Application of nano-selenium biological particle as protein vaccine carrier or preparation of antibacterial infection medicine or antioxidant medicine

Abstract

The invention discloses an application of nano-selenium biological particles as a protein vaccine carrier or preparation of an antibacterial infection drug or an antioxidant drug, and belongs to the technical field of vaccine delivery carriers. The invention aims to provide a novel microbial particle which is used as an anti-infection and anti-oxidation drug, can induce training immunity, and has multiple functions of antibiosis, anti-infection, anti-oxidation and antigen delivery. The invention provides an application of a nano-selenium biological particle as a protein vaccine carrier, or preparation of a bacterial-like particle for preventing bacterial infection, or an application of the nano-selenium biological particle as an antioxidant drug. In-vitro RAW264.7 cells and in-vivo mouse peritoneal macrophages are induced to generate a training immunophenotype, and the vaccine can be used as a vaccine delivery vector to be connected with a staphylococcus aureus mode antigen to design a novel multifunctional biological material by utilizing training immunity to resist multi-drug-resistant bacterium infection, which is a novel strategy.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine delivery carriers, and specifically relates to the use of nano-selenium bioparticles as protein vaccine carriers or in the preparation of antibacterial infection or antioxidant drugs. Background Art

[0002] Bacterial infections, particularly those caused by multidrug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), urgently require new antimicrobial agents or strategies to combat drug-resistant bacterial infections. With the development of advanced materials technologies, researchers have recently shifted their focus to various biomaterials. While nano-antimicrobial materials (such as silver and zinc oxide) offer new avenues for combating drug-resistant infections through direct bactericidal effects, their limitations are becoming increasingly apparent: they can cause tissue damage and, more importantly, overlook the dynamic regulation of the immune microenvironment during infection. Notably, the infection process exhibits a phased immune response—the acute phase requires a proinflammatory response to control pathogen spread, while the chronic phase shifts to an anti-inflammatory repair response to promote tissue regeneration. Unfortunately, only a few immunomodulatory biomaterials, through their physicochemical properties or as drug delivery vehicles, can adaptively regulate responses at different stages of infection, limiting their effectiveness. Furthermore, biomaterials can mediate the activation of innate and adaptive immune memory by bacterial vaccines. For example, by constructing biomaterial-based vaccine delivery systems, the duration of antigen release can be prolonged to enhance adaptive immune responses. Furthermore, by developing biomaterials that can induce trained immunity, they can achieve broad-spectrum protection against infections without the need for pathogen-specific components. However, training immune inducers (such as β-glucan, BCG, and oxLDL) increase the production of reactive oxygen species (ROS), and the concentration of ROS exhibits a double-edged sword effect: low levels promote cell survival and proliferation, while high levels induce DNA damage and cell death.

[0003] Current strategies to combat antibiotic-resistant infections are hampered by insufficient immune modulation and adverse side effects, highlighting the need for novel multifunctional biomaterials that combine broad-spectrum antimicrobial and antioxidant activities with the induction of robust immune memory. Summary of the Invention

[0004] The purpose of this invention is to develop new immunomodulatory biomaterials to achieve multifunctional synergy, with antioxidant, rapid broad-spectrum antibacterial and immunomodulatory activities, while being able to deliver antigens to achieve efficient immune activation, and by inducing the host innate immune system to produce sustained trained immune memory, providing a new strategy for the immune prevention and control of multidrug-resistant (MDR) bacterial infections.

[0005] The present invention provides a use of nano-selenium bioparticles as a protein vaccine carrier, or in the preparation of a drug for preventing bacterial infection or as an antioxidant drug. Further defined, the method for obtaining nano-selenium bioparticles is as follows: sodium selenite with a final concentration of 30 μg / ml is added to Lactobacillus brevis, the culture time is 18 hours, washed three times with phosphate-buffered saline (PBS), added to 10% trichloroacetic acid and boiled for 30 minutes, washed three times with PBS, and resuspended in PBS to obtain nano-selenium bioparticles.

[0006] It is further defined that the bacteria are Staphylococcus aureus, Escherichia coli and Listeria monocytogenes.

[0007] Further definition, antioxidant is the scavenging of ABTS free radicals and DPPH free radicals, and T-SOD activity.

[0008] It is further defined that the protein vaccine carrier displays the antigen on the surface of nano-selenium bioparticles.

[0009] The present invention provides a nano-selenium bacteria-like particle displaying an antigen, which is obtained by displaying the antigen on the surface of the nano-selenium biological particle.

[0010] It is further defined that the sequence of the antigen is shown as SEQ ID NO.1.

[0011] The present invention provides an application of the above-mentioned nano-selenium bioparticles in preparing vaccines for preventing diseases.

[0012] It is further defined that the disease is a bacterial infection.

[0013] It is further defined that the bacteria is methicillin-resistant Staphylococcus aureus.

[0014] Beneficial Effects: This study developed a novel immunomodulatory biomaterial that achieves synergistic multiple functions, exhibiting antioxidant, rapid broad-spectrum antimicrobial, and immunomodulatory activities. It can also deliver antigens for efficient immune activation and induce sustained trained immune memory in the host innate immune system, providing a novel strategy for the immune prevention and control of multidrug-resistant (MDR) bacterial infections. This study innovatively prepared nanoselenium bacteria-like particles (SeBLPs) by simply treating selenium-enriched probiotics with thermal acid. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared spectroscopy (FT-IR) confirmed the successful preparation. Further SDS-PAGE and immunofluorescence experiments verified that SeBLPs could successfully bind to multiple epitopes and exhibited excellent antigen delivery capabilities. In vivo mouse immunization experiments further validated the biosafety of SeBLPs. In vitro experiments demonstrated the antioxidant and broad-spectrum antimicrobial activity of SeBLPs by measuring DPPH and ABTS scavenging capacity, T-AOC and SOD activities, and by plotting time-kill curves against Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli. In vivo, a single intraperitoneal injection of SeBLP significantly improved the survival of infected mice and was effective against a variety of Gram-positive and Gram-negative bacteria. Concurrently, serum GSH-Px activity was significantly elevated, further demonstrating its excellent antioxidant capacity. To evaluate whether SeBLP enhances mouse survival through trained immunity, we employed in vitro, in vivo, and ex vivo trained immunity models. Results demonstrated that SeBLP effectively activated peritoneal macrophages (PMs) and induced a typical and balanced trained immune phenotype. Transcriptome analysis further revealed that SeBLP-induced trained immunity primarily relied on the coordinated action of the GSH-ROS-mTOR and NF-κB signaling pathways, involving the regulation of metabolic reprogramming, redox homeostasis, and inflammatory balance. Finally, we evaluated the immunopotentiation potential of SeBLP as a vaccine delivery vehicle. Results showed that SeBLP, when conjugated to a Staphylococcus aureus multi-epitope antigen (SAMEA), induced a more rapid and robust adaptive immune response than BLP, with a more balanced Th response. In summary, this study combines trained immune mechanisms with multifunctional biomaterials to provide an integrated solution for drug-resistant bacterial infections: immune mobilization, bactericidal treatment, and balanced inflammation. Furthermore, it provides new insights and opportunities for the design of future biomaterials, paving the way for the development of more effective strategies to combat such infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Characterization of SeBLP; (A) Diagram of the preparation process of BLP and SeBLP; (B) Agarose gel electrophoresis, M: DL10,000 DNA Marker, 1: Nano-selenium-enriched Lactobacillus 23017 genomic DNA, 2: SeBLPs genomic DNA; (C) SDS-PAGE profile, M: Protein Marker, 1: Nano-selenium-enriched Lactobacillus brevis 23017 protein, 2: SeBLPs protein; (D) Quantification of DNA, RNA, and protein in SeBLPs after hot acid treatment; (E) SEM images of BLPs (left) and SeBLPs (right); Scale bar: 1 μm; (F) Ultrathin section transmission electron microscopy observation of the internal structure of SeBLPs; Scale bar: 1 μm (left); 200 nm (right); Figure 2 SeBLP has strong biocompatibility; (A) Body weight of mice treated intraperitoneally (ip) with PBS or SeBLPs on day 0; (B) Representative H&E-stained images of major organs of mice treated with PBS or SeBLPs; Scale bars: 200 μm, 40 μm (n=10 mice per group); Figure 3 SeBLP can be used as an antigen delivery vector; (A) SDS-PAGE profile, M:: protein marker, 1: SeBLP, 2: SeBLP-OACD-SAMEA; (B) Immunofluorescence detection of SeBLP-OACD-SAMEA and SeBLP, scale bar: 5 μm; (C) Size distribution of BLP, SeBLP, and SeBLP-OACD-SAMEA; (D) Zeta potential of BLP, SeBLP, and SeBLP-OACD-SAMEA; (E) FT-IR spectra of SeBLP and SeBLP-OACD-SAMEA; Figure 4 SeBLP has excellent in vitro antioxidant and antibacterial activities; Antioxidant activities of SeBLP and BLP: (A) DPPH free radical scavenging activity, (B) ABTS free radical scavenging ability, (C) T-SOD activity, and (D) T-AOC activity; Time-dependent killing curves of SeBLP or BLP against Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus; (F) Escherichia coli, (E) Listeria monocytogenes, and (G) Staphylococcus aureus were grown to the exponential phase and treated with BLP (1 OD) or SeBLP (1 OD) alone and then cultured for 7 h; *: BLP / SeBLP group compared with control group, #: SeBLP group compared with BLP group; Data represent three independent experiments and are shown as mean ± SD; Figure 5SeBLP exhibits rapid, broad-spectrum anti-infective properties. (A) Mouse models immunized and infected with different bacteria. On days 1, 2, 3, and 7 after immunization, control and treated mice were intraperitoneally infected with (B) Staphylococcus aureus, (C) Escherichia coli, and (D) Listeria monocytogenes. Additionally, mice were challenged with (E) Enterococcus faecalis on day 3 and (F) Clostridium perfringens on day 7 after immunization, and survival was monitored 3 and 7 days after infection. Figure 6 SeBLP has rapid and broad-spectrum anti-infective properties; (A) weight changes and (B) bacterial loads of mice infected with Staphylococcus aureus were recorded on days 1, 2, 3, and 7 after immunization; (C) weight changes and (D) bacterial loads in the liver and spleen of mice infected with Escherichia coli were recorded; (E) weight changes after infection with Listeria monocytogenes were recorded; In addition, mice were infected with Enterococcus faecalis on day 3 and Clostridium perfringens on day 7 after immunization; for Enterococcus faecalis infection, (F) weight changes and (G) bacterial loads were recorded on day 3 after infection; for Clostridium perfringens infection, (H) weight changes were recorded on day 7 after infection; Figure 7 SeBLP induces enhanced trained immunity in Raw264.7 cells. (A) Experimental design of the in vitro trained immunity model. (B) Relative mRNA expression levels of IL-6, IL-1β, and TNF-α on days 1, 7, and 8 (n = 5). (C) Heat map of relative mRNA expression levels of CCL2, CCL7, IL-10, IL-12, IFN-β, TGF-β, and TLR-2 on day 8. (D) ROS in the cytoplasm and (E) NO in the cell supernatant on day 8 (n = 5). (F) Raw264.7 cells were harvested on day 7 and allowed to rest overnight before being cocultured with E. coli pET28a-GFP / Rosetta for 1 hour. Phagocytosis of Raw264.7 cells in each group was measured by fluorescence microscopy. (G) Relative mRNA expression levels of HDAC7, mTORC2, and HlF1-α were measured by RT-qPCR on day 8 (n = 5). (H) Concentrations of glucose (GLU) and lactate (LD) in cell supernatants on day 8. Scale bar, 100 µm. (*p < 0.05, **p < 0.01, ***p < 0.001). Figure 8 (A) Experimental design of trained immunity: In vitro inhibition of glycolysis by 2-DG, histone methylation by MTA, or mTOR signaling by RAPA model; 2-DG, MTA, or RAPA inhibited (B) relative mRNA expression levels of IL-6 and IL-1β on day 1; Figure 9SeBLP-induced trained immunity protects mice from S. aureus infection faster than BLP; (A) Experimental design of the in vivo trained immunity model; (B) Survival curve of mice after S. aureus challenge; (C) Body weight change of mice after S. aureus challenge (n = 10); (D) Lesion score of mice after S. aureus challenge; (E) Bacterial load of S. aureus in the spleen, kidney, and liver was measured on day 10. Histological HE staining of (F) spleen and (G) kidney of mice; RT-PCR analysis of IL-6, IL-1β, TNF-α, CCL2, CCL7, IL-10, IL-12, IFN-β, TGF-β, and TLR-2 expression levels in the spleen; (I) RT-qPCR detection of HDAC7, mTORC2, and HIF1-α mRNA expression in the spleen; (J) Serum glucose (GLU) and lactate (LD) levels were measured on day 10 (n = 10). (*p < 0.05. **p < 0.01. ***p < 0.001). Figure 10 Macrophages isolated from mice trained with SeBLP exhibit innate immune memory; (A) Schematic diagram of the experimental design for in vitro trained immunity; RT-qPCR measurements show the relative mRNA expression levels of (B) IL-1β, TNF-α, and IL-6; (C) HDAC7, HIF1-α, and mTORC2; (D) GLU and LD levels in cell supernatants; (n = 5), (*p < 0.05, **p < 0.01, ***p < 0.001); Figure 11 RNA-seq analysis of mouse peritoneal macrophages activated with LPS for 6 hours; (A) Principal component analysis (PCA) of the transcriptome of SeBLP- and PBS-trained macrophages (n = 3); (B) Volcano plot showing differentially expressed genes in trained and restimulated macrophages compared with PBS-stimulated cells. Differential expression of epigenetic-related genes is indicated; bubble plot between the control and SeBLP groups shows upregulated genes. (C) KEGG pathway enrichment analysis; (D) GSEA enrichment analysis. (E) Heatmap of upregulated genes related to cytokines, chemokines, PRRs, and glutathione metabolism; (F) Training of immune-related metabolic pathways; differentially expressed genes for key enzymes in immune-related metabolic pathways, red: upregulated genes. Figure 12 (A) Bubble plot showing GO enrichment analysis, (B) GSEA enrichment analysis, (C) metabolism-related genes, and (D) expression of genes related to antioxidant and inflammation regulation of upregulated genes between the control group and the SeBLP group; Figure 13SeBLP-induced training immunity enhances the protection of a single-dose vaccine against methicillin-resistant Staphylococcus aureus (MRSA) infection; (A) Experimental design of training immunity combined with subunit vaccine immunization against MRSA; (B) Serum-specific IgG, (C) IgG1, IgG2a, and IgG2a / IgG1 levels by ELISA at 7, 14, and 21 days after immunization; (D) Fecal-specific antibody SIgA levels at 7, 14, and 21 days after immunization; (E) Survival rate of mice after MRSA challenge; (F) Body weight change of mice after MRSA challenge; (G) MRSA bacterial load in the kidneys at day 36; (H) Heat map showing the relative mRNA expression levels of IL-6, IL-1β, TNF-α, IL-10, IL-12, IFN-β, TGF-β, CCL2, CCL7, TLR2, TLR4, HDAC7, mTORC2, and HIF1-α in kidneys at D36 based on RT-qPCR; (I) Histopathological sections of kidneys (n = 10). (*p < 0.05, **p < 0.01, and ***p < 0.001). DETAILED DESCRIPTION

[0016] Female Kunming mice (SPF grade) weighing 20 ± 2 g were used in the experiments. Mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). Mice were housed under specific pathogen-free conditions with a 12-h light-dark cycle and free access to rodent chow and tap water. RAW264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (Biosharp, China) supplemented with 10% (v / v) fetal bovine serum (CLARK Bioscience, USA) and 1% (v / v) Protease Inhibitor (P / S) (Biotopped, Beijing, China) at 37°C and 5% CO₂.

[0017] Clostridium perfringens type A (C57-8) was purchased from the China Medical Bacteria Collection. The China Medical Bacteria Collection provided Staphylococcus aureus strain CMCC26003.

[0018] Escherichia coli (CVCC230) was purchased from the China Center for the Collection of General Microorganisms, and Listeria monocytogenes (ATCC19111) and Enterococcus faecalis (ATCC29212) were purchased from the American Type Culture Collection.

[0019] Dr. Li Yanhua from Northeast Agricultural University generously provided the methicillin-resistant Staphylococcus aureus strain USA300 (ATCC BAA-1717). Methicillin-resistant Staphylococcus aureus colonies were cultured in LB medium stored at 37°C.

[0020] Escherichia coli pET28a-GFP / Rosetta was maintained in our laboratory.

[0021] Barley β-glucan (CAS: 904122-9) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0022] Lactobacillus brevis 23017 strain is recorded in the article Ren Hongkun. Construction of a novel bacterial-like particle surface display platform and its application in displaying multi-epitope antigens[D]. Heilongjiang: Northeast Agricultural University, 2023.

[0023] HiSeL is described in the literature Nano selenium-enriched probiotic Lactobacillus senhances alum adjuvanticity and promotes antigen-specific systemic and mucosal immunity, which describes a method for obtaining heat-killed nano-selenium lactic acid bacteria and using them as an adjuvant for an inactivated Clostridium perfringens type A vaccine. Mice (1 × 10 9 CFU), once a day from day 1 to day 5 for 5 consecutive days, and 200 mL of vaccine was injected subcutaneously on day 6 (5). HiSeL was not treated with trichloroacetic acid, but only heat-inactivated, and contained proteins and nucleic acids, which could not bind to antigens.

[0024] Example 1. Preparation of Selenium Nanobacteria-Like Particles (SeBLP) 1. Inoculate 8% of the fresh culture of Lactobacillus brevis 23017 into 1L of MRS liquid medium and culture it statically in a 37°C incubator. 600 When the pH reached 0.25, sodium selenite was added to a final concentration of 30 μg / ml and further cultured at 37 °C for 18 hours to obtain selenium-enriched Lactobacillus brevis 23017 culture. Bacteria were harvested from fresh overnight cultures and washed three times with PBS. The resulting bacteria were then suspended and boiled in 200 mL of 10% trichloroacetic acid for 30 minutes. Subsequently, the cells were washed three times with 50 mL of PBS and resuspended in PBS to obtain SeBLP. OD was measured in the initial culture. 595 This was used to adjust the concentration of BLP particles per ml. Finally, the cells were resuspended in PBS and stored at -80°C.

[0025] 2. SeBLPs were characterized using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) to determine their size, shape, and surface elemental composition.

[0026] The results showed that: (1) Characterization of SeBLP Several analyses were performed to demonstrate that SeBLP contained no protein or DNA that could pose a potential biosafety risk. Figure 1 B, lane 1), no DNA band was detected in TCA-treated SeBLP ( Figure 1 B, lane 2). In addition, SDS-PAGE analysis showed that the intracellular protein band in SeBLP seemed to be released or degraded ( Figure 1 C). In addition, NanoDrop microvolume spectrophotometer measurements showed that the DNA, RNA, and protein contents in SeBLPs were negligible ( Figure 1 D), which further supports its biosafety. SEM images confirmed these findings ( Figure 1 E) shows nanoselenium synthesized on the cavity wall of a BLP. SEM results also show a distinct extra-dot on the SeBLP. The extra-dot exhibits a spherical morphology, similar to nanoselenium. The surface of the SeBLP shows wrinkles and collapse, indicating structural changes due to heat and acid treatment ( Figure 1 E). In addition, ultrathin transmission electron microscopy sections showed that the internal structure of SeBLPs was hollow and surrounded by particle-like substances ( Figure 1 F). Severe acid treatment exposes peptidoglycan, the main cell wall component of lactobacilli, which maintains their structural integrity.

[0027] (2) In vivo toxicity assessment: Female Kunming mice (4-6 weeks old) were divided into two groups, with 10 mice in each group, and intraperitoneally injected with PBS or 1 × 10 8 CFU SeBLPs. The body weight of mice was monitored daily. On day 10, the mice were euthanized and their major organs were collected for histopathological evaluation using H&E staining at Wuhan Saiwei Biotechnology Co., Ltd. Assessing the potential tissue and organ damage of nanomaterials is crucial for evaluating their biocompatibility in in vivo experiments. In vivo experiments with SeBLP were conducted using adult mice. The body weight ( Figure 2 A) and tissue sections ( Figure 2 B) The absence of adverse differences provides evidence of the excellent safety and biocompatibility of SeBLP in mice.

[0028] Example 2. Application of SeBLP with excellent antioxidant properties in vivo and in vitro SeBLP was immunized 1 OD 2 days after rapid and broad-spectrum anti-infection ability. Mice were randomly divided into four groups, 10 in each group. Infected with Staphylococcus aureus (CMCC 26003, 1×10 9 CFU / 200 μL).

[0029] Detection of oxidative stress substances and enzymes: The levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) in serum and jejunum samples were measured using ELISA kits from Nanjing Jiancheng Bioengineering Institute, China, to evaluate the in vitro antioxidant capacity.

[0030] ABTS free radical scavenging ability was assessed by adding potassium persulfate (2.6 mM) to ABTS (7.4 mM) to obtain an ABTS stock solution. The solution was then incubated at room temperature in the dark for 12 hours. The solution was diluted with deionized water to prepare an ABTS working solution (absorbance at 734 nm, 0.70 ± 0.02). Four mL of the ABTS working solution was then mixed with 1 mL of the sample in the dark for 6 minutes. The absorbance of the mixture was then recorded at a wavelength of 734 nm using a TU-1901 dual-beam UV-visible spectrophotometer (Persee, Beijing, China). Ascorbic acid was used as a positive control.

[0031] DPPH free radical scavenging activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. Vc (100 μM), BLP (1 OD), and SeBLP (1 OD) were added to a 0.2 mM DPPH methanol solution. After incubation in the dark for 30 minutes, the color change of the reaction mixture was measured at 517 nm using a TU-1901 dual-beam UV-visible spectrophotometer (Persee, Beijing, China). Ascorbic acid was used as a positive control in this assay.

[0032] The antioxidant activity of SeBLP was evaluated using DPPH and ABTS assays with ascorbic acid as the standard. SeBLP inhibited the oxidation of DPPH and ABTS, respectively. + In contrast, the DPPH and ABTS free radical scavenging activities of BLP were significantly reduced, indicating poor antioxidant capacity ( Figure 4 A, 4B). These findings indicate that SeBLP can effectively scavenge DPPH and ABTS free radicals. However, the scavenging activity of SeBLP was lower than that of ascorbic acid, especially in the DPPH assay. In addition, our results showed that the SOD activity of SeBLP was higher than that of BLP ( Figure 4 C). However, the scavenging activities of BLP and SeBLP were lower than those of ascorbic acid, especially in the DPPH and T-AOC assays ( Figure 4 B, 4D).

[0033] To further evaluate the in vivo antioxidant potential, we assessed the antioxidant capacity of mouse serum, as shown in Table 1. Two days after immunization, serum T-SOD, T-AOC, and GSH-Px levels were increased, while MDA content was decreased, in the BLP and SeBLP groups compared with the control group (p < 0.05). Furthermore, serum GSH-Px levels were significantly higher in the SeBLP group than in the BLP group (p < 0.05). However, there were no significant differences in serum MDA and T-AOC activities between the SeBLP groups (p > 0.05). Three days after S. aureus infection, serum MDA levels were decreased in both the BLP and SeBLP groups compared with the control group. Correspondingly, serum T-SOD, T-AOC, and GSH-Px activities were increased in both treatment groups compared with the control group. Serum GSH-Px activity was significantly increased in the SeBLP group compared with the BLP group (p < 0.05). However, there were no significant differences in serum T-SOD and T-AOC levels between the groups (p > 0.05). In conclusion, three days after infection, the difference between the BLP and SeBLP groups decreased, and the redox balance in the mouse serum was relatively restored. SeBLP immunization promoted the restoration of the balance of the oxidative and antioxidant systems in infected mice. Our results suggest that SeBLP is a biomaterial capable of regulating oxidative stress.

[0034] Table 1

[0035] Serum and tissue samples were collected from mice on day 2 after immunization and day 3 after S. aureus infection to assess antioxidant function. a: BLP / SeBLP group versus control group; b: SeBLP group versus BLP group. Each experimental group consisted of 10 mice. Data are expressed as mean ± SD (*p < 0.05 and **p < 0.01). Statistical analysis was performed using one-way ANOVA.

[0036] Example 3. Application of SeBLP with excellent in vitro antibacterial and in vivo anti-infection properties 1. Time-dependent killing (in vitro): Escherichia coli, Listeria, and Staphylococcus aureus were incubated at 37°C for 4 hours (exponential phase). The cells were then resuspended in BLP (1 OD) and SeBLP (1 OD). A 100 μL aliquot was removed after 0, 1, 3, 5, and 7 hours, centrifuged, and resuspended in 100 μL of sterile PBS. Subsequently, 10-fold serial dilutions of the suspension were plated on LB plates and incubated overnight at 37°C. The bacterial colonies were counted and the CFU / mL was calculated. All experiments were performed with at least three biological replicates.

[0037] The antibacterial activity of BLP or SeBLP against Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus was evaluated by counting colony-forming units (CFU). Figure 4 As shown in Figures E, 4F, and 4G, BLP (1 OD) or SeBLP (1 OD) was co-cultured with Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus for 0, 1, 3, and 7 hours, then plated on LB agar plates, and the resulting colonies were counted. We found that the bacterial load in the SeBLP-treated group was significantly reduced at 5 and 7 hours compared to the control and BLP groups, indicating that SeBLP is more effective against E. coli, Listeria monocytogenes, and Staphylococcus aureus. Furthermore, we found that BLP and SeBLP exhibited more significant antibacterial activity against Staphylococcus aureus within 1 hour. In summary, the SeBLP group exhibited more pronounced antioxidant and bactericidal effects in vitro, suggesting its effectiveness against both Gram-positive and Gram-negative bacteria.

[0038] 2. Anti-infection experiment (in vivo): To evaluate the rapid and broad-spectrum anti-infection ability of the mice at 1, 2, 3, and 7 days after immunization, mice were randomly divided into four groups, with 10 mice in each group. On day 0, mice were intraperitoneally injected with PBS (control group and infection group), BLP (1 OD), or SeBLP (1 OD). On days 1, 2, 3, and 7 after immunization, mice were intraperitoneally injected with different pathogens, such as Staphylococcus aureus (CMCC26003, 1 × 10 9 CFU / 200 μL), 1.25 × 10 8 CFU / 200 μL Escherichia coli (CVCC230), 1.5 × 10 9 CFU / 200μL Listeria monocytogenes (ATCC19111), 2 × 10 8 CFU / 200 μL Enterococcus faecalis (ATCC29212) or 2 × 10 9CFU / 200 µL of Clostridium perfringens (C57-8). Survival rate and body weight were monitored until day 5. Subsequently, all mice were euthanized by cervical dislocation to collect liver, spleen, or kidney tissues, as well as blood for further analysis.

[0039] 3. Bacterial Load Measurement Tissues were weighed and homogenized in sterile PBS. Ten-fold dilutions of spleen, liver, and lung tissues infected with Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes were plated onto Luria-Bertani (LB) plates (AOBOX Biotechnology, China). Colon and kidney tissues infected with Enterococcus faecalis were plated onto bile salt esculin agar plates (AOBOX Biotechnology, China), with 10 µL of sample per plate. After incubating the plates at 37°C for 18 hours, colonies were counted.

[0040] 4. Histopathological Examination Liver, spleen, and kidney tissue samples from mice were fixed with 10% paraformaldehyde at room temperature for at least 48 hours. Pathological sections were prepared by a contract company (Wuhan Co., Ltd., Hubei Province, China).

[0041] 5. Injury score According to the method of the reference [Enhanced Mucosal Antibody Production and Protectionagainst Respiratory Infections Following an Orally Administered BacterialExtract], mice with a score of 0 or 1 were considered healthy. Mice with a score of 3 showed signs of discomfort, such as slight erection of hair, a slight change in stride, and increased walking ability. If a mouse showed severe erection of hair, abdominal contraction, gait change, or prolonged inactivity, it would receive a score of 5. A mouse that showed the enhanced characteristics of the previous group, was minimally active, and was dying, received a score of 7. Dead mice received a score of 10.

[0042] Results: SeBLP has rapid and broad-spectrum antibacterial properties in vitro and anti-infective properties in vivo Having demonstrated that SeBLP has good in vitro antimicrobial activity, we sought to assess the duration of its protective effect and determine how quickly vaccine-mediated efficacy sets in in mice. Mice were immunized with BLP or SeBLP and subsequently challenged with Escherichia coli, Listeria monocytogenes, or Staphylococcus aureus 1, 2, 3, or 7 days after immunization ( Figure 5 A).

[0043] For S. aureus infection, BLP and SeBLPS vaccination conferred complete protection ( Figure 5 B). We observed that mice in the SeBLP group regained weight faster than those in the BLP group on the second day after challenge, suggesting that SeBLP facilitates rapid recovery from S. aureus (CMCC 26003) infection ( Figure 6 A). The bacterial load in the liver, spleen, and kidney of mice immunized with SeBLP was lower than that in the BLP group ( Figure 6 B), indicating accelerated bacterial clearance. For E. coli infection, SeBLP-treated mice had a higher survival rate two days after immunization than BLP-treated mice (100% vs. 30%) ( Figure 5 C). Notably, after E. coli infection, SeBLP showed similar effects on body weight changes as the control group ( Figure 6 C). Next, we examined the bacterial load in mice immunized with SeBLP, BLP, or PBS. Figure 6 As shown in D, SeBLP immunization reduced the bacterial load of Escherichia coli in liver and spleen tissues compared with the BLP group, indicating that these pathogens were effectively cleared after challenge.

[0044] For Listeria monocytogenes, immunization with SeBLP improved survival when mice were challenged 3 or 7 days after immunization ( Figure 5 D). However, mice immunized with SeBLP or BLP did not show less weight loss compared with the infected group ( Figure 6 E).

[0045] To further evaluate the broad-spectrum anti-infective activity of SeBLP and BLP, we administered Enterococcus faecalis and Clostridium perfringens to mice 3 and 7 days after immunization, respectively. The E. faecalis infection experiment showed that the weight change rate was similar to that of Staphylococcus aureus infection ( Figure 5 E, 6F). For Clostridium perfringens infection, the results showed that SeBLP treatment increased survival and body weight change compared with untrained and BLP-treated mice ( Figure 5 F, 6H).

[0046] Because Listeria monocytogenes, Enterococcus faecalis, and Clostridium perfringens are Gram-positive bacteria, and Escherichia coli is a Gram-negative bacterium, we speculated that SeBLP treatment significantly enhanced host immunity and might be an effective strategy for preventing certain Gram-positive and Gram-negative bacteria.

[0047] Example 4. Inducing trained immunity to acquire anti-infective function in in vitro, in vivo, and ex vivo models 1. In vitro immune training: Raw264.7 cells were treated with PBS, β-glucan (30 µg), BLP (0.01 U), or SeBLP (0.01 U) for 24 hours. Cells were then washed three times with PBS and cultured in fresh medium for 6 days before use. Subsequently, cells were stimulated with LPS (100 ng / mL) for 24 hours. After stimulation, the supernatant and cells were collected and stored at -80°C.

[0048] 2. Inhibitor Experiments: To inhibit the induction step of trained immunity, Raw264.7 cells were preincubated with 5'-deoxy-5'-(methylthio)adenosine (3 mM, [MTA], S880063, Macklin, Shanghai, China), 2-deoxy-D-glucose (15 mM, [2-DG], D807272, Macklin), or rapamycin (10 µmol / L, [RAPA], S115842, Aladdin, Shanghai, China) for 1 hour and then pretreated with β-glucan (30 µg), BLP (0.01 U), or SeBLP (0.01 U). After preincubation, the cells were inducible for a 24-hour period according to the standard training protocol without washing. After 24 hours, the supernatant and cells were collected and stored at −80°C for subsequent analysis by RT-qPCR.

[0049] 3. Phagocytic activity of RAW 264.7 cells Trained macrophages were incubated overnight and then co-cultured with E. coli pET28a-GFP / Rosetta for one hour. Gentamycin and ampicillin were combined to form a dual antibiotic to eradicate bacteria that could not be phagocytosed. Macrophages were labeled with DAPI, and fluorescence microscopy was used to assess the phagocytic capacity of each group of Raw264.7 cells.

[0050] 4. Glucose and Lactate Determination Lactate and glucose levels in cell supernatants and serum were quantified using lactate and glucose assay kits (Nanjing Jiancheng, Nanjing, China), respectively.

[0051] 5. ROS and NO Determination Fluorescence intensity was measured at 525 nm using a fluorescence spectrophotometer. Total NO2 in the culture supernatant was determined using a nitrate reductase NO assay kit (Nanjing Jiancheng, Nanjing, China). -Levels were determined by ELISA at 550 nm using the manufacturer's instructions. RAW264.7 cells and positive control samples were incubated with 2',7'-dichlorofluorescein diacetate probe for 20 min using a ROS detection kit (Nanjing Jiancheng, Nanjing, China). The culture medium was then removed and the samples were resuspended in PBS to quantify cytoplasmic ROS.

[0052] 6. RNA Extraction and RT-qPCR Total RNA was extracted using Trizol reagent (Invitrogen, Sigma-Aldrich, St. Louis, MO). RNA was then reverse transcribed into cDNA using ReverTra-Ace® (TRT-101, TOYOBO, Shanghai, China) according to the manufacturer's protocol. Each 40 μL reverse transcription reaction contained 2 μg of RNA template. cDNA was quantified and expression analyzed using 2 × S6 SYBR Green qPCR Mix (EnzyArtisan, Shanghai, China). Real-time quantitative PCR reactions were performed using the Applied Biosystems® 7500 Real-Time PCR System according to the manufacturer's instructions. 2 -ΔΔCT Methods: Relative gene expression was quantified, with β-actin serving as a housekeeping control. Table S1 lists the primers used in this study.

[0053] Table S1 PCR primer sequences

[0054] Results: SeBLP induced stronger trained immunity in Raw264.7 cells To further investigate whether the rapid and broad-spectrum protection induced by BLP or SeBLP was due to the phenotype of trained immunity, we trained Raw264.7 cells with BLP (0.01 U) or SeBLP (0.01 U) for 24 h, rested them for 6 days, and then stimulated them with LPS to observe the macrophage response ( Figure 7A). Cells exhibiting trained immunity displayed a memory-like phenotype and showed an enhanced response to secondary stimulation. The transcript abundance of the hallmark cytokines IL-6, IL-1β, and TNF-α was assessed at different time points. After β-glucan induction on day 1, the levels of all three cytokines increased and returned to baseline levels before secondary stimulation on day 7. Their expression levels were higher than those in the control group under LPS stimulation. SeBLP training resulted in increased levels of IL-1β, TNF-α, and IL-6 after the initial stimulation, which returned to normal by day 7. However, after the secondary stimulation, their expression increased significantly again (IL-1β: p<0.0001, TNF-α: p<0.001, IL-6: p<0.01). For BLP induction, IL-1β (p<0.0001) and IL-6 (p<0.01) were significantly increased after the initial stimulation and returned to normal levels by day 7. After the secondary stimulation, only IL-1β (p<0.0001) showed a significant increase ( Figure 7 B), indicating that both BLP and SeBLP can induce trained immunity, and SeBLP induces a stronger trained immune response ( Figure 7 B).

[0055] To comprehensively characterize the inflammatory cytokine profile elicited by SeBLP and BLP, we expanded the scope of monitoring to include other cytokines ( Figure 7 C). During β-glucan training, only CCL7 expression increased among the other tested cytokines after secondary stimulation. In contrast, after BLP training, expression levels of all tested cytokines (IL-10, IL-12, IFN-β, TGF-β, CCL2, and CCL7) increased, and in the SeBLP group, expression levels of all cytokines except IL-12 and IFN-β were significantly elevated. Importantly, increased expression of the anti-inflammatory cytokines IL-10 and TGF-β, as well as chemokines, suggests a modulation of the inflammatory response, indicating that SeBLP promotes a balanced trained immune response. Furthermore, both BLP and SeBLP increased TLR2 expression after secondary stimulation.

[0056] The distribution of phagocytic bacteria in activated macrophages can reflect the effect of trained immunity. We evaluated the activation state of macrophages after trained immunity by measuring the levels of reactive oxygen species (ROS), NO, and phagocytic activity in macrophages. Compared with untrained macrophages, exposure to SeBLP and BLP resulted in a decrease in ROS levels and a significant increase in NO production in trained macrophages ( Figure 7D, 7E). However, the decrease in ROS levels and increase in NO levels induced by SeBLP in macrophages were greater than those induced by BLP. In addition, immunofluorescence images showed that the content of E. coli in macrophages was higher in BLP and SeBLP groups compared with the β-glucan group ( Figure 7 F) shows that the phagocytic capacity of macrophages was significantly enhanced in both treatment groups. These findings suggest that BLP- and SeBLP-induced immunity may be mediated by regulating cellular function.

[0057] Epigenetic and metabolic reprogramming mechanisms are crucial for inducing trained immunity and play a crucial role. β-glucan, a classic activator of trained immunity, enhances glycolysis via the Akt-mTOR-HIF1-α pathway, indicating activation of innate immune memory. Histone deacetylase 7 (HDAC7) is a key marker of β-glucan-induced epigenetic reprogramming. HDAC7 expression in macrophages increased after induction with BLP and SeBLP; however, SeBLP-induced HDAC7 expression levels were only slightly increased ( Figure 7 G), indicating the potential role of other epigenetic modifications. BLP (p < 0.0001) or SeBLP (p < 0.01) significantly increased the expression of mTORC2 and HIF1-α in Raw264.7 cells ( Figure 7 Glucose and lactate concentrations in the cell supernatant indicated that BLP or SeBLP induced enhanced glycolytic capacity of macrophages ( Figure 4 H). In conclusion, SeBLP induced stronger trained immunity in Raw264.7 cells.

[0058] To further confirm the key role of metabolic reprogramming and epigenetics in SeBLP-induced trained immunity, we pretreated macrophages with the glycolysis inhibitor 2-DG, the histone methylation inhibitor MTA, and the mTOR pathway inhibitor RAPA ( Figure 8 A). In the presence of these three inhibitors, BLP- and SeBLP-induced macrophages showed significantly lower expression of IL-6 (p<0.0001) and IL-1β (p<0.00001) after secondary stimulation compared to those without inhibitors ( Figure 8 B). In conclusion, SeBLP induces trained immunity in macrophages through glycolysis and epigenetic reprogramming.

[0059] 7. In vivo training immunization: Forty female Kunming mice were randomly divided into four groups, with 10 mice in each group. On days 0 and 3, the mice were intraperitoneally injected with PBS (control group and infection group), β-glucan (500 μg), BLP (1 OD), or SeBLP (1 OD). Then, on day 7, they were intraperitoneally injected with Staphylococcus aureus CMCC26003 (1 × 10 9 CFU / 200 µL). Body weight, survival rate, and injury scores were monitored 3 days after infection. Finally, all mice were euthanized by cervical dislocation to collect liver, spleen, lung tissue, and serum for further analysis.

[0060] Results: SeBLP-induced trained immunity protected mice against Staphylococcus aureus infection faster than BLP We hypothesized that SeBLP effectively protected mice by inducing stronger trained immunity. Therefore, we immunized mice twice with SeBLP or BLP on days 0 and 3, and intravenously injected them 4 days after immunization with S. aureus CMCC26003. Their survival and recovery were monitored over 5 days to assess the protective effect ( Figure 9 A). The results showed that the survival rate of the group immunized with SeBLP or BLP (100% survival rate) was significantly higher than that of the group infected with Staphylococcus aureus CMCC26003 (0%) ( Figure 9 B). However, the SeBLP group showed significantly reduced weight loss on the first day after infection compared with the BLP group, and a similar trend was observed in the BLP group only after 2 days, as evidenced by faster recovery and reduced weight injury scores ( Figure 9 Consistent with the survival curves, this superiority of SeBLP was further confirmed by the significant reduction in lung, spleen, and liver burden compared to the BLP group three days after infection ( Figure 9 E).

[0061] We also observed lesions in the spleen and kidneys of mice. Compared with the control group, the number of inflammatory cells in the spleen of mice in the BLP group was slightly increased ( Figure 9 F). Histopathological analysis of spleen sections also showed that the spleens of the infected group were congested and hemorrhaged significantly, while the spleen damage in the BLP group was alleviated, and the spleens of the SeBLP-trained mice were almost undamaged ( Figure 9 F). Infected mice showed glomerular proliferation, tubular vacuolar degeneration, epithelial cell edema, and significant inflammatory infiltration in the kidneys, whereas BLP-treated mice exhibited partial tubular vacuolar degeneration and glomerular congestion. No obvious lesions were observed in the SeBLP group ( Figure 9G). 3 days after S. aureus infection, decreased cytokines and chemokines were detected in the spleens of mice immunized with SeBLP, confirming the reduced bacterial load and tissue damage in trained mice ( Figure 9 H). Specifically, the expression of IL-6, IL-1β, CCL2, and TGF-β in spleen tissues of the BLP group was significantly increased compared with the infection group. However, in the SeBLP-treated group, the cytokine levels were decreased compared with the BLP group or the infection group ( Figure 9 H). These findings suggest that SeBLP-induced trained immunity enables mice to recover more quickly from S. aureus infection.

[0062] Trained immunity is associated with epigenetic modifications and metabolic changes, both of which are the molecular basis for gene expression modifications during restimulation. In addition, analysis of metabolic and epigenetic factors associated with trained immunity showed significant changes in HIF1-α levels (p < 0.00001) in the spleens of SeBLP-treated mice compared to the infected group ( Figure 9 I). However, SeBLP-treated mice showed decreased levels of mTORC2 and HDAC7 (p>0.05), while BLP-treated mice showed the opposite trend. Next, we investigated whether the enhanced glycolysis after SeBLP or BLP training coincided with increased cytokine production. Serum lactate and glucose measurements revealed that mice treated with BLP or SeBLP had significantly increased glucose consumption (p<0.001), as evidenced by significant changes in lactate production (p<0.05). Figure 9 In conclusion, consistent with the initial hypothesis, the SeBLP group induced stronger trained immunity than the BLP group, resulting in less damage and faster recovery in mice infected with Staphylococcus aureus.

[0063] 8. In vitro trained immune model: Mice were intraperitoneally injected with PBS, β-glucan (500 µg), BLP (1 OD), or SeBLP (1 OD) twice on days 0 and 3. On day 6, peritoneal macrophages were isolated and cultured for 2 hours before being stimulated with LPS (100 ng / mL). Supernatants and cells were collected 1, 6, 12, and 24 hours after stimulation for subsequent analysis.

[0064] Results: Macrophages isolated from mice trained with SeBLP exhibit innate immune memory Both in vitro and in vivo models have shown that SeBLP training induces a robust cytokine response, including IL-1β, TNF-α, and IL-6. We undertook a more comprehensive analysis of innate immune parameters associated with SeBLP-induced trained immunity. Therefore, peritoneal macrophages (PMs) were isolated from trained mice and stimulated with LPS and PBS to assess whether these macrophages exhibit trained responses and to evaluate their immune response status ( Figure 10 A). Supernatants were analyzed for IL-1β, IL-6, and TNF-α levels produced by PM after 1, 6, 12, and 24 hours of continuous LPS stimulation, which are typically monitored in training cytokine profiles. Both the BLP and SeBLP groups significantly increased the mRNA expression of these inflammatory factors ( Figure 10 B). In addition, the levels of TNF-α, IL-1β, and IL-6 in SeBLP-treated cells were significantly higher than those in BLP-treated cells ( Figure 10 B).

[0065] Regarding metabolic and epigenetic indicators, a significant increase in HDAC7 persisted in the SeBLP group 1 hour after secondary LPS stimulation. Since mTOR-dependent HIF1α accumulation is crucial for profound metabolic reprogramming in trained innate immunity, we also analyzed HIF1α, mTORC2 levels, and glycolysis after BLP or SeBLP initiation. Both mTORC2 and HIF1-α levels in the SeBLP group increased rapidly but returned to baseline levels 12 hours after LPS stimulation ( Figure 10 C). Notably, 6 hours after the second stimulation, the levels of mTORC2 and HIF1α in the SeBLP group were higher than those in the BLP group. To evaluate macrophage metabolism after BLP or SeBLP training, we measured lactate and glucose levels in the cell supernatant at 1, 6, 12, and 24 hours after LPS stimulation ( Figure 10 D). Six hours after LPS stimulation, glucose levels decreased in both groups (p < 0.05) and lactate levels increased (p > 0.05). These results indicate that BLP- and SeBLP-induced training immunity enhances glycolytic activity in macrophages, further confirming the induction of trained immunity in vivo. In summary, after SeBLP induction, mouse macrophages were trained to exhibit a stronger response to secondary stimulation.

[0066] 9. Transcriptome Sequencing Data Analysis Using the STRING database and Cytoscape, we analyzed and constructed a protein-protein interaction (PPI) network and identified differentially expressed genes (DEGs) based on p < 0.05 and |log2FC| > 1. We then performed gene function annotation, differential expression analysis, KEGG enrichment analysis, and GO enrichment analysis to ensure the integrity of the data.

[0067] Results: Transcriptomic analysis revealed the molecular mechanism of SeBLP-induced macrophage immune training To further explore the potential mechanisms behind this phenomenon, peritoneal macrophages (isolated from mice) were immunized intraperitoneally with SeBLP and stimulated with LPS 6 h later to investigate the transcriptional profiles of PMs. Principal component analysis 6 h after LPS stimulation showed that macrophages treated with SeBLP were transcriptionally distinct from those treated with PBS ( Figure 11 A). RNA sequencing analysis (RNA-seq) revealed significantly differentially expressed genes (DEGs) between the transcriptional profiles of macrophages treated with SeBLP and those treated with PBS. A total of 4070 DEGs were found between the two groups; 1947 genes were upregulated and 2123 genes were downregulated in the SeBLP training set using DESeq2 ( Figure 11 B). A volcano plot of epigenetically related DEGs shows changes in epigenetically upregulated genes, such as histone deacetylase (Hdac4), lysine (K)-specific demethylases (KDM3A, KDM5A, KDM2B, etc.), lysine acetyltransferase (KAT6B), and DNA (cytosine-5) methyltransferase (DNMT3B). Decreased KDM7a expression weakens the function of H3K27me3 in compacting chromatin and inducing gene silencing, while increased expression promotes H3K27Ac, ​​leading to an open chromatin structure and promoting gene transcription.

[0068] After SeBLP training, pathway activation was analyzed using the KEGG pathway database, and biological processes were analyzed by gene ontology enrichment analysis. Examination of commonly upregulated GO (biological process) pathways reflected activation of antimicrobial activity pathways, including inflammatory response, immune system processes, cell adhesion, positive regulation of interferon gamma production, and response to lipopolysaccharide ( Figure 12 A). Pathways related to intracellular signaling, such as intracellular signal transduction, T cell receptor signaling pathway, positive regulation of α-β T cell proliferation, and T cell differentiation ( Figure 12 A). Macrophages also reflected the expression of pathways related to antioxidant pathways during glutathione biosynthesis ( Figure 11E). KEGG analysis of the SeBLP and PBS groups mainly revealed identified immune pathways, such as the hematopoietic cell lineage pathway, NF-κB signaling pathway, C-type lectin receptor signaling pathway, and MAPK signaling pathway ( Figure 11 C). Gene set enrichment analysis (GSEA) also showed that SeBLP stimulation induced a significant metabolic reprogramming of macrophages, characterized by enhanced glycine, serine, threonine metabolism, and glutathione metabolism, while impaired OXPHOS and TCA ( Figure 11 D).

[0069] Transcriptomic data showed that SeBLP treatment of PM induced the transcription of multiple PRRs, cytokines, and chemokines. After secondary stimulation, the fold changes of PRRs (Tlr1, Tlr6, Tlr9, Clec1b, etc.), pro-inflammatory cytokines (Tnf, Il12a, Il12b, etc.), anti-inflammatory cytokines (Il10, Socs7, Socs4), chemokines (Ccl3, Ccl4, Cxcl2, etc.), and genes involved in glutathione metabolism (Gclc, Gclm, Gss, and PHGDH) were significantly increased ( Figure 11 E). In addition, we observed upregulation of metabolism-related genes, including Pdk1, Acod1, and Slc16a3, which collectively indicate enhanced glycolysis. ( Figure 12 C). Increased expression of Acod1, encoding aconitate decarboxylase, is associated with multi-level glycolysis activation, while Pdk1 inhibits the pyruvate dehydrogenase complex (PDH), thereby blocking the conversion of pyruvate to acetyl-CoA, preventing its entry into the TCA cycle and promoting lactate production through Slc16a3, which exports lactate. Further analysis of differentially expressed genes (DEGs) in metabolic pathways showed that SeBLP-trained macrophages downregulated genes encoding key enzymes in the citric acid cycle (TCA cycle) and oxidative phosphorylation (OXPHOS), while upregulated genes encoding key enzymes in glycolysis ( Figure 11 E). Gene set enrichment analysis (GSEA) also showed that SeBLP stimulation induced a significant metabolic reprogramming of macrophages, characterized by enhanced glutathione metabolism and impaired OXPHOS and TCA ( Figure 12 B).

[0070] We further analyzed the links between training immunity, inflammatory regulation, and redox regulation. Genes involved in antioxidant defense were significantly altered, with GSH biosynthesis components and the transcription factor Nrf2 significantly upregulated ( Figure 12D), indicating enhanced antioxidant capacity. Simultaneously, an increase in mTOR pathway activity was observed, although not significantly, supporting its role in metabolic reprogramming. Proinflammatory cytokines TNF-α and IL-12 were significantly upregulated, while expression of the anti-inflammatory cytokine IL-10 was increased, suggesting a dual regulation of the inflammatory response. IκBα expression was also increased ( Figure 12 D), indicating that NF-κB signaling is activated and negative feedback regulation is induced. In the JAK-STAT signaling pathway, cytokines IL-12 and TNF-α activate the corresponding receptors, Jak1 and Jak3 are upregulated, while Stat3 expression is downregulated ( Figure 12 D). Notably, SOCS4 was strongly induced ( Figure 12 D), which may help inhibit the STAT pathway. The anti-apoptotic gene Bcl2 was significantly upregulated ( Figure 12 D), indicating improved cell survival. Activation of the FoxO pathway, along with upregulation of Nrf2 and PI3K-AKT signaling, further supports the involvement of oxidative stress regulation in SeBLP-induced responses. Together, these findings suggest that SeBLP triggers a coordinated transcriptional program that incorporates metabolic reprogramming, redox control, and inflammatory modulation as part of its conditioned immune phenotype.

[0071] Example 4. Application of Nano-Selenium Bacteria-like Particles to Deliver Antigens for Immunity Enhancement 1. Express the fusion protein SAMEA-OACD to facilitate binding of the SAMEA protein to the SeBLP surface. (SAMEA is an epitope group of key Staphylococcus aureus antigens; OACD is a novel anchoring protein that connects the model antigen SAMEA to the BLP surface.) The recombinant SAMEA protein is referred to as SAMEA-OACD. The SAMEA antigen is a multi-epitope antigen from Staphylococcus aureus (SAMEA sequence), and OACD is the anchoring protein that facilitates SAMEA attachment to the SeBLP surface.

[0072] 2. Attach the SAMEA-OACD sequence to the pET24a plasmid to obtain the recombinant plasmid and transform it into Escherichia coli BL21. Select positive clones for recombinant protein expression. Amplify the positive clones and culture them overnight in 5 ml of LB liquid medium containing kanamycin at 37°C with shaking at 180 rpm. Subsequently, inoculate the clones at a ratio of 1:100 into 100 ml of LB liquid medium containing kanamycin and continue culturing. When the OD600 value reaches 0.6-0.8, induce the cells with IPTG (0.5 mM) and shake at 180 rpm at 25°C for 24 hours. Centrifuge the cells, resuspend the induced cells in 20 ml of PBS, and disrupt them with a 200 W ultrasonic wave in ice water for 20 minutes. After sonication and centrifugation, the supernatant contains soluble protein.

[0073] 3. SeBLP-SAMEA: Add 4 ml of prepared SeBLP to 20 ml of the sonicated supernatant of SAMEA-OACD, induced for soluble expression, and gently shake at 37°C for 30 minutes. Wash the sample three times with PBS, resuspend it in PBS, designate it SeBLP-SAMEA, and store it at -80°C until further use. SAMEA-OACD sequence: SEQ ID NO. 1.

[0074] SDS-PAGE was used to analyze the expression of SAMEA-OACD protein and proteins binding to SeBLPs.

[0075] 4. SeBLP-SAMEA was stained using standard immunofluorescence techniques to facilitate detection of antigen-BLP binding. After incubation with an anti-His tag antibody overnight at 4°C, fluorescent labeling was performed using a FITC secondary antibody (Biosharp Biotechnology, China). FITC images were captured using a super-resolution microscope (Deltavision OMX SR; GE Healthcare UK Limited).

[0076] 5. Preparation and Identification of SeBLP-Based Antigen Display Systems. Cytotoxic T lymphocyte epitopes, helper T lymphocyte epitopes, and B cell epitopes were created by screening and combining different virulence-associated components of Staphylococcus aureus to create a multivalent epitope antigen (SAMEA). The synthetic gene was expressed in recombinant Escherichia coli to produce SAMEA. OACD is the peptidoglycan binding site of E. coli outer membrane protein A.

[0077] To bind SAMEA-OACD to SeBLPs, the prepared SeBLPs were added to the supernatant containing the SAMEA-OACD fusion protein and incubated at 37°C and 180 rpm for 5 hours. Unbound protein was removed by washing the pellet three times with PBS. Finally, the binding product was resuspended in sterile PBS. To confirm the binding of the antigen to SeBLPs, SAMEA-displaying SeBLPs were verified by SDS-PAGE and immunofluorescence, both of which were performed according to standard methods.

[0078] result: To evaluate whether SeBLP could be linked to SAMEA as a surface display system, SDS-PAGE and immunofluorescence analysis confirmed the binding of the antigen to SeBLP. Compared with the SeBLP control group, SDS-PAGE analysis showed that a distinct protein band at 77.8 kDa appeared after the SAMEA-OACD protein bound to the bacteria-like particles, confirming the presence of the SAMEA-OACD protein ( Figure 3 A). Meanwhile, bright green fluorescence was detected on the surface of SeBLP SAMEA by IFA ( Figure 3 B). These findings suggest that SeBLP can serve as a potential antigen display system.

[0079] To confirm the data obtained from SEM analysis, we measured BLP, SeBLP, and SeBLP-SAMEA using dynamic light scattering (DLS), a technique used to determine particle size and size distribution. BLP exhibited a hydrodynamic size of 1406 ± 5.00 nm, while SeBLP had a hydrodynamic diameter of 1901 ± 6.00 nm. However, the size of SeBLP-SAMEA remained consistent with that of SeBLP ( Figure 3 C). Compared with BLP, the zeta potential of SeBLP decreased from -10.71 mV to -27.52 mV ( Figure 3 D). After binding to the model antigen, the zeta potential increased. This indicates that selenium nanoparticles reduce the zeta potential, and the presence of the antigen protein weakens this effect. Fourier transform infrared spectroscopy analysis shows that SeBLP has a peak at 699.53 cm -1 There is an obvious characteristic peak in SeBLP SAMEA, but not in SeBLP SAMEA ( Figure 3 E). This indicates that SeBLP-SAMEA contains both SeBLP and SAMEA components, confirming the successful binding of SAMEA to SeBLP.

[0080] 5. Vaccine Model: In the single-dose vaccination study, 50 female Kunming mice were randomly divided into five groups of 10 mice each. On day 0, both the infection and control groups received an intraperitoneal injection of PBS. The SAMEA+ALU group was given PBS and SAMEA (50 μg) with an equal volume of alum adjuvant. Mice in the BLP-SAMEA and SeBLP-SAMEA groups received an intraperitoneal injection of 50 μg on day 0. Serum samples were collected from all groups on days 7, 14, and 21 for antibody analysis. On day 21, all groups except the control group received live MRSA (5 × 10 8 CFU / 100 μL) were injected intravenously. Body weight changes and survival rates were monitored until day 36. Serum and kidney tissue were collected from surviving mice on day 36 for further analysis.

[0081] In the single-dose vaccination study, 50 female Kunming mice were randomly divided into five groups of 10 mice each. On day 0, both the infection group and the control group received an intraperitoneal injection of PBS. The SAMEA+ALU group was given SAMEA (50 μg, soluble protein expressed in E. coli) and an equal volume of alum adjuvant. Mice in the BLP-SAMEA group (BLP was added to 20 ml of SAMEA-OACD ultrasonic supernatant to induce soluble expression and then slowly shaken at 37 °C for 30 minutes. The sample was washed three times with PBS, resuspended in PBS, and then named BLP-SAMEA) and SeBLP-SAMEA group received an intraperitoneal injection of 50 μg on day 0. Serum samples were collected from all groups on days 7, 14, and 21 for antibody analysis. On day 21, all groups except the control group received live MRSA (5 × 10 8 CFU / 100 μL) were injected intravenously. Body weight changes and survival rates were monitored until day 36. Serum and kidney tissue were collected from surviving mice on day 36 for further analysis.

[0082] Results: SeBLP-induced training immunity enhanced the protective effect of a single dose of SAMEA vaccine This model aims to explore whether the combination of SeBLP and multi-epitope vaccine can further enhance the protective immune response against MRSA by inducing trained immunity.

[0083] To investigate the ability of BLP-SAMEA and SeBLP-SAMEA to induce humoral responses in vivo, blood samples were collected from immunized mice on days 7, 14, and 21, and the levels of SAMEA-specific antibodies (IgG, IgG1, and IgG2a) were assessed using an indirect ELISA method ( Figure 13 A). Antigen-specific antibody titers stimulated by SeBLP combined with SAMEA showed that the specific IgG level at each observation point was higher than that of aluminum gel (p>0.05) or BLP (p<0.05) ( Figure 13 B). Humoral immune responses can indicate T lymphocyte polarization by measuring the IgG1 / IgG2a antibody isotype ratio ( Figure 13 C). 7 days after vaccination, SeBLP-SAMEA, BLP-SAMEA, and ALU+SAMEA induced similar IgG1 titers (4 × 10 3 , 3 × 10 3 , 1.7 × 10 4On day 14 after immunization, SeBLP-SAMEA induced significantly higher IgG1 titers compared to mice receiving ALU+SAMEA (p<0.01) or BLP-AMEA (p<0.001). A similar trend was observed on day 21 after immunization. Regarding IgG2a, the SeBLP-SAMEA group exhibited 8-fold and over 10-fold higher IgG2a responses over 7 days compared to the BLP-AMEA and ALU+SAMEA groups, respectively (p<0.05 and p<0.001), indicating that SeBLP accelerated antibody production in response to the vaccine. Overall, alum induced a strongly polarized, Th2-biased immune response. BLP exhibited a Th2-biased immune response on day 7, followed by a balanced Th1 / Th2 response on day 14. SeBLP induced a Th1-biased immune response for the first 14 days, achieving a balanced Th1 / Th2 response by day 21. In contrast, SeBLP induced a more balanced Th immune response. In addition, SeBLP significantly enhanced the mucosal immune response to the vaccine. By day 21, the specific SIgA level in the SeBLP-SAMEA group was significantly higher than that in the ALU+SAMEA and BLP-SAMA groups, indicating that SeBLP enhanced the mucosal immune effect ( Figure 13 D).

[0084] Subsequently, the mice were monitored for 15 days after intravenous injection of MRSA following the third blood draw. Thus, the survival rates were 40% in the ALU+SAMEA group, 80% in the SeBLP-SAMEA group, and 60% in the BLP-AMEA group ( Figure 13 E). Changes in body weight further highlight the benefits of SeBLP in combating MRSA infection. The SeBLP-SAMEA group reduced weight loss in the early stages of infection, and SeBLP promoted faster weight recovery in mice during the later stages of observation ( Figure 13 F). The body weight of mice in the SeBLP-SAMEA group recovered to 85% of their initial body weight, while the body weights of the ALU+SAMEA and BLP-SMAEA groups did not reach the same level as that of the SeBLP-SAMEA group ( Figure 13 F). At the end of the monitoring period, bacterial loads in the kidneys of mice showed that the SeBLP-SAMEA group (p<0.05) significantly reduced bacterial loads compared to the ALU+SAMEA group ( Figure 13 G). H&E staining of the kidney tissue in the PBS group showed normal structure and no pathological changes. MRSA infection (4 × 10 8CFU / mouse), the kidneys of sepsis model mice showed swelling and necrosis of renal tubular epithelial cells, as well as excessive bleeding and inflammatory cell infiltration. In the SAMEA+ALU group, mice showed severe renal damage, including vacuolar degeneration of renal tubules and extensive infiltration of inflammatory cells. In the BLP-SAMEA group, although some vacuolar degeneration of renal tubules was present, no obvious renal damage was observed. Mice in the SeBLP-SAMEA group did not show obvious renal damage ( Figure 13 I).

[0085] To determine the changes in factors related to renal inflammation in mice, we also analyzed the mRNA expression of key cytokines (IL-6, IL-1β, TNF-α, IL-10, IL-12, IFN-β, TGF-β, CCL2, CCL7) and TLR (TLR2) 15 days after MRSA challenge. In the mouse model of MRSA infection, SeBLP-SAMEA was found to have an anti-inflammatory effect by downregulating the secretion of inflammatory cytokines in the kidney. RT-qPCR analysis showed that SeBLP-SAMEA significantly reduced the expression level of IL-6 compared with the infection group (p<0.05). For the detection of TLRs, the SAMEA+ALU, BLP-SAMEA and SeBLP-SAMEA groups showed increased expression of TLR2 compared with the infection group, with the highest level observed in the SeBLP SAMPA group ( Figure 13 H), indicating the immunopotentiating effect of SeBLP and its ability to improve vaccine performance. We then examined changes in epigenetic and metabolic markers. Compared with the ALU+SAMEA group, the SeBLP-SAMEA group showed increased levels of HDAC7, mTORC2, and HIF1-α, while there was no significant difference in HDAC7 levels (p>0.05) ( Figure 13 H). In conclusion, SeBLP-induced training immunity can induce an immune enhancement effect after a single dose of immunization.

Claims

1. Application of nano-selenium bioparticles as protein vaccine carriers or in the preparation of drugs for preventing bacterial infections or as antioxidant drugs.

2. The use according to claim 1, characterized in that Method for obtaining nano-selenium bioparticles: sodium selenite with a final concentration of 30ug / ml is added to Lactobacillus brevis, cultured for 18h, washed three times with phosphate-buffered saline, added to 10% trichloroacetic acid and boiled for 30 minutes, washed three times with PBS, and resuspended in PBS to obtain nano-selenium bioparticles.

3. The use according to claim 1, characterized in that The bacteria were Staphylococcus aureus, Escherichia coli and Listeria monocytogenes.

4. The use according to claim 1, characterized in that Antioxidation is the removal of ABTS free radicals and DPPH free radicals or the increase of T-SOD activity.

5. The application according to claim 1, characterized in that: Protein vaccine carriers display antigens on the surface of nano-selenium bioparticles.

6. A nano-selenium bacteria-like particle displaying an antigen, characterized in that: The antigen is displayed on the surface of the nano-selenium bioparticles described in claim 1.

7. The antigen-displaying nano-selenium bacteria-like particles according to claim 6, characterized in that The sequence of the antigen is shown in SEQ ID NO.

1.

8. Use of the nano-selenium bioparticles according to claim 6 or 7 in the preparation of vaccines for preventing diseases.

9. The use according to claim 8, characterized in that The disease is an antibacterial infection.

10. The use according to claim 9, characterized in that The bacteria is methicillin-resistant Staphylococcus aureus.

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