between its fused SpyTag tag and the SpyCatcher protein.

3. The monkeypox virus nanoparticle vaccine based on the HBc-VLP vector according to claim 2, characterized in that, The amino acid sequence of the monkeypox virus antigen polypeptide fused with the SpyTag tag is shown in SEQ ID NO.2; the amino acid sequence of the hepatitis B virus core protein virus-like particle vector fused with SpyCatcher protein is shown in SEQ ID NO.

3.

4. A polynucleotide, characterized in that, The polynucleotide comprises: The first nucleotide sequence encodes the hepatitis B virus core protein virus-like particle vector fused with SpyCatcher protein; and / or The second nucleotide sequence encodes the monkeypox virus antigenic epitope polypeptide fused with the SpyTag tag.

5. An expression carrier, characterized in that, It comprises the polynucleotide of claim 4.

6. A host cell, characterized in that, The host cell comprises the expression vector of claim 5, wherein the host cell is used to express the hepatitis B virus core protein virus-like particle backbone or the monkeypox virus antigenic epitope polypeptide.

7. A method for preparing a monkeypox virus nanoparticle vaccine based on an HBc-VLP vector as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Construct a virus-like particle vector for hepatitis B virus core protein fused with SpyCatcher protein; (2) Synthesize and purify an antigenic epitope polypeptide derived from monkeypox virus M1R protein fused with a SpyTag tag; (3) The hepatitis B virus core protein virus-like particle vector obtained in step (1) is mixed with the antigenic epitope polypeptide obtained in step (2), and covalently reacted through the SpyTag / SpyCatcher system to obtain a monkeypox virus nanoparticle vaccine displaying the antigenic polypeptide.

8. The method for preparing a monkeypox virus nanoparticle vaccine based on an HBc-VLP vector according to claim 7, characterized in that, In step (1), the hepatitis B virus core protein virus-like particle vector fused with SpyCatcher protein is prepared by a prokaryotic expression system; the molar ratio of the hepatitis B virus core protein virus-like particle vector to the antigen peptide is 1:1 to 1:

5.

9. An immune composition, characterized in that, The vaccine comprises the monkeypox virus nanoparticle vaccine according to any one of claims 1-3, and a pharmaceutically acceptable adjuvant or carrier.

10. The use of a monkeypox virus nanoparticle vaccine based on an HBc-VLP vector as described in any one of claims 1 to 3, or the polynucleotide as described in claim 4, or the expression vector as described in claim 5, or the host cell as described in claim 6 in the preparation of a medicament for the prevention or treatment of monkeypox virus infection.

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