An engineered probiotic outer membrane vesicle vaccine co-expressing multiple antigens of monkeypox virus, and a preparation method and application thereof

Abstract

This invention discloses an engineered probiotic outer membrane vesicle vaccine co-expressing multiple antigens of monkeypox virus, its preparation method, and its application. The outer membrane vesicle vaccine is based on... lpxm Using the gene-deleted probiotic Escherichia coli Nissle 1917 as the host, ABM@OMV was constructed by genetically engineering and co-expressing and displaying three key monkeypox virus antigens, A9R, B6R, and M1R, on the surface of its secreted outer membrane vesicles. Leveraging the natural adjuvant and highly efficient mucosal delivery characteristics of OMV, combined with a multi-antigen synergistic strategy, nasal immunization can simultaneously activate strong systemic humoral immunity (high-titer neutralizing IgG) and cellular immunity (CD4+). + / CD8 + T cells are involved in the vaccine, which induces high levels of specific sIgA antibodies in the respiratory and distal genital tract mucosa, forming an effective immune barrier. Challenge experiments showed that the vaccine completely protects mice from lethal doses of vaccinia virus, significantly reduces tissue damage, and provides a novel solution for preventing monkeypox virus transmission via mucosa.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to an engineered probiotic outer membrane vesicle vaccine co-expressing multiple antigens of monkeypox virus, its preparation method, and its application. Background Technology

[0002] Monkeypox virus (MPXV) belongs to the genus Orthopoxvirus in the family Poxviridae. Its transmission routes are diverse, including respiratory droplets, close contact, and sexual contact, posing a persistent threat to global public health. The World Health Organization has repeatedly declared monkeypox outbreaks a Public Health Emergency of International Concern, highlighting the urgency of prevention and control efforts. Currently approved monkeypox vaccines are mainly administered via injection. While they can induce a systemic immune response, they have significant limitations: they are difficult to effectively activate specific immune responses in key mucosal sites such as the respiratory and reproductive tracts, and they are not effective against CD8+. + T-cell immunity has limited activation capacity, while CD8 + T cells play an irreplaceable role in clearing intracellular viruses.

[0003] Outer membrane vesicles (OMVs), naturally secreted nanoscale lipid vesicles (20–200 nm in diameter) by Gram-negative bacteria, have been considered a highly promising vaccine delivery platform in recent years due to their unique biological characteristics. OMVs are rich in various pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), outer membrane proteins, and lipoproteins. They can efficiently initiate innate immunity and enhance antigen-specific adaptive immune responses by activating the Toll-like receptor (TLR2 / TLR4) signaling pathway, exhibiting excellent natural adjuvant effects. More importantly, OMVs possess multiple immune activation advantages: firstly, their nanoscale size and surface adhesion molecules (such as OmpA) can prolong their residence time at mucosal sites, promoting antigen-presenting cells (APCs) capture; secondly, the specific binding of surface components to APCs enables efficient antigen internalization; and thirdly, OMVs can promote antigen entry into the cytoplasm through endosome escape mechanisms mediated by porins (such as ClyA), thereby significantly enhancing the presentation efficiency of MHC-I class I antigens and strongly activating CD8. + T-cell immune response. These properties make OMV an ideal carrier for inducing a comprehensive immune response, particularly mucosal and cellular immunity. However, there are currently no reports of OMV being used in monkeypox vaccine. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned defects and shortcomings in the prior art and provide an engineered probiotic outer membrane vesicle. Using probiotic-derived OMV as a smart delivery carrier, it collectively displays three key monkeypox virus antigens: A9R, B6R, and M1R.

[0005] A second objective of this invention is to provide a method for preparing the engineered probiotic outer membrane vesicles.

[0006] A third objective of this invention is to provide the application of the engineered probiotic outer membrane vesicles.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: An engineered probiotic outer membrane vesicle, the outer membrane vesicle being secreted by an engineered probiotic Escherichia coli stably expressing the Trx-A9R fusion protein and the ClyA-B6R-M1R fusion protein, the surface of which displays monkeypox virus A9R antigen, B6R antigen and M1R antigen.

[0008] This invention utilizes engineered probiotic *E. coli* strains stably expressing three key monkeypox virus antigens—A9R, B6R, and M1R—as outer membrane vesicle production strains. By fusing the A9R antigen with thioredoxin (Trx) and the B6R and M1R antigens with porin (ClyA), the three antigens are synergistically displayed and accurately localized on the surface of the outer membrane vesicles. Leveraging the natural adjuvant and highly efficient mucosal delivery characteristics of OMV, this multi-antigen synergistic strategy can be applied to the subsequent development of novel monkeypox OMV vaccines.

[0009] Furthermore, the probiotic Escherichia coli is Escherichia coli Nissle 1917. Escherichia coli Nissle 1917 (EcN) is a non-pathogenic Gram-negative Escherichia coli strain that has been used clinically as a probiotic for over a century. It is one of the few representative Escherichia coli probiotics in the field, possessing unique physiological characteristics and therapeutic value. It exhibits good safety and immunomodulatory functions.

[0010] Furthermore, to improve safety while ensuring strong immunogenicity, the probiotic E. coli is a knockout strain. lpxM The gene in E. coli Nissle 1917, i.e. Δ lpxM EcN. This was achieved by selecting and knocking out attenuated probiotics, specifically EcN. lpxM Genes that reduce the inflammatory toxicity of OMV; lpxM The absence of lipid A acylation leads to incomplete reduction of the inflammatory toxicity of LPS in OMV, while retaining its effective immunostimulatory capacity, laying a solid foundation for the development of safe and effective OMV vaccines.

[0011] Furthermore, the particle size of the engineered probiotic outer membrane vesicles is 50–150 nm (preferably 80–120 nm).

[0012] Furthermore, the zeta potential of the engineered probiotic outer membrane vesicles is approximately -12 mV.

[0013] The present invention also provides a method for preparing any of the engineered probiotic outer membrane vesicles described above, which involves transforming a recombinant plasmid encoding the Trx-A9R and ClyA-B6R-M1R fusion protein into probiotic Escherichia coli and collecting the outer membrane vesicles by ultracentrifugation.

[0014] Specifically, the method includes the following steps: S1. The encoding genes of the three key antigens of monkeypox virus, A9R, B6R and M1R, were used to construct fusion expression genes Trx-A9R and ClyA-B6R-M1R, which were then tandemly cloned into expression vectors to construct recombinant plasmids. These plasmids were then introduced into probiotic Escherichia coli to construct engineered probiotic strains. S2. The engineered probiotic strain obtained in step S1 is cultured in a large scale, and IPTG is added to induce protein expression to obtain bacterial culture. S3. Centrifuge the bacterial culture, collect the supernatant, concentrate by ultrafiltration, and then centrifuge again to obtain the precipitate. Resuspend the precipitate to obtain the engineered probiotic outer membrane vesicles.

[0015] Furthermore, the expression vector described in step S1 is pThioHisA.

[0016] Furthermore, the fusion gene also carries a His tag and an anti-FLAG tag, namely Trx-A9R-His and ClyA-B6R-M1R-FLAG, respectively, which facilitates the subsequent use of anti-His tag and anti-FLAG tag antibodies to verify the expression and display of the A9R antigen and the B6R-M1R fusion antigen.

[0017] The engineered probiotic outer membrane vesicles of this invention utilize the natural adjuvant and highly efficient mucosal delivery characteristics of OMV, combined with a multi-antigen synergistic strategy, to simultaneously activate strong systemic humoral immunity (high-titer neutralizing IgG) and cellular immunity (CD4+) after nasal instillation. + / CD8 + It induces high levels of specific sIgA antibodies in the respiratory and distal genital tract mucosa, forming an effective immune barrier. Challenge experiments showed that it can completely protect mice from lethal doses of vaccinia virus, significantly reduce tissue damage, and effectively prevent monkeypox virus infection.

[0018] Therefore, the present invention also provides the use of any of the engineered probiotic outer membrane vesicles described above in the preparation of vaccines against monkeypox virus.

[0019] This invention also provides a vaccine containing any of the engineered probiotic outer membrane vesicles described above. Specifically, it provides a monkeypox multivalent mucosal vaccine (ABM@OMV) based on engineered probiotic outer membrane vesicles (OMV).

[0020] Furthermore, the vaccine is in the form of nasal drops.

[0021] As a preferred embodiment, the preparation method of the ABM@OMV vaccine includes the following steps: (1) Construction of engineered strains First, an engineered strain, ABMΔEcN, capable of secreting the target OMV, was constructed. This strain used the well-regarded probiotic *Escherichia coli* Nissle 1917 (EcN) as a chassis, and was developed by knocking out… lpxM Gene (Δ) lpxM EcN was used to attenuate the virus, and then the recombinant plasmid pThioHisA-ABM carrying the encoding genes for monkeypox virus antigens A9R, B6R, and M1R was introduced. In this design, the A9R antigen was fused with thioredoxin (Trx), and the B6R and M1R antigens were fused with porin (ClyA), thereby achieving the synergistic display and accurate localization of the three antigens on the OMV surface.

[0022] (2) Vaccine preparation a) Fermentation and induced expression: ABMΔEcN engineered bacteria were cultured to the logarithmic growth phase, and IPTG was added to induce the expression of the target antigen under low temperature conditions.

[0023] b) Collection and purification of OMV: The induced bacterial culture was subjected to a series of differential centrifugations (300×g, 2,000×g, 10,000×g) to remove bacterial cells and cell debris. The supernatant was collected, concentrated by ultrafiltration, and then settled by ultracentrifugation (125,000×g, 2 h) to obtain crude ABM@OMV.

[0024] c) Refining and Finished Product: The ABM@OMV precipitate was resuspended in PBS and washed again by ultracentrifugation. Finally, it was resuspended in sterile PBS and filtered through a 0.45 μm filter membrane to obtain the refined ABM@OMV vaccine product, which was then aliquoted and stored at -80°C.

[0025] (3) Quality control of vaccines The prepared ABM@OMV vaccine was systematically characterized to ensure its quality: Morphology: The vesicle morphology was confirmed by transmission electron microscopy (TEM), and the particle size distribution (mainly 50–150 nm) and zeta potential were analyzed by dynamic light scattering (DLS) to evaluate particle uniformity and stability.

[0026] Antigen display validation: Western blotting was used to validate the successful display of A9R antigen and B6R-M1R fusion antigen on OMV using anti-His and anti-FLAG tag antibodies, respectively.

[0027] Aseptic and concentration control: The asepticity of the formulation was ensured by plate culture and the total protein concentration was standardized by BCA method.

[0028] The ABM@OMV vaccine is administered to subjects via nasal drops after passing quality inspection. The preferred immunization schedule involves two immunizations (e.g., on day 0 and day 15) at a dose of 20 μg total OMV protein per dose, thereby efficiently inducing specific humoral, cellular, and mucosal immunity in the respiratory mucosa and systemic system, achieving prevention against monkeypox virus.

[0029] This invention utilizes the natural adjuvant and mucosal delivery properties of OMV (Oxygen Flow Mucosa) to directly target the respiratory mucosa-associated lymphoid tissue via nasal drops, effectively inducing systemic immunity and mucosal immunity in the respiratory and distal genital tracts. It overcomes the shortcomings of existing vaccines in effectively activating mucosal immunity, providing a vaccine that can simultaneously induce sIgA antibody responses in the distal mucosa of the respiratory and genital tracts. It also addresses the limitations of existing vaccines in activating CD8. + To address the problem of insufficient T-cell immune activation, a multi-antigen co-expression strategy (especially the introduction of the A9R antigen) and an OMV-mediated endosome escape mechanism were employed to significantly enhance CTL responses. While ensuring strong immunogenicity, safety was improved by selecting attenuated probiotics EcN and knocking out… lpxM Genes can reduce the inflammatory toxicity of OMV. This provides a vaccine form with clearly defined components and controllable quality, avoiding the potential biosafety risks of live bacterial preparations. It offers a convenient needle-free vaccination method, achieving effective protection through nasal drops, avoiding the need for specialized procedures and facilitating widespread adoption; and it constructs a vaccine platform that is easy to standardize and scale up, providing a rapid vaccine development strategy for responding to emerging infectious diseases.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The outer membrane vesicle vaccine of this invention uses probiotic *Escherichia coli* as a host, and through genetic engineering, co-expresses and displays three key monkeypox virus antigens, A9R, B6R, and M1R, on the surface of its secreted outer membrane vesicles (OMV). By utilizing the natural adjuvant and highly efficient mucosal delivery characteristics of OMV, combined with a multi-antigen synergistic strategy, after nasal immunization, it can simultaneously activate strong systemic humoral immunity (high-titer neutralizing IgG) and cellular immunity (CD4+). + / CD8 +This vaccine (contains T cells) and induces high levels of specific sIgA antibodies in the respiratory and distal genital tract mucosa, forming an effective immune barrier. In other words, it not only efficiently activates APCs and promotes endosome escape, thus exhibiting excellent immune activation potential in vitro; it also induces a comprehensive and robust mucosal, humoral, and cellular immune response in vivo, providing complete protection against viral challenge while maintaining good safety. Challenge experiments showed that this vaccine completely protects mice against lethal doses of vaccinia virus, significantly reducing tissue damage, and providing a novel solution for preventing monkeypox virus transmission via mucosa. Attached Figure Description

[0031] Figure 1 The design, preparation, and characterization of ABM@OMV were carried out. Figure 1 Figure A shows a schematic diagram of the ABM@OMV preparation process. Figure B shows bacterial lysates analyzed by SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue. Lanes: Marker; Uninduced ΔEcN; ΔEcN+PTG; Uninduced BMΔEcN; BMΔEcN+IPTG; Uninduced ABMΔEcN; ABMΔEcN+IPTG; Black boxes indicate ClyA-B6R-M1R and Trx-A9R fusion proteins. Figure C shows Western blot analysis of the target proteins expressed in ABMΔECN bacteria. Trx-A9R was detected with anti-His tag antibody; ClyA-B6R-M1R was detected with anti-Flag tag antibody. The lane order is the same as in Figure B. Figure D shows ΔEcN. lpxM Dynamic light scattering (DLS) particle size distribution curves for OMVs, BM@OMV, and ABM@OMV. E represents Δ... lpxM Zeta potential measurements of outer membrane vesicles, BM@OMV, and ABM@OMV (n=3). F represents Δ lpxM Transmission electron microscopy (TEM) images of outer membrane vesicles, BM@OMV, and ABM@OMV, scale bar: 100 nm. G represents Western blot analysis of the target protein expressed in ABM@OMV, band: Δ lpxM Outer membrane vesicles, BM@OMV, ABM@OMV. H is the Δ measured by the Limulus amebocyte lysate (LAL) assay. lpxM Endotoxin levels in EcN-derived outer membrane vesicles and wild-type EcN-derived outer membrane vesicles (n=3). I is a representative Western blot pattern showing the target protein at Δ... lpxM The stability of OMVs, BM@OMV and ABM@OMV after storage at 4°C (left) or -80°C (right) for a specified time. J and K represent the changes in particle size (J) and zeta potential (K) of OMVs stored at 4°C and -80°C (n=3).

[0032] Figure 2This refers to antigen processing and presentation mediated by antigen-presenting cells (APCs) that drives adaptive immunity. Among these, Figure 2 Image A shows confocal microscopy images of ABM@OMV endocytosis in RAW 264.7 cells, including the cytoskeleton (phalloidin, green), cell nucleus (DAPI, blue), and vaccine (Cy5-labeled, red), scale bar = 30 μm. Image B shows flow cytometry analysis of vaccine internalization. Image C shows quantitative analysis of fluorescence intensity in image A (n=3). Image D shows statistical analysis of endocytosis efficiency in image B (n=3). Image E shows lysosomal escape of ABM@OMV (Cy5-labeled, red) at 2, 4, and 12 hours, with lysosomes (LysoTracker, green), scale bar = 10 μm. Image F shows CD86 expression (M1 marker) in RAW 264.7 cells after treatment with the following substances for 24 hours: free ABM, Δ... lpxM OMVs, ABM@OMV (10 μg·mL) -1 ) or LPS (1 μg·mL -1 (Positive control). G represents CD86 in F. + Statistical analysis of cells, n=3. H represents the result of CD11c assay under the same treatment conditions as F. + Maturity of bone marrow-derived dendritic cells (BMDCs) as determined by CD80 / CD86 expression on cells. I represents CD80 in Figure H. + CD86 + Statistical analysis of cells (n=3). J represents the ELISA detection of TNF-α, IL-6, and IL-10 in RAW264.7 supernatant (n=3). K represents the ELISA detection of TNF-α, IL-6, and IFN-γ in BMDC supernatant (n=3). For all figures A–K, unless otherwise stated, cells are associated with ABM, Δ lpxM OMVs or ABM@OMV (10 μg·mL) -1 The cells were cultured for 6 hours. The ABM dosage in the above experiments was determined based on the ABM loading on ABM@OMV. Groups: G1, PBS; G2, ABM; G3, Δ lpxM OMVs; G4, ABM@OMV; G5, LPS.

[0033] Figure 3 It prolongs the residence time of BM@OMV in the lungs and can activate innate immunity. Among these, Figure 3 In the middle, A represents ABM-Cy5 and Δ lpxMOMVs-Cy5 or ABM@OMV-Cy5 was administered intranasally to mice, and in vitro lung imaging was performed at 2, 12, and 24 hours post-administration. B shows the relative fluorescence intensity of the lungs analyzed using molecular imaging software (n=3). C–H show the lung immune cell profile analysis 24 hours after primary immunization: C represents a flow cytometry atlas showing activated M1 macrophages (F4 / 80) in total macrophages. + CD80 + D represents mature dendritic cells (CD80). + CD86 + A representative atlas of dendritic cells in total dendritic cells. E represents dendritic cells (cDCs, CD11c). + CD103 + Representative atlases of total dendritic cells. F represents the statistical analysis of the proportion of M1 macrophages (n=5). G represents the statistical analysis of the proportion of mature dendritic cells (n=5). H represents the statistical analysis of the proportion of classical dendritic cells (n=5). I represents the ELISA detection of pro-inflammatory cytokines (TNF-α, IFN-γ) and type I interferon (IFN-β) in lung tissue homogenate (n=5). For all figures A-I, female BALB / c mice (6-8 weeks old) were initially administered PBS, Δ lpxM OMVs (20 μg protein per 40 μL volume for each mouse), ABM (5 μg each of A9R, B6R and M1R per mouse, dissolved in 40 μL), and ABM@OMV (20 μg protein per mouse, dissolved in 40 μL), unless otherwise stated.

[0034] Figure 4 Intranasal administration of ABM@OMV induced high levels of humoral and mucosal immunity in mice. Figure 4 Figure A shows the primary-boost immunization strategy and sample collection schedule: Six-week-old female BALB / c mice received two intranasal immunizations on day 0 and day 15, respectively, using PBS, ABM (5 μg A9R, B6R, and M1R per mouse, dissolved in 40 μL volume), and Δ lpxM Two intranasal immunizations were administered: OMVs (20 μg protein per mouse, dissolved in 40 μL volume), BM@OMV, or ABM@OMV (20 μg protein per mouse, 40 μL volume). Serum, bronchoalveolar lavage fluid (BALF), and vaginal lavage fluid were collected on days 0 and 15 to determine antibodies; spleen and lymph nodes were harvested on day 29 for immunological analysis. Figure B shows the neutralizing antibody titer against vaccinia virus (VACV) in mouse serum 14 days after booster immunization, and PRNT was calculated. 50 Value. C is the infected serum-VACV mixture (200-fold diluted serum with 100 TCID).50 Representative immunofluorescence images of VeroE6 cells (co-incubated with VACV). Viral antigens were detected using anti-vaccinia virus antibody, followed by color development with CoraLite® Plus 488-labeled goat anti-mouse IgG secondary antibody, scale bar = 100 μm (control group: PBS with 100 TCID55). 50 Co-culture with vaccinia virus; Other groups: Serum from immunized mice diluted 200-fold and mixed with 100 TCID50. 50 (Vaccinia vaccinia virus co-culture). D-F represent the antigen-specific IgG antibody titers in serum samples on day 14 post-boost immunization (n=6). A9R-specific (D), B6R-specific (E), and M1R-specific (F) IgG titers were detected by indirect ELISA. G-I represent the antigen-specific IgA antibody titers in bronchoalveolar lavage fluid (BALF) on day 14 post-boost immunization (n=6). J-L represent the antigen-specific IgA levels in vaginal lavage fluid on day 14 post-boost immunization (n=6). The vaginal lavage fluid was diluted 5-fold, and OD was measured by ELISA. 450 Values. M represents the level of IgG2a in mouse serum. N represents the level of IgG1 in mouse serum. O represents the IgG2a / IgG1 ratio in mouse serum.

[0035] Figure 5 The intranasally administered ABM@OMV induced a systemic cellular immune response in mice. Figure 5 In the middle A, follicular helper T cells (PD-1) were collected from mediastinal lymph nodes (mLNs) 14 days after booster immunization. + CXCR5 + Representative flow cytometry atlases of GL7 cells (n=6). B cells are mesodermal B cells (GL7) collected 14 days after booster immunization from mediastinal lymph nodes. + Fas + Representative flow cytometry atlases of 14 days post-boost immunization (n=6). C represents spleen CD3 counts 14 days after booster immunization. + CD4 + Representative flow cytometry atlas of T cells (n=3). D represents spleen CD3 on day 14 after booster immunization. + CD8 + Representative flow cytometry atlas of T cells (n=3). E represents follicular helper T cells (PD-1) in mediastinal lymph nodes. + CXCR5 + Quantitative analysis of ) (n=6). F represents germinal center B cells (GL7) in mediastinal lymph nodes. + Fas + Quantitative analysis of ) (n=6). G~L represents quantitative analysis of spleen T cell subsets on day 14 after booster immunization (n=3): G represents CD3+. + CD4+ T cells; H is CD3 + CD8 + T cells; I is CD3 + CD4 + IFN-γ + T cells; J represents CD3. + CD4 + IL-4 + T cells; K is CD3 + CD8 + IFN-γ + T cells; L represents CD3. + CD8 + IL-4 + T cells. Experimental groups: G1, PBS; G2, free monkeypox antigen mixture (ABM); G3, Δ lpxM OMVs; G4, BM@OMV; G5, ABM@OMV.

[0036] Figure 6 ABM@OMV induced durable protective immunity against orthopoxvirus challenge in mice. Figure 6 Figure A illustrates the ABM@OMV vaccination protocol, VCV challenge (nasal infection), and subsequent analysis. Immunized mice received 1×10⁻⁶ mice. 6 TCID 50 Mice were challenged with VAV and sacrificed 7 days post-infection to collect lung tissue for viral load testing and pathological examination. B shows the weight change of vaccinated mice within 14 days post-VAV challenge (n=4). C shows the survival analysis of vaccinated mice within 14 days post-VAV challenge (n=4). D shows the viral titer in lung tissue 7 days post-challenge (n=4). E shows the viral load in lung tissue on day 7 post-challenge (measured by qPCR, n=4), (values ​​below the limit of detection (LOD) are plotted as LOD values). F shows representative H&E staining and immunohistochemical (IHC) detection of VAV antigen in lung, spleen, and lymph node tissues on day 7 post-challenge. G–I show the quantitative analysis of IHC-positive area from G: lung, H: spleen, and I: lymph node tissues in Figure F (n=4). Experimental groups: G1, PBS; G2, free monkeypox antigen mixture (ABM); G3, Δ lpxM OMVs; G4, BM@OMV; G5, ABM@OMV.

[0037] Figure 7 The in vitro and in vivo safety evaluation of ABM@OMV was conducted. Figure 7 In Figures A and B, the results of CCK-8 assays were obtained for (A) RAW 264.7 macrophages and (B) Vero E6 monkey kidney epithelial cells in relation to different concentrations of WTOMVs and Δl. pxMCytotoxicity of OMVs or ABM@OMV after 24 hours of co-culture. C represents fresh healthy red blood cells with different concentrations of wild-type OMV and Δ. lpxM Hemolysis analysis after 2 hours of co-incubation with OMVs or ABM@OMV (n=3). ddH2O and PBS served as positive and negative controls, respectively. The inset shows the macroscopic observation of hemolysis. D and E show the body weight of mice in group D and the body temperature of mice in group E during immunization (n=5). F shows representative H&E stained sections of the mouse heart, liver, spleen, lung, and kidney, scale bar = 100 μm. G–J show serum biochemical parameters of different groups of mice, including G: ALT, H: AST, I: CR, and J: BUN (n=5). The gray area represents the normal range of each indicator. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CR, creatinine; BUN, blood urea nitrogen.

[0038] Figure 8 This is a schematic diagram of the invention. Wherein, Figure 8 Figure A shows a schematic diagram of the engineering and preparation of ABM@OMV. Figure B illustrates how, after the ABM@OMV vaccine is taken up by antigen-presenting cells (APCs) such as macrophages and dendritic cells and migrates to mucosa-associated lymphoid tissue, it can induce strong IgG / IgA production and enhanced multifunctional systemic T cell responses, thereby activating mucosal and systemic immunity and providing effective protection against monkeypox virus infection. ΔEcN: lpxM Gene-deleted Escherichia coli Nissle 1917 strain; APC: antigen-presenting cells; Tfh: follicular helper T cells; Th1: type 1 helper T cells; Th2: type 2 helper T cells; CTL: cytotoxic T lymphocytes. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0040] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0041] Example 1: Construction, preparation and physical characterization of ABM@OMV vaccine 1. Construction of engineered strains and vaccine preparation like Figure 1 As shown, the ABM@OMV preparation process involves transforming the pThioHisA plasmid encoding the Trx-A9R and ClyA-B6R-M1R fusion protein into Δ... lpxMIn Escherichia coli Nissle 1917 (ΔEcN), outer membrane vesicles (ABM@OMV) were collected by ultracentrifugation. Details are as follows: The ABM@OMV vaccine was constructed using the following steps: First, using the pThioHisA plasmid as a backbone, a recombinant plasmid pThioHisA-ABM was constructed via molecular cloning, co-expressing monkeypox virus antigens A9R (fused with thioredoxin (Trx) and tagged with His, i.e., Trx-A9R-His) and B6R-M1R (fused with porin (ClyA) and tagged with a Flag, i.e., ClyA-B6R-M1R-Flag). This plasmid was then electroporated into a vaccine with knockout antigens... lpxM Attenuated E. coli Nissle 1917 (Δ) lpxM The engineered strain ABMΔEcN was obtained from EcN. This strain was inoculated into LB medium containing ampicillin and cultured at 37°C until OD. 600 When the concentration was 0.6–0.8, 1 mM IPTG was added, and expression was induced at 27°C for 14 hours. After removing bacterial cells and debris by differential centrifugation, the supernatant was concentrated by ultrafiltration, and then ultracentrifuged at 125,000×g for 2 hours to obtain OMV precipitate. The precipitate was resuspended in PBS and filtered to remove bacteria, thus obtaining the ABM@OMV vaccine.

[0042] At the same time, using a similar method as described above, Δ lpxM EcN strain obtained Δ lpxM OMVs were used as a control, and an engineered bacterium expressing only B6R-M1R was constructed to obtain BM@OMV (without A9R) as a control.

[0043] 2. Characterization results of engineered strains and vaccines Antigen expression validation: SDS-PAGE and Western Blot analysis confirmed that the engineered bacterium ABMΔEcN successfully expressed Trx-A9R (~35 kDa) and ClyA-B6R-M1R (~70 kDa) fusion proteins after IPTG induction, and these proteins were effectively loaded into purified ABM@OMV. Figure 1 B, 1C, 1G).

[0044] Physical properties: Dynamic light scattering (DLS) revealed that the hydrodynamic diameter of ABM@OMV particles is mainly concentrated in the range of 80–120 nm, with a uniform particle size distribution. The zeta potential is approximately -12 mV, indicating that the particle system is stable. Figure 1 D, 1E).

[0045] Morphological observation: Transmission electron microscopy (TEM) confirmed that ABM@OMV exhibits a typical spherical or near-spherical vesicle structure. Figure 1 F).

[0046] Safety and stability: Detection using the Limulus amebocyte lysate (LAL) reagent method showed that Δ lpxM The endotoxin content of OMV from EcN was significantly lower than that of wild-type OMV strains. Figure 1 H). Stability tests showed that after 5 weeks of storage at -80°C, ABM@OMV exhibited no significant changes in particle size, zeta potential, or antigen protein expression, indicating good stability. Figure 1 I, 1J, 1K).

[0047] Example 2: In vitro immunological efficacy evaluation of ABM@OMV vaccine I. Methods 1. OMV uptake and endosome escape in RAW 264.7 cells Candidate vaccines (Δ lpxM OMV, BM@OMV, and ABM@OMV were labeled with Cy5-NHS, purified, and resuspended in PBS to obtain labeled samples. RAW 264.7 cells (1×10⁻⁶) were used. 6 Culture the samples in each well until they reach 60-70% confluence, then label with a sample (10 μg·mL⁻¹). - ¹) or PBS treatment for 6 hours. After washing the cells, scrape them and analyze them by flow cytometry, using untreated cells as a control. Mean fluorescence intensity (MFI) and phagocytosis rate were calculated using FlowJo software.

[0048] During imaging, cells were fixed and permeabilized, the cytoskeleton was labeled with phalloidin and the nuclei were stained with DAPI, and the slides were mounted for imaging. The Cy5 signal (red) indicates the location of the vaccine.

[0049] To assess endosome escape, cells were co-cultured with Cy5-ABM@OMV for 2, 4, and 24 hours, washed, and then stained with LysoTracker Green for lysosome staining and imaging. Colocalization of red (ABM@OMV) and green (lysosome) signals was evaluated.

[0050] 2. Polarization of RAW 264.7 cells and maturation of BMDCs RAW 264.7 cells and isolated BMDCs were co-cultured with different labeled samples for 24 hours (37°C, 5% CO2) to assess their effect on cell maturation. Cells were collected after stimulation and stained for surface markers. RAW 264.7 cells were stained with PE-labeled anti-mouse CD86 antibody. BMDCs were stained using a multicolor method with APC-eFluor™ 780-labeled anti-mouse CD11c antibody, Super Bright™ 436-labeled anti-mouse CD80 antibody, and PE-labeled anti-mouse CD86 antibody. After staining, all cells were fixed with 4% glutaraldehyde and analyzed by flow cytometry to detect the expression levels of maturation-related surface markers.

[0051] II. APC-mediated antigen processing and presentation drive adaptive immunity outcomes 1. Cellular uptake and endosome escape Flow cytometry analysis showed that, compared with free antigen (ABM), Cy5-labeled ABM@OMV was more efficiently taken up by mouse macrophages (RAW 264.7) and bone marrow-derived dendritic cells (BMDC), with significantly improved mean fluorescence intensity (MFI) and phagocytosis rate. Figure 2 (A-2D) indicates that ABM@OMV possesses excellent antigen delivery efficiency. Further confocal microscopy revealed that after co-incubation with APC, ABM@OMV effectively escapes from the lysosomal compartment into the cytoplasm. This process is attributed to the ClyA porin displayed on its surface, which disrupts the stability of the endosome membrane, thereby releasing the antigen into the cytoplasm. This facilitates subsequent antigen presentation via the MHC-I pathway and activation of CD8. + The T cell response laid a crucial foundation. Lysosomal colocalization experiments revealed that ABM@OMV could effectively escape lysosomes and enter the cytoplasm after 4 hours of incubation, indicating that ABM@OMV possesses highly efficient endosome escape capabilities. Figure 2 E).

[0052] 2. Activation and maturation of antigen-presenting cells (APCs) Flow cytometry analysis showed that ABM@OMV significantly promoted the expression of the M1 macrophage marker CD86 in RAW 264.7 cells. Figure 2 F, 2G). Simultaneously, ABM@OMV effectively induced the maturation of bone marrow-derived dendritic cells (BMDCs) and significantly upregulated the expression levels of their surface co-stimulatory molecules CD80 and CD86. Figure 2 H, 2I). ELISA detection of cytokines revealed that the secretion levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IFN-γ in the cell supernatant of the ABM@OMV treatment group were significantly higher than those in the control group. Figure 2These results collectively indicate that ABM@OMV can effectively activate APCs and initiate adaptive immunity.

[0053] Example 3: In vivo immunogenicity and challenge protection experiment of ABM@OMV vaccine I. Methods 1. Mouse immunization Female BALB / c mice aged 6-8 weeks were randomly divided into five groups and treated as follows: phosphate-buffered saline (PBS), empty saline, and phosphate-buffered saline (PBS). ΔlpxM OMVs (20 μg protein per mouse, 40 μL volume), free ABM monkeypox antigen mixture (5 μg each of A9R, B6R, and M1R per mouse, 40 μL volume), BM@OMV (carrying B6R and M1R, 20 μg protein per mouse in a 40 μL volume, containing 15 μg of antigens B6R and M1R), and ABM@OMV (carrying A9R, B6R, and M1R, 20 μg protein per mouse in a 40 μL volume, containing 15 μg of antigens A9R, B6R, and M1R). All groups were immunized via the nasal route on days 0 and 15. Specifically, mice were first anesthetized with sodium pentobarbital, followed by administration of 40 μL of the corresponding preparation via the nasal cavity (20 μL per nostril). Serum, bronchoalveolar lavage fluid, and vaginal lavage fluid samples were collected one day before the second immunization and 14 days after immunization. Fourteen days after the last immunization, some mice were euthanized and their spleen, lung tissue, and mediastinal lymph nodes (mLNs) were harvested for subsequent lymphocyte separation.

[0054] 2. Flow cytometry analysis Fourteen days after booster immunization, mice were euthanized; lung and spleen tissues were collected to prepare single-cell suspensions. Lung tissue was cut into 1 mm pieces. 3 Fragments of this size, used at 1 mg / mL -1Type V collagenase was digested at 37°C for 10 minutes, then filtered through a 70 μm filter. Spleen was homogenized in PBS, filtered, and centrifuged at 1800×g for 5 minutes. Cells from both tissues were subjected to erythrocyte lysis, washed twice with PBS (500×g, 5 minutes), and blocked with 1% BSA for 30 minutes. Subsequently, they were stained with fluorescein-labeled antibody at 4°C in the dark for 30 minutes. The antibodies used included: FITC anti-mouse CD3e; APC-eFluor™ 780 anti-mouse CD8a; APC anti-mouse CD4; PerCP-Cyanine 5.5 anti-mouse IFN-γ; PE-Cyanine 7 anti-mouse IL-4; eFluor™ 450 anti-mouse CD62L (L-selectin); Brilliant Violet™ 605 anti-mouse CD44; FITC anti-mouse CD45R (B220); PE anti-mouse CD95 (APO-1 / Fas); eFluor™ 660 anti-mouse GL7; PE anti-mouse CD185 (CXCR5); Super Bright™ 600 anti-mouse CD279 (PD-1); FITC anti-mouse F4 / 80; APC-eFluor™ 780 anti-mouse CD11c; Super Bright™ 436 anti-mouse CD80 (B7-1); and PE anti-mouse CD86 (B7-2). After staining, the cells were analyzed using flow cytometry, and the data were processed using FlowJo software.

[0055] 3. Enzyme-linked immunosorbent assay (ELISA) The antibody titers of monkeypox-specific IgG, its subclasses (IgG1 and IgG2a), and secretory IgA (SIgA) in serum, bronchoalveolar lavage fluid (BALF), and vaginal lavage fluid were determined using an indirect ELISA method. Briefly, 1 μg / mL of the antibody was coated onto a 96-well polystyrene plate. -1 Recombinant A9R, B6R, or M1R proteins were added and incubated overnight at 4°C. After discarding the coating solution, the plate was blocked with 1% BSA for 2 hours at room temperature and washed twice with PBST. Appropriately diluted samples were added to the wells and incubated for 2 hours at room temperature. After washing four times with PBST, HRP-labeled goat anti-mouse IgG, IgG1, IgG2a, or IgA secondary antibody was added and incubated for 2 hours at room temperature. The plate was washed four times with PBST, followed by the addition of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate.

[0056] 4. Plaque Reduction Neutralization Test (PRNT) The activity of neutralizing antibodies against vaccinia virus (VACV, Tiantan strain) was evaluated using a micro-neutralization assay. Vero E6 cells were cultured at 1.0 × 10⁶ cells per cell line. 5Virus suspension was inoculated into 96-well plates at a density of 100 cells / well and incubated at 37°C for 2 hours. Serum samples were pre-diluted 20-fold and then serially diluted 2-fold; 2% fetal bovine serum (FBS) / DMEM medium was used as a negative control. Virus suspension (100 TCID50) was added to each well. 50 The serum-virus mixture was incubated with 2% rabbit complement and 5% FBS medium. After incubation at 37°C for 2 hours, the serum-virus mixture was transferred to the cells and incubated for 1 hour. The supernatant was replaced with fresh 2% FBS medium and cultured at 37°C and 5% CO2 for 4 days.

[0057] Cytopathic effects were assessed on day 4. Neutralizing titer was defined as the highest serum dilution that resulted in plaque reduction; PRNT 50 The values ​​were calculated using GraphPad Prism 8 software.

[0058] 5. Immunofluorescence detection of the virus An immunofluorescence-based neutralization assay was performed using the Tiantan strain of vaccinia virus (VACV). Vero E6 cells were cultured at 1.0 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 100 cells / well in 96-well plates and incubated at 37°C for 2 hours to allow adherence. Serum samples were diluted 1:200 and inoculated with vaccinia virus (100 TCID50). 50 The serum-virus complex was mixed with 5% rabbit complement. After incubation at 37°C for 2 hours, the serum-virus complex was added to Vero E6 cells, and incubation continued for 12 hours. The supernatant was discarded; the cells were fixed with 4% paraformaldehyde (at room temperature for 30 minutes), permeabilized with PBS containing 0.5% Triton X-100 (protected from light for 30 minutes), and blocked with 10% normal goat serum for 30 minutes. The cells were incubated with anti-vaccinia virus antibody at 4°C for 16 hours, followed by incubation at room temperature in the dark with CoraLite® Plus 488-labeled goat anti-mouse IgG (H+L). Images were acquired and analyzed using an inverted fluorescence microscope.

[0059] 6. Vaccinepox virus challenge On day 15 after booster immunization, all immunized mice were intranasally inoculated with vaccinia virus (1 × 10⁶ mmol / L per mouse). 6 TCID 50 (50 μL). Body weight and survival were monitored daily for the following 14 days. On day 7 post-inoculation, mice were euthanized, and lung, spleen, and axillary lymph node tissues were collected for pathological and immunological analysis.

[0060] 7. Quantitative analysis of viral genome Viral nucleic acid was extracted from lung tissue using a viral RNA / DNA extraction kit and quantified using a vaccinia virus probe-based qPCR kit. Positive standards were serially diluted 10-fold (1×10⁻⁶).1 Up to 1×10 6 copy·μL -1 Plot a standard curve. Extract sample DNA using the kit, and simultaneously add negative and positive preparative controls. Perform probe-based qPCR in a 20 μL reaction system. Each run includes a standard curve, a template-free negative control, and a positive quality control. After confirming that all control results meet expectations, quantify the samples according to the standard curve.

[0061] 8. Immunohistochemistry (IHC) Following standard histological procedures, mouse tissue samples were fixed in 4% formaldehyde for over 48 hours and then embedded in paraffin. 3 μm thick sections were excised from the paraffin-embedded tissue blocks and fixed onto glass slides. Histological observation was performed after hematoxylin and eosin (H&E) staining. For immunohistochemical (IHC) detection, sections were first dewaxed, rehydrated, and subjected to antigen retrieval, followed by blocking non-specific binding sites with 5% bovine serum albumin (BSA). The sections were then incubated with primary antibody (1:500 dilution, anti-vaccinia virus antibody, ab35219), and subsequently with goat anti-rabbit secondary antibody. Staining was performed using an alkaline phosphatase detection system combined with Fast Red chromogenic reagent. Finally, full-slide images were acquired using a whole-slide scanner, and quantitative analysis was performed using ImageJ software.

[0062] II. In vivo immunogenicity and protection results of ABM@OMV vaccine 1. Lung retention and innate immune activation Following intranasal administration of the Cy5-labeled vaccine to mice, in vitro imaging showed that ABM@OMV had a significantly longer retention time in the lungs than the free antigen, with fluorescence signals persisting for up to 24 hours. Figure 3 A, 3B). Flow cytometry analysis of lung tissue lymphocytes 72 hours after inoculation showed that the ABM@OMV group mice had activated M1 macrophages (F4 / 80) in their lungs. + CD80 + Mature dendritic cells (CD80) + CD86 + ) and CD103 + The proportion of cDC was significantly higher in the PBS control group than in the control group. Figure 3 C~H). ELISA detection revealed that the levels of TNF-α, IFN-γ, and IFN-β in the lung tissue of the ABM@OMV group were also significantly increased. Figure 3 I) indicates that it effectively activates the innate immune system in the lungs.

[0063] 2. Specific antibody response Mice were immunized twice via intranasal drops ( Figure 4A), ELISA detection showed that the serum of the ABM@OMV group produced high-titer, high-affinity specific IgG antibodies against A9R, B6R, and M1R (anti-A9R, B6R, M1R). Figure 4 D-F) and high levels of virus-neutralizing antibodies (PRNT50) Figure 4 B, 4C). More importantly, high levels of antigen-specific IgA (sIgA) antibodies were detected in both bronchoalveolar lavage fluid (BALF (respiratory mucosa)) and vaginal douches (genital mucosa). Figure 4 The presence of G~L indicates that a distal mucosal immune barrier was successfully established, providing direct protection against viral invasion via the mucosa. Serum IgG subclass analysis showed a significantly elevated IgG2a / IgG1 ratio (G~L). Figure 4 M~O) suggests that the vaccine induced a strong Th1 immune bias.

[0064] 3. Cellular immune response Flow cytometry analysis showed that, compared with BM@OMV (without A9R), the spleen of mice immunized with ABM@OMV contained antigen-specific IFN-γ. + CD4 + T cells and IFN-γ + CD8 + The proportion of T cells increased significantly ( Figure 5 (C, D, I, K), confirming that the introduction of A9R significantly enhanced Th1 and CTL responses. Simultaneously, the proportions of follicular helper T cells (Tfh) and germinal center B cells (GC B) in the thymic lymph nodes of mice in the ABM@OMV group were significantly higher than in other groups. Figure 5 A, B, E, F), indicating that it can effectively promote the maturation of humoral immunity.

[0065] 4. Virus attack protection efficacy Mice challenged with a lethal dose of vaccinia virus (VACV) showed a 100% survival rate in the ABM@OMV immunized group, with minimal weight loss and rapid recovery. Figure 6 B, C). On day 4 post-challenge, the viral titer and viral genome copy number in the lung tissue of this group of mice both decreased to their lowest levels. Figure 6 D, E). Histopathological (H&E) and immunohistochemical analyses further confirmed that the ABM@OMV group mice had the mildest pathological damage in their lungs, spleen, and lymph nodes, showing only mild inflammatory cell infiltration in the lungs, intact alveolar structure, no severe pathological damage, and almost no detectable viral antigens. Figure 6 (F~I). The above results indicate that ABM@OMV can provide complete protection.

[0066] Example 4: Safety evaluation of ABM@OMV vaccine I. Methods 1. Cell viability determination ABM@OMV was prepared at different concentrations (0, 3.12, 6.25, 12.5, 25, 50, 100 μg / mL, based on total protein). The serially diluted OMVs were then inoculated at a concentration of 1×10⁻⁶. 4 We cultured RAW 264.7 or Vero E6 cells at 37°C and 5% CO2 for 24 hours. Then, 10 μL of CCK-8 solution was added to each well, and the cells were cultured for another 3 hours. Cell viability was assessed by measuring absorbance at 450 nm using a microplate reader.

[0067] 2. Hemolysis test Fresh anticoagulated whole blood from mice was collected and centrifuged at 3,000 rpm for 10 min at 4°C to remove plasma and the white membrane layer. Red blood cells were resuspended in sterile PBS, and the centrifugation and washing were repeated three times until the supernatant was clear. The supernatant was then removed by centrifugation, and the precipitate was the red blood cells. ABM@OMV was prepared at different concentrations (0, 3.12, 6.25, 12.5, 25, 50, 100 μg / mL, based on total protein). 1 mL of the nanomaterial at different concentrations was added to each Eppendorf tube, followed by 20 μL of the red blood cell precipitate. The mixture was incubated at 37°C for 2 h. PBS was used as the negative control, and sterile deionized water was used as the positive control. After incubation, the mixture was centrifuged at 3000 rpm for 10 min, and the supernatant was transferred to a 96-well plate. The absorbance was measured at 540 nm using a microplate reader. Hemolysis rate (%) = (Experimental group OD - Negative control OD) / (Positive control OD - Negative control OD) × 100%.

[0068] 3. Biosafety Analysis After two rounds of immunization, blood was collected from mice in each group using the heart puncture method. Whole blood was centrifuged at 500×g for 10 minutes at 4°C to separate plasma. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) in plasma were measured using biochemical assays to assess the in vivo biosafety of each formulation. Simultaneously, major organ tissues, including the heart, liver, spleen, lungs, and kidneys, were harvested from mice in each group. These tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) for histomorphological analysis. Finally, full-section images of the sections were acquired under bright-field scanning using a whole-slide scanning system, and organ structure and pathological changes were evaluated.

[0069] II. Safety evaluation results of ABM@OMV vaccine 1. In vitro safety CCK-8 assay showed that, within the effective concentration range, ABM@OMV had no significant cytotoxicity against RAW 264.7 and Vero E6 cells. Figure 7 A~B). Hemolysis tests confirmed that ABM@OMV did not cause significant erythrocyte hemolysis at different concentrations. Figure 7 C) indicates that it has no hemolytic activity.

[0070] 2. In vivo safety Throughout the immunization process, the body weight and body temperature of mice in the ABM@OMV group remained normal, with no abnormal fluctuations observed. Figure 7 D, E). Histopathological examination of major organs (heart, liver, spleen, lungs, kidneys) also revealed no vaccine-related abnormalities. Figure 7 The F indicates that the vaccine has no adverse effects on major organs. Serum biochemical indicators (ALT, AST, BUN, CREA) are all within the normal range. Figure 7 The data (G-J) indicate that the vaccine has no adverse effects on liver and kidney function. These data collectively demonstrate that the attenuated OMV vaccine has good in vivo biocompatibility.

[0071] Conclusion: The above embodiments fully demonstrate that the ABM@OMV vaccine preparation process provided by the present invention is feasible and the product properties are stable. In vitro, the vaccine can be efficiently taken up by APCs, promoting endosome escape and driving APC maturation; in vivo, it can be administered via nasal drops, simultaneously activating strong systemic humoral and cellular immunity, and inducing high levels of sIgA antibodies in the respiratory and reproductive tract mucosa, thereby providing complete protection against viral attack while exhibiting good biocompatibility.

[0072] like Figure 8 This invention presents a needle-free inhaled nanovaccine against monkeypox. The vaccine utilizes engineered bacterial vesicles whose surface displays three key viral antigens. Nasal administration induces potent systemic and mucosal immune responses in the respiratory and genital tracts, providing comprehensive protection against lethal viral infections. This outer membrane vesicle (OMV)-based platform offers a novel and effective strategy for combating the spread of mucosal pathogens.

Claims

1. An engineered probiotic outer membrane vesicle, characterized in that, The outer membrane vesicles are secreted by engineered probiotic E. coli that stably expresses the Trx-A9R fusion protein and the ClyA-B6R-M1R fusion protein, and the surface of the outer membrane vesicles displays monkeypox virus A9R antigen, B6R antigen and M1R antigen.

2. The engineered probiotic outer membrane vesicles according to claim 1, characterized in that, The probiotic Escherichia coli is Escherichia coli Nissle 1917.

3. The engineered probiotic outer membrane vesicles according to claim 1, characterized in that, The probiotic E. coli is knocked out. lpxM The gene is from the Escherichia coli Nissle 1917.

4. The engineered probiotic outer membrane vesicles according to claim 1, characterized in that, The engineered probiotic outer membrane vesicles have a particle size of 50–150 nm.

5. The method for preparing engineered probiotic outer membrane vesicles according to any one of claims 1 to 4, characterized in that, The recombinant plasmid encoding the Trx-A9R and ClyA-B6R-M1R fusion protein was transformed into probiotic E. coli, and the outer membrane vesicles were collected by ultracentrifugation.

6. The preparation method according to claim 5, characterized in that, Includes the following steps: S1. The encoding genes of the three key antigens of monkeypox virus, A9R, B6R and M1R, were used to construct fusion expression genes Trx-A9R and ClyA-B6R-M1R, which were then tandemly cloned into expression vectors to construct recombinant plasmids. These plasmids were then introduced into probiotic Escherichia coli to construct engineered probiotic strains. S2. The engineered probiotic strain obtained in step S1 is cultured on a large scale, and protein expression is induced by IPTG to obtain bacterial culture. S3. Centrifuge the bacterial culture, collect the supernatant, concentrate by ultrafiltration, and then centrifuge again to obtain the precipitate. Resuspend the precipitate to obtain the engineered probiotic outer membrane vesicles.

7. The preparation method according to claim 6, characterized in that, The expression vector described in step S1 is pThioHisA.

8. The use of the engineered probiotic outer membrane vesicles according to any one of claims 1 to 4 in the preparation of a vaccine to prevent monkeypox virus.

9. A vaccine, characterized as follows: Contains the engineered probiotic outer membrane vesicles as described in any one of claims 1 to 4.

10. The vaccine according to claim 9, characterized in that, The vaccine is in the form of nasal drops.

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