Liposome-based toxoid vaccine, preparation method thereof, and application thereof in preventing bacterial infection

Abstract

The present invention discloses a liposome-based toxoid vaccine, a preparation method thereof and an application thereof in preventing bacterial infection. The vaccine comprises a lipid PS formed by fusing egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000, adsorbing the virulence factors sMV or sPV of MRSA or CRPA to form PSV, and encapsulating a polylactic acid-glycolic acid copolymer nanoparticle CNP containing CpG to form a toxoid vaccine PSV-CNP. Specifically, S1 fuses egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000 to form a lipid PS; S2 encapsulates the lipid PS with a polylactic acid-glycolic acid copolymer nanoparticle CNP containing CpG to form PS-CNP; S3 incubates PS-CNP with the virulence factors sMV or sPV of MRSA or CRPA to construct and obtain a toxoid vaccine PSV-CNP. The vaccine can have broad adaptability, avoid the immune risk of allogeneic substances, can significantly increase the abundance of virulence factors, can more effectively activate the anti-virulence factor immune response of the body, and enhance the body's ability to resist bacterial infection.

Description

Technical Field

[0001] The present invention relates to biomedicine, specifically to a liposome-based toxoid vaccine, a preparation method thereof, and its application in preventing bacterial infections. Background Art

[0002] Antibiotics are one of the most conventional and effective means for treating bacterial infections. However, the unreasonable use of antibiotics is accelerating the global bacterial resistance process, resulting in the emergence of Gram-positive bacteria represented by methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria represented by carbapenem-resistant Pseudomonas aeruginosa (CRPA). The problem of bacterial resistance significantly increases the treatment difficulty and cost of bacterial infections and adds to the global medical burden. Staphylococcus aureus , MRSA) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) Pseudomonas aeruginosa . The problem of bacterial resistance significantly increases the treatment difficulty and cost of bacterial infections and adds to the global medical burden.

[0003] Bacterial vaccines are an effective means for preventing bacterial infections, with characteristics such as no drug resistance selection pressure, excellent safety, and high efficiency. They can induce protective and long-lasting antibacterial immune responses in the host. Different from the simple structure of viruses, bacteria contain complex antigens, and the immunogenic potential of these antigens is usually unclear, which makes the research and development of bacterial vaccines more difficult than that of viral vaccines.

[0004] Currently, the research and development of bacterial vaccines mainly rely on attenuated or inactivated whole bacteria, but there are balance problems between safety and effectiveness. For example, live attenuated vaccines can induce strong immune responses but have an infection risk; while inactivated vaccines may lose key immunogenic components during the inactivation process, thus reducing the effectiveness of the vaccine.

[0005] In addition, whole proteins derived from bacteria have also been directly used for vaccination, but the risk of their side effects (such as severe inflammation or allergic reactions) is often greater than the benefits of the vaccine. Another feasible method is to use known virulence factors to construct virulence factor vaccines for vaccination, such as the tetanus vaccine and diphtheria vaccine currently in clinical use. Many bacteria secrete a large number of virulence factors, such as pore-forming toxins (PFTs), to help them survive in the host, making them important antibacterial immune materials. Although vaccines based on virulence factors have broad prospects, existing strategies face two major defects: (1) When preparing toxoid vaccines based on virulence factors, processes such as denaturation are required, making it difficult to achieve a balance between effectiveness and safety and affecting the immune effect; (2) The developed vaccines based on virulence factors lack multiple virulence factor antigens involved in the infection process, and the immune breadth is limited.

[0006] Cell membrane-based nano-toxoids have attracted much attention in recent years due to their applications in bacterial vaccine development. They capture and inactivate toxins using the plasma membrane of target cells, thereby improving the safety of toxoid vaccines without affecting immunogenicity, and better solving the defects of the above-mentioned toxoid vaccine strategies. However, some unnecessary membrane proteins on the cell membrane surface, such as the "don't eat me" signal protein CD47, not only occupy the space for toxins to bind to lipids, but may also reduce the recognition by antigen-presenting cells. Therefore, the efficiency of capturing bacterial virulence factors based on the cell membrane is limited, and its potential for anti-virulence factor immunotherapy cannot be fully exerted. In addition, the construction of vaccines based on the adsorption of virulence factors by the cell membrane is limited by individual differences, and vaccines must be prepared using the cells of the cell membrane provider itself, otherwise it may bring the risk of allogeneic immunity. In summary, it is imperative to develop a new type of toxoid vaccine platform that can enhance the capture of bacterial virulence factors and is more suitable for clinical translation. Summary of the Invention

[0007] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a liposome-based toxoid vaccine, its preparation method and its application in preventing bacterial infections, which can have broad adaptability, avoid the risk of allogeneic immunity, can significantly increase the abundance of virulence factors, can more effectively activate the anti-virulence factor immune response of the body, and enhance the body's ability to resist bacterial infections.

[0008] To achieve the above purpose, the present invention provides the following technical solution: A liposome-based toxoid vaccine, comprising a lipid PS formed by the fusion of egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000, adsorbing the virulence factors sMV or sPV of MRSA or CRPA to form PSV, and encapsulating a polylactic-co-glycolic acid copolymer nanoparticle CNP containing CpG to form a toxoid vaccine PSV-CNP.

[0009] A preparation method of a liposome-based toxoid vaccine, comprising the following steps

[0010] S1 Fuse egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000 to form a lipid PS;

[0011] S2 Encapsulate the lipid PS with a polylactic-co-glycolic acid copolymer nanoparticle CNP containing CpG to form PS-CNP;

[0012] S3 Incubate PS-CNP with the virulence factors sMV or sPV of MRSA or CRPA to construct and obtain the toxoid vaccine PSV-CNP.

[0013] As a further improvement of the present invention, in S1, the mass ratio of egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000 is 6:3:1 (w / w / w).

[0014] As a further improvement of the present invention, egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000 in S1 are mixed and dissolved in chloroform. After removing chloroform by rotary evaporation, the lipid film is hydrated with 10% sucrose solution to form a lipid PS suspension.

[0015] As a further improvement of the present invention, in S2, the lipid PS suspension and CNP are mixed at a lipid:PLGA mass ratio of 2:1 (w / w), and then PS-CNP is prepared by combining water bath ultrasound and probe ultrasound.

[0016] As a further improvement of the present invention, in S3, PS-CNP and virulence factor sMV or sPV are mixed and incubated at 37°C for 30 min to construct the toxoid vaccine PSV-CNP.

[0017] An application of a liposome-based toxoid vaccine in preventing bacterial infection, wherein the liposome-based toxoid vaccine platform adopts the liposome-based toxoid vaccine platform as described above.

[0018] An application of a liposome-based toxoid vaccine in preventing bacterial infection, wherein the liposome-based toxoid vaccine platform adopts the liposome-based toxoid vaccine obtained by the preparation method of the liposome-based toxoid vaccine according to any one of the above improvement schemes.

[0019] The beneficial effects of the present invention are as follows: a lipid structure imitating the cell membrane can be obtained for more effectively adsorbing bacterial virulence factors, so that the abundance of virulence factors in the toxoid vaccine is significantly increased, and the anti-virulence factor immune response of the body can be more effectively activated, enhancing the body's ability to resist bacterial infection. Since it does not involve the cell membrane, the lipid-based toxoid vaccine has broad adaptability and avoids the immune risk of allogeneic. To further enhance the response intensity of the immune system, CpG oligodeoxynucleotide (CpG ODN) is also introduced into the system as an immune adjuvant. CpG is a synthetic short single-stranded DNA fragment containing a specific non-methylated CpG dinucleotide sequence, which can specifically activate TLR9, thereby causing a strong immune response. Description of the Drawings

[0020] Figure 1 It is a diagram showing the difference in the ability of PS-NP and RM-NP to adsorb sMV and sPV;

[0021] Figure 2 It is a diagram showing the preparation and characterization of the PSV-CNP toxoid vaccine;

[0022] Figure 3 Evaluation diagram of PSV-CNP toxoid vaccine for enrichment in the lymph nodes of ICR mice;

[0023] Figure 4 Diagram of antibody titers against sMV, sPV, MRSA lysate, and CRPA lysate produced in ICR mice subcutaneously immunized with PSV-CNP toxoid vaccine;

[0024] Figure 5 Comparison diagram of subcutaneous and systemic infections of ICR mice subcutaneously immunized with PSV-CNP toxoid vaccine for the prevention of MRSA and CRPA. Detailed implementation mode

[0025] The present invention will be further described in detail below with reference to the embodiments given in the accompanying drawings.

[0026] This solution involves a lipid (PS) formed by fusing egg yolk phosphatidylcholine (PC-98T), sphingomyelin (Sm), and lipid-modified polyethylene glycol (DSPE-mPEG2000) for adsorbing virulence factors of MRSA and CRPA (referred to as sMV and sPV respectively) to form (PSV), and simultaneously encapsulating a poly (lactic-co-glycolic acid) nanoparticle (CNP) containing CpG to construct a novel toxoid vaccine (PSV-CNP). The prepared PSV-CNP toxoid vaccine can activate ICR mice to produce neutralizing antibodies against sMV and sPV, and enhance the ability of mice to resist MRSA and CRPA infections.

[0027] In this solution, PC-98T, Sm, and DSPE-mPEG2000 are mixed in a ratio of 6:3:1 (w / w / w) and dissolved in chloroform. The chloroform is removed by rotary evaporation, and the lipid membrane is hydrated with a 10% sucrose solution to form a PS hybrid lipid suspension. Subsequently, the PS suspension and CNP are mixed at a lipid:PLGA mass ratio of 2:1 (w / w), and PS-CNP is prepared by first using water bath ultrasound combined with probe ultrasound. Subsequently, the extracted and separated sMV and sPV are respectively mixed with PS-CNS, incubated at 37°C for 30 min, and then centrifuged. After removing the supernatant, the toxoid vaccine PSV-CNP is obtained. The enrichment of the toxoid vaccine can be detected in the nearby lymph nodes 24 h after subcutaneous injection of the toxoid vaccine into ICR mice. After three subcutaneous immunizations with the toxoid vaccine, a significant increase in the antibody titers against sMV and sPV in the mouse serum can be detected. Finally, the preventive effect of the toxoid vaccine against MRSA and CRPA infections in ICR mice is detected through MRSA and CRPA subcutaneous and systemic infection models (bacteremia models).

[0028] Specifically:

[0029] 1. Preparation of CNP

[0030] CNP was prepared by encapsulating CpG into PLGA nanoparticles using the double emulsion method. 0.5 mL of a 50 mg / mL PLGA dichloromethane solution was prepared. 50 μL of a 1×TE buffer containing CpG was mixed with 50 μL of a 400 mM Tris buffer and then added to the 0.5 mL PLGA solution. The mixture was emulsified by ultrasound (80 W, 1 min, 2 sec on / 1 sec off) to form a primary emulsion. Subsequently, the primary emulsion was added to 5 mL of a 10 mM Tris buffer (pH = 8.0) and sonicated again to form a double emulsion. Then, the double emulsion was added to 10 mL of a 10 mM Tris buffer (pH = 8.0), and the dichloromethane was removed by stirring and evaporation for 180 min. The final volume of the liquid was about 15 mL, and the Tris solution of CNP was obtained. PLGA nanoparticles (NP) without CpG were prepared using Tris buffer without CpG according to the above procedure.

[0031] 2. Preparation of virulence factors (sMV, sPV) of MRSA and CRPA

[0032] MRSA USA300 was streaked on a TSB solid medium and cultured for 30 h. Then, a single colony was picked and transferred to 4 mL of TSB liquid medium and cultured for another 12 h. Subsequently, the 4 mL bacterial solution was transferred to 200 mL of TSB liquid medium and cultured at a speed of 200 rpm / min for 24 h. The bacterial supernatant was collected by centrifugation and filtered through a 0.2 μm filter membrane for later use. Subsequently, the collected supernatant was freeze-dried and then resuspended in 1 / 10 volume of deionized water to prepare a 10× supernatant, which was the MRSA virulence factor (sMV) and stored at -80 °C for later use.

[0033] CRPA was streaked on a TSB solid medium and cultured for 24 h. Then, a single colony was picked and transferred to 5 mL of TSB liquid medium and cultured at a shaking speed of 200 rpm / min for 12 h. Then, the bacterial solution was transferred to 500 mL of fresh TSB liquid medium and cultured at a speed of 200 rpm / min for 24 h. The bacterial supernatant was collected by centrifugation and filtered through a 0.2 μm filter membrane for later use. Subsequently, the collected supernatant was freeze-dried and then resuspended in 1 / 10 volume of deionized water to prepare a 10× supernatant, which was the CRPA virulence factor (sPV) and stored at -80 °C for later use.

[0034] 3. Study on the difference in the ability of PS-NP and RM-NP to adsorb sMV and sPV

[0035] Prepare the PS liposome suspension using the thin film hydration method. Dissolve 60 mg of PC-98T (PC), 30 mg of sphingomyelin (Sm), and 10 mg of lipid-modified PEG (DSPE-mPEG2000) in chloroform. Evaporate the chloroform completely by rotary evaporation. Subsequently, add 4.5 mL of sterile 10% sucrose solution and hydrate the lipid film by washing it off at 60 °C to obtain a 20 mg / mL PS liposome suspension. Then, add 1 mL of the 20 mg / mL PS liposome suspension to the Tris solution of NP such that the ratio of lipid to NP is 2:1 (w / w). First, use a water bath sonicator and then a probe sonicator (80 W, 5 min, 2 sec on / 3 sec off) to completely encapsulate NP with liposomes to form PS-NP. Meanwhile, in this application, red blood cell membrane (RM) is used to replace PS liposomes to prepare RM-NP as a control group.

[0036] Subsequently, determine the dose of sMV or sPV that causes HD100 (100% hemolysis dose) of ICR mouse red blood cells. Isolate RBC from the whole blood of ICR mice and dilute the red blood cells to 5% (v / v) with PBS for later use. Add different doses of sMV or sPV to 100 μL of 5% RBC suspension (v / v) respectively, and at the same time, make up the total volume of the system to 160 μL with PBS. After incubating at 37 °C for 30 min, centrifuge at 13,000 rpm for 5 min, and collect the supernatant to measure the absorbance at 540 nm. Among them, the RBC group disrupted by sonication serves as a positive control, and the group without any treatment serves as a negative control. The results show that as the dose of sMV or sPV increases, the hemolytic toxin continuously enhances. sMV can cause complete hemolysis of 100 μL of 5% RBC at a ratio of 2.5% (v / v), while sPV can cause complete hemolysis of 100 μL of 5% RBC at a ratio of 1.67% (v / v). The doses of the above two virulence factors that cause complete hemolysis are called HD100 ( Figure 1 a).

[0037] The sMV or sPV dose of ICR mouse RBC HD100 was incubated with different concentrations of RM-NP or PS-NP at 37 °C for 30 min, and then added to 100 μL of 5% RBC suspension (v / v), and the total volume was made up to 160 μL. After incubation at 37 °C for 30 min, it was centrifuged at 13,000 rpm for 5 min, and the supernatant was collected to measure the absorbance at 540 nm to determine the neutralizing effect of different doses of RM-NP or PS-NP on the virulence factors sMV or sPV. The results showed that as the added dose of RM-NP or PS-NP increased, the hemolytic ability caused by sMV (HD100) decreased continuously, and it could be completely neutralized until the lipid concentration reached 0.78 mg / mL, while the lipid concentration of RM-NP required to completely neutralize sPV was 0.83 mg / mL and that of PS-NP was 0.21 mg / mL ( Figure 1 b). The dose that completely neutralizes the hemolytic toxicity of sMV and sPV is called IC100 (100% inhibitory concentration). The group of RBCs disrupted by ultrasound was used as the positive control, and the group without any treatment was used as the negative control.

[0038] According to IC100:HD100, the RM-NP or PS-NP:sMV or sPV dose was incubated at 37 °C for 30 min, and then the dose of sMV or sPV (made up to 100 μL) was continuously increased and added to the 96-well plate of endothelial cells HUVEC (80% confluence rate), and after continued culture for 24 h, the cell viability was detected using CCK-8. This experiment detected the neutralizing and adsorbing effects of RM-NP or PS-NP on other types of toxins such as endothelial cells in addition to adsorbing hemolytic-related toxins at the cell level. The results showed that although the dose of RM-NP could completely neutralize the hemolytic toxins in sMV or sPV, it could not neutralize the toxins against endothelial cells; while PS-NP could not only effectively neutralize the hemolytic toxins in sMV or sPV, but also effectively neutralize the toxins against endothelial cells ( Figure 1 c), that is, it was proved at the cell level that the neutralizing ability of PS-NP to sMV or sPV was significantly higher than that of RM-NP.

[0039] Subsequently, the present invention used proteomics technology to analyze the types and abundances of different virulence factors in RM-NP or PS-NP adsorbing sMV or sPV (the ratio was still their respective IC100:HD100). The results showed that the adsorption ability of PS-NP for virulence factors in sMV or sPV was much greater than that of RM-NP.

[0040] 4. Preparation and characterization of PSV-CNP toxoid vaccine

[0041] First, determine the dose relationship between PS-CNP and sMV in the vaccine. Mix PS-CNP (1 mg / mL) with different doses of sMV and incubate at 37 °C for 30 min to allow PS-CNP to adsorb sMV to form the PSV-CNP toxoid vaccine. The results show that as the dose of sMV increases, the protein content in PSV-CNP also continuously increases (indicating that sMV is adsorbed onto the surface of PS-NP). At the same time, the particle size of PSV-CNP under physiological conditions also continuously climbs, meaning that the disruption of the PS membrane structure by sMV may lead to the exposure of internal PLGA nanoparticles and aggregation ( Figure 2 a). This speculation was confirmed by observing the mixture of PSV-CNP and different doses of sMV through a transmission electron microscope, that is, as the dose of sMV increased from 2.5 to 10 μL, the disruption of PS-CNP became more severe, resulting in the aggregation or degradation and deformation of internal PLGA under physiological conditions ( Figure 2 b). Based on the above results, determine the ratio of PS-CNP to sMV as 1 mg / mL: 2.5‰ (v / v). Use a particle size and zeta potential analyzer to measure the hydrodynamic size of PSV-CNP to be approximately 200 nm and the surface potential to be approximately -15 mV. At the same time, measure the particle size and potential of control groups such as PS-Lipo, CNP, and PS-CNP ( Figure 2 c). Meanwhile, in this application, a nano-vaccine without CpG (PSV-NP) is prepared, and RMV-CNP and RMV-NP without CpG are prepared for subsequent research. Similarly, use the above ratio to determine the ratio of PS-CNP to sPV and prepare the PSV-CNP toxoid vaccine against CRPA infection.

[0042] 5. Evaluation of the enrichment of PSV-CNP toxoid vaccine in the lymph nodes of ICR mice

[0043] Anesthetize ICR mice (6 - 8 weeks old) by inhaling isoflurane. Inject 125 μL of the PSV-CNP toxoid vaccine prepared from sMV (IR780 near-infrared dye-labeled lipid or cell membrane, 5 mg / mL) subcutaneously into the neck of the mice. After 24 h, euthanize the mice and open the skin of the mice to fully expose the systemic lymph nodes of the mice. Use an IVIS small animal in vivo imaging system to perform fluorescence imaging on the lymph nodes of the mice, and perform quantitative statistical analysis on the fluorescence at the lymph nodes of the mice. The experimental settings include experimental groups such as PSV-CNP, RMV-CNP, PSV-NP, RMV-NP, and an equal volume of PBS. The results show that PSV-CNP can enter the lymph node site more efficiently, which is beneficial for the maturation of B cells in the lymph nodes to produce neutralizing antibodies ( Figure 3 a - b).

[0044] 6. Study on the antibody titers against sMV, sPV, MRSA lysate and CRP lysate produced by subcutaneous immunization of ICR mice with PSV-CNP toxoid vaccine

[0045] ICR mice (6 - 8 weeks old) were anesthetized with isoflurane. 125 μL of the toxoid vaccine PSV-CNP (5 mg / mL) prepared from sMV or sPV was injected subcutaneously into the necks of the mice. The vaccination frequency was as follows: the first vaccination was completed on day 0, and then two booster vaccinations were carried out on days 7 and 14 respectively. On day 21, whole blood of ICR mice was collected, and serum was collected by centrifugation at 2000 g for 10 min. Antibody titer analysis was performed by ELISA. First, sMV (100 μL / well), sPV (100 μL / well), MRSA lysate and CRP lysate (10 μg / mL) were coated onto a 96-well ELISA plate using ELISA coating buffer and incubated overnight at 4°C. Subsequently, after washing the ELISA plate 3 times with PBS, it was blocked with 1% BSA for 1 h, and then mouse serum samples with different dilution factors (10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 ) were added and incubated for 2 h at room temperature. After washing 5 times with PBS, HRP-conjugated secondary antibody at a dilution of 1:200 was added and incubated for 1 h at room temperature. After rinsing 5 times with PBS, 100 μL of TMB substrate was added to each well and incubated for 8 min. Subsequently, 100 μL of ELISA stop solution was added to end the reaction. The absorbance was measured at 450 nm using a microplate reader. In this experiment, RMV-CNP, PSV-NP, RMV-NP and an equal volume of PBS were set as control groups. The results showed that PSV-CNP could immunize ICR mice to produce higher antibody titers against sMV, sPV, MRSA lysate and CRP lysate ( Figure 4 a - b).

[0046] 7. Use of PSV-CNP toxoid vaccine for subcutaneous immunization of ICR mice to prevent subcutaneous and systemic infections of MRSA and CRP

[0047] ICR mice were immunized according to the experimental procedure described in 4 to produce antibodies against sMV, MRSA lysate, sPV and CRP lysate. On day 21 after the primary immunization, 2 × 10 6CFU of MRSA bacteria or CRPA bacteria, and record the survival of the infected mice within 7 days and analyze the number of bacteria in the skin at the infection site on the 7th day. In this experiment, RMV-CNP, PSV-NP, RMV-NP and an equal volume of PBS were set as the control groups. The results showed that immunizing ICR mice with PSV-CNP could significantly reduce the number of bacteria in the infected skin site and prolong the survival rate of ICR mice, effectively preventing MRSA and CRPA infections ( Figure 5 ).

[0048] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the idea of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and retouches made without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A liposome-based toxoid vaccine, characterized in that, It includes lipid PS formed by the fusion of egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000, adsorbs the virulence factors sMV or sPV of MRSA or CRPA to form PSV, and encapsulates the polylactic acid-glycolic acid copolymer nanoparticles CNP containing CpG to form the toxoid vaccine PSV-CNP.

2. A preparation method of a liposome-based toxoid vaccine, characterized in that, It includes the following steps S1: Fuse egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000 to form lipid PS; S2: Encapsulate the polylactic acid-glycolic acid copolymer nanoparticles CNP containing CpG with lipid PS to form PS-CNP; S3: Incubate PS-CNP with the virulence factors sMV or sPV of MRSA or CRPA to obtain the toxoid vaccine PSV-CNP.

3. The preparation method of the liposome-based toxoid vaccine according to claim 2, wherein In S1, the mass ratio of egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000 is 6:3:1 w / w / w.

4. The preparation method of the liposome-based toxoid vaccine according to claim 3, wherein In S1, egg yolk phosphatidylcholine PC-98T, sphingomyelin Sm, and lipid-modified polyethylene glycol DSPE-mPEG2000 are mixed and dissolved in chloroform, the chloroform is removed by rotary evaporation, and the lipid film is hydrated with 10% sucrose solution to form a lipid PS suspension.

5. The preparation method of the liposome-based toxoid vaccine according to claim 2, wherein In S2, the lipid PS suspension and CNP are mixed at a lipid:PLGA mass ratio of 2:1 w / w, and then PS-CNP is prepared by combining water bath ultrasound and probe ultrasound.

6. The preparation method of the liposome-based toxoid vaccine according to claim 2, wherein In S3, PS-CNP and the virulence factor sMV or sPV are mixed and incubated at 37°C for 30 min to construct the toxoid vaccine PSV-CNP.

7. Use of a liposome-based toxoid vaccine platform in the preparation of a medicament for preventing MRSA and CRPA bacterial infections, characterized in that, The liposome-based toxoid vaccine platform adopts the liposome-based toxoid vaccine platform as described in claim 1.

8. Use of a liposome-based toxoid vaccine platform in the preparation of a medicament for preventing MRSA and CRPA bacterial infections, characterized in that, The liposome-based toxoid vaccine platform adopts the liposome-based toxoid vaccine obtained by the preparation method of the liposome-based toxoid vaccine as described in any one of claims 2 to 6.

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