A monkeypox virus polyepitope nanoparticle vaccine based on an industrial modular platform and a preparation method and application thereof

Abstract

This invention discloses a monkeypox virus multi-epitope nanoparticle vaccine based on an industrial modular platform, its preparation method, and its applications, belonging to the field of biomedical technology. The vaccine is formed by covalently coupling a recombinant multi-epitope-SpyTag fusion protein with recombinant AP205-SpyCatcher virus-like particles via a SpyTag / SpyCatcher system. The fusion protein contains a SpyTag tag, a PADRE helper T cell epitope, and six dominant monkeypox virus B / T cell epitopes, and is industrially produced using stable CHO cell lines. The AP205-SpyCatcher VLP is prepared on a large scale through engineered bacterial fermentation. The modular assembly achieves a coupling rate of approximately 70%. Animal experiments show that the vaccine induced a significantly higher titer of specific IgG antibodies than the positive control group and the tandem epitope protein group, and activated a potent IFN-γ+ T cell immune response. This invention solves the core bottlenecks of insufficient conformational immunogenicity and industrialization difficulties in multi-epitope vaccines, providing a complete, robust, and scalable vaccine production process with promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and vaccine technology, specifically relating to a monkeypox virus multi-epitope nanoparticle vaccine based on an industrial modular platform, its preparation method, and its application. Background Technology

[0002] The global spread of monkeypox virus (MPXV) has become a major public health threat, and developing safe, effective, and accessible vaccines is crucial for its control. Existing vaccines (such as those based on live attenuated viruses) have limitations in terms of production scale, safety, and duration of immunity. Subunit multiepitope vaccines, due to their precise design and high safety profile, are considered an important direction for next-generation vaccine development. However, their development has long been constrained by two interrelated core bottlenecks: first, how to design and produce complex multiepitope antigens that can mimic the natural conformation of the virus and effectively elicit a protective immune response; and second, how to establish a stable, controllable, and scalable complete production process system to transform laboratory concepts into industrialized products.

[0003] Early explorations of multi-epitope vaccines, such as strategies based on chemically synthesized peptides and self-assembled vectors, have initially verified the feasibility of multi-epitope immunization and screened out potential antigenic epitopes. However, their inherent defects have severely restricted the industrialization process: First, chemically synthesized linear short peptides are difficult to mimic the complex spatial conformation of viral antigens, especially conformation-dependent epitopes, resulting in insufficient immunogenicity; second, the synthesis process of long-chain peptides is complex, purification is difficult, costs are high, and batch-to-batch consistency is poor, making large-scale production neither economical nor feasible.

[0004] To overcome the aforementioned limitations, utilizing virus-like particles (VLPs) as vectors to display antigens is a recognized and effective strategy for enhancing the immunogenicity of subunit vaccines. Currently, VLP vaccines based on platforms such as human papillomavirus (HPV) and hepatitis B virus (HBV) have been successfully applied clinically, validating the reliability of this technological approach. Among them, AP205 VLP, as a structurally well-defined and easily genetically engineered nanoparticle vector, has shown great potential in novel vaccine development and has been explored for preclinical studies of candidate vaccines such as porcine epidemic diarrhea virus (PEDV). Meanwhile, the SpyTag / SpyCatcher system, as a highly efficient and specific bioorthogonal coupling tool, provides a mature technical solution for the site-specific and quantitative display of antigens on the vector surface.

[0005] However, a clear technological gap remains in the specific development of monkeypox virus multiepitope vaccines. While the aforementioned VLP vectors and conjugation tools are known, there are currently no publicly reported successful solutions for creatively and systematically integrating them to address the unique challenges of transitioning monkeypox multiepitope vaccines from the laboratory to industrialization: "how to construct a complete process system integrating high-quality antigen industrial production, standardized VLP vector controllable preparation, and high-precision point assembly." Existing technologies either remain at the level of simple laboratory proof-of-concept, lacking comprehensive process research and pharmaceutical-grade quality control; or are limited by existing antigen production platforms (such as chemically synthesized peptides), failing to simultaneously meet the requirements of conformational accuracy, high yield, and low-cost production.

[0006] In summary, the long-standing technical challenge in this field lies in how to design a novel monkeypox virus multi-epitope antigen containing key auxiliary epitopes, and to match it with a standardized "plug-and-play" production and assembly platform based on the AP205VLP and SpyTag / SpyCatcher system that has undergone sufficient process development and validation, so as to obtain a vaccine product that achieves breakthrough improvements in both immunogenicity and production process feasibility. Summary of the Invention

[0007] To address the two major technical bottlenecks of existing monkeypox multi-epitope vaccines—insufficient conformational immunogenicity due to the use of chemically synthesized peptides and the inability to achieve industrial-scale production (high cost and poor batch consistency)—this invention aims to provide a monkeypox virus multi-epitope nanoparticle vaccine based on an industrial modular platform, along with its preparation method and applications. This vaccine can significantly improve immunogenicity and possesses a complete, robust, and scalable production process.

[0008] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a monkeypox virus multi-epitope nanoparticle vaccine, which is formed by covalently coupling recombinant multi-epitope-SpyTag fusion protein and recombinant AP205-SpyCatcher virus-like particles through the SpyTag / SpyCatcher system.

[0009] The polypeptide antigenic epitope portion of the recombinant multi-epitope-SpyTag fusion protein is formed by the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 in sequence.

[0010] Preferably, the polypeptide antigen epitopes are connected by linkers.

[0011] More preferably, the connector is selected from one or more of KK, AYY, GGPPG, or GS.

[0012] The recombinant multi-epitope-SpyTag fusion protein consists of, from N-terminus to C-terminus, the following components: T-cell helper epitope PADRE protein, a multi-epitope fragment composed of the polypeptide antigen epitopes described in claim 2, and a SpyTag tag.

[0013] The novel design employing a 6-epitope combination plus the PADRE helper epitope significantly induced specific IgG antibody titers higher than the tandem epitope protein group and the mixed protein positive control group, all three significantly higher than the saline control group. This demonstrates the excellent application potential of the vaccine in preventing monkeypox virus infection. PADRE (Pan HLA DR-binding Epitope) is a broad-spectrum, potent artificial helper T cell epitope that can be recognized by multiple MHC class II molecules. Its introduction aims to provide B cells with more effective helper signals, promoting germinal center formation, antibody class switching, and the establishment of immune memory.

[0014] Preferably, the amino acid sequence of the recombinant multi-epitope-SpyTag fusion protein is shown in SEQ ID NO.1.

[0015] This invention provides a method for preparing a monkeypox virus multi-epitope nanoparticle vaccine, which involves covalently coupling recombinant multi-epitope-SpyTag fusion protein with recombinant AP205-SpyCatcher virus-like particles to obtain the monkeypox virus multi-epitope nanoparticle vaccine.

[0016] The molar ratio of the recombinant multi-epitope-SpyTag fusion protein to the recombinant AP205-SpyCatcher virus-like particles is 1:1.

[0017] The recombinant AP205-SpyCatcher virus-like particles are formed by expressing the recombinant AP205-SpyCatcher fusion protein in Escherichia coli and then self-assembling.

[0018] Preferably, the recombinant AP205-SpyCatcher virus-like particle is formed by the self-assembly of AP205 phage capsid protein and SpyCatcher protein through the fusion expression of a flexible linker peptide, wherein the flexible linker peptide is preferably GGSGS.

[0019] More preferably, the preparation of the recombinant AP205-SpyCatcher virus-like particles includes: constructing the gene encoding the AP205-SpyCatcher fusion protein into an expression vector, transforming it into Escherichia coli, and establishing a three-level seed bank; inducing expression at 30°C and 1.5 mM IPTG; after cell lysis, precipitating the supernatant with 10% saturated ammonium sulfate; redissolving the precipitate in Tris buffer (pH 8.5) containing 50 mM NaCl to drive self-assembly; and purifying by size exclusion chromatography.

[0020] The preparation of the recombinant multi-epitope-SpyTag fusion protein includes: constructing a multi-epitope-SpyTag fusion gene expression vector, transforming it into CHO cells, screening to obtain a stable high-expression cell line, and obtaining the fusion protein through Fed-batch culture and purification.

[0021] The CHO cell expression system is used, which has excellent post-translational modification capabilities (such as glycosylation and disulfide bond formation) to ensure the correct folding and native conformation of multi-epitope fusion proteins, which is crucial for maintaining the immunogenicity of conformation-dependent epitopes.

[0022] The optimized process parameters—1:1 molar ratio, 4°C, pH 7.4, and overnight reaction—ensure efficient coupling (approximately 70% coupling rate) while maximizing the structural stability of the antigen and VLP. The bioorthogonal properties of the SpyTag / SpyCatcher system guarantee the directional and uniform display of the antigen on the VLP surface, ensuring structural uniformity and batch-to-batch consistency of the vaccine particles. SDS-PAGE analysis revealed a characteristic covalent complex band at 50 kDa in the coupling product, confirming the formation of the covalent link.

[0023] The present invention provides an anti-monkeypox virus pharmaceutical composition, characterized in that it comprises the monkeypox virus multi-epitope nanoparticle vaccine.

[0024] This invention provides the use of the aforementioned monkeypox virus multi-epitope nanoparticle vaccine or the aforementioned monkeypox virus multi-epitope nanoparticle vaccine in the preparation of a medicament for the prevention or treatment of monkeypox virus infection.

[0025] Compared with the prior art, the present invention achieves the following technical effects: The monkeypox virus multi-epitope nanoparticle vaccine provided by this invention is formed by covalently coupling a recombinant multi-epitope-SpyTag fusion protein and a recombinant AP205-SpyCatcher VLP via a SpyTag / SpyCatcher system. The SpyTag / SpyCatcher system enables the site-specific, directional, and covalent display of antigens on the VLP surface, overcoming the drawbacks of traditional chemical coupling, which may disrupt antigen conformation and result in random display orientation. This ensures the structural uniformity and stability of the vaccine particles and mimics the repeating array structure of virus particles, laying the foundation for subsequent potent immune activation.

[0026] Furthermore, by introducing the PADRE universal helper T cell epitope and six monkeypox virus dominant epitopes covering B cells / T cells, this vaccine successfully induced high levels of specific antibodies (humoral immunity) and potent IFN-γ+ T cell responses (cellular immunity), achieving a dual defense mechanism against the virus and solving the problem of insufficient single immune responses. The novel 6-epitope combination and the design incorporating the PADRE helper epitope are key features. The addition of the PADRE epitope aims to strongly activate CD4+ T cells, providing more effective helper signals to B cells, potentially inducing a stronger and more durable humoral immune response. The introduction of the linker provides spatial flexibility, effectively preventing mutual interference or misfolding of tandem epitopes during fusion expression, helping to maintain the independent natural conformation of each epitope, thus ensuring that the specific immune responses induced by each epitope remain unaffected. The PADRE epitope introduced at the N-terminus is designed to strongly and broadly activate CD4++ T cells and provide auxiliary signals to B cells to enhance the strength, affinity, and persistence of antibody responses. The position of the SpyTag at the C-terminus ensures its accessibility on the surface of the fusion protein, facilitating efficient conjugation with SpyCatcher, which is key to the "plug-and-play" modular design.

[0027] The preparation method provided by this invention integrates the SpyTag / SpyCatcher system with the AP205 VLP platform system, forming a standardized process for the entire chain from antigen production and vector preparation to final assembly. This successfully solves the core contradiction in the background art of the difficulty in balancing the "process scalability" and "research and development timeliness" of multi-epitope vaccines.

[0028] Furthermore, the SpyCatcher-modified AP205 protein was expressed using the E. coli system, which can spontaneously and correctly assemble into a VLP. The production process is mature, low-cost, and easy to scale up, solving the problem of large-scale vector production and providing a reliable guarantee for the industrialization of vaccines.

[0029] Furthermore, the stable and high-yield CHO cell line (>50 µg / mL) and the scaled-up VLP production system respectively solved the challenges of large-scale, low-cost, and high-quality production of antigens and vectors. These two components are assembled through a standardized conjugation and purification process, forming a complete, robust, and scalable pharmaceutical manufacturing process with the potential for direct clinical and production translation. The established master cell bank (MCB) underwent rigorous testing (sterility, mycoplasma, STR identification, and viral safety), and all indicators met the requirements of the Chinese Pharmacopoeia, providing a reliable guarantee for continuous vaccine production and batch-to-batch consistency. A two-step purification strategy of affinity chromatography + molecular exclusion was employed to obtain recombinant protein with a purity >98%, meeting the quality requirements for injectable vaccines and avoiding batch-to-batch variations common in chemically synthesized peptides.

[0030] The anti-monkeypox virus pharmaceutical composition provided by this invention provides a legal basis for preparing the vaccine into a final pharmaceutical form that can be used for clinical administration (such as injection) by combining the vaccine with pharmaceutically acceptable excipients (such as adjuvants, buffer solutions, etc.), and has clear industrial application value.

[0031] The pharmaceutical application provided by this invention is that the vaccine can induce significantly higher titers of specific IgG antibodies (a key indicator of humoral immunity) and potent IFN-γ than the control group. + Direct experimental evidence of T-cell response (a key indicator of cellular immunity) establishes its dual application potential and solid technical efficacy in the prevention and treatment of monkeypox virus infection.

[0032] Furthermore, the dual activation of humoral and cellular immunity in this vaccine not only blocks viral infection but also eliminates infected cells, forming comprehensive immune protection. The application provided by this invention is based on a complete, robust, and scalable production process. The antigen production, VLP preparation, and vaccine assembly processes ensure the quality control and batch-to-batch consistency of the vaccine products used, providing a reliable guarantee for the clinical translation and industrial production of the aforementioned pharmaceutical applications. It exhibits significantly superior protective efficacy compared to existing technologies and has a clear prospect for industrial application. Attached Figure Description

[0033] Figure 1 For the preparation of the recombinant multi-epitope-SpyTag fusion protein of the present invention, wherein A is the titer screening result of OJ027-Nhis-B in a 96-well plate, B is the titer screening result of OJ027-Nhis-B in a 24-well plate, C is the titer screening result of OJ027-Nhis-B in a 6-well plate, D is the expression result of OJ027-Nhis-B in a shake flask, and E is the SDS-PAGE detection result; Figure 2This invention relates to the large-scale preparation of recombinant AP205-SpyCatcher virus-like particles; wherein, A represents the screening of SpyCatcher-AP205 VLP engineered bacteria, B represents the passage stability of SpyCatcher-AP205 VLP engineered bacteria, C represents the passage stability of SpyCatcher-AP205 VLP engineered bacteria, D represents the determination of the SpyCatcher-AP205 VLP fermentation process, E represents the determination of the SpyCatcher-AP205 VLP purification process, F represents the SpyCatcher-AP205 VLP recovery rate, and G represents the quality identification of SpyCatcher-AP205 VLP. Figure 3 For the modular assembly and identification of the vaccine nanoparticles of the present invention, A is SDS-PAGE analysis, and B is a transmission electron microscope image of the nanoparticles (uncoupled epitope AP205-VLP, electron microscope comparison after coupled tandem epitope). Figure 4 This is a bar chart showing the key results of the animal immunization experiments of this invention—serum-specific IgG titers. Figure 5 The key results of the animal immunization experiment of this invention are antigen-specific T cell responses, where A is the immunospot scan of each group and B is the statistical results of the spot data of each group. Detailed Implementation

[0034] 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.

[0035] In this invention, CHO-K1Q cells were purchased from QuaCell (catalog number A13101), and Escherichia coli BL21(DE3) strain was purchased from TSINGKE (catalog number TSC-E01).

[0036] In this invention, the linkers used to tandem the six dominant epitopes of monkeypox virus are selected from KK, AYY, or GGPPG.

[0037] In this invention, E. coli DH5α (used for plasmid amplification) was 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.

[0038] In this study, the GSV0 expression vector (Sanyou Biotechnology) and restriction endonuclease SalI were purchased from New England Biolabs (NEB), model R0138L; methionine sulfoxide imide (MSX) (Millipore, model GSS-1015-F), IPTG (Sangon Biotech, model B541007-0001), kanamycin (Sangon Biotech, model A100408-0005), anti-SpyCatcher antibody (Abcam), BCA protein concentration assay kit (Beyotime, model P0398), aluminum adjuvant (InvivoGen Alhydrogel® adjuvant 2% (catalog number: vac-alu), and IFN-γ ELISPOT assay (CTL mouse IFN-γ) were performed. The ELISPOT kit (catalog number: mIFNgp-2M / 2) and HRP-labeled affinity-purified goat anti-mouse secondary antibody (H+L) were purchased from Beyotime Biotechnology Co., Ltd. (catalog number: A0210). The TMB substrate was NEOGEN's Custom TMB Substrate (catalog number: 309176). The QS-21 adjuvant was purchased from Beijing Huanotai Biopharmaceutical Technology Co., Ltd. The H3L protein was purchased from Beyotime Biotechnology, A29L and E8L were purchased from Yeasen, and A35R and B6R were purchased from Vazyme.

[0039] The present invention will now be described in further detail with reference to the accompanying drawings: Example 1: Prediction and screening of dominant B and T cell epitopes of monkeypox virus This embodiment is used to screen dominant B-cell and T-cell epitopes of monkeypox virus suitable for multi-epitope vaccine design, providing antigen components for the subsequent construction of fusion proteins.

[0040] 1) Target protein selection: Based on the infection characteristics of monkeypox virus (MPXV), key immunogenic proteins from both intracellular mature virus (IMV) and extracellular enveloped virus (EEV) were selected as targets for epitope prediction. The amino acid sequences of proteins including M1R, A29L, E8L, H3L, A35R and B6R were downloaded (referencing the 2022 epidemic monkeypox strain MPXV_USA_2022_MA001, GenBank accession number ON563414.2).

[0041] 2) T-cell epitope prediction: The T-cell epitope prediction tool from the Immune Epitope Database (IEDB, http: / / www.iedb.org / ) was used to predict MHC-I and MHC-II class-restricted T-cell epitopes in the above protein sequences. The prediction parameters were set as follows: IC50...50 Threshold ≤ 200 nM, epitope length 12-20 amino acids, with preference given to top overlapping epitopes that bind to multiple HLA alleles.

[0042] 3) B-cell epitope prediction: Linear B-cell epitope prediction was performed using the BCpred server (http: / / crdd.osdd.net / raghava / bcpred / ), with the threshold set to 0.8 (default parameter). Linear epitopes capable of inducing antibody responses in B lymphocytes were screened.

[0043] 4) Epitope Screening and Optimization: Overlap analysis was performed on the predicted B-cell and T-cell epitopes to select overlapping regions that simultaneously contain B-cell and T-cell epitope activity. The IEDB population coverage tool (http: / / tools.iedb.org / population / ) was used to evaluate the MHC coverage of the selected epitopes in different populations, prioritizing epitopes with high coverage.

[0044] 5) Immunogenicity and biochemical characteristics analysis: Antigenicity was predicted using VaxiJen v2.0 software (http: / / www.ddg-pharmfac.net / vaxijen / ), with a default threshold >0.4.

[0045] Use the AllerTOP2.0 tool (https: / / www.ddg-pharmfac.net / AllerTOP / ) to predict the sensitization of epitopes and screen for non-sensitizing epitopes.

[0046] The toxicity of epitopes was assessed using the ToxinPred software (https: / / webs.iiitd.edu.in / raghava / toxinpred / ), and non-toxic epitopes were screened.

[0047] The physicochemical properties of epitopes were analyzed using the ExPASy-ProtParam tool (https: / / web.expasy.org / protparam / ).

[0048] The MOE (Molecular Operating Environment) software was used to simulate the location of the epitope in the original protein structure, ensuring that the epitope was exposed on the protein surface.

[0049] 6) Screening results: Through the above multi-step screening, six dominant epitopes of monkeypox virus that simultaneously possess B cell and T cell immunogenicity, high MHC coverage, non-sensitization, and non-toxicity were finally obtained. Their specific information is shown in Table 1.

[0050] Table 1: Epitope sequence information and related sequences screened from monkeypox virus

[0051] Example 2: Preparation of recombinant multi-epitope-SpyTag fusion protein Based on Example 1, this embodiment constructs a recombinant multi-epitope-SpyTag fusion protein. The specific steps are as follows: (1) Gene synthesis and expression vector construction The amino acid sequence of the novel multi-epitope-SpyTag fusion gene designed according to this invention is shown in SEQ ID NO.1. Its structure, from N-terminus to C-terminus, consists of: a PADRE helper T cell epitope (as shown in SEQ ID NO.2), six monkeypox virus dominant B / T cell epitopes screened in Example 1 linked by linkers (KK, AYY, GGPPG), and a SpyTag tag linked by the linker GGPPGGPGPPGGPGPG. After codon optimization for CHO cells, a gene encoding the above amino acid sequence was synthesized. This gene was cloned into the GSV0 vector, and the sequence was confirmed to be correct by enzyme digestion and sequencing.

[0052] Table 2: Related sequences

[0053] (2) Construction and screening of stable cell lines CHO-K1Q cells in good logarithmic growth phase were transfected with linearized plasmids using electroporation. After transfection, the cells were revived and passaged in selective medium containing 50 μM MSX at 37°C and 5% CO2. Two weeks later, resistant clones were observed to grow.

[0054] Cells were seeded into 96-well plates using a limiting dilution method to screen for single clones. After approximately 14 days of culture, single clones or well-growing small clone pools were selected under a microscope and transferred to 24-well plates for expansion culture. The supernatant was used to determine protein expression levels (titer). High-expression pools were selected and transferred to 6-well plates, and finally, the cells were transferred to shake flasks for 9-14 days of Fed-batch culture for validation. Through this stepwise selection, the optimal clone with the highest expression level and stable growth was obtained and named OJ027-NHis-B-CHO-K1Q-A1. Figure 1 (Chinese AD).

[0055] (3) Establishment and testing of cell banks The selected OJ027-NHis-B-CHO-K1Q-A1 clone was amplified and cultured at a rate of 5 × 10⁻⁶ cells / year. 6The cells were aliquoted and frozen at a density of cells / mL to establish a master cell bank (MCB). In accordance with the relevant requirements of the Chinese Pharmacopoeia, the MCB underwent sterility, mycoplasma, and cell identification (STR analysis) tests, as well as safety tests for endogenous and exogenous viral factors. All results met the requirements.

[0056] (4) Protein expression and purification Cells were resuscitated from MCBs and scaled up stepwise to a 5L bioreactor for fed-batch production. During cultivation, pH was controlled at 7.0±0.2, dissolved oxygen at 40-60%, and temperature at 37℃ (later reduced to 32℃). The feeding strategy involved daily addition of 5% Feed A and 0.1% Feed B. After 12-14 days of cultivation, the culture medium was harvested. The harvested medium was then centrifuged (4000 rpm, 30 min) and clarified by filtration through a 0.22 μm filter before being loaded onto a pre-equilibrated Ni-NTA affinity chromatography column and a Superdex 200 size exclusion chromatography column (Cytiva, formerly GE Healthcare Life Sciences) for purification. The purified protein was analyzed by SEC-HPLC, with a purity greater than 98%. Figure 1 (E).

[0057] Example 3: Large-scale preparation of recombinant AP205-SpyCatcher virus-like particles This embodiment, based on Embodiments 1-2, details the standardized production process of the universal vaccine chassis, covering the entire chain from strain construction to the release of qualified products.

[0058] (1) Construction of engineered bacteria and establishment of seed bank The fusion gene encoding the AP205 capsid protein and SpyCatcher was synthesized after codon optimization. The gene structure is: SpyCatcher-flexible linker peptide GGSGS-AP205 capsid protein, and the specific amino acid sequence is shown in Table 3. This fusion gene was cloned into the pET-28a(+) vector to obtain a recombinant plasmid, named pET-28a(+)-SpyCatcher-AP205. The recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C.

[0059] Table 3: Relevant sequences of recombinant AP205-SpyCatcher virus-like particles

[0060] Single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking until OD reached. 600≈0.6, add IPTG to a final concentration of 0.5 mM to induce expression for 4 hours. Expression levels were analyzed by SDS-PAGE, high-expression clones were screened and verified by DNA sequencing to confirm sequence accuracy. Figure 2 (A)

[0061] The correctly sequenced clones were used as the original bacterial strain. After expansion culture, 200 original cell banks were established using the 50% glycerol cryopreservation method. Subsequently, the original cell banks were streaked and revived, and single clones were picked and expanded to establish the master cell bank (MCB) and working cell bank (WCB). The WCB strains were subjected to comprehensive testing as shown in Table 4. The results showed that the colonies were white, round, and uniform in morphology; Gram staining was red (Gram negative); they grew well on kanamycin-containing plates; electron microscopy showed no mycoplasma or virus-like particle contamination; VITEK2 identification was consistent with Escherichia coli BL21(DE3); after 30 passages, the plasmid restriction enzyme pattern was correct, and SDS-PAGE showed no significant decrease in protein expression. Only those that passed all indicators were used for production. Figure 2 (Chinese BC).

[0062] Table 4: Testing Items and Standards for Working Banks of Engineered Microorganisms (WCB) for Production

[0063] (2) Fermentation process optimization and production Bacteria were taken from WCB and inoculated into 50 mL LB medium (containing 50 μg / mL kanamycin), and cultured overnight at 37°C with shaking at 220 rpm; then transferred to 500 mL LB medium at a 1% inoculum size and cultured until OD500. 600 The seed culture was approximately 0.8 μg / mL. A 5% inoculum was added to a 5L fermenter using modified TB medium (containing 50 μg / mL kanamycin). Through system optimization, the following optimal fermentation parameters were determined and adopted: temperature 37℃, pH 7.0±0.1 (adjusted with 25% ammonia and phosphoric acid), dissolved oxygen 30-40% (controlled by agitation speed and aeration rate); cultured to OD... 600 After reaching the logarithmic growth phase (≈4.0), adjust the temperature to 30℃ and add IPTG to a final concentration of 1.5 mM for induction. Monitor and adjust pH, dissolved oxygen (DO), and feeding strategy throughout the process, inducing culture for 4-6 hours. These conditions maximize the yield of soluble target protein (…). Figure 2 (D).

[0064] (3) Purification process The bacterial cells were resuspended in lysis buffer (25 mM Tris-HCl, 50 mM NaCl, pH 8.5) at a ratio of 1:10 (w / v). The cells were homogenized three times using a high-pressure homogenizer at 10–12 MPa. The lysis buffer was centrifuged (12,000 rpm, 30 min, 4°C), and the supernatant was collected and clarified by filtration through a 0.45 μm filter membrane.

[0065] Slowly add solid ammonium sulfate to the clarified supernatant until 10% saturation, let stand at 4°C for 2 hours, then centrifuge (12000 rpm, 30 min, 4°C) and collect the precipitate. This step effectively concentrates the protein and removes a large number of impurities.

[0066] The precipitate was reconstituted in the above Tris-NaCl buffer. This 50 mM NaCl buffer condition was experimentally confirmed as the optimal environment for the correct self-assembly of the AP205-SpyCatcher protein into the VLP. The reconstituted solution was purified using a Sepharose 4FF size exclusion column (Konosai Biotechnology, Chromrose 4FF) (300 mL column volume, equilibration buffer: 25 mM Tris-HCl, 50 mM NaCl, pH 8.5) at a flow rate of 1.5 mL / min. The precipitate was monitored under UV light. 280 Collect the main peak (retention time corresponds to the complete VLP particle) Figure 2 (E). Concentrate the target peak collection solution using an ultrafiltration tube (MWCO 100 kDa) and dialyze or replace it to the final storage buffer (PBS).

[0067] (4) Quality inspection Batch testing was performed on the purified AP205-SpyCatcher VLP: a. Purity and Identification: SDS-PAGE ( Figure 2 (E) and HPLC ( Figure 2 Analysis of the chromium (G) content showed a purity of ≥95%.

[0068] b. Specificity identification: Western blotting was performed using anti-SpyCatcher antibody, which showed a single specific band at 23.4 kDa.

[0069] c. Particle characteristics: Observation by transmission electron microscopy (TEM) Figure 2 The results (G) show that the product consists of well-dispersed, hollow spherical particles with regular morphology.

[0070] d. Safety: Bacterial endotoxin test results were less than 1 EU / mL.

[0071] e. Concentration: determined by the BCA method or A 280The protein concentration was determined to be 2.6 mg / mL. Five consecutive pilot-scale production batches showed stable process recovery (>1.17%). Figure 2 The F indicates that the process is robust and the consistency between batches is good.

[0072] The AP205-SpyCatcher VLP that has passed the above tests can be used as a ready-to-use universal vaccine chassis, stored at 2-8℃, for subsequent modular conjugation with different SpyTag antigens.

[0073] Example 4: Modular assembly and identification of vaccine nanoparticles This embodiment demonstrates in detail how to rapidly prepare vaccine formulations that can be directly used for animal immunization using optimized processes, and how to systematically characterize their quality.

[0074] (1) Coupling reaction The AP205-SpyCatcher VLP (molecular weight 23.4 kDa, concentration 2.6 mg / mL) produced in Example 3 and the recombinant monkeypox multi-epitope-SpyTag fusion protein (molecular weight 26.3 kDa, concentration 6.21 mg / mL) produced in Example 2 were precisely measured and mixed in a 1:1 molar ratio in a basic PBS buffer at pH 7.4. The mixture was placed in a temperature-controlled shaker and reacted overnight (16 hours) at 4°C with gentle shaking at 150 rpm. The entire reaction was performed under aseptic conditions.

[0075] (2) Key quality characterization of coupling products After the reaction is complete, the resulting mixture (i.e., vaccine stock solution) is subjected to the following key quality attribute tests to ensure its usability for subsequent immunization studies: a. Molecular linkage and coupling rate analysis: Samples were subjected to non-reducing SDS-PAGE analysis ( Figure 3 (A). The results showed that, compared with the individual VLP subunit band (~24 kDa) and antigen band (~26 kDa), the conjugated product exhibited a new, high-intensity characteristic band at approximately 50 kDa, whose molecular weight matched the theoretically predicted molecular weight of the covalent heterodimer. The conjugation rate was calculated to be approximately 70% by calculating the band grayscale using gel image analysis software.

[0076] b. Particle morphology identification: Transmission electron microscopy: Observation of samples after negative staining ( Figure 3 (B) As can be seen, the original AP205-SpyCatcher VLP particles are typical hollow spherical particles with a regular morphology. In the conjugated product, the nanoparticles still maintain their intact morphology, and the surface contours of the particles are rougher than those of the original VLPs, indicating that the antigen was successfully modified on the particle surface.

[0077] (3) Preparation of ready-to-use immunomodulators Based on the dosage required for the animal experimental design, the characterized vaccine stock solution was precisely diluted to the working concentration with sterile PBS. The diluted formulation can be used directly or gently mixed with an equal volume of aluminum adjuvant for mouse immunization.

[0078] (4) Explanation of technological advantages The "characterization-ready-to-use" process described in this embodiment is the result of systematic optimization. A 1:1 molar ratio and overnight reaction at 4°C ensure effective coupling while maximizing product stability. This process eliminates the time-consuming purification steps of traditional processes, ensuring that formulations directly used for immunization have consistent and definable quality properties, providing crucial technical support for efficient and reliable preclinical pharmacodynamic studies.

[0079] Example 5: Evaluation of vaccine immunogenicity This embodiment demonstrates the immune advantages of the present invention through rigorous animal experiments and verifies the robustness of the manufacturing process.

[0080] (1) Animal immunization experiments Six- to eight-week-old female BALB / c mice were randomly divided into four groups of six mice each. Group G1: Saline control; Group G2: Mixed protein positive control group (M1R 15μg + A29L 15μg + A35R 15μg + B6R 15μg + E8L 15μg + H3L 15μg + adjuvant QS-21 15μg); G3 group: Tandem epitope protein group (100 μg / animal); Group G4: Tandem epitope protein-VLP conjugated group (100 μg / animal, based on the amount of fusion protein). Immunization regimen: Multiple subcutaneous injections were administered via the back of the neck on days 0, 14, and 28. Blood samples were collected and spleen cells were isolated 10 days after the third immunization. Blood was collected from the eyeballs of mice. The obtained blood was left at room temperature for 2 hours, then centrifuged at 12000g for 20 minutes. The supernatant was used as mouse antiserum and stored at -20℃.

[0081] (2) Preparation of spleen cells Spleens from immunized mice were collected, ground in a 70 μm filter, and washed with PBS to obtain a single-cell suspension. The suspension was centrifuged at 400g for 5 min, and the supernatant was discarded. Red blood cell lysis buffer was added, and the cells were lysed on ice for 5 min. After lysis was terminated with PBS, the suspension was centrifuged again. The cells were washed twice with PBS, resuspended in 1640 complete culture medium, and counted for later use.

[0082] (3) Antibody titer detection The level of specific IgG antibodies against monkeypox virus multi-epitope antigens in serum was detected by ELISA. The specific steps were as follows: the antigen protein was diluted to 0.5 μg / mL with coating buffer and coated onto a 96-well ELISA plate, incubated overnight at 4 °C; blocking buffer was added and the plate was incubated at room temperature for 2 h; serially diluted mouse antiserum was added and the plate was incubated at room temperature for 1 h; HRP-labeled anti-mouse secondary antibody was added and the plate was incubated at room temperature for 1 h; TMB substrate was added and the plate was developed for 10-20 min; after termination, the absorbance was measured at 490 nm.

[0083] The results show that ( Figure 4 The titer of specific IgG antibodies induced by group G4 (tandem epitope protein-VLP conjugated group) was significantly higher than that of group G3 (tandem epitope protein group) and group G2 (mixed protein positive control group) (p<0.01), and all three were significantly higher than that of group G1 (saline control group) (p<0.001). This indicates that displaying the screened epitopes through the VLP platform significantly enhances the humoral immune response induced by them.

[0084] (4) The ELISPOT method was used to detect the level of IFN-γ secreted by antigen-specific T cells. Specific steps: Take an ELISPOT plate pre-coated with anti-mouse IFN-γ antibody, add spleen cell suspension (2×10⁻⁶) 5 Cells / well) and stimulants were added and cultured at 37 °C for 32 h; after washing, biotin-labeled IFN-γ detection antibody was added and incubated at room temperature for 2 h; HRP-labeled streptavidin was added and incubated at room temperature for 1 h; after TMB substrate color development, the number of cells forming spots was counted using an ELISPOT plate reader.

[0085] The results show that ( Figure 5 The number of IFN-γ secreting cells induced in group G4 was significantly higher than that in groups G3 and G2 (p<0.01), and all three were significantly higher than that in group G1 (p<0.001). This indicates that the vaccine of the present invention can effectively activate antigen-specific T cell immune responses.

[0086] The above results show that the ability of the epitopes obtained by screening to induce humoral and cellular immunity is significantly enhanced after being displayed through the VLP platform, which confirms the technical advantages of the present invention.

[0087] This invention represents a systematic integration and in-depth process development across the entire chain, from innovative antigen design to industrial production. Its creativity lies in the construction and synergy of three core subsystems: An innovative antigen design and production system: It abandons the limitations of traditional chemically synthesized peptides and has pioneered the design and expression of a recombinant multi-epitope-SpyTag fusion protein driven by the PADRE helper epitope and containing six novel advantageous epitopes. It has also established an industrial production system based on stable CHO cell lines, ensuring the correctness of antigen conformation, scalability of production, and batch consistency from the source.

[0088] A VLP chassis production system with clearly defined process parameters: The AP205-SpyCatcher VLP is transformed from a laboratory concept into a standardized "drug chassis" with completely defined process parameters and controllable quality (including key processes such as engineered bacterial library, 30℃ / 1.5mM IPTG fermentation, and reconstitution self-assembly at specific salt concentrations), providing a stable and reliable carrier basis for the standardized display of antigens.

[0089] A highly efficient and controllable modular assembly system: By optimizing the established SpyTag / Catcher coupling conditions (1:1 molar ratio, overnight at 4°C), the system achieved site-specific, quantitative, and covalent assembly of antigens on the VLP surface. It also established a rapid quality control method based on "non-reducing SDS-PAGE 50 kDa characteristic bands" and "electron microscopy morphology observation," forming a highly efficient R&D process that is "characterized and ready to use."

[0090] This complete technical solution successfully resolves the core contradiction in the field of multi-epitope vaccines: the difficulty in simultaneously achieving "conformative immunogenicity," "process scalability," and "development timeliness." Key animal experimental data show that the vaccine induced a significantly higher titer of specific IgG antibodies than the positive control group and the tandem epitope protein group, and activated a potent IFN-γ+ T cell response. This is not due to a single component, but rather an unexpected and superior technical effect resulting from the synergistic effect of the aforementioned three subsystems. Therefore, this invention not only provides a highly effective candidate vaccine, but more importantly, it provides a clear and patentable industrialization technical path from gene design and process development to product characterization, possessing outstanding substantive characteristics and significant industrial application value.

[0091] 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 multi-epitope nanoparticle vaccine, characterized in that, The monkeypox virus multi-epitope nanoparticle vaccine is formed by covalently coupling recombinant multi-epitope-SpyTag fusion protein and recombinant AP205-SpyCatcher virus-like particles through the SpyTag / SpyCatcher system.

2. The monkeypox virus multi-epitope nanoparticle vaccine according to claim 1, characterized in that, The polypeptide antigenic epitope portion of the recombinant multi-epitope-SpyTag fusion protein is formed by the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 in sequence.

3. The monkeypox virus multi-epitope nanoparticle vaccine according to claim 2, characterized in that, Each polypeptide antigen epitope is connected by a linker.

4. The monkeypox virus multi-epitope nanoparticle vaccine according to claim 3, characterized in that, The connector is selected from one or more of KK, AYY, GGPPG, or GS.

5. The monkeypox virus multi-epitope nanoparticle vaccine according to claim 1, characterized in that, The recombinant multi-epitope-SpyTag fusion protein consists of, from N-terminus to C-terminus, the following components: T-cell helper epitope PADRE protein, a multi-epitope fragment composed of the polypeptide antigen epitopes described in claim 2, and a SpyTag tag.

6. A method for preparing a monkeypox virus multi-epitope nanoparticle vaccine according to any one of claims 1 to 5, characterized in that, The monkeypox virus multi-epitope nanoparticle vaccine was obtained by covalently coupling recombinant multi-epitope-SpyTag fusion protein with recombinant AP205-SpyCatcher virus-like particles.

7. The method for preparing a monkeypox virus multi-epitope nanoparticle vaccine according to claim 6, characterized in that, The molar ratio of the recombinant multi-epitope-SpyTag fusion protein to the recombinant AP205-SpyCatcher virus-like particles is 1:

1.

8. The method for preparing a monkeypox virus multi-epitope nanoparticle vaccine according to claim 6, characterized in that, The recombinant AP205-SpyCatcher virus-like particles are formed by expressing the recombinant AP205-SpyCatcher fusion protein in Escherichia coli and then self-assembling.

9. An anti-monkeypox virus drug composition, characterized in that, Including the monkeypox virus multi-epitope nanoparticle vaccine according to any one of claims 1-5.

10. The use of the monkeypox virus multi-epitope nanoparticle vaccine according to any one of claims 1-5 or the anti-monkeypox virus pharmaceutical composition according to claim 9 in the preparation of a medicament for the prevention or treatment of monkeypox virus infection.

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