Monkey pox virus self-assembled nano-particles as well as preparation method and application thereof

Abstract

The invention discloses a monkey pox virus self-assembled nanoparticle and a preparation method and application thereof, and relates to the technical field of monkey pox virus vaccine research and development, the monkey pox virus nanoparticle (NP) is a DAM-NP nanoparticle vaccine formed by covalent coupling of a DAM antigen and SD-Ferritin, the DAM antigen comprises a fusion sequence of monkey pox virus M1 protein and A35 protein, the N end is connected with a GvTagOpti tag, the D-Ferritin is connected with a GvTagOpti tag, and the D-Ferritin is connected with a GvTagOpti tag. The middle parts are connected through a glycine-serine-glycine spacer region; the SD-Ferritin is formed by fusing an SdCatcher fragment and the N end of the helicobacter pylori ferritin, and the SdCatcher fragment and the N end of the helicobacter pylori ferritin are connected through a GSG spacer region; the coupling ratio of the DAM antigen to the SD-Ferritin is 3: 1, and covalent binding is formed after incubation in an assembly buffer solution at 4 DEG C for 24 hours. The monkey pox DAM-NP vaccine is constructed by fusing M1 and A35 antigens of the monkey pox virus in a bivalent form and displaying the M1 and A35 antigens on the surfaces of self-assembled 24-polymer ferritin nanoparticles by using a GvTagOpti / SdCatcher covalent coupling system, antigen-specific body fluid and cellular immune response can be stimulated, and protective immunity can be induced by a single dose of the monkey pox DAM-NP vaccine.

Description

Technical Field

[0001] This invention belongs to the field of monkeypox virus technology, and in particular relates to a monkeypox virus self-assembled nanoparticle, its preparation method and application. Background Technology

[0002] Monkeypox virus (MPXV), a member of the orthopoxvirus genus, is highly similar to smallpox virus and cowpox virus in genetics and antigens. Infection with or immunity to one of these viruses can provide cross-protection against other viruses in the same genus. Therefore, traditional smallpox vaccines were once used to prevent monkeypox.

[0003] Since 2022, monkeypox outbreaks have spread to many parts of the world, and the World Health Organization has repeatedly listed it as a Public Health Emergency of International Concern. MPXV is divided into branch I and branch II, with branch I being more virulent, and the currently prevalent branch Ib posing a more significant threat to human health.

[0004] Most existing vaccines for preventing monkeypox are live virus vector vaccines, which pose risks of adverse reactions, and their immune targets are not fully understood. Furthermore, existing vaccines based on MPXV-specific antigens suffer from insufficient immunogenicity, require multiple immunizations, and have limited duration of protection, making it difficult to meet the needs for effective control of monkeypox, especially the highly virulent Ib branch. Therefore, there is an urgent need to develop safe, effective, and specific novel vaccines. Summary of the Invention

[0005] The purpose of this invention is to provide a monkeypox virus self-assembled nanoparticle, its preparation method, and its application. By fusing the M1 and A35 antigens of monkeypox virus in a bivalent form, a DAM-NP vaccine is constructed and its preparation process is optimized. This vaccine can stimulate a strong antigen-specific humoral and cellular immune response, and a single dose can induce protective immunity, providing effective protection against monkeypox virus and other orthopox viruses. This invention solves the problems of adverse reaction risks, unclear immune targets, insufficient immunogenicity, need for multiple immunizations, and limited protective effects of existing live virus vector vaccines.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution:

[0007] This invention relates to a monkeypox virus self-assembled nanoparticle, wherein the nanoparticle is formed by covalent coupling of DAM antigen and SD-Ferritin, wherein:

[0008] The DAM antigen contains a fusion sequence of monkeypox virus M1 protein (amino acids 3-181) and A35 protein (amino acids 90-181 or 64-181), with a GvTagOpti tag attached to the N-terminus and connected in the middle by a glycine-serine-glycine (GSG) spacer region.

[0009] The SD-Ferritin is formed by fusing the SdCatcher fragment with the N-terminus of Helicobacter pylori ferritin (HPF), linked by the GSG spacer region, and contains eight C-terminal histidine tags.

[0010] The DAM antigen and SD-Ferritin were coupled at a molar ratio of 3:1 and covalently bound in an assembly buffer (50 mM HEPES (pH 8.0), 300 mM NaCl, 5% glycerol, 10% trehalose) for 24 hours.

[0011] Furthermore, the coding sequence of the DAM antigen was optimized with humanized codons and cloned into the VRC8405 mammalian expression vector, which contains an N-terminal tissue plasminogen activator signal peptide and eight C-terminal histidine tags.

[0012] Furthermore, the coding sequence of SD-Ferritin was optimized using E. coli codons and cloned into the pTO-T7 prokaryotic expression vector.

[0013] A method for preparing monkeypox virus self-assembled nanoparticles, the method comprising the following steps:

[0014] Plasmid construction: The optimized DAM antigen coding sequence was cloned into the VRC8405 vector, and the SD-Ferritin coding sequence was cloned into the pTO-T7 vector;

[0015] Protein expression and purification: on Expi293F TM DAM antigen was expressed in cells and purified by Ni Sepharose affinity chromatography and Superdex 200 gel filtration; SD-Ferritin was expressed in E. coli Rosetta competent cells, purified by CaptoDEAE column and Superdex 200, and endotoxin was removed by Triton X-114 phase separation.

[0016] Conjugation and assembly: The purified DAM antigen was incubated with SD-Ferritin at a molar ratio of 3:1 in assembly buffer for 24 hours. The conjugated products were separated by Capto Core 700 chromatography combined with Superose 6 10 / 300GL gel column, concentrated and stored at -80°C.

[0017] Furthermore, the purification of the DAM antigen includes washing with 50 mM HEPES (pH 8.0), 300 mM NaCl, and 30 mM imidazole, followed by elution with 300 mM imidazole.

[0018] Furthermore, the purification of SD-Ferritin involves equilibration with 20 mM Tris (pH 7.5) and 50 mM NaCl, followed by elution with 400 mM NaCl.

[0019] The application of a monkeypox virus self-assembled nanoparticle in the preparation of a vaccine for the prevention or treatment of monkeypox virus infection, the application comprising:

[0020] Immunization procedure: Mix the nanoparticles with FH002C adjuvant and immunize experimental animals (such as mice and rabbits) by intramuscular injection at a dose of 0.2-10 μg DAM, and repeat the immunization twice at 2-week intervals.

[0021] Immunogenicity assessment: M1 and A35 specific IgG and subtype titers were detected by ELISA, and neutralizing activity was detected by plaque reduction neutralization test (PRNT).

[0022] Challenge protection: After immunization, experimental animals were infected with VCV or MPXV via intranasal infection, and changes in body weight, survival rate, and viral load in tissues were monitored.

[0023] The present invention has the following beneficial effects:

[0024] 1. The monkeypox virus self-assembled nanoparticle (DAM-NP) vaccine of this invention can effectively stimulate the body to produce a strong antigen-specific humoral and cellular immune response. By covalently binding the DAM antigen to the surface of the self-assembled ferritin nanoparticles, it enhances the immunogenicity of the antigen and can induce the production of high-titer specific antibodies against M1 and A35 antigens, including IgG and its subtypes. At the same time, it can stimulate a balanced Th1 / Th2-related immune response, promote the increase of the proportion of follicular helper T cells and germinal center B cells, accelerate antibody response and affinity maturation, and induce a stronger Th1-type and cytotoxic T lymphocyte response, thereby enhancing the body's ability to clear the virus.

[0025] 2. The DAM-NP vaccine of this invention has cross-protective efficacy against multiple orthopoxviruses. Since monkeypox virus and vaccinia virus are highly similar in genetics and antigens, the immune response induced by this vaccine can not only act against monkeypox virus, but also protect against vaccinia virus and other orthopoxviruses. The neutralizing antibodies it induces can effectively inhibit related viral infections, reduce viral replication in the body, alleviate tissue lesions, thereby reducing the risk of disease after infection and providing broad cross-immune protection for the body.

[0026] 3. The DAM-NP vaccine of this invention can induce effective protective immunity with a single dose. Compared with the traditional strategy that requires multiple immunizations, this vaccine improves antigen presentation efficiency through a nanoparticle platform. A single immunization can stimulate a sufficient immune response, producing protective neutralizing antibodies and cellular immune responses. It can prevent disease and death in deadly viral attacks, reduce the inconvenience caused by multiple immunizations, improve vaccination compliance, and meet the needs of rapid epidemic prevention and control.

[0027] 4. The immunogenicity and protective efficacy of the DAM-NP vaccine of this invention are not affected by prior antibodies. For individuals who have previously been vaccinated against smallpox or have been infected with related orpox viruses and have pre-existing antibodies, the vaccine can still effectively stimulate an immune response and may even have a synergistic effect with pre-existing immunity, enhancing neutralizing antibody titers and viral clearance capacity. This characteristic expands the applicable population of the vaccine, especially for individuals with a history of prior immunization, ensuring that it can play a protective role in all types of populations.

[0028] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 A schematic diagram illustrating the design and molecular properties of the DAM-NP nanoparticle vaccine;

[0031] Figure 2 A schematic diagram illustrating the immunization program of the DAM-NP vaccine in mice and its protective effect against VACV-WR challenge;

[0032] Figure 3 This is a schematic diagram illustrating the immunization program and induced protective antibody response of the DAM-NP vaccine in New Zealand rabbits.

[0033] Figure 4 A schematic diagram illustrating the immunogenicity and protective efficacy of the DAM-NP vaccine in mice immunized with pre-stored VCV-VTT.

[0034] Figure 5 This is a schematic diagram illustrating the immunization program and protective effect against MPXV challenge of a single-dose DAM-NP vaccine in mice.

[0035] Figure 6A schematic diagram of SDS-PAGE analysis of the in vitro binding efficiency of SD-NPs and GvO-DAM at different ratios.

[0036] Figure 7 A schematic diagram of the biolayer interference antigen characterization analysis of M1, A35, DAM, and DAM-NP;

[0037] Figure 8 A schematic diagram of the Th1 / Th2-related humoral immune response (IgG subtype) induced by the DAM-NP vaccine in mice;

[0038] Figure 9 A schematic diagram illustrating the immunization schedule of the DAM-NP vaccine in mice and its protective effect against VACV-VTT infection;

[0039] Figure 10 A schematic diagram of a gating strategy for flow cytometry of T cells and B cells in mice immunized with DAM or DAM-NP.

[0040] Figure 11 This is a schematic diagram of the cellular immune response against MPXVA35 and M1 induced in mice by the DAM-NP vaccine. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0042] This invention relates to a monkeypox virus self-assembled nanoparticle, which is formed by covalent coupling of DAM antigen and SD-Ferritin, wherein:

[0043] The DAM antigen contains a fusion sequence of monkeypox virus M1 protein (amino acids 3-181) and A35 protein (amino acids 90-181 or 64-181), with a GvTagOpti tag attached to the N-terminus and connected in the middle by a glycine-serine-glycine (GSG) spacer region.

[0044] SD-Ferritin is formed by fusing the SdCatcher fragment with the N-terminus of Helicobacter pylori ferritin (HPF), linked by the GSG spacer region, and contains eight histidine tags at the C-terminus.

[0045] The DAM antigen was conjugated to SD-Ferritin at a molar ratio of 3:1, and covalently bound was formed after incubation for 24 hours in assembly buffer (50 mM HEPES (pH 8.0), 300 mM NaCl, 5% glycerol, 10% trehalose).

[0046] The coding sequence of the DAM antigen was optimized with humanized codons and cloned into the VRC8405 mammalian expression vector, which contains an N-terminal tissue plasminogen activator signal peptide and eight C-terminal histidine tags.

[0047] The coding sequence of SD-Ferritin was optimized with E. coli codons and cloned into the pTO-T7 prokaryotic expression vector.

[0048] A method for preparing monkeypox virus self-assembled nanoparticles, the method comprising the following steps:

[0049] Plasmid construction: The optimized DAM antigen coding sequence was cloned into the VRC8405 vector, and the SD-Ferritin coding sequence was cloned into the pTO-T7 vector;

[0050] Protein expression and purification: on Expi293F TM DAM antigen was expressed in cells and purified by Ni Sepharose affinity chromatography and Superdex 200 gel filtration; SD-Ferritin was expressed in Escherichia coli Rosetta (DE3), purified by Capto DEAE column and Superdex 200, and endotoxin was removed by Triton X-114 phase separation.

[0051] Conjugation and assembly: The purified DAM antigen was incubated with SD-Ferritin at a molar ratio of 3:1 in assembly buffer for 24 hours. The conjugated products were separated by Capto Core 700 chromatography combined with Superose 6 10 / 300GL gel column, concentrated and stored at -80°C.

[0052] Purification of DAM antigen involved washing with 50 mM HEPES (pH 8.0), 300 mM NaCl, and 30 mM imidazole, followed by elution with 300 mM imidazole.

[0053] Purification of SD-Ferritin involved equilibration with 20 mM Tris (pH 7.5) and 50 mM NaCl, followed by elution with 400 mM NaCl.

[0054] The application of a monkeypox virus self-assembled nanoparticle in the preparation of vaccines for the prevention or treatment of monkeypox virus infection, including:

[0055] Immunization procedure: Mix the nanoparticles with FH002C adjuvant and immunize animals (such as mice and rabbits) by intramuscular injection at a dose of 0.2-10 μg DAM, and immunize twice at an interval of 2 weeks.

[0056] Immunogenicity assessment: M1 and A35 specific IgG and subtype titers were detected by ELISA, and neutralizing activity was detected by plaque reduction neutralization test (PRNT).

[0057] Challenge protection: After immunization, experimental animals were infected with VCV or MPXV via intranasal infection, and changes in body weight, survival rate, and viral load in tissues were monitored.

[0058] One specific application of this embodiment is:

[0059] I. Implementation Materials:

[0060] 1) Cells and Viruses

[0061] African green monkey kidney epithelial cells (VeroE6 cells, ATCC, CRL1586) used for virus production and infection were cultured in Dulbecco modified Eagle medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, ExCellBio) and 1% penicillin-streptomycin (Gibco) at 37°C and 5% CO2. Expi293F TM Cells (Thermo Fisher Scientific, A14527) were cultured in a 37°C, 8% CO2 incubator with shaking at 115 rpm in serum-free medium (OPMBiosciences). Mycoplasma contamination was detected by PCR in all cells weekly, and the results were all negative.

[0062] Engineered wild-type vaccinia virus (VACV) strain Tiantan (VTT), VACVVTT strain expressing green fluorescent protein (GFP), and wild-type vaccinia virus Western Reserve (VACVWR) strain were used. The MPXVIb branch M25050 strain was isolated from a 30-year-old female patient presenting with a rash. All operations involving live viruses were performed in a BSL-3 laboratory. All viruses were passaged in Vero-E6 cells in complete DMEM medium.

[0063] 2) Convalescent serum

[0064] In a prospective cohort study at Shenzhen Third People's Hospital, 20 plasma samples were collected. MPXV DNA copy number was detected using a commercial qRT-PCR kit manufactured by GeneoDX Co., Ltd., confirming the diagnosis. This study was approved by the Ethics Committee of Shenzhen Third People's Hospital (Approval No.: 2023-006-02), and all subjects infected with MPXV signed written informed consent forms. There was no self-selection bias in this study; the sole inclusion criterion for serum samples was a confirmed case of MPXV infection. Furthermore, due to the limitations of this study, no sex-based or gender-related analyses were performed.

[0065] II. Implementation Methods:

[0066] 1) Plasmid construction

[0067] The MPXV DAM expression construct consists of the following parts: a GvTagOpti (GvO) fragment, a glycine-serine-glycine (GSG) spacer, an MPXV (hMpxV / China / SZ-SZTH41 / 2023,EPI_ISL_18213375) M1 (amino acids 3-181) dimer, and an A35 (amino acids 90-181 and 64-181, respectively), which is inserted at the N-terminus of the M1 domain. Similarly, the SdCatcher (SD) fragment is fused to the N-terminus of Helicobacter pylori ferritin (HPF) via the GSG spacer, thereby enabling multivalent antigen display on the nanoparticle vaccine. The GvO / SD covalent coupling system produces the MPXV nanoparticle vaccine by spontaneously forming isopeptide bonds. The GvO-DAM, M1 (amino acids 1-181), and B6R (amino acids 20-275) constructs were optimized for humanized codons and synthesized by Synbio Technologies. They were then cloned into the VRC8405 mammalian expression vector using BamHI restriction endonuclease sites and Gibson assembly technology. This vector contains an N-terminal tissue plasminogen activator signal peptide (for protein secretion) and an eight-histidine tag at the C-terminus (for protein purification). The MPXV A35 (amino acids 90-181), A29L (amino acids 1-110), E8L (amino acids 1-264), and SD-Ferritin constructs were synthesized and codon-optimized for *E. coli*. Subsequently, they were cloned into the pTO-T7 prokaryotic expression vector containing an eight-histidine tag at the C-terminus using NdeI and XhoI restriction endonuclease sites.

[0068] For monoclonal antibodies, the heavy and light chain coding sequences of 7D11 and A27D7 were retrieved from a protein database, codons were optimized for mice, and then synthesized by GeneCreate. The VH sequences of 7D11 and A27D7 were fused to the N-terminus of the constant regions of mouse IgG1 and IgG2a, while the VK sequence was fused to the constant region of mouse Igκ, and then cloned into the pCAGGS antibody expression vector.

[0069] 2) Instantaneous transfection

[0070] MPXV DAM (with or without GvO tag), M1 and B6R recombinant proteins, and 7D11 and A27D7 monoclonal antibodies were prepared independently in suspensions of the Expi293FTM cell expression system. In short, plasmids were transformed into DH5α competent cells (AlpaLifeBio), and single clones were cultured in TB medium supplemented with 50 mg / L kanamycin. After overnight incubation at 37°C, bacterial cultures were collected by centrifugation, and transfection-grade plasmids were extracted using the NucleoBond Xtra Midi kit (Macherey-Nagel) according to the manufacturer's standard protocol. Expi293FTM cells were cultured in suspension in Erlenmeyer flasks at 37°C with 8% CO2 humidity at a rotation speed of 115 rpm. Cells were cultured until a density of approximately 3 × 10⁶ cells / year was reached. 6 At 1 cell / mL, use Expi293F containing 1 mg plasmid and 3 mg polyethyleneimine "Max" (PEI-MAX, Polyscience). TM Suspension cells were transfected using a growth medium mixture. Twenty-four hours after transfection, preheated medium was added, and valproic acid was supplemented to a final concentration of 2.5 mM to increase the yield of the target protein. Cells were then cultured for another 5 days. The supernatant was collected and clarified by centrifugation at 13845 g for 1 hour.

[0071] For M1-specific 7D11 and M1H11, and A35-specific A27D7 monoclonal antibodies, heavy chain and light chain expression plasmids were transiently co-transfected into Expi293F using PEI-MAX transfection reagent at a ratio of 1:1.2. TM In the cells. After 5 days, the cell supernatant was clarified as previously described.

[0072] 3) Expression and purification of recombinant proteins

[0073] Proteins fused with eight histidine tags were purified using immobilized metal affinity chromatography. For DAM, M1, and B6R soluble proteins, the clarified supernatant was first filtered through a 0.22 μm filter using vacuum filtration and then transferred to a PD-10 gravity column (Cytiva) pre-packed with Ni Sepharose excel histidine-tagged protein purification resin (Cytiva) or Ni Sepharose 6FastFlow resin (Cytiva). The sample was washed with 10 column volumes of 50 mM HEPES (pH 8.0), 300 mM NaCl, and 30 mM imidazole, followed by elution with the same solution. The elution fraction was collected and purified using a 10 kDa MWCO... The protein was concentrated using ultracentrifuge tubes (Millipore) and purified by gel filtration chromatography using a Superdex 200 increase 10 / 300GL column (Cytiva). Expression and purification of MPXV A29L, A35, and E8L proteins were performed using an *E. coli* expression system. In short, plasmids were transformed into Rosetta competent cells (Tiangen) for protein expression. After induction at 16°C for 16 to 20 hours in 3L Erlenmeyer flasks with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Takara), cell lysates were clarified by centrifugation and filtration as previously described, and purified by immobilized nickel affinity chromatography and... Purification was performed using gel filtration chromatography on the Pure25m system (GE Healthcare).

[0074] 4) Expression and purification of industry-standard SD-Ferritin

[0075] The clinical-grade SD-Ferritin nanoparticle scaffold was manufactured by Shenzhen Biomedical Technology Co., Ltd. In short, SD-Ferritin is independently expressed in a prokaryotic expression system. The plasmid was transformed into Rosetta competent cells (Tiangen) and cultured on LB agarose plates containing 30 mg / L chloramphenicol and 50 mg / L kanamycin. After overnight incubation, single clones were picked and cultured in TB medium at 37°C with magnetic stirring at 220 rpm in a 10 L LB IO-10 GJGGC fermenter (Bailun) at 37°C. When the OD600 of the bacterial culture reached 0.6 to 0.8, the cells were cooled to 16°C, and then 1 mM IPTG was added to induce target protein expression for 16 to 20 hours. After centrifugation, the cells were resuspended in pre-chilled buffer containing 20 mM Tris (pH 7.5) and 50 mM NaCl. Subsequently, lysis was performed using an ultra-high pressure continuous flow cell disruptor at 4°C. The crude extract was then centrifuged at 26533g for 1 hour at 4°C to remove cell debris, yielding a clear solution. This clear solution was then filtered through a 0.45μm nitrocellulose membrane using a Millipore vacuum filter. To denature and remove heat-resistant impurities, the supernatant of SD-Ferritin was transferred to a 5L flask, treated at 56°C for 30 minutes, centrifuged, and then placed in a 70°C constant temperature reaction bath with stirring at 300rpm for 30 minutes. After passing through a 0.22μm filter membrane, the filtered supernatant was loaded onto a Capto DEAE column (Cytiva) pre-equilibrated with 20mM Tris (pH 7.5) and 50mM NaCl. The nanoparticles were then washed with 1 L of equilibration buffer and separated with 2 L of elution buffer containing 20 mM Tris (pH 7.5) and 400 mM NaCl. For further purification of the target protein, the purified SD-Ferritin nanoparticles were concentrated using a 100 kDa hollow fiber assembly via tangential flow filtration (Repligen) and then fractionally loaded onto HiLoad buffer pre-equilibrated with 50 mM HEPES (pH 8.0) and 300 mM NaCl. TM 26 / 600 Superdex TM In a 200 pg size exclusion (SEC) column (Cytiva). After validating the peak components by SDS-PAGE, a 100 kDa MWCO was used. Ultrafiltration centrifuge tubes (Millipore) were used for pooling and concentration; bacterial endotoxins in the SD-Ferritin nanoparticle scaffold were removed using the Triton X-114 phase separation method (as previously reported 49), and residual endotoxins were quantified using the ToxinSensor™ colorimetric LAL endotoxin assay kit (GenScript).

[0076] 5) In vitro conjugation and assembly of nanoparticle vaccines

[0077] Following the manufacturer's operating procedures, the concentration of freshly purified protein was determined using a standard BCA protein assay kit (Thermo Fisher Scientific). In sterile Eppendorf tubes, the conjugation and assembly process was reversed by gentle inverting the mixture for 24 hours at 40 rpm and 4°C. The purified GvO-DAM protein was incubated with SD-Ferritin NPs at a 3:1 molar ratio in an assembly buffer containing 50 mM HEPES (pH 8.0), 300 mM NaCl, 5% glycerol, and 10% trehalose. After 24 hours of incubation and conjugation, the unassembled subunits were separated from the conjugated NP vaccine mixture in PBS using Capto Core 700 chromatography combined with size exclusion chromatography on a Superose 6-increase 10 / 300GL column (Cytiva). The elution peak was collected and subjected to SDS-PAGE electrophoresis to verify purity and homogeneity, followed by analysis using a 100 kDa MWCO... The mixtures and concentrations were then combined using ultrafiltration centrifugal filters, subsequently dispensed, rapidly frozen in liquid nitrogen, and finally stored at -80°C.

[0078] III. Physicochemical Properties and Structural Analysis Methods:

[0079] 1) SDS-PAGE analysis

[0080] Mix a total of 2 μg of purified protein sample (SD-NP, DAM, and DAM-NP) with 5x loading buffer (with or without 20 μM MDTT), and then transfer the mixture to a 0.2 mL Eppendorf tube. Use Veriti TMThe test tubes were incubated at 100°C for 6 minutes using a thermal cycler (ThermoFisher) to denature the proteins. Protein samples were then separated using a FuturePAGE™ 4%–20% pre-prepared polyacrylamide gel (ACE) at 120V electrophoresis, with protein standards used as molecular weight indicators. The gels were first stained with Coomassie Brilliant Blue R-250 (Meilunbio), and then destained in a buffer solution of water, methanol, and glacial acetic acid in a 45:45:10 volume ratio. Heating aided the destaining process. Images were captured using an automated digital gel imaging analysis system (Tanon).

[0081] 2) Dynamic light scattering

[0082] The hydrodynamic diameter and polydispersity index of the purified protein were determined by dynamic light scattering analysis. The protein was diluted with PBS to a final concentration of 0.5 mg / mL, filtered through a 0.22 μm syringe filter, and then added to a 40 μL disposable solvent-resistant microcuvette (Malvern PANalytical). After two minutes, the hydrodynamic diameter and polydispersity index were obtained using a Zetasizer Ultra instrument (Malvern PANalytical) at 25 °C and an optimal scattering angle of 173 °C, and analyzed using Zetasizer Ultra ZS Xplorer version 2.0.

[0083] 3) Negative staining transmission electron microscopy

[0084] Before imaging the assembled nanoparticles using a negative staining electron microscope, the protein sample was diluted with PBS to a final concentration of 0.1 mg / mL. After centrifugation, a 300-mesh copper grid was subjected to glow discharge. Then, 10 μL of sample was carefully placed on the copper grid and incubated for 1 minute. Excess sample was then gently blotted away using filter paper (Whatman). To ensure thorough staining, the grid was immersed in 10 mL of 2% phosphotungstic acid solution for 30 seconds, blotted dry with filter paper, and then immersed in another staining solution for 45 seconds. After drying for 1 minute, the grid was imaged and photographed at 150,000x magnification using a FEITecnai T12 transmission electron microscope.

[0085] 4) Molecular docking

[0086] Based on the chimeric immunogen structure of M1R and A35R, predictions were made using AlphaFold 3.51. The dimer formed by M1R and A35R in the predicted model is similar to the crystal structures of vaccinia virus L1 protein (PDB 1YPY) and vaccinia virus A33R (PDB 4M1G). Due to the flexibility of the linker, the linker region connecting M1R and A35R is largely disordered. According to the binding model predicted by AlphaFold 3, two copies of the variable region (Fv) of M1H11 were docked to the M1R dimer of the immunogen; based on the crystal structure of the L1-7D11 complex (PDB 2I9L), two copies of the 7D11 Fv domain were modeled onto the M1R dimer; and based on the crystal structure of the A33R-A27D7 complex (PDB 4M1G), the A27D7 Fab region was modeled onto the A35R dimer. Relevant images were prepared using PyMOL 52.

[0087] IV. Detection and Experimental Methods:

[0088] 1) Antigen capture enzyme-linked immunosorbent assay (ELISA)

[0089] To assess the antigenic properties of the purified proteins, SD-NP, M1, A35, DAM, and DAM-NP (10 ng / well) were pre-coated onto ELISA microplates (NEST) and washed with PBS (PBST) containing 0.05% v / v Tween-20. The wells were then blocked for 2 hours at room temperature with PBS containing 5% w / v skim milk. After washing, the wells were detected with 100 μL of 4-fold serially diluted M1-specific (7D11 and MM1H11) or A35-specific (A27D7) mAbs at an initial concentration of 10 μg / mL, followed by the addition of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody. The reaction was developed with TMB substrate (Tiangen) and terminated with ELISA stop solution (Solarbio). Absorbance at 450 nm was measured using a multi-functional microplate reader (TECAN Spark).

[0090] 2) Mouse immunization and VCV challenge

[0091] Six- to eight-week-old female BALB / c mice purchased from the Guangdong Provincial Center for Medical Laboratory Animal Science were randomly divided into five groups. Before immunization, the purified immunogen was diluted with PBS and gently mixed with an equal volume of bisphosphonate-modified zinc-aluminum adjuvant (FH002C). The mixture was incubated overnight at 4°C with shaking at 40 rpm to ensure complete adsorption of the antigen onto the adjuvant particles. Each group of mice was immunized twice intramuscularly at two-week intervals, with 100 μL of the vaccine formulation injected into the hind limb muscles of both hind legs each time. The immunization dose was 0.2 μg or 1 μg of DAM, or an equimolar ratio of DAM-bound nanoparticle immunogen (at an equivalent dose). SD-NP was used as a negative control. Blood samples were collected 14 days after each immunization and allowed to coagulate fully at 37°C for 30 minutes. Subsequently, the samples were centrifuged at 16,250 × g for 10 minutes at 4°C, and the supernatant serum was carefully removed. The serum was incubated at 56°C for 30 minutes to inactivate complement factors and pathogens, and then stored at -80°C for later use.

[0092] On day 42 post-immunization, mice were anesthetized with isoflurane and then administered 5 × 10 4 TCID 50 (50% tissue culture infection dose) or 1×10 6 PFU (35LD) 50 Mice were infected nasally with either VACV VTT-GFP or lethal VACV-WR at appropriate doses. Body weight and survival rates were monitored daily. On day 3 post-infection, mice were euthanized and autopsied to collect lung tissue for histopathological analysis and viral titer determination. Lung tissue was weighed, homogenized in serum-depleted culture medium, and then stored at -80°C.

[0093] To investigate whether prior VCV-VTT infection interferes with the immunogenicity of the MPXV DAM nanoparticle vaccine, a pre-existing antibody assay was conducted in mice. Thirty-six mice were divided into two groups (n=18). One group of mice, under isoflurane anesthesia, received 50 μL of VCV-VTT intranasally at a dose of 5 × 10⁻⁶. 3 PFU. The remaining mice were intranasally inoculated with PBS as a control. Blood was collected from these animals 21 days later to detect pre-existing antibody titers. Subsequently, mice in the VCV-VTT and PBS groups were re-divided into four groups (n=9 per group). Mice from one group in each of the VCV-VTT and PBS groups were immunized intramuscularly with 0.2 μg DAM-NP prepared with FH002C adjuvant. Ten days later, blood was collected again from the mice before intranasal infection. On the same day, 4 × 10⁻⁶ PFU was administered. 5All mice were infected with lethal VAV-WR at a dose of PFU. Weight loss and survival were monitored from day 1 to day 14 post-infection. Animals were euthanized on day 3 post-infection, and nasal turbinates, lungs, and trachea tissues were collected for viral titer determination and histopathological analysis of the lung tissue.

[0094] 3) Challenge and protection of MPXV in mice

[0095] Six-week-old female BALB / c mice were purchased from the Guangdong Provincial Center for Laboratory Animal Science and Technology. After a week of quarantine, they were randomly divided into three groups of 16 mice each. Before immunization, immunogen suspensions (SD-NP, DAM, and DAM-NP) were gently mixed with FH002C adjuvant at a 1:1 volume ratio to achieve final antigen concentrations of 2, 2, or 2.86 μg / mL, respectively. After anesthesia with isoflurane, the immunogen was injected intramuscularly into the gastrocnemius muscle using a 1 mL syringe, 50 μL into each hind leg, for a total of 100 μL. Two weeks after vaccination, serum samples were collected to measure the neutralizing antibody titer against MPXV. Some mice (n=6 per group) were sacrificed, and spleens, inguinal lymph nodes, and mesenteric lymph nodes were collected for flow cytometry analysis. The remaining mice (n=10 per group) were transported from the Guangdong Provincial Center for Laboratory Animal Science and Technology to the Animal Biosafety Level 3 (ABSL3) laboratory of the Third People's Hospital of Shenzhen. After a one-week acclimatization period, mice were anesthetized with isoflurane and infected 5 × 10⁶ mice via nasal cavity. 5 The MPXV IIb clade SZTH41 strain, representing focal forming units (FFUs), was used to evaluate the efficacy of the nanoparticle vaccine. Body weight and survival were monitored daily until the end of the study, two weeks post-infection.

[0096] On the third day after challenge, the mice were sacrificed and lung tissue was collected (n=4 per group) to detect viral load.

[0097] 4) Rabbit immunization

[0098] Fifteen New Zealand rabbits were purchased from the Guangdong Provincial Experimental Animal Center and randomly divided into three groups. The immunogen was prepared as described previously. Each group of rabbits was anesthetized with isoflurane and then injected intramuscularly into the deltoid muscle of each hind leg at a dose of 0.5 mL per injection site. Four weeks later, the animals were given a booster immunization. The immunization dose was 10 μg DAM, or an equimolar ratio of DAM-bound nanoparticles. PBS was used as a negative control. Blood samples were collected 14 days after the primary and booster immunizations and allowed to stand at 37°C for 30 minutes to allow complete coagulation. The blood sample processing method was the same as for mouse blood samples.

[0099] 5) Determination of IgG binding antibody titer

[0100] The titers of MPXV M1, A35, and DAM-specific total IgG and subtype-specific binding antibodies were determined using ELISA. In short, high-binding-strength ELISA microplates (NEST) were coated overnight at 4°C with 1 μg / mL of purified MPXV M1, A35, and DAM proteins (100 μL per well).

[0101] The ELISA plate was washed once with PBST and then blocked in PBS containing 5% w / v skim milk powder at room temperature for 2 hours. Heat-inactivated serum samples from mice and rabbits in the immunization and control groups were serially diluted 4-fold with PBS and added to the coated plates, then incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of HRP-labeled secondary antibody was added. For mouse serum, goat anti-mouse IgG, IgG1, and / or IgG2a antibodies were used; for rabbit serum, anti-rabbit IgG was used; and for MPXV human convalescent serum, anti-human IgG was used, all at a dilution ratio of 1:20000. After incubation at 37°C for 30 minutes, the ELISA plate was washed five more times, and TMB substrate (Tiangen) was added. The plate was incubated at room temperature for 15 minutes, and then the reaction was terminated with ELISA stop solution (Solarbio). Data were immediately read at 450 nm using a TECAN Spark microplate reader. The antibody binding endpoint titer was defined as the maximum dilution of serum, where the absorbance value was greater than 2.5 times the background value. Antibody titers below the detection limit are calculated as 50% of the detection limit.

[0102] 6) Preparation of MPXV and VCV standard viruses

[0103] Vero-E6 cells were placed in fresh DMEM containing 10% FBS and cultured in 150 mm TC-treated dishes (Corning). When cell confluence reached 90%, MPXV I and II strains, VACV WR, and / or VTT were infected at a multiplicity of infection (MOI) of 0.03 for 1 hour, with occasional rotation during incubation. The viral inoculum was removed and replaced with complete DMEM medium. After 48 hours of incubation, the supernatant containing viral particles was collected by centrifugation at 500 g for 10 minutes and then stored at -80°C. The viral titers of MPXV and VACV were determined in Vero E6 cells.

[0104] 7) MPXV and VCV-VTT neutralization test

[0105] Serum samples were diluted 8-fold and then serially diluted 2-fold in DMEM supplemented with 2% FBS and 10% fresh guinea pig serum or 5% young rabbit serum as complement. These dilutions were then mixed with an equal volume of VACV-VTT-GFP containing 100 TCID. 50Live virus (50% tissue culture infection dose) was used, with a final sample volume of 75 μL, and each sample was retested.

[0106] After incubating the mixture at 37°C for 2 hours, it was added to pre-prepared 96-well plates containing a monolayer of BHK-21 or Vero E6 cells (prepared 24 hours prior to incubation), followed by the addition of a mixture of virus and diluted serum. One hour later, the supernatant containing virus and serum was removed, the plate was washed once with PBS, and then replaced with DMEM containing 2% FBS. The plate was then incubated at 37°C in a 5% CO2 atmosphere for another 24 hours, and cytopathic effects and green fluorescent protein (GFP) were observed at 40x magnification. Subsequently, virus titers were back-tied for each plate. Complete protection was defined as no cytopathic effects observed in any single well. Data analysis was performed using GraphPad Prism 9.5.1.

[0107] In a certified biosafety level 3 laboratory, the neutralizing activity of mouse serum against MPXV strains of clades VIb and IIb was evaluated using a micro-neutralization assay. Serum samples were serially diluted 2-fold at ten different gradients and mixed with an equal volume of MPXV (containing 100 TCID50). 50 The mixture was prepared in 96-well plates and supplemented with 5% young rabbit complement (Pel-Freez Biologicals) or 10% fresh guinea pig serum. The mixture was incubated at 37°C for 60 minutes. Then, the mixture was transferred to 96-well plates pre-seeded with Vero E6 cells, incubated at 37°C for 1 hour, and then removed. The cells were subsequently cultured in a cell culture incubator at 37°C with 5% CO2. Cytopathic effects were observed 4–6 days post-infection, and the results were statistically analyzed using GraphPad Prism 9.5.1.

[0108] 8) VCV-WR Plaque Reduction and Neutralization Test (PRNT)

[0109] First, serum samples were heat-inactivated at 56°C for 30 minutes using an Eppendorf ThermoMixer to remove complement. Then, serum samples were serially tripled from 1:10 to 1:21,870 in DMEM containing 2% FBS, with two aliquots of each dilution, for a total volume of 100 μL. Each dilution was mixed with an equal volume of diluted VCV-WR to achieve a final viral concentration of 60 PFU per well in a 48-well plate (this concentration allows for the formation of 30–50 plaques per well). The mixture was incubated at 37°C for 2 hours. A total volume of 200 μL of the virus-serum mixture was added to a culture plate pre-coated with a Vero E6 cell monolayer and incubated at 37°C for 1 hour in a 5% CO2 incubator. After incubation, the virus-containing medium was removed, and the cells were washed once with PBS. Finally, 0.25 mL of preheated cover medium was added, consisting of DMEM supplemented with 2% heat-inactivated FBS, 1.2% Avicel (RC-591, FMC Biopolymer), and / or 2% penicillin-streptomycin. Forty-eight hours post-infection, the culture plates were washed twice with PBS, fixed with 4% (v / v) neutral buffered formalin, and stained with 0.5% (w / v) crystal violet for 30 minutes. The plates were then rinsed with PBS to observe viral plaques. Plaque counts from viral samples not co-incubated with serum were set to 100% as a positive control. Antibody titers (PRNT) were measured. 50 The value is defined as the highest serum dilution that reduces the number of plaques by 50% or more.

[0110] 9) Biomembrane Interference Technology

[0111] Biomembrane interference (BLI) assays were performed and analyzed on an Octet R8 (Sartorius) platform according to the manufacturer's instructions. All assays were performed at 30°C in 96-well microplates (Greiner Bio-One) with stirring at 1000 rpm. Assays were performed in PBS buffer containing 0.05% Tween 20 and 1% BSA as the kinetic buffer.

[0112] Before analyzing the binding kinetics of antibodies and antigens in solution on the sensor (lasting 200 seconds), a 60-second baseline step was performed on the sensor. To determine the binding affinity of purified MPXV antigen to antibodies, a second-generation anti-mouse IgG Fc capture (AMC2) biosensor (Sartorius) was used to capture M1-specific antibodies MM1H11 and 7D11, and A35-specific antibody A27D7. After a 10-minute equilibration period, the analytes (M1, A35, DAM, and DAM-NP) were serially 2-fold diluted from an initial concentration of 200 nM (equimolar with A35 or M1 in the analyte) and bound to the AMC2 sensor. Interaction monitoring of the dissociation process lasted 300 seconds. Baseline drift was corrected by subtracting the following shift values: sensors with antibodies but without antigen incubation, and sensors without antibodies but with antigen incubation. The raw curves from each experiment were aligned at the baseline and fitted using a 1:1 binding model to calculate the kinetic parameters. Use Octet BLIAnalysis 12.2 to process and collect data.

[0113] To assess the competitive protective efficacy of serum samples, a serum competitive BLI assay was performed according to previously reported methods. Briefly, equal volumes (20 μL) of serum collected from New Zealand rabbits immunized with SD-NP, DAM, and DAM-NP two weeks prior were mixed within each group to obtain a total volume of 100 μL. Purified M1 and A35 proteins were biotinylated using NHS-LC-LC-Biotin (ThermoFisher) and diluted to a final concentration of 10 μg / mL, then immobilized onto the SA biosensor. Rabbit serum was serially 2-fold diluted, starting at a 50-fold dilution, or a blank control was loaded onto the SA biosensor to saturate M1 or A35 protein for 300 seconds. Then, anti-M1 antibodies (including M1H11 and 7D11) and anti-A35 antibody A27D7 (each at a concentration of 100 nM) were bound to the sensor for 300 seconds to assess competitive binding at each serum dilution level in the presence of primary saturation. Ro represents the maximum response value of the non-competitive binding curve when there is no antibody competition; Rc represents the maximum response value of competitive antibody binding under serum saturation. The competitive inhibition rate is calculated as (Ro-Rc)*100 / Ro.

[0114] 10) Flow cytometry

[0115] To evaluate the response of antigen-specific T cells in mice vaccinated with the MPXV nanoparticle vaccine, spleens were collected 14 days post-vaccination and prepared into single-cell suspensions using a 70 μm cell filter (BD Bioscience). Erythrocyte lysis was performed using erythrocyte lysis buffer (Elabscience) at room temperature for 3 minutes. Cells were then washed twice with complete RPMI 1640 medium containing 10% FBS and 2% penicillin-streptomycin, resuspended, and counted using a cell analyzer (JIBIO). Splenic cells were cultured at 2.5 × 10⁻⁶ cells / year. 6 Cells were seeded in 24-well plates at a concentration of [cells / mL] and treated with BD GolgiPlug (BD Bioscience) to inhibit intracellular protein transport. Subsequently, cells were stimulated with 50 μg / mL MPXV M1 and A35 recombinant protein for 7 hours, or with 4 × 10 [cells / mL] [recombinant protein]. 6 PFU was used to stimulate cells with VAV-WR virus for 24 hours. PBS and leukocyte activation mixture (BDPharmingen) were used as negative and positive controls, respectively. After stimulation, cells were stained for 30 minutes at 4°C in the dark with PE-Cy7-conjugated anti-CD3e antibody, BV510-conjugated anti-CD4 antibody, and / or Per-CP5.5-conjugated anti-CD8 antibody. After washing twice with PBS containing 2% BSA, cells were fixed and permeabilized using the Cytofix / Cytoperm kit (BD Bioscience) according to the manufacturer's protocol, followed by washing twice with PBS. Subsequently, cells were stained for 30 minutes at 4°C in the dark with BV421-labeled anti-IL4 antibody, FITC-labeled anti-IFN-γ antibody, and PE-labeled anti-TNF-α antibody. After washing twice more, cells were resuspended in PBS and analyzed using a BD FACS flow cytometer (BD FACSymphony S6).

[0116] Inguinal and mesenteric lymph nodes were collected from each mouse and homogenized in complete RPMI medium containing 10% FBS and 2% antibiotics. The homogenized tissue was passed through a 70 μm cell sieve (BD Bioscience) to prepare a single-cell suspension. Cells were then washed twice with PBS, blocked with anti-CD16 / 32Fc antibody (BD Pharmingen), and stained with appropriate markers for subsequent analysis. Germinal center (GC) B cells (B220) + IgD low GL7 + FAS +Staining with Per-CP5.5-labeled anti-B220 antibody, PE-labeled anti-IgD antibody, AF647-labeled anti-GL7 antibody, and FITC-labeled anti-FAS antibody; Follicular helper T cells (TFH, CD3) were stained. + CD4 + CXCR5 + PD-1 + Then staining was performed using PE-Cy7 labeled anti-CD3 antibody, BV510 labeled anti-CD4 antibody, Per-CP5.5 labeled anti-CD8 antibody, PE labeled anti-CXCR5 antibody and APC labeled anti-PD-1 antibody.

[0117] 11) Plaque titration test

[0118] Viral titers in tissue samples and viral stock were determined by a standard plaque assay performed on a monolayer of Vero E6 cells. Following the manufacturer's (Qiagen) instructions, tissue was first collected and homogenized using a tissue homogenizer, then clarified by centrifugation at 12,400 g for 10 minutes at 4°C. 1×10⁻⁶ 5 Vero E6 cells were seeded in 24-well Corning plates. One day later, cells were infected with a 10-fold serial dilution of 250 μL of tissue homogenate supernatant. Infectious particles were removed 1 hour post-infection, followed by washing twice with PBS. Then, 0.5 mL of pre-warmed DMEM-containing covering medium (2% heat-inactivated FBS, 1.2% Avicel (RC-591, FMC biopolymer), and 2% penicillin-streptomycin) was added. Forty-eight hours after infection, cells were fixed with 4% (v / v) neutral buffered formalin and stained with 0.5% (w / v) crystal violet for 30 minutes to observe and quantify plaques. Cells were then washed with PBS at room temperature. Viral titers are expressed as PFU / g per tissue sample.

[0119] 12) Histopathological analysis

[0120] Histopathological evaluation was performed using H&E staining. Mouse lung specimens were fixed in 4% paraformaldehyde at room temperature for 24 hours, then dehydrated and embedded in molten paraffin. The embedded tissue blocks were cut into thin sections and stained with hematoxylin and eosin alcohol. The stained sections were scanned using a Pannoramic MIDI tissue scanner (3DHISTECH) and analyzed using SlideViewer. TM The software records the images, which are then analyzed by veterinary pathologists.

[0121] 13) Detection of viral genome in lung tissue

[0122] VAV DNA copy number was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In short, lung tissue samples were collected and weighed, and then 1 mL of serum-free DMEM was added. The samples were homogenized using a low-temperature homogenizer (LUKYM-I, 70 Hz, 5 min), centrifuged, and the supernatant was collected for viral copy number detection. Nucleic acid was extracted using the FastPure Viral DNA / RNA Mini Kit Pro (Vazyme, China). The primer sequences used for qPCR were as follows: vaccinia virus forward primer, 5′-ACATCTGGAGAATCCACAACA-3′; vaccinia virus reverse primer, 5′-CATCATCGGTGGTTGATTTA-3′; VAV probe, 5′-FAM-GAGACTCCGGAACCAAT-TAMRA-3′. All samples were prepared in triplicate. The results were analyzed and plotted using GraphPadPrism 9.5.1 software.

[0123] Please see Figure 1-11 As shown, Figure 1 (A) illustrates the design of the DAM-NP vaccine: this vaccine is based on a self-assembled iron nanoparticle platform with 24-mer DAM antigens anchored on its surface. The bivalent construct GvO-DAM is formed by fusing MPXV antigens M1 and A35 into a "two-in-one" configuration and adding a GvTagOpti (GvO) tag at the N-terminus. Simultaneously, SD-NP is fused at its N-terminus with an SdCatcher (SD) tag and ferritin; the structures of both were predicted using AlphaFold3. Co-incubation of GvO-DAM and SD-NP in vitro leads to spontaneous covalent binding and self-assembly, ultimately forming the DAM-NP nanoparticle vaccine.

[0124] Figure 1 (B) shows the SDS-PAGE analysis results of SD-NP, DAM, and DAM-NP stained with Coomassie Brilliant Blue under reducing and non-reducing conditions. SD-NP, unmodified DAM subunits, and NPs with DAM are visible on the gel.

[0125] Figure 1 (C) shows the SEC spectra of DAM, SD-NP, and DAM-NP on a Superdex 200 increase 10 / 300GL. Compared with SD-NP and DAM, DAM-NP showed a significant forward shift in SEC chromatography.

[0126] Figure 1 (D) Hydrodynamic diameters and particle distributions of DAM, SD-NP, and DAM-NP as measured by dynamic light scattering. Results are expressed as mean ± standard deviation (SD) of three replicate experiments.

[0127] Figure 1 (E) Negative staining TEM micrographs of purified SD-NP scaffolds and DAM-NP immunogens.

[0128] Figure 1 (F) ELISA analysis of the binding of purified immunogen to M1-specific monoclonal antibodies 7D11 and M1H11, and A35-specific monoclonal antibody A27D7. Each symbol in the figure represents the mean ± SEM.

[0129] Figure 2 The study demonstrated that the DAM-NP nanoparticle vaccine elicited a robust and protective neutralizing antibody response against lethal VCV-WR challenge in mice.

[0130] Figure 2 (A) BALB / c mice were immunized using purified proteins (SD-NP, DAM, and DAM-NP) as immunogens. Each group of BALB / c mice (n=6) was quarantined for 7 days, and then immunized intramuscularly on days 0 and 21, with equal volumes of soluble DAM immunogen (0.2 μg and 1 μg) mixed with an equal volume of FH002C adjuvant. The control group received 1 μg of SD-NP. Serum samples were collected 14 days after the first immunization (primary immunization, day 14) and the second immunization (booster immunization, day 35). 21 days after the booster immunization, mice were intranasally inoculated with 1×10⁻⁶ DAM immunogen under isoflurane anesthesia. 6 PFU (35LD) 50 Mice were infected with the live virus, and after 3 days, they were sacrificed, lung tissue was collected, and weight changes were monitored over 14 days. This illustration was created by Biorender.com.

[0131] Figure 2 (B) The binding titers of MPXV-specific M1, A35 and DAM in the serum of BALB / c mice immunized with SD-NP, DAM or DAM-NP were determined by ELISA. The tests were performed 14 days after the initial immunization and the second booster immunization (n=6 / group).

[0132] Figure 2 (C) Serum neutralizing antibody titers were assessed by the VCV-WR specific PRNT50 test 14 days after primary and booster immunization (n = 6 / group).

[0133] Figure 2 (D and E) Monitor continuously for 14 days using VCV-WR (35LD) 50 Survival rate (D) (n=6 / group) and weight change (E) of attacked mice.

[0134] Figure 2 (F) Viral titer was quantified in lung tissue collected on day 3 post-VACV-WR infection using a standard plaque assay, and the results were expressed as PFU / g lung tissue (n = 3 / group).

[0135] Figure 2 (G) The lungs taken from mice on day 3 after VCV-WR infection were sectioned and H&E stained (n=3 / group).

[0136] Each symbol represents one mouse. Data are presented as mean ± SEM. HCS refers to MPXV human convalescent serum. Data in B, D, and F were calculated using one-way ANOVA and statistically analyzed using Turkey's multiple comparison test. Data were calculated using one-way ANOVA, and statistical analysis was performed using Turkey's multiple comparison test (B, D, and F) and the two-tailed Mann-Whitney test (C). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Horizontal dashed lines indicate the detection limit of the assay, and vertical dashed lines indicate that different secondary antibodies were used for HCS and mouse serum.

[0137] Figure 3 The results showed that the DAM-NP vaccine exhibited high immunogenicity and could effectively induce New Zealand rabbits to produce potent protective antibodies.

[0138] Figure 3 (A) Immunization schedule for New Zealand rabbit DAM-NP vaccine: Each group of rabbits (n=5) underwent a 7-day quarantine period. On days 0 and 28, each rabbit received an intramuscular injection of a mixture of equal volumes of soluble DAM immunogen (10 μg) and FH002C adjuvant. The control group received 10 μg of SD-NP. Serum samples were collected on day 14 after the first immunization (primary immunization on day 14) and the second immunization (booster immunization on day 42) for serological and virological analysis. This figure is from Biorender.com.

[0139] Figure 3 (B) The binding titers of MPXV-specific M1, A35 and DAM in the serum of BALB / c mice were determined by ELISA. These mice were inoculated with SD-NP, DAM or DAM-NP, respectively. The measurement time point was 14 days after the primary immunization and booster immunization (n=5 / group).

[0140] Figure 3 (C) The titers of neutralizing antibodies in serum were assessed using real MPXVI branch strains, VCV-WR, and VCV-VTT. The assessment time point was also day 14 after the primary immunization and booster immunization (n=5 / group).

[0141] Figure 3 (D) Serum samples collected 14 days after the initial immunization were mixed and then subjected to competitive binding assays with M1-specific monoclonal antibodies M1H11 and 7D11, and A35-specific mAb A27D7. Rc represents the signal value of the neutralizing antibody competing for binding with rabbit serum. The initial dilution of rabbit serum was 1:50, followed by serial dilutions of 2-fold. The target was either M1 or A35. O This indicates the maximum signal value at which the neutralizing antibody binds to M1 or A35 in the absence of rabbit serum.

[0142] Figure 3 (E) The heatmap shows the relative intensity of competition between rabbit serum and neutralizing antibodies. The darker the brown, the more intense the competition for a particular neutralizing antibody, suggesting that more antibodies are binding to similar antigenic target epitopes.

[0143] Each symbol represents an individual rabbit, and data are presented as mean ± SEM. Data from groups B and C were analyzed using one-way ANOVA and Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. The horizontal dashed line indicates the detection limit of the method, while the vertical dashed line distinguishes between primary and booster immunizations.

[0144] Figure 4 The results demonstrated that the DAM-NP vaccine exhibited high immunogenicity and efficacy in mice previously infected with the VACV-VTT vaccine.

[0145] Figure 4 (A) DAM vaccine immunization regimen in BALB / c mice. First, BALB / c mice were infected with 5 × 10 3 PFU-VACV-VTT was used to establish a pre-existing antibody model; 21 days later, mice were immunized with DAM-NP vaccine via intramuscular injection.

[0146] Serum samples were collected 10 days post-immunization, and mice were then infected intranasally with a viral dose of 4 × 10⁻⁶. 5 Lethal VAV-WR of PFU. Four mice were sacrificed 3 days after infection, and the weight change and survival of each mouse were continuously monitored for 14 days. This chart was created by Biorender.com.

[0147] Figure 4 (B and C) use ELISA and PRNT 50The analytical method measured the antibody titers (B) against MPXVE8, A29 and B6 (n=18 / group) and the neutralizing titer (C) against VACV-WR in serum collected 21 days after VACV-VTT infection.

[0148] Figure 4 (D and E) Mice were immunized with DAM-NP three weeks after establishing pre-existing antibodies via VCV-VTT infection. Serum samples were collected 14 days after immunization, and antibody titers against MPXVE8, A29, and B6 were analyzed by ELISA (D) (n = 9 / group); and neutralization was performed using a CEP-based neutralization assay or PRNT. 50 Analyze the neutralizing titers (E) against MPXVcladeIb strain or VAV-WR (n = 9 / group).

[0149] Figure 4 (FI) Fourteen days after immunization, nine BALB / c mice in each group were injected with 4×10 5 Mice were challenged with PFU-VACV-WR. Body weight changes were monitored for 14 days (F) (n=9 / group), and survival rate was recorded (G) (n=9 / group). Three days after infection, viral titers in lung tissue (n=4 / group) and nasal turbinates were assessed (H) (n=4 / group), and lung sections were stained with hematoxylin and eosin (HE) for histopathological evaluation (I) (n=6 / group).

[0150] Each symbol represents an individual mouse. Data are presented as mean ± SEM. Two-tailed Mann-Whitney test (BC) was used to calculate data, followed by one-way ANOVA and Turkey's multiple comparison test (DF and / or H) for statistical analysis. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. The horizontal dashed line indicates the detection limit of the detection method.

[0151] Figure 5 This demonstrates that a single dose of the DAM-NP vaccine provides complete protection against lethal MPXV infection in BALB / c mice;

[0152] Figure 5 (A) BALB / c mouse DAM-NP vaccine immunization program: BALB / c mice were intramuscularly injected with 0.2 μg DAM and an equimolar concentration of DAM-NP, with 0.2 μg SD-NP as a control. After 14 days, serum samples were collected, and the titer of neutralizing antibodies against MPXV was detected using a micro-neutralization assay. On day 21 post-immunization, six mice from each group were sacrificed, and spleens were harvested for intracellular cytokine staining; lymph nodes were also collected to assess the proportions of TFH and GCB cells. On the same day, ten mice from each group were intranasally inoculated with 5 × 10⁵ DAM-NP vaccine.5 Live, lethal MPXV virus with TCID50. Four mice in each group were sacrificed on day 3 post-viral challenge, and lungs were collected for viral titer determination and HE staining.

[0153] Weight changes and survival rates were monitored daily for 14 days.

[0154] Figure 5 (B) 14 days after the initial immunization, the serum neutralizing antibody titer was assessed by the MPXV micro-neutralization test (n=6 / group).

[0155] Figure 5 (C) Percentage of germinal center B (GCB) cells (top) and follicular helper T (Tfh) cells (bottom) in DAM- and DAM-NP-induced lymph nodes 21 days after a single immunization (n = 6 / group).

[0156] Figure 5 (D) On day 21 after primary immunization, CD4 levels in the spleen were measured using intracellular cytokine staining. + (Above image) and CD8 + (See image below) IFNγ in cells + TNF-α + and IL4 + The proportion of this measurement was determined after restimulation with VCV-WR (n = 6 / group).

[0157] Figure 5 (E and F) Monitored body weight change (E) (n=6 / group) and survival rate (F) (n=6 / group) of mice attacked with lethal MPXV for 14 consecutive days.

[0158] Figure 5 (G) The viral titer of lung tissue collected on the third day after MPXV infection was quantified using the standard plaque assay, and the results were expressed as PFU / g lung tissue (n=4 / group).

[0159] Each symbol represents one mouse. Data are presented as mean ± SEM. Data from groups BE and G were analyzed using one-way ANOVA followed by Tukey's multiple comparison test for statistical analysis.

[0160] *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. The horizontal dashed line indicates the detection limit of this detection method.

[0161] Figure 6 To investigate the in vitro binding efficiency of SD-NPs and GvO-DAM under different ratios in reducing conditions;

[0162] Figure 6This study investigated the in vitro binding efficiency of SD-NPs and GvO-DAM at different ratios under reducing conditions. The reactions were carried out overnight at 4°C, and the results were analyzed by Coomassie Brilliant Blue staining and SDS-PAGE gel electrophoresis, showing the percentage of unreacted SD-NPs. These reactions were repeated multiple times in the subsequent preparation and immunization of DAM-NP vaccines.

[0163] Figure 7 This is a schematic diagram illustrating the antigen characterization of M1, A35, DAM, and DAM-NP using biological layer interference (BLI) technology.

[0164] Figure 7 (A) The binding capacity of neutralizing antibodies (7D11, M1H11, and A27D7) was determined by biolayer interferometry, and the original signal curves and fitted curves of binding and dissociation are shown. KD: dissociation equilibrium constant; kon: binding rate constant; koff: dissociation rate constant.

[0165] Figure 7 (B) Competitive binding assays of 7D11 and A27D7 on MPXVDAM and DAM-NP were performed using BLI technology. First, neutralizing antibodies 7D11 or A27D7 (100 nM) were immobilized on the AMC2 sensor and then incubated with 100 μM MPXVDAM or DAM-NP. Subsequently, exposure to A27D7 or 7D11 was performed to assess the competitive binding of DAM-NPM1 and A35 epitopes.

[0166] Figure 8 The DAM-NP vaccine demonstrated that it can induce a more balanced Th1 / Th2-related humoral immune response;

[0167] Figure 8 The study shows the titers of anti-A35R, M1R, and DAM-specific IgG1 and IgG2a subtypes, as well as the IgG1:IgG2a ratio (n=6 / group), collected from the serum of mice vaccinated with SD-NP, DAM, and DAM-NP vaccines 14 days after the first immunization.

[0168] Figure 9 A schematic diagram illustrating the immunization schedule of the DAM-NP vaccine in mice and its protective effect against VACV-VTT infection;

[0169] Figure 9(A) BALB / c mice were immunized using purified SD-NP, DAM, and DAM-NP proteins as immunogens. Each group of BALB / c mice (n=6) was quarantined for 7 days and then immunized intramuscularly on days 0 and 21, respectively. The immunogens were equal volumes of soluble DAM (0.2 μg and 1 μg) mixed with an equal volume of FH002C adjuvant. The control group received 1 μg of SD-NP. Serum samples were collected 14 days after the first immunization (primary immunization, day 14) and the second immunization (booster immunization, day 35). 21 days after the booster immunization, 5 x 10^5 SD-NP proteins were administered intranasally under isoflurane anesthesia. 4 TCID 50 One VCV-VTT virus was detected; mice were euthanized 3 days after infection, lung tissue was collected, and body weight changes were continuously monitored for 14 days. (Image created by Biorender.com)

[0170] Figure 9 (B) Serum neutralizing antibody titers were assessed by the VCV-VTT micro-neutralization assay (n = 6 / group).

[0171] Figure 9 (C) Monitor the weight changes of VCV-VTT challenged mice for 14 consecutive days (n=6 / group).

[0172] Figure 9 (D) Quantification of viral DNA copy number in lung tissue collected on day 3 after VCV-VTT infection by qPCR (n = 3 / group).

[0173] Figure 9 (E) Lung sections were taken from mice on day 3 after VCV-VTT infection and H&E staining was performed (n=3 / group).

[0174] Figure 10 This study demonstrates the presence of CD4+ in the spleen and lymph nodes of mice immunized with DAM or DAM-NP in an independent animal experiment. + CD8 + T cell gating strategies;

[0175] Figure 10 (A) Shows GCB cells (B220) + IgDlowGL7 + FAS + ) and TFH cells (CD3) + CD4 + CXCR5 + PD-1 + Representative phenotypes and sorting strategies.

[0176] Figure 10 (B) showed CD4+ (CD3 + CD4 + ) and CD8 + (CD3 + CD8 + Representative gating strategies of T cells. Data in the figure are from single spleen cell suspensions of mice immunized with SD-NP, DAM, and DAM-NP vaccines, these suspensions were sized at 4 × 10⁻⁶ cells / mL. 6 PFU was stimulated with VAV-WR virus for 24 hours. The middle and bottom rows show further analysis results for these cells, with the middle row representing CD4. + Cytokine secretion status, with CD8 being a representative cytokine at the bottom. + Cytokine secretion status.

[0177] Figure 11 The study demonstrated that the DAM-NP vaccine elicited a significant cellular immune response against MPXV infection in mice.

[0178] On day 21 after the initial immunization, antigen-specific CD4 in the spleen was assessed using intracellular cytokine staining. + and CD8 + T cell response. The assessment method was as follows: restimulation with 50 μg / mL A35(A) (n=6 / group) and M1(B) (n=6 / group) for 7 hours, respectively. The values ​​represent the CD4+ response after restimulation with MPXV specific antigen in vitro. + or CD8 + TNF-α in T cells + IFN-γ + and / or IL-4 + The percentage of cells, with each symbol representing one mouse.

[0179] Data are presented as mean ± SEM. One-way ANOVA was used to analyze the data, followed by Tukey's multiple comparison test for statistical analysis. The significance levels were: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0180] V. Results:

[0181] 1) Construction and expression of MPXV DAM-carrying nanoparticle vaccines;

[0182] To develop a self-assembled nanoparticle vaccine based on an MPXV-specific antigen, this embodiment selected an immunogen (DAM) composed of the tandem fusion of A35 and M1 proteins. This immunogen has been previously reported as a structure-antigen-guided "two-in-one" monkeypox protein vaccine. This embodiment also utilizes an irreversible covalently coupled GvTagOpti / SdCatcher (GvO / SD) conjugation system to display the immunogen on the surface of the nanoparticles (NPs). The structural model of the DAM-NP was predicted using AlphaFold3, as shown below. Figure 1 Figure A shows the optimal conformation for antigen capture and recognition. To optimize the coupling efficiency between the immunogen and NPs, SD-NPs were co-incubated with different concentrations of GvO-DAM to evaluate the covalent coupling ability and determine the optimal concentration. Figure 6 The results showed that 3 μM GvO-DAM effectively bound to SD-NPs, leaving only a small amount of residual SD-NPs. Furthermore, size exclusion chromatography (SEC) and SDS-PAGE electrophoresis confirmed the high purity of the purified DAM-NPs. Different peak positions in the chromatograms indicated that the NPs had different valences, including SD-NPs and DAM-NPs. Figure 1 B and 1C). To further characterize the purified immunogens, their size and polydispersity were also assessed using dynamic light scattering (DLS) in this embodiment. Figure 1 D). The results showed that the hydrodynamic diameter of DAM-NP was significantly larger than that of SD-NP and free DAM protein; however, the polydispersity index (PDI) of both nanoparticle formulations was less than 20%, indicating a narrow and uniform particle size distribution. To further verify the homogeneity of DAM-NP, their morphology was observed using negatively stained transmission electron microscopy (TEM) in this embodiment. TEM micrographs showed that both SD-NP and DAM-NP exhibited highly uniform granular morphology. In addition, prominent peaks were observed on the surface of DAM-NP in this embodiment, indicating that DAM protein is displayed on the self-assembled surface of SD-NP ( Figure 1 E).

[0183] Next, neutralizing antibodies targeting M1 specifically, 7D11 and M1H11, and neutralizing antibody targeting A35 specifically, A27D7, were expressed and purified. The antigenicity of the purified DAM, DAM-NP, SD-NP, and soluble MPXV antigens (M1 and A35) was characterized by enzyme-linked immunosorbent assay (ELISA). ELISA results showed that, similar to the binding patterns of specific antigens, the M1-specific monoclonal antibodies 7D11 and M1H11, and the A35-specific monoclonal antibody A27D7, all exhibited dose-dependent binding to DAM-NP and DAM. Figure 1F). Furthermore, the binding kinetics of DAM-NP and DAM with M1-specific monoclonal antibodies 7D11 and M1H11, and A35-specific monoclonal antibody A27D7 were further investigated using biomembrane interference technology. For example... Figure 7 As shown in Figure A, the calculated dissociation constants (KD(M)) for the binding of M1, DAM, and DAM-NP to antibody 7D11 are 6.729 × 10⁻⁶. -9 3.292×10 -10 and 2.750×10 -12 Similarly, the KD(M) values ​​for antibody M1H11 binding to M1, DAM, and DAM-NP were 4.407 × 10⁻⁶. -9 1.834×10 -9 and <1.0×10 -12 The KD(M) values ​​for antibody A27D7 binding to A35, DAM, and DAM-NP were 5.168 × 10⁻⁶. -9 4.478×10 -10 and 1.476×10 -12 These results indicate that the KD(M) value of each antibody binding to DAM-NP is more than 100 times lower than that binding to DAM, suggesting that DAM-NP has a stronger affinity for these specific antibodies. Furthermore, consistent with previous reports that DAM can simultaneously bind to the A35 and M1 epitopes, competitive binding experiments showed that DAM-NP can bind to both 7D11 and A27D7 simultaneously without interference, indicating that the design of DAM-NP did not alter the M1 and A35 epitopes, and confirming its higher affinity. Figure 7 B).

[0184] 2) The DAM-NP vaccine elicited high levels of protective antibodies in BALB / c mice;

[0185] To assess the immunogenicity of the DAM-NP vaccine, BALB / c mice were intramuscularly injected with DAM, DAM-NP, or SD-NP, and adjuvanted with FH002C (…). Figure 2 A and Figure 9 A). Studies have shown that this adjuvant, when co-immunized with the SARS-CoV-2 spike protein in cynomolgus monkeys, can continuously induce high titers of neutralizing antibodies and cellular immune responses. Serum samples were collected on day 14 after the primary and secondary immunizations, and the binding titers of A35 and M1 specific antibodies in the serum were determined by ELISA. Figure 2As shown in Figure B, after primary immunization, compared with the model group, DAM vaccine group, and MPXV convalescent serum, the serum of mice in the DAM-NP vaccine group produced higher specific IgG binding titers against A35, M1, and DAM. Notably, mice vaccinated with one or two doses of the DAM-NP vaccine showed significantly higher levels of binding antibody titers against M1, A35, and DAM than those in MPXV convalescent serum. Figure 2 B). Furthermore, this embodiment comprehensively evaluated the binding titers of IgG isoforms in mouse serum after a single immunization. As expected, the ELISA results showed a trend consistent with the total IgG titer, indicating that DAM-NP induced higher IgG1 and IgG2a isoform antibody titers against M1, A35, and DAM with increasing DAM density on SD-NP. To preliminarily assess the dominance of humoral or cellular immune responses during the dynamics of the immune response, this embodiment also calculated the IgG1 / IgG2a ratio. The results showed that, compared to the control and DAM vaccine groups, the DAM-NP vaccine primarily induced a balanced TH1 / TH2-related immune response in mice after primary immunization. Figure 8 However, after the first booster immunization, no significant differences were observed in the titers of total M1, A35, and DAM-specific IgG induced in mice from the DAM and DAM-NP groups. Furthermore, this example also used real VCV-WR and VCV-VTT viruses to assess the neutralizing activity of serum samples using plaque reduction neutralization assays (PRNT) and micro-neutralization assays. Figure 2 As shown in C, after the initial immunization, a single injection of a low-dose DAM-NP vaccine (0.2 μg, PRNT) was administered. 50 4.2×10 2 The antibody titer of neutralizing VACV-WR in the serum of mice was significantly higher than that in the mimic group (PRNT). 50 1×10 1 ) and DAM vaccine group (0.2μg, PRNT) 50 6×10 1 More importantly, after the first booster immunization with the DAM-NP vaccine, serum antibody titers for neutralizing VACV-WR and VACV-VTT significantly increased. Following the booster immunization, the neutralizing antibody titers for VACV-WR and VACV-VTT induced by the low-dose (0.2 μg) DAM-NP vaccine were 5.3 times and 11.1 times higher, respectively, than those in the DAM vaccine group (0.2 μg). Figure 2 C and 9B). The results showed that both high-dose (1 μg) and low-dose (0.2 μg) DAM-NP vaccines induced neutralizing antibodies, with titers (PRNT) 50 Between 1.26×10 3 Up to 1.89×10 3The titer of neutralizing antibodies (PRNT) in convalescent serum of human VACV-WR after booster immunization (between) and (into) 50 2.14×10 2 Basically unchanged Figure 2 (C) This enhanced neutralizing activity may be attributed to the high immunogenicity of NPs, which enhances the immunogenicity of DAM, thereby promoting more effective uptake and processing of antigens by immune cells, ultimately leading to higher levels of neutralizing antibodies. In summary, these results indicate that the DAM-NP vaccine can elicit a robust humoral immune response in mice, resulting in high levels of both cross-reactive and protective antibodies.

[0186] 3) The DAM-NP vaccine provides cross-protection against VCV-VTT and lethal VCV-WR challenges in BALB / c mice;

[0187] To verify the protective efficacy of DAM and DAM-NP vaccines in vivo, mice were intranasally infected with VCV-VTT and VCV-WR on day 21 after the second immunization. Figure 2 (A and 9A). Following VACV-WR infection, the control group began to die on day 5 post-infection, reaching 100% mortality by day 6. In contrast, mice in the DAM-NP group experienced less weight loss and recovered to baseline weight more quickly than mice in the DAM-immunized group, indicating that the vaccine provides complete protection against VACV-WR virus. Figure 2 (D and 2E). Lung viral load analysis showed that while both the DAM and DAM-NP vaccine groups maintained relatively low viral levels, the active viral count in the DAM-NP vaccine group showed a decreasing trend, particularly in RPNT. 50 The value only reaches 1×10 2 Detection limit ( Figure 2 F). In addition, hematoxylin and eosin (H&E) staining was performed to assess the extent of lung lesions following viral infection. Pathological features of lung sections correlated with observed viral load: the control group showed significant inflammatory infiltration and alveolar septal thickening; conversely, the lesions in the DAM and DAM-NP vaccine groups were milder, with the DAM-NP vaccine group showing the least pathological changes, only a small accumulation of eosinophils and neutrophils. The results indicate that a large amount of live virus was present in the control group mice; while mice vaccinated with DAM-NP developed a strong protective antibody response, rapidly clearing the virus and thus protecting the body from viral infection. Figure 2 G). Furthermore, after VACV-VTT infection, mice in all groups experienced varying degrees of weight loss, but no deaths were observed. Figure 9C). Lung virus detection and H&E staining results showed that, compared with the control group, the viral DNA copy number in the lungs of mice in the DAM and DAM-NP groups was extremely low, indicating that mice vaccinated with DAM and DAM-NP vaccines were effectively protected. Figure 9 (D and 9E). In summary, these results further confirm that the neutralizing antibodies induced by the DAM-NP vaccine can provide cross-protection against VACV-VTT and lethal VACV-WR infection in BALB / c mice.

[0188] 4) The DAM-NP vaccine can induce high levels of neutralizing antibodies in New Zealand rabbits;

[0189] Consistency in responses across different animal models is a key criterion for validating the effectiveness of next-generation vaccines. Therefore, this embodiment evaluated the immunogenicity of the DAM-NP vaccine in New Zealand rabbits. New Zealand rabbits were intramuscularly injected with 10 μg of SD-NP, DAM, and DAM-NP (equimolar amounts of DAM) on days 0 and 28; serum samples were collected on day 14 after each immunization. Figure 3 A). For example Figure 3 As shown in B and 3C, compared with rabbits vaccinated only with DAM, rabbits vaccinated with DAM-NP showed a stronger humoral immune response after the initial immunization, with IgG titers against MPXVA35, M1, and DAM increasing by 2.2-fold, 2.0-fold, and 1.7-fold, respectively. Furthermore, based on the inhibition of infection with the real MPXVIb branch M25050 strain, VACV-WR, and VACV-VTT, the antibody neutralizing efficacy increased by 1.3-fold, 3.8-fold, and 1.4-fold, respectively. A significant upward trend in antibody response was observed after the first booster immunization in this example. Serum collected from rabbits vaccinated with DAM-NP showed significantly higher titers of M1-specific and DAM-specific IgG binding antibodies, increasing by 3.5-fold and 6.1-fold, respectively. In addition, DAM-NP induced higher levels of neutralizing antibodies against MPXV, VACV-WR, and VACV-VTT, increasing by 4.1-fold, 7.2-fold, and 2.3-fold, respectively. However, no significant difference was observed in the titer of A35-specific binding antibodies compared to the group that received only the DAM vaccine.

[0190] Furthermore, to better understand the diversity of antibody recognition between DAM and DAM-NP and to elucidate the differences in antibody neutralizing potency, this embodiment utilizes biomembrane interferometry (BLI) to perform competitive analysis on well-characterized neutralizing antibodies against MPXVM1 and A35 in rabbit serum, thereby roughly mapping the epitope recognition spectrum of serum antibodies. Figure 3As shown in Figure D, elevated serum concentrations reduce secondary binding of antibodies (M1-specific antibodies M1H11 and 7D11, or A35-specific antibody A27D7), indicating that both DAM and DAM-NP can induce the production of antibodies targeting epitopes associated with these antibodies. The accessibility of M1 and A35 on the nanoparticle surface differs from that of unmodified M1 and A35; theoretically, the presence of nanoparticles may increase the exposure of M1 and A35 epitopes and enhance their binding affinity. Structural analysis shows that the 7D11 binding epitope of M1 is the most readily identifiable region, potentially leading to the production of more effective antibodies targeting vulnerable sites. Figure 3 E, left). Furthermore, this embodiment also measured the relative competitive binding of serum to specific antibodies at different concentrations, expressed as a percentage of (R0-Rc) / R0. The results showed that serum from rabbits immunized with DAM-NP exhibited the strongest competition for 7D11, indicating stronger neutralizing potency of 7D11-class antibodies and higher abundance of M1H11-class antibodies, consistent with the hypothesis of enhanced accessibility to the 7D11 binding epitope on DAM-NP. Figure 3 (E, right). Consistent with the trend of A35-specific binding titers, this example found no difference in competition between serum induced by DAM and DAM-NP vaccines and A35-specific A27D7 antibodies, suggesting that the A35 antigen in DAM and DAM-NP vaccines may not induce an overt humoral immune response. Figure 3 E). The results show that there are significant differences in the antigenic epitope presentation patterns of DAM and DAM-NP, and nanoparticles play a decisive role in these differences.

[0191] 5) Previous antibodies do not affect the immunogenicity and protective efficacy of the DAM-NP vaccine;

[0192] Prophylactic MPXV vaccines are intended for individuals who have not previously been infected with MPXV or smallpox. Many of these individuals were vaccinated against smallpox before 1980, which may affect the immunogenicity and protective efficacy of the DAM-NP vaccine. To investigate whether prior VACV-VTT infection interferes with the immunogenicity and protective efficacy of the DAM-NP vaccine, experiments were conducted using a mouse model. Specifically, 18 mice in each group were intranasally infected with VACV-VTT, while the control group was treated with PBS; serum samples were collected 21 days post-infection to detect antigen-specific IgG binding antibodies and neutralizing antibodies against VACV-WR infection. Figure 4 A). The results showed that all 18 mice infected with VCV-VTT were seropositive for E8, A29, and B6, and their IgG titers against MPXVE8, A29, and B6 increased by 330.5-fold, 6.1-fold, and 4.5-fold, respectively. Figure 4B); Compared with the control group, the antibody neutralizing efficacy against real VACV-WR infection was increased by 8 times ( Figure 4 C) indicates that all 18 animals successfully developed prior immunity against MPXV and other orthopoxviruses. Mice (n=18) treated with PBS and infected with VACV-VTT were randomly assigned to a non-immunized group and a group receiving a single dose of DAM-NP vaccine. Serum antibody levels in the four groups of mice were measured 14 days after a single injection of DAM-NP vaccine. The results showed that the DAM-NP vaccine significantly increased serum antibody titers against MPXVM1, A35, and DAM-specific ELISA, as well as neutralizing antibodies against MPXVIb branch M25050 strain or VACV-WR infection previously infected with VACV-VTT. Figure 4 (D and 4E). In particular, mice previously infected with VACV-VTT via the nasal cavity and then boosted with the DAM-NP vaccine showed significantly elevated neutralizing antibody titers against MPXVIb branch and VACV-WR infection. Specifically, antibody titers were 237-fold, 55-fold, and 21.6-fold higher than the control group, respectively; and 129-fold, 14.3-fold, and 9.7-fold higher than mice infected with VACV-VTT only and mice vaccinated with the DAM-NP vaccine only, respectively. Figure 4 E). These results strongly demonstrate that pre-existing immunity does not affect the immunogenicity of the DAM-NP vaccine.

[0193] Based on the above findings, using 4×10 5 Lethal VACV-WR infection was performed on mice using PFU. On day 3 post-infection, four mice from each group were randomly sacrificed, and nasal and lung tissues were collected. Furthermore, weight change and survival were monitored for 14 days, and viral load in tissues was assessed. Results showed that mice in the control group experienced a sharp decline in weight, reduced activity, and other adverse symptoms, ultimately dying on day 7 post-infection. Conversely, all other treatment groups provided complete protection against VACV-WR infection. Notably, mice previously infected with VACV-VTT intranasally showed only mild weight loss after vaccination with DAM-NP vaccine (VACV-VTT & DAM-NP), and rapidly recovered to baseline levels after infection. Figure 4 (F and 4G); Consistent with body weight measurements, the control group showed the highest viral load in the lungs and nasal turbinates, up to 10. 7 The PFU / g level was lower in the VACV-VTT&DAM-NP group, while the lowest levels of live virus were detected in the lung and nasal turbinate tissues, indicating a stronger viral clearance capacity, which is related to a robust immune response. Figure 4H). Furthermore, histological examination of the lungs in the control group revealed extensive inflammatory infiltration, thickening of the alveolar septa, and multiple hemorrhagic lesions, indicating massive viral replication causing severe, irreversible damage and ultimately leading to mouse death. Additionally, pathological analysis showed significant changes in all treatment groups. The groups vaccinated with VACV-VTT & DAM-NP vaccines also exhibited a large accumulation of neutrophils and eosinophils, abundant pink exudate in the alveolar spaces, and thickening of the alveolar septa. These histopathological changes may have been caused by repeated viral attacks, leading to a strong immune response; even after viral clearance, significant tissue damage remained. In conclusion, the pre-existing immune response did not interfere with the immunogenicity of the DAM-NP vaccine and may even have a synergistic effect, resulting in a stronger humoral immune response and thus completely protecting mice from poxvirus infection.

[0194] 6) The DAM-NP vaccine elicited a strong T-cell response in mice;

[0195] To investigate the differences between DAM and DAM-NP vaccines in inducing cellular immune responses, this study examined the proportions of follicular helper T cells (TFH) and germinal center B cells (GCB) in lymph nodes 21 days after a single immunization in mice. Figure 5 A and 10A). The results showed that, compared with the control group, the proportions of GCB and TFH cells were significantly increased in the DAM-NP vaccine group, which is consistent with the faster antibody response and earlier antibody affinity maturation observed after DAM-NP immunization. Figure 5 C).

[0196] To further evaluate antigen-specific T cell responses, this embodiment first stimulated the mouse spleen with VACV-WR ( Figure 5 D), then intracellular cytokine staining (ICS) revealed that DAM-NP could induce a stronger TH1 and cytotoxic T lymphocyte (CTL) response, manifested as virus-specific INF-γ. + CD4 + and INF-γ + CD8 + The proportion of cells was higher. Compared with the control group and the DAM vaccine group, the DAM-NP group induced TNF-α. + CD8 + The proportion of T cells increased significantly, while TNF-α + CD4 + The proportion of T cells showed no significant difference, suggesting that different T cell subsets may participate in the immune response at different time points after in vitro VACV-WR virus stimulation. Furthermore, IL-4 levels varied among the groups. + CD4 +T cells and IL-4 + CD8 + There were no statistically significant differences in the proportion of T cells.

[0197] Next, in this embodiment, in vitro MPXV antigens (A35 and M1) were used. Figure 10 and 11 A35 stimulation stimulated spleen cells. Results showed that, compared to the control group, A35 stimulation significantly induced a higher frequency of antigen-specific TNF-α. + CD4 + and TNF-α + CD8 + T cells, and INF-γ + CD4 + and INF-γ + CD8 + T cells, indicating that the DAM-NP vaccine can elicit a stronger TH1 and CTL response. Notably, induced IL-4 + CD4 + T cell frequency is higher, while IL-4 + CD8 + The T cell frequency did not change significantly, suggesting that the Th2 response may play a role in the early stages after A35 stimulation, while CTLs do not produce IL-4 at this stage. Similarly, the results of spleen stimulation with M1 were similar to those of A35 stimulation; DAM-NP induced a stronger Th1 and CTL response, which could be attributed to a higher frequency of antigen-specific TNF-α. + CD4 + and TNF-α + CD8 + T cells have been demonstrated. In summary, DAM-NP can induce a more balanced Th1 and Th2 immune response.

[0198] 7) A single dose of DAM-NP vaccine protects mice from MPXV challenge;

[0199] Vaccine dosage is one of the key factors that must be considered in vaccine development. The aforementioned study found that a single dose of DAM-NP vaccine can induce high levels of neutralizing antibodies and effectively protect mice from VAV-WR challenge. Therefore, to further investigate the preventive potential of the DAM-NP vaccine against MPXV, this embodiment conducted a challenge experiment using real MPXV in an ABSL-3 laboratory to determine whether a single dose of DAM-NP vaccine can provide protection against MPXV infection in mice. BALB / c mice were immunized with a single dose of DAM, DAM-NP, or PBS, respectively. Fourteen days after immunization, serum samples were collected, followed by intranasal infection with a lethal MPXVIb branch strain. Three days after infection, four mice from each group were randomly selected and sacrificed, and lung tissue was collected to assess viral load. The weight of the remaining mice was monitored for 14 days; a weight loss of 25% or more was considered death. Figure 5 A). On the one hand, this embodiment conducted a real MPXV micro-neutralization test to assess the neutralizing titer in mouse serum. The results showed that, compared with the control group, both the DAM and DAM-NP immunization groups exhibited significant antiviral effects ( Figure 5 B). On the other hand, similar to the symptoms observed in mice infected with VACV-WR, the control group exhibited persistent weight loss and clinical symptoms such as piloerection and reduced activity, ultimately dying on day 8 post-infection. In contrast, all DAM and DAM-NP immunization groups showed complete protection against MPXV infection, with the DAM-NP group showing the smallest weight loss and the fastest recovery. Figure 5 E and 5F). Furthermore, lung viral load results showed that the DAM-NP vaccine group had the lowest viral titer compared to the control group and the DAM immunization group, demonstrating superior viral clearance ability. Figure 5 G). The results showed that a single dose of DAM-NP can elicit a strong immune response, thereby providing complete protection against MPXV infection.

[0200] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0201] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A monkeypox virus self-assembled nanoparticle, characterized in that, The nanoparticles are formed by covalent coupling of DAM antigen and SD-Ferritin, wherein: The DAM antigen contains a fusion sequence of monkeypox virus M1 protein and A35 protein, with a GvTagOpti tag attached to the N-terminus and connected in the middle by a glycine-serine-glycine spacer region. The SD-Ferritin is formed by fusing the SdCatcher fragment with the N-terminus of Helicobacter pylori ferritin, linked by the GSG spacer region, and contains eight histidine tags at the C-terminus. The DAM antigen and SD-Ferritin were coupled at a molar ratio of 3:1 and covalently bound after incubation in assembly buffer for 24 hours.

2. The monkeypox virus self-assembled nanoparticle according to claim 1, characterized in that, The coding sequence of the DAM antigen was optimized with humanized codons and cloned into the VRC8405 mammalian expression vector, which contains an N-terminal tissue plasminogen activator signal peptide and eight C-terminal histidine tags.

3. The monkeypox virus self-assembled nanoparticle according to claim 1, characterized in that, The coding sequence of SD-Ferritin was optimized with E. coli codons and cloned into the pTO-T7 prokaryotic expression vector.

4. A method for preparing monkeypox virus self-assembled nanoparticles according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: Plasmid construction: The optimized DAM antigen coding sequence was cloned into the VRC8405 vector, and the SD-Ferritin coding sequence was cloned into the pTO-T7 vector; Protein expression and purification: on Expi293F TM DAM antigen was expressed in cells and purified by Ni agarose gel affinity chromatography and Superdex 200 gel filtration; SD-Ferritin was expressed in Escherichia coli Rosetta competent cells, purified by CaptoDEAE column and Superdex 200, and endotoxin was removed by Triton X-114 phase separation. Conjugation and assembly: The purified DAM antigen was incubated with SD-Ferritin at a molar ratio of 3:1 in assembly buffer for 24 hours. The conjugated products were separated by Capto Core 700 chromatography combined with Superose 6 10 / 300GL gel column, concentrated and stored at -80°C.

5. The method for preparing monkeypox virus self-assembled nanoparticles according to claim 4, characterized in that, The purification of the DAM antigen included washing with 50 mM HEPES, 300 mM NaCl, and 30 mM imidazole at pH 8.0, followed by elution with 300 mM imidazole.

6. The method for preparing monkeypox virus self-assembled nanoparticles according to claim 4, characterized in that, The purification of SD-Ferritin involved equilibration with 20 mM Tris and 50 mM NaCl at pH 7.5, followed by elution with 400 mM NaCl.

7. The use of the monkeypox virus self-assembled nanoparticles as described in claim 1 in the preparation of vaccines for the prevention or treatment of monkeypox virus infection.

8. The application according to claim 7, characterized in that, The applications include: Immunization procedure: Mix the nanoparticles with FH002C adjuvant and immunize the animals intramuscularly at a dose of 0.2-10 μg DAM, and repeat the immunization twice at 2-week intervals. Immunogenicity assessment: M1 and A35 specific IgG and subtype titers were detected by ELISA, and neutralizing activity was detected by plaque reduction neutralization assay; Challenge protection: After immunization, experimental animals were infected with VCV or MPXV via intranasal infection, and changes in body weight, survival rate, and viral load in tissues were monitored.

Images