Anti-pseudomonas aeruginosa vaccine as well as preparation method and application thereof

Abstract

The invention provides an anti-pseudomonas aeruginosa vaccine as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) guiding Ca < 2 + > and PO4 < 3-> to generate heterogeneous nucleation on the surface of the pseudomonas aeruginosa by utilizing the electrostatic adsorption effect of polyacrylic acid on the surface of the pseudomonas aeruginosa so as to form a complete calcium phosphate mineral shell and obtain PA (at) Ca; (2) carrying out ultraviolet irradiation on the PA (at) Ca obtained in the step (1), and removing pseudomonas aeruginosa which is not completely wrapped by a calcium phosphate mineral shell to obtain PA (at) Ca-UV; and (3) encapsulating the PA (at) Ca-UV obtained in the step (2) in hydrogel, so as to obtain the pseudomonas aeruginosa resistant vaccine. The problem of contradiction between the virulence risk of a viable vaccine and the weak immunogenicity of an inactivated vaccine is solved, through the synergistic effect of the mineral shell and the hydrogel, the extremely high safety is ensured, meanwhile, strong body fluid and cellular immunity can be stimulated, and lasting protective memory is generated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-Pseudomonas aeruginosa vaccine, its preparation method, and its application. Background Technology

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa Pseudomonas aeruginosa is a common opportunistic pathogen in hospital-acquired infections. Due to its inherent broad-spectrum drug resistance mechanism, it is difficult to treat clinically and often leads to high morbidity and mortality. Some strains even develop resistance to last-line antibiotics such as carbapenems, evolving into multidrug-resistant Pseudomonas aeruginosa infections that are difficult to cure, posing a serious threat to global public health.

[0003] The pathogenic mechanism of this bacterium is complex, involving numerous virulence factors, a precisely regulated secretion system, a quorum sensing network, and highly efficient antibiotic resistance mechanisms. Furthermore, the diversity of its interactions with the host presents significant obstacles to the development of an effective vaccine. Despite substantial efforts, the development of vaccines against Pseudomonas aeruginosa remains slow.

[0004] For example, flagellated antigen-based vaccines offer limited protection against clinical isolates (such as strains isolated from patients with cystic fibrosis); subunit vaccines targeting a single antigen (such as lipopolysaccharide O antigen, alginate, or a specific outer membrane protein) struggle to induce broad-spectrum immune protection. While whole-cell inactivated vaccines can present multiple antigens, they generally suffer from weak immunogenicity, short duration of protection, and potentially high reactivity. For instance, patent application CN104189898A discloses a method for preparing a Pseudomonas aeruginosa vaccine, which includes the following steps: first, obtaining Pseudomonas aeruginosa cells, and then irradiating the bacteria with radiation to obtain the Pseudomonas aeruginosa bacterial vaccine. Live attenuated vaccines constructed through gene editing can elicit strong cellular immune responses, but their complex production processes, stringent quality control, and high costs limit their clinical translation potential. Currently, there are no approved Pseudomonas aeruginosa vaccines globally; therefore, developing a safe, efficient, and easily prepared novel vaccine strategy is urgently needed.

[0005] In recent years, biomineralization technology has shown broad application prospects in the biomedical field due to its unique biocompatibility and functional tunability. Various metal ions, such as calcium and manganese, have been used in biomimetic synthetic mineral coatings to achieve purposes such as antigen protection, sustained release, or immunomodulation. Among them, calcium phosphate, as a classic adjuvant, has had its safety verified through long-term practice and is recommended by the World Health Organization for use in various traditional vaccines (such as diphtheria, tetanus, pertussis, and polio vaccines). Its mechanism of action includes protecting antigens from degradation, enhancing antigen delivery efficiency, and effectively activating the innate immune system.

[0006] For example, patent application GB1546035A discloses a mixed vaccine against infection caused by *Pseudomonas aeruginosa*, comprising an elastase toxoid from *Pseudomonas aeruginosa* and a protease toxoid from *Pseudomonas aeruginosa*, further including an adjuvant such as aluminum hydroxide, aluminum phosphate, calcium phosphate, alum, or Freund's incomplete adjuvant. This approach simply uses calcium phosphate as an adjuvant in a simple mixture, and the efficacy of the prepared vaccine needs further improvement. Summary of the Invention

[0007] To address the aforementioned technical problems in the prior art, this invention provides an anti-Pseudomonas aeruginosa vaccine, its preparation method, and its application.

[0008] This invention provides a method for preparing an anti-Pseudomonas aeruginosa vaccine, comprising the following steps: (1) Using polyacrylic acid in Pseudomonas aeruginosa ( Pseudomonas aeruginosa The electrostatic adsorption of the surface guides Ca 2+ and PO4 3- Heterogeneous nucleation occurs on the surface of Pseudomonas aeruginosa to form a complete calcium phosphate mineral shell, thus obtaining PA@Ca; (2) The PA@Ca obtained in step (1) is subjected to ultraviolet irradiation to remove Pseudomonas aeruginosa that is not completely covered by the calcium phosphate mineral shell, and PA@Ca-UV is obtained. (3) The PA@Ca-UV obtained in step (2) is encapsulated in a hydrogel to obtain the anti-Pseudomonas aeruginosa vaccine (abbreviated as PCUF).

[0009] The reaction principle in step (1) is as follows: a calcium phosphate mineral shell is constructed on the bacterial surface using biomimetic mineralization technology. First, polyacrylic acid (PAA) electrostatically adsorbs onto the bacterial surface. Subsequently, the carboxylic acid groups exposed on the PAA serve as nucleation sites, reacting with calcium... 2+ Ion coordination. This synergistic effect leads to a rearrangement of the internal structure of the PAA, resulting in localized enrichment of Ca. 2+ The concentration of phosphate ions increases, leading to the formation of ordered pre-nucleation clusters. The attraction of phosphate ions to these clusters significantly lowers the nucleation energy barrier, directly driving preferential heterogeneous nucleation on the cell surface. Guided by the PAA template, Ca... 2+ and PO4 3- The gradual assembly formed a crystalline calcium phosphate structure that grew uniformly along the bacterial surface, ultimately achieving complete encapsulation of Pseudomonas aeruginosa (PA).

[0010] Preferably, in step (1), the Ca 2+ Derived from calcium chloride, the PO4 3- It comes from disodium hydrogen phosphate or sodium dihydrogen phosphate.

[0011] More preferably, in step (1), the *Pseudomonas aeruginosa* suspension is mixed evenly with sodium polyacrylate aqueous solution, calcium chloride aqueous solution, and disodium hydrogen phosphate aqueous solution or sodium dihydrogen phosphate aqueous solution to form a complete calcium phosphate mineral shell on the surface of *Pseudomonas aeruginosa*, thus obtaining PA@Ca; wherein the concentration of the *Pseudomonas aeruginosa* suspension is 1×10⁻⁶. 8 ~2×10 9 The concentrations of the sodium polyacrylate aqueous solution (CFU / mL) are 25wt%~30wt%, the concentrations of the calcium chloride aqueous solution (0.1~1 mol / L), the concentrations of the disodium hydrogen phosphate aqueous solution or sodium dihydrogen phosphate aqueous solution (0.1~1 mol / L), the volume ratios of Pseudomonas aeruginosa suspension to sodium polyacrylate aqueous solution and calcium chloride aqueous solution (1:15~10:50~10), and the volume ratios of Pseudomonas aeruginosa suspension to disodium hydrogen phosphate aqueous solution or sodium dihydrogen phosphate aqueous solution (1:20~25).

[0012] More preferably, in step (1), the reaction temperature for forming a complete calcium phosphate mineral shell is 10~20 ℃, and the reaction time is not less than 1 hour.

[0013] More preferably, in step (1), the reaction temperature for forming a complete calcium phosphate mineral shell is 20 °C and the reaction time is 2 hours.

[0014] Preferably, in step (2), the wavelength of the ultraviolet light is 254~280 nm, and the irradiance is 0.2~20 mW / cm. 2 The irradiation time shall not be less than 0.5 hours.

[0015] More preferably, in step (2), the wavelength of the ultraviolet light is 254 nm and the irradiance is 20 mW / cm². 2 The irradiation time is 1 hour.

[0016] Preferably, in step (3), the hydrogel is Planck F-127 hydrogel. Planck F127 hydrogel is a thermoresponsive polymer used to enhance the biosafety and sustained antigen release of vaccines.

[0017] More preferably, in step (3), the PA@Ca-UV obtained in step (2) is mixed with Planck F-127 hydrogel solution at 4 °C for gelation, and the gelation time is not less than 1 hour, to obtain the anti-Pseudomonas aeruginosa vaccine.

[0018] The present invention also provides an anti-Pseudomonas aeruginosa vaccine, which is prepared by the above-described method for preparing an anti-Pseudomonas aeruginosa vaccine.

[0019] The present invention also provides the use of the above-mentioned anti-Pseudomonas aeruginosa vaccine in the preparation of a drug for preventing Pseudomonas aeruginosa infection.

[0020] Preferably, the Pseudomonas aeruginosa infection is pneumonia.

[0021] This invention utilizes biomimetic mineralization technology to construct a calcium phosphate mineral shell on the surface of bacteria, inducing bacterial dormancy and weakening its virulence. Simultaneously, selective ultraviolet inactivation ensures biosafety and maintains antigen integrity. Finally, encapsulation with thermosensitive hydrogel F127 achieves controlled antigen release and enhanced immune response. Compared with existing technologies, this invention has the following advantages: (1) High safety: The combination of mineral shell-induced dormancy and selective ultraviolet inactivation fundamentally avoids the potential infection risk of live bacterial vaccines.

[0022] (2) Strong immunogenicity: While reducing virulence, it retains a variety of bacterial antigens, which can stimulate synergistic innate and adaptive immunity.

[0023] (3) Long-lasting protection: Hydrogel enables controlled release of antigens, which can induce germinal center reactions, long-lived plasma cells, memory B / T cells (such as TEM / TCM) and multifunctional effector T cells (Th1 / Th2 / Th17 / CTLs) in vivo, providing comprehensive and long-lasting immune protection.

[0024] (4) Platform universality: The biomimetic mineralization strategy described herein has universality, providing a general technical platform for developing vaccines against other multidrug-resistant pathogens. Animal experiments have shown that the PCUF vaccine can effectively resist Pseudomonas aeruginosa pneumonia, significantly reduce lung injury, decrease bacterial load, and improve survival rate, demonstrating great potential for translational applications. Attached Figure Description

[0025] Figure 1 This represents the results of detection and characterization of calcifying bacteria. Among them, Figure 1 A in the diagram represents the synthesis of a mineral coating on the bacterial surface. Figure 1 Scanning electron microscope images of B:PA@Ca and PA in the image; Figure 1 Transmission electron microscope images of C:PA@Ca and PA in the image; Figure 1 Elemental distribution diagrams of D: PA@Ca and PA in the diagram; Figure 1 E: EDX spectrum of PA@Ca in E; Figure 1 Fourier transform infrared spectra of F: PA, CAP, and PA@Ca; Figure 1 G in the figure: flow cytometry analysis results of calcein-labeled PA@Ca and PA; Figure 1 Confocal microscopy image of H:PA@Ca; Figure 1 In the figure, I represents the change in Zeta potential during calcification. Figure 1 Particle size distribution diagram of J:PA@Ca and PA.

[0026] Figure 2 This contributes to the cell compatibility and functional diversity of the calcified coating. Among them, Figure 2 Confocal microscopy images of the three-stained bacteria (SYTO9, green / live; PI, red / dead; Calcein Blue, blue / mineral) and the results of colocalization and quantitative analysis of each group; Figure 2 A schematic diagram of the selective scavenging mechanism of B:PA@Ca under ultraviolet light; Figure 2 C in the diagram represents the pathway of mineralization-induced dormancy and demineralization-triggered reactivation. Figure 2 The results of ATP concentration determination in the D:PA, PA@Ca and PA@Ca-UV groups; Figure 2 Representative colony formation experimental images and colony count statistics for EF: PA, PA-UV, PA@Ca, PA@Ca-UV and demineralized PA@Ca-UV; Figure 2 G in OD 600 Values ​​were used to monitor the growth kinetic curves of PA, PA@Ca-UV, and demineralized PA@Ca-UV. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0027] Figure 3 This refers to the immunostimulatory effect of PA@CA-UV on dendritic cells. Among them, Figure 3 A: Cell viability assay results of DC2.4 cells after 24 hours of incubation with PA@Ca-UV or the control group; Figure 3 Confocal microscopy image of B:DC2.4 cells after 3 hours of co-incubation with fluorescently labeled PA-UV and PA@Ca-UV; Figure 3 Time-dependent uptake kinetics of PA-UV and PA@Ca-UV in C:DC2.4 cells; Figure 3 DG in the figure: the secretion levels of cytokines IL-12p40, IL-1β, IL-6, and TNF-α 24 hours after stimulation; Figure 3 HK: Flow cytometry analysis of the expression and quantification of surface maturation markers MHC-II, CD40, CD80, and CD86 in DC2.4 cells after 24 hours of treatment. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

[0028] Figure 4 The characteristics of the PCUF vaccine and its vector F127. Among them, Figure 4 Image A in the image shows the sol-gel transition of the hydrogel as temperature changes. Figure 4 Rheological properties of BC:F127 and PCUF as a function of temperature; Figure 4Cryo-scanning electron microscopy images of D:F127 and PCUF.

[0029] Figure 5 This describes the biodistribution of the PCUF vaccine in vivo. Among other things, Figure 5 Whole-body fluorescence imaging and quantification of average fluorescence intensity of AB:Cy7 labeled PA-UV, PA@Ca-UV and PCUF at different time points after subcutaneous injection; Figure 5 CD: Distribution of Cy7 signal in ex vivo organs over 24 hours, MFI analysis and lymph node-specific MFI quantification; Figure 5 F in the table represents the changes in mouse body weight during the initial and booster immunization periods. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0030] Figure 6 This refers to the adaptive immune response induced by the PCUF vaccine. Among them, Figure 6 The AC in the sample refers to CD11c in the draining lymph nodes 72 hours after injection. + Expression of dendritic cell maturation markers CD86, CD80, and CD40; Figure 6 DE in the text refers to the proportion of germinal center B cells in draining lymph nodes (B220). + GL7 + ); Figure 6 FH in the text refers to the serum anti-PA IgG antibody titer on days 7, 10, and 14 post-immunization. Figure 6 The IK in this context refers to the analysis of memory T cell subsets in lymph nodes, including CD4+. + and CD8 + The proportion of central memory T cells, effector memory T cells, and naive T cells in the population; Figure 6 JL in the text refers to spleen antigen-specific T cell responses, including IFN-γ. + CD4 + (Th1), IL-4 + CD4 + (Th2), IL-17 + CD4 + (Th17) and IFN-γ + CD8 + (Cytotoxic T cells); Figure 6 M in the figure represents the Th1 / Th2 polarization ratio of the spleen (IFN-γ). + CD4 + / IL-4 + CD4 + * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0031] Figure 7 The study aimed to assess the in vivo protective effect of the PCUF vaccine against Pseudomonas aeruginosa pneumonia. Figure 7 In this diagram, A represents the experimental timeline. Figure 7 B in the figure represents the survival curve of mice infected with PA after vaccination. Figure 7 The CF in the figure represents the quantitative results of cytokines IL-1β, IL-6, IL-12p40 and TNF-α in lung tissue. Figure 7 GH in the text refers to: bacterial CFU culture plates and quantitative statistics of mouse lung homogenate; Figure 7 In this context, "I" stands for: Lung tissue damage score; Figure 7 J in the text stands for: MPO + area; Figure 7 K in the table represents: H&E stained sections and MPO immunohistochemical staining images of representative lung tissues from each group. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0032] Figure 8 For the in vivo safety evaluation of the PCUF vaccine. Figure 8 AB in the figure represents the hemolysis test results of red blood cells after treatment with PA-UV, CAP, and PA@Ca-UV, respectively. Figure 8 The CL in this context refers to the complete blood count (including WBC, white blood cells; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin concentration; MCHC, mean corpuscular hemoglobin concentration; PLT, platelets) and blood biochemistry analysis (ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CREA, creatinine) of mice on day 28 after immunization with PA-UV, CAP, PA@Ca-UV, F127, and PCUF. Figure 8 M in the table represents representative H&E stained tissue sections of the heart, liver, spleen, lungs, and kidneys from each group on day 28. Detailed Implementation

[0033] Example 1: Preparation and characterization of calcified Pseudomonas aeruginosa (1) Pseudomonas aeruginosa (purchased from the American Type Culture Collection, ATCC) was cultured in LB liquid medium at 37°C and 200 rpm for 12 hours.

[0034] (2) Wash the *Pseudomonas aeruginosa* obtained in step (1) with physiological saline. According to the Lambert-Beer law, when a beam of light passes through a suspension, suspended particles (such as bacteria) absorb and scatter the light, resulting in a decrease in transmitted light. Bacterial cells do not have major light-absorbing pigments near a wavelength of 600 nm, so the absorbance here is mainly caused by the scattering of light by the bacterial cells. The more concentrated the bacterial solution, the more light is scattered, the weaker the light signal received by the detector, and the higher the measured absorbance value. Within a certain absorbance range, the absorbance value (OD) of the bacterial solution at a wavelength of 600 nm is... 600 The OD value is directly proportional to the bacterial concentration. This is achieved by measuring and adjusting the OD value. 600 The value was adjusted to make the bacterial suspension concentration 2×10⁻⁶. 9 CFU / mL, bacterial suspension volume is 100 μL.

[0035] (3) Disperse the bacterial suspension obtained in step (2) in 1.5 mL of an aqueous solution containing 30% sodium polyacrylate.

[0036] (4) Add 1 mL of 1 mol / L calcium chloride solution to the mixed solution obtained in step (3) and stir at room temperature for 20 minutes.

[0037] (5) Add 2.5 mL of 1 mol / L disodium hydrogen phosphate solution to the mixed solution obtained in step (4), react for 60 minutes at a temperature of 20 °C. Centrifuge and discard the supernatant to obtain calcified Pseudomonas aeruginosa, i.e., PA@Ca.

[0038] Using polyacrylic acid as a template polyelectrolyte, a calcium phosphate mineral coating was successfully formed on Pseudomonas aeruginosa. Figure 1 (A in the original text). The structure and composition of the obtained PA@Ca were thoroughly characterized. Scanning electron microscopy revealed that the complete bacteria were encased within a mineral layer. Figure 1 (B in the text), while transmission electron microscopy further refined the morphology, revealing increased surface roughness and enhanced electron density contrast (B in the text). Figure 1 The C in the image is shown. Energy-dispersive X-ray spectroscopy elemental diagrams reveal a uniform distribution of calcium on the PA@Ca surface, confirming the coverage of the mineral coating. Figure 1 (D and E in the text). Fourier transform infrared spectroscopy analysis determined the characteristic absorption peaks corresponding to the bacterial components and calcium phosphate, verifying the complex nature of the structure. Figure 1 Flow cytometry analysis of calcein-labeled PA@Ca showed that the bacterial encapsulation efficiency was as high as 88.5% (F in the text). Figure 1 The finding (G in the image) was visually confirmed by confocal laser scanning microscopy images showing uniform bright green fluorescence associated with the mineral coating. Figure 1The H in the text. Analysis of surface charge and particle size using dynamic light scattering revealed that the zeta potential exhibits a progressive change (H). Figure 1 In the I), the diameter increases significantly ( Figure 1 (J in the text). Importantly, this mineralization strategy was successfully extended to bacteria such as Corynebacterium martensii, highlighting the broad applicability and adaptability of the PAA template method in encapsulating a wide range of bacteria.

[0039] Example 2: Calcification induces bacterial dormancy, limiting their growth while maintaining their vitality. The obtained PA@Ca was subjected to irradiation at 254 nm and 20 mW / cm². 2 Irradiation under ultraviolet light for 60 minutes selectively removes bacteria not fully encased in a mineral shell, yielding PA@Ca-UV. The calcium phosphate mineral shell of PA@Ca provides strong physical protection and maintains permeability to oxygen and small molecule nutrients through its unique eggshell-like structure. This selective permeability induces bacterial dormancy by modulating cellular metabolic gradients, characterized by a significant reduction in ATP levels that enable bacteria to survive long-term under stress. Crucially, the combination of biomimetic mineralization and targeted ultraviolet irradiation forms a key biosafety strategy that selectively removes unencapsulated active bacteria while maintaining mineral-encapsulated bacterial populations in a dormant state. The recovery of bacterial activity after hydrochloric acid demineralization confirms the reversibility of this dormancy and demonstrates that the intact mineral coating physically prevents essential nutrient exchange, thereby inhibiting cell division.

[0040] Calcium phosphate mineral coatings exhibit important functional properties, including UV shielding and induction of reversible dormancy. When exposed to 100 μW / cm², [the coating exhibits these properties]. 2 After 60 minutes of ultraviolet radiation, the mineralizing bacteria (PA@Ca) remained viable with no significant decrease in survival rate, while the unmineralized control group almost completely died. Figure 2 (A, B in the original text). To selectively remove ~11.5% of unencapsulated viable bacteria, we used ultraviolet irradiation. Tri-staining (SYTO9 / PI / calcein blue) confirmed the viability of mineralized bacteria after ultraviolet irradiation (green fluorescence) and the death of unencapsulated cells (red fluorescence). Figure 2 (A in the text). Agar plate assays confirmed this selectivity; PA@Ca produced sparse colonies from incompletely mineralized bacteria, while UV-treated samples (PA@Ca-UV) showed a complete loss of proliferative capacity. Figure 2 In addition, compared with the control, the ATP level of mineralized PA was significantly reduced (E, F). Figure 2 The D in the text is consistent with induced hibernation (D). Figure 2(C in the text). UV treatment further reduced total ATP by eliminating residual uncoated bacteria. Crucially, HCl demineralization reversed PA@Ca-UV dormancy and restored growth kinetics in liquid culture (C in the text). Figure 2 In the G group, compared with the unmineralized control group, the colony count in the demineralized group was reduced by approximately 11.5%, which partially matched the results of UV elimination. Figure 2 (E and F in the figure). Undemineralized PA@Ca-UV did not show proliferation in growth curves and plate experiments, confirming that the mineralized shell can effectively physically inhibit cell division.

[0041] Example 3: In vitro dendritic cell activation and immune enhancement effects of PA@Ca-UV.

[0042] DC maturation is crucial for initiating an effective antigen-specific immune response. In vitro assessment of the immunostimulatory effects of PA@Ca-UV began with a cytotoxicity evaluation of DC2.4 cells, establishing a cytotoxic concentration range for subsequent experiments. Analysis of cellular uptake kinetics using fluorescently labeled samples showed that, compared to PA-UV, internalization of PA@Ca-UV in DC2.4 cells was significantly accelerated within the first 5 hours. Figure 3 (A in the original text). This enhanced uptake efficiency was confirmed by confocal microscopy images at the 3-hour time point, showing a significant increase in intracellular accumulation of PA@Ca-UV compared to PA-UV, indicating its superior immunostimulatory potential. Figure 3 (B in the text). Furthermore, compared to the PA-UV and CAP groups, PA@Ca-UV stimulation significantly enhanced the secretion of key pro-inflammatory cytokines in DC2.4 cells, including IL-12p40, IL-1β, IL-6, and TNF-α (…). Figure 3 Consistent with the cytokine profile, PA@Ca-UV treatment also significantly upregulated the surface expression of dendritic cell maturation markers, including CD40, CD80, and CD86. Figure 3 (GJ in the text). These findings suggest that mineralized vaccine formulations enhance dendritic cell uptake and activation.

[0043] Example 4: PCUF enables continuous lymph node targeted delivery To improve the injectability, biocompatibility, and sustained-release properties of the vaccine, the PCUF vaccine is prepared by mixing mineral-coated bacteria (PA@Ca-UV) with a heat-responsive F127 hydrogel matrix and then gelling at 4 °C.

[0044] Specific operating procedure: Under aseptic conditions, mix pre-cooled 20% (w / v) Pluronic F127 hydrogel stock solution with an equal volume of PA@Ca-UV bacterial suspension (2×10⁻⁶). 9The CFU / mL solution was mixed at 4 °C, and the mixture was placed in an ice bath and magnetically stirred at 500 rpm for 1 h to form a uniform milky white suspension. After centrifugation at 2000×g for 5 minutes to remove air bubbles, the suspension was dispensed into sterile containers and finally stored at 4 °C to obtain a liquid PCUF vaccine formulation. This liquid PCUF vaccine formulation turned into a gel-like semi-solid state after injection into mice.

[0045] Both F127 and PCUF exhibit temperature-dependent phase behavior, remaining in solution at 4 °C and forming a stable gel at 37 °C. Figure 4 (A) Rheological analysis confirmed the gelation transition of F127 and PCUF through crossover points, at which the storage modulus (G') of F127 and PCUF exceeded the loss modulus (G”). Figure 4 Cryo-electron microscopy (Cryo-EM) images validated the successful encapsulation of PA@Ca-UV in the hydrogel matrix, revealing the distribution of mineralized bacteria in the porous PCUF network. Figure 4 (D in the middle).

[0046] F127 hydrogel is a thermoresponsive polymer designed to enhance vaccine biosafety and sustained antigen release. Upon injection, the solution gels in situ, forming a reservoir that prolongs the residence time of the PCUF, ensuring controlled release kinetics. It also exhibits targeted aggregation in lymph nodes with reduced off-target distribution. This lymphotropic targeting is crucial because lymph nodes play a central role in adaptive immunity (immune cell aggregation and antigen processing). Simultaneously, the hydrogel creates a local inflammatory microenvironment, promoting bacterial uptake, enhancing antigen processing and presentation within lymph nodes, and ultimately leading to robust humoral and cellular immunity through germinal center B cell expansion and improved cross-presentation. In conclusion, the combination of the mineral coating and F127 hydrogel makes PCUF a promising spatially controlled vaccine delivery system with enhanced safety.

[0047] In vivo tracking was performed by subcutaneous injection of cy7-labeled PCUF, PA-UV, and PA@Ca-UV. Within 24 hours, compared to the control group, PCUF showed significantly enhanced fluorescence retention at the injection site. Figure 5 In A), semi-quantitative mean fluorescence intensity (MFI) analysis showed minor inter-animal variations ( Figure 5 (B in the text). Further 24-hour immune assessment showed that PCUF exhibited targeted aggregation in lymph nodes compared to the control. Figure 5 (C and E in the middle), and the off-target distribution in major organs was significantly reduced ( Figure 5 (C and D in the text).

[0048] Example 5: PCUF can induce strong humoral and cellular adaptive immunity in vivo.

[0049] Within 72 hours after subcutaneous PCUF administration, the expression of maturation markers CD40, CD80, and CD86 in draining lymph nodes was significantly upregulated. Figure 6 (AC in the middle). Subsequent analysis on day 14 after the initial booster immunization showed that, compared with the control, GL7 in the draining lymph nodes of PCUF-immunized mice was significantly higher. + The proportion of B cells in the germinal centers was significantly higher ( Figure 6 DE in the middle). Serum analysis showed that, compared with the control group, the PCUF group had significantly higher titers of anti-Pseudomonas aeruginosa specific IgG antibodies on days 7, 10 and 14 post-immunization ( Figure 6 Despite PA-UV and PA@Ca-UV inducing a moderate response, PCUF consistently produced higher titers. T-cell immune analysis showed an expanded memory population in the draining lymph nodes, including CD4+. + and CD8 + CD44 in subgroup + / CD62L effect memory (TEM) and CD44 + / CD62L + The frequency of central memory (TCM) T cells increases, while the proportion of naïve T cells decreases. Figure 6 The spleen response on day 14 indicated a significant expansion of antigen-specific effector T cells, including Th1 (IFN-γ). + CD4 + ), Th2 (IL-4) + CD4 + ), Th17 (IL-17) + CD4 + ) and cytotoxic T lymphocytes (IFN-γ) + CD8 + () Figure 6 Flow cytometry analysis confirmed Th1 / Th2 mixed polarization and moderate Th1 bias (J and L in the data). Figure 6 M in (the middle part).

[0050] Example 6: PCUF can effectively prevent acute pneumonia in mice.

[0051] Mouse immunization regimen: Mice were divided into groups on day 0 and day 7, and administered PBS and PA-UV (2×10⁻⁶) respectively. 9 CFU / mL), CAP (Ca 2+ Equal volume: 13000 μg / mL), PA@Ca-UV (2×10 9CFU / mL), F127 hydrogel (20%, w / v) or PCUF (20% w / v F127 hydrogel, containing 2×10⁻⁶ CFU / mL), F127 hydrogel (20%, w / v), or PCUF (20% w / v F127 hydrogel, containing 2×10⁻⁶ CFU / mL). 9 CFU / mL PA@Ca-UV), 100 μL per dose.

[0052] According to the experimental time axis ( Figure 7 The A in the formula was administered subcutaneously on days 0 and 7 as a primary and booster dose. Following immunization, an acute pneumonia model was established in mice. Animals were housed in a standard environment, and the entire procedure was performed on a sterile table. Mice were shaved and disinfected one day prior to immunization. After anesthesia, a long incision was made in the middle of the neck, and muscle tissue was carefully dissected. Finally, blunt dissection was used to fully expose the trachea. 50 μL of PA (5 × 10⁻⁶) resuspended in PBS was added. 7 CFU was slowly injected into the trachea of ​​mice, and the mice were then upright and gently shaken to ensure even distribution of the bacteria in the lungs. Mice were sacrificed after 48 hours. Lung tissue was dissected and photographed for subsequent experiments. In the survival study, mice were given a lethal dose of Pseudomonas aeruginosa (50 μL, 1×10⁻⁶). 8 CFU infection. Histological staining and analysis: Lungs were collected, fixed in 4% paraformaldehyde, and embedded in paraffin. H&E staining and MPO immunohistochemical staining were performed using standard methods. Images and sections were blinded and counted using ImageJ. All scores were performed independently by two professionals not involved in the animal experiments.

[0053] Mice immunized with PCUF showed strong protection against lethal intratracheal challenge. Compared with the CAP, PA-UV, and PA@Ca-UV control groups, the survival rate of PCUF-immunized mice was significantly improved. Figure 7 (B in the text). Lung homogenate cytokine analysis showed that in PCUF-treated mice, the levels of inflammatory factors (IL-1β, IL-6, IL-12p40, TNF-α) were significantly reduced. Figure 7 CF in the lungs). Quantitative CFU assay confirmed a significant reduction in pulmonary bacterial load (CF). Figure 7 Histopathological evaluation using H&E staining showed that the PCUF group had mild inflammation, preserved alveolar structure, and no hemorrhage or edema, in stark contrast to the control group, which exhibited severe alveolar wall thickening, consolidation, inflammatory infiltration, and tissue damage. Figure 7 The significant reduction in semi-quantitative lung injury scores in PCUF mice confirmed these observations. Figure 7 Immunohistochemistry further showed that myeloperoxidase expression was extremely low in the lungs of the PCUF group, demonstrating reduced neutrophil inflammation. Figure 7 (K and J in the text). In summary, these findings suggest that the PCUF vaccine can promote effective bacterial clearance and suppress excessive inflammation.

[0054] Example 7: PCUF has good biocompatibility.

[0055] In vitro erythrocyte hemolysis analysis confirmed that Pa@Ca-UV has good biocompatibility. Figure 8 (AB in the text). To comprehensively assess the safety of PCUF, in vivo toxicity assessment was performed. Blood was collected from the eyeballs on day 28, and major organs (heart, lungs, liver, spleen, and kidneys) were isolated, fixed in 10% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and stained with H&E for histological examination. Finally, complete blood count and blood biochemistry tests were performed. Serum and plasma were collected from mice on day 28 after treatment in each group, and the results of blood biochemistry and complete blood count were analyzed. The results showed that the blood biochemistry results of each group showed that aspartate transaminase (AST), alanine transaminase (ALT), creatinine (CREA), and blood urea nitrogen (BUN) were within the normal range. Figure 8 The presence of CF in the samples indicates that the treatments did not affect the liver and kidney function of the mice. Furthermore, the white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin concentration (HGB), platelet (PLT) count, lymphocyte percentage, neutrophil percentage, macrophage percentage, and mean corpuscular hemoglobin concentration (MCHC) were all within the normal range. Figure 8 The presence of GL (globulin level) indicates that the treatments did not affect the hematopoietic system of the mice. Finally, on day 28 after treatment, we dissected the hearts, lungs, livers, spleens, and kidneys of the mice in each group for H&E staining. No significant inflammatory response was observed in any of the vital organs, and they maintained normal tissue structure. These results demonstrate that the PCUF vaccine has high biocompatibility in animals and holds promise for clinical translation.

[0056] Example 8: Preparation of anti-Pseudomonas aeruginosa vaccine (PCUF) (1) Pseudomonas aeruginosa was cultured in LB liquid medium at 37 °C and 200 rpm for 12 hours.

[0057] (2) Wash the Pseudomonas aeruginosa obtained in step (1) with physiological saline, and measure and adjust the OD. 600 The value was adjusted to make the bacterial suspension concentration 2×10⁻⁶. 9CFU / mL, bacterial suspension volume is 100 μL.

[0058] (3) Disperse the bacterial suspension obtained in step (2) in 1 mL of an aqueous solution containing 30% sodium polyacrylate.

[0059] (4) Add 1 mL of 1 mol / L calcium chloride solution to the mixed solution obtained in step (3) and stir at room temperature for 20 minutes.

[0060] (5) Add 2 mL of 1 mol / L sodium dihydrogen phosphate solution to the mixed solution obtained in step (4), and react for 2 hours at a temperature of 20 °C. Centrifuge and discard the supernatant to obtain calcified Pseudomonas aeruginosa, i.e., PA@Ca. The formed calcium phosphate mineral shell coating is complete and uniform, with a higher coverage than that in Example 1.

[0061] (6) The obtained PA@Ca was irradiated under the conditions of 254 nm and 20 mW / cm². 2 Irradiation under ultraviolet light for 60 minutes selectively removes bacteria not completely encased in a mineral shell, yielding PA@Ca-UV.

[0062] (7) Under sterile conditions, the pre-cooled 20% (w / v) Pluronic F127 hydrogel stock solution was mixed with an equal volume of the above PA@Ca-UV bacterial suspension at 4 °C. The mixture was placed in an ice bath and magnetically stirred at 300 rpm for 2 h to form a uniform milky white suspension. After centrifugation at 2000×g for 5 minutes to remove air bubbles, the suspension was dispensed into sterile containers and finally stored at 4 °C to obtain the liquid PCUF vaccine preparation.

[0063] Example 9: Preparation of Anti-Pseudomonas aeruginosa Vaccine (PCUF) The difference from Example 8 is that: In step (2), the concentration of the bacterial suspension is made to be 1×10⁻⁶. 9 CFU / mL; In step (3), the bacterial suspension obtained in step (2) is dispersed in 1.5 mL of an aqueous solution containing 30% sodium polyacrylate; In step (5), 2.5 mL of 1 mol / L sodium dihydrogen phosphate solution is added to the mixed solution obtained in step (4), and the reaction is carried out for 2 hours at a temperature of 10°C. In step (6), the obtained PA@Ca is irradiated under the conditions of 280 nm and 0.2 mW / cm². 2 Expose to ultraviolet light for 60 minutes.

[0064] Example 10: Preparation of Anti-Pseudomonas aeruginosa Vaccine (PCUF) The difference from Example 8 is that: In step (2), the concentration of the bacterial suspension is made to be 1×10⁻⁶. 8 CFU / mL; In step (3), the bacterial suspension obtained in step (2) is dispersed in 1 mL of an aqueous solution containing 25% sodium polyacrylate; In step (4), 5 mL of 0.1 mol / L calcium chloride solution is added to the mixed solution obtained in step (3), and the mixture is stirred at room temperature for 20 minutes.

[0065] In step (5), 2 mL of 0.1 mol / L sodium dihydrogen phosphate solution is added to the mixed solution obtained in step (4), and the reaction is carried out for 1 hour at a temperature of 20°C.

[0066] Comparative Example 1: Preparation of Anti-Pseudomonas aeruginosa Vaccine (PCUF) The difference from Example 8 is that in step (5), the reaction temperature is 40 °C and the reaction time is 2 hours.

[0067] ATP levels were detected using an ATP assay kit, revealing that the bacteria's dormancy state was incomplete or poorly irreversible, with only a slight decrease in ATP levels and high residual proliferative capacity.

[0068] Comparative Example 2: Preparation of Anti-Pseudomonas aeruginosa Vaccine (PCUF) The difference from Example 8 is that in step (7), 20% (w / v) Pluronic F127 hydrogel stock solution and an equal volume of the above PA@Ca-UV bacterial suspension are mixed in an environment of 20 °C and stirred magnetically at 300 rpm for 1 h.

[0069] It was found that the Pluronic F127 hydrogel stock solution partially gelled and could not be fully mixed with the PA@Ca-UV bacterial suspension.

Claims

1. A method of preparing a vaccine against Pseudomonas aeruginosa, characterized by, Comprising the following steps: (1) Using polyacrylic acid in Pseudomonas aeruginosa ( Pseudomonas aeruginosa The electrostatic adsorption of the surface guides Ca 2+ and PO4 3- Heterogeneous nucleation occurs on the surface of Pseudomonas aeruginosa to form a complete calcium phosphate mineral shell, thus obtaining PA@Ca; (2) The PA@Ca obtained in step (1) is irradiated with ultraviolet rays to remove Pseudomonas aeruginosa that is not completely wrapped by the calcium phosphate mineral shell, and PA@Ca-UV is obtained; (3) The PA@Ca-UV obtained in step (2) is encapsulated in a hydrogel to obtain the anti-Pseudomonas aeruginosa vaccine.

2. The method for preparing the anti-Pseudomonas aeruginosa vaccine according to claim 1, characterized in that, In step (1), the Ca 2+ from calcium chloride, the PO4 3- from sodium hydrogen phosphate or sodium dihydrogen phosphate.

3. The method of claim 2, wherein the anti-P. aeruginosa vaccine is prepared by, In step (1), the P. aeruginosa suspension is uniformly mixed with a sodium polyacrylate aqueous solution, a calcium chloride aqueous solution, and a sodium hydrogen phosphate aqueous solution or a sodium dihydrogen phosphate aqueous solution to form a complete calcium phosphate mineral shell on the surface of the P. aeruginosa to obtain PA@Ca; wherein the concentration of the P. aeruginosa suspension is 1×10 8 ~2×10 9 CFU / mL, the mass concentration of the sodium polyacrylate aqueous solution is 25wt%-30wt%, the concentration of the calcium chloride aqueous solution is 0.1-1 mol / L, the concentration of the sodium hydrogen phosphate aqueous solution or the sodium dihydrogen phosphate aqueous solution is 0.1-1 mol / L, the volume ratio of the P. aeruginosa suspension to the sodium polyacrylate aqueous solution and the calcium chloride aqueous solution is 1:15-10:50-10, and the volume ratio of the P. aeruginosa suspension to the sodium hydrogen phosphate aqueous solution or the sodium dihydrogen phosphate aqueous solution is 1:20-25.

4. The method for preparing the anti-Pseudomonas aeruginosa vaccine according to claim 2, characterized in that, In step (1), the reaction temperature for forming the complete calcium phosphate mineral shell is 10-20 ℃, and the reaction time is not less than 1 hour.

5. The method for preparing the anti-Pseudomonas aeruginosa vaccine according to claim 1, characterized in that, In step (2), the wavelength of the ultraviolet light is 254-280 nm, the irradiance is 0.2-20 mW / cm 2 , and the irradiation time is not less than 0.5 hours.

6. The method of claim 1, wherein the anti-P. aeruginosa vaccine is prepared by, In step (3), the hydrogel is a Pluronic F-127 hydrogel.

7. The method of claim 6, wherein the anti-P. aeruginosa vaccine is prepared by, In step (3), the PA@Ca-UV obtained in step (2) is mixed with a Pluronic F-127 hydrogel solution at 4 ℃ to gel, the gelation time is not less than 1 hour, and the anti-Pseudomonas aeruginosa vaccine is obtained.

8. A vaccine against Pseudomonas aeruginosa, characterized in that, The anti-Pseudomonas aeruginosa vaccine is prepared by the method of any one of claims 1-7.

9. The use of the anti-Pseudomonas aeruginosa vaccine of claim 8 in the preparation of a drug for preventing Pseudomonas aeruginosa infection.

10. Use of the anti-Pseudomonas aeruginosa vaccine according to claim 9 for the preparation of a medicament for the prevention and / or treatment of Pseudomonas aeruginosa infections, characterized in that, The Pseudomonas aeruginosa infection is pneumonia.

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