Application of pseudomonas aeruginosa membrane vesicles in prevention of acute pneumonia

Abstract

The invention relates to the field of biological medicine, in particular to application of pseudomonas aeruginosa membrane vesicles to preparation of medicine for treating and / or preventing acute pneumonia caused by pseudomonas aeruginosa in subjects. The pseudomonas aeruginosa membrane vesicle provided by the invention can effectively eliminate pseudomonas aeruginosa, eliminate inflammatory response caused by the pseudomonas aeruginosa, relieve the symptom of acute pneumonia caused by the pseudomonas aeruginosa and protect the lung function of a subject.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to the use of Pseudomonas aeruginosa membrane vesicles in the preparation of a drug for treating and / or preventing acute pneumonia caused by Pseudomonas aeruginosa in a subject. Background Art

[0002] Pseudomonas aeruginosa is an obligate aerobic Gram-negative bacillus widely distributed in nature. Pseudomonas aeruginosa is an acquired opportunistic pathogen that widely exists in the environment and can be obtained from human skin, medical devices, air, water, and food. Pseudomonas aeruginosa has very low nutritional requirements for survival and can survive in a variety of environments. This strong environmental adaptability and survival ability help it survive on dry, inanimate surface environments such as hospitals for 6 hours to 6 months. Pseudomonas aeruginosa can infect almost any part and organ of the human body, and severe acute or chronic infections will lead to the death of the body.

[0003] Infection with Pseudomonas aeruginosa is considered to be one of the most difficult pathogenic infections to treat, and clinical treatment of lower respiratory tract infections (such as acute pneumonia) caused by Pseudomonas aeruginosa faces increasing challenges. On the one hand, Pseudomonas aeruginosa itself has low sensitivity to existing antibiotics and is also prone to developing resistance to a variety of antibiotics. On the other hand, Pseudomonas aeruginosa is extremely easy to colonize in the lower respiratory tract (such as the lungs) and form a biofilm with extremely strong antibiotic resistance, which seriously increases the difficulty of clearing Pseudomonas aeruginosa in patients infected with Pseudomonas aeruginosa, and even directly causes Pseudomonas aeruginosa in some patients to be unable to be completely cleared.

[0004] The prior art believes that, compared with other pathogenic bacteria, once Pseudomonas aeruginosa colonizes in the lower respiratory tract (such as the lungs), it is very difficult to be cleared. Therefore, acute pneumonia caused by Pseudomonas aeruginosa (especially in patients with weakened immunity, such as patients with metabolic diseases, blood diseases, and malignant tumors, as well as postoperative or certain treated patients (such as cancer, extensive burns, HIV, etc.)) has extremely high morbidity and mortality rates. If the Pseudomonas aeruginosa colonizing in the respiratory tract cannot be effectively cleared, the lower respiratory tract infection of the patient will recur repeatedly, seriously affecting the physical and mental health of the patient and even threatening death.

[0005] For Pseudomonas aeruginosa infection, antibiotic drugs are still widely used clinically at present. Although antibiotics are effective in treating various bacterial infections, the abuse and improper use of antibiotics are likely to cause drug side effects and the emergence of multi-drug resistant bacteria. Moreover, the development of new antibiotics is not only time-consuming but also has very limited development potential. After Pseudomonas aeruginosa colonizes, the formed biofilm not only enhances the vitality of Pseudomonas aeruginosa but also greatly reduces the effectiveness of conventional treatment methods. In addition, the problem of drug resistance of Pseudomonas aeruginosa is constantly worsening, leading to a decrease or even complete failure of the efficacy of antibiotics. In recent years, new antibacterial drugs and new treatment methods have been developed to attempt to treat and / or prevent Pseudomonas aeruginosa infection (such as acute pneumonia caused by Pseudomonas aeruginosa). However, due to reasons such as their far lower effects in in vivo experiments than in in vitro experiments, production difficulties, and administration methods, their clinical applications are subject to considerable limitations. In addition, although many new antibacterial drugs and new treatment methods show a certain degree of antibacterial effect, they can only transiently reduce the number of Pseudomonas aeruginosa colonizing the lungs to a certain extent and cannot achieve their true clearance. For example, CN113134004A discloses the use of 3-methyladenine in the preparation of a drug for preventing Pseudomonas aeruginosa-induced acute pneumonia. Although it can reduce the number of Pseudomonas aeruginosa colonizing the lungs to a certain extent and transiently improve the survival rate of experimental animals, the survival rate of experimental animals will further decrease 3 days after infection, indicating that it does not substantially clear or effectively control the Pseudomonas aeruginosa colonizing the lungs, and the Pseudomonas aeruginosa infection will instead worsen again. Summary of the Invention

[0006] The present invention provides a use of Pseudomonas aeruginosa membrane vesicles in the preparation of a drug for treating and / or preventing acute pneumonia caused by Pseudomonas aeruginosa in a subject, characterized in that the drug is used for treating and / or preventing the colonization and / or infection of Pseudomonas aeruginosa in the lungs of the subject.

[0007] In some embodiments, the Pseudomonas aeruginosa membrane vesicles (abbreviated as XMVs in the present invention) are obtained by subjecting a Pseudomonas aeruginosa suspension to X-ray irradiation treatment and then centrifuging by ultra-high speed centrifugation and eluting by size exclusion chromatography.

[0008] In some embodiments, the Pseudomonas aeruginosa strain includes Pseudomonas aeruginosa PAO1. The present invention attempts to purify the Pseudomonas aeruginosa membrane vesicles produced by the relatively common Pseudomonas aeruginosa PAO1 strain in current research by other methods (such as differential centrifugation and density gradient centrifugation) and evaluate their immunoprotective effects. The results show that the immunoprotective effects of the Pseudomonas aeruginosa membrane vesicles purified by other methods are inferior to those of the Pseudomonas aeruginosa membrane vesicles (XMVs) of the present invention.

[0009] In some embodiments, the specific method of the X-ray irradiation treatment includes: irradiating the Pseudomonas aeruginosa suspension with X-rays, and the total irradiation dose includes 980–1000 Gy. In some embodiments, the OD 600 value of the Pseudomonas aeruginosa suspension includes approximately 40–60. In some embodiments, the OD 600 value of the Pseudomonas aeruginosa suspension includes approximately 50.

[0010] In some embodiments, the method of the ultra-high-speed centrifugation method specifically includes: centrifuging the Pseudomonas aeruginosa suspension irradiated with the X-rays at an ultra-high-speed centrifugation rate of 5000–150000 g for 60–600 min.

[0011] In some embodiments, the Pseudomonas aeruginosa suspension also sequentially undergoes a centrifugation step and a high-speed centrifugation step before undergoing the ultra-high-speed centrifugation, specifically including: centrifuging the Pseudomonas aeruginosa suspension irradiated with the X-rays at 100–10000 g for 10–60 min and collecting the first supernatant (to remove Pseudomonas aeruginosa cells); centrifuging the first supernatant at a high speed of 5000–25000 g for 10–100 min and collecting the second supernatant (to remove flagella and most cell debris). Next, centrifuging the second supernatant at an ultra-high-speed centrifugation rate of 5000–150000 g for 60–600 min to obtain a Pseudomonas aeruginosa membrane vesicle precipitate. To better remove the Pseudomonas aeruginosa cells, a first filtration step may also be performed between the centrifugation step and the high-speed centrifugation step or after the high-speed centrifugation step. In some embodiments, the first filtration step includes filtering the first supernatant with a 0.3–0.5 μM filter (such as a 0.45 μM filter).

[0012] In some embodiments, the size exclusion chromatography method specifically includes: loading the Pseudomonas aeruginosa membrane vesicle suspension (prepared by resuspending the Pseudomonas aeruginosa membrane vesicle precipitate with a buffer solution) onto a qEV chromatographic column equilibrated with a buffer solution, and collecting the components eluted after the void volume. In some embodiments, the buffer solution includes phosphate buffer or physiological saline. In some embodiments, the volume ratio of the Pseudomonas aeruginosa suspension to the Pseudomonas aeruginosa membrane vesicle suspension includes 25–35:1. In some embodiments, the volume ratio of the Pseudomonas aeruginosa suspension to the Pseudomonas aeruginosa membrane vesicle suspension includes 30:1. In some embodiments, the separation range of the qEV chromatographic column includes 35 nm.

[0013] In some embodiments, before the size exclusion chromatography step, a second filtration step may also be performed on the Pseudomonas aeruginosa membrane vesicle suspension. In some embodiments, the second filtration step includes filtering the Pseudomonas aeruginosa membrane vesicle suspension through a 0.3 - 0.5 μM filter (such as a 0.45 μM filter).

[0014] The present invention elutes a Pseudomonas aeruginosa membrane vesicle suspension at an appropriate concentration using a qEV chromatography column with an appropriate separation range (such as 35 nm) to prepare the Pseudomonas aeruginosa membrane vesicles (XMVs) of the present invention. Unexpectedly, the method of the present invention can prepare Pseudomonas aeruginosa membrane vesicles mainly composed of intracellular proteins (i.e., most proteins are located in the cytoplasm rather than outer membrane proteins), and the Pseudomonas aeruginosa membrane vesicles of the present invention show excellent immune protection in in vivo experiments. On the contrary, a qEV chromatography column with a separation range of 70 nm may exclude the Pseudomonas aeruginosa membrane vesicles (XMVs) of the present invention, and the Pseudomonas aeruginosa membrane vesicles (XMVs) of the present invention cannot be obtained.

[0015] In addition, the present invention also found that filtering the Pseudomonas aeruginosa membrane vesicle suspension first and then purifying it by size exclusion chromatography can synergistically exclude inappropriate-sized impurities (such as macromolecular proteins) from the Pseudomonas aeruginosa membrane vesicles (XMVs), further ensuring that the proteins of the eluted Pseudomonas aeruginosa membrane vesicles (XMVs) are mainly composed of cytoplasmic proteins.

[0016] In some embodiments, the volume of each component is 500 μL.

[0017] In some embodiments, the drug is used for treating and / or preventing the colonization of Pseudomonas aeruginosa in the lungs of the subject.

[0018] In some embodiments, the drug is used for treating and / or preventing the colonization of Pseudomonas aeruginosa in the lungs of the subject at a level of approximately 10 8 CFU / mL.

[0019] In some embodiments, the drug is used for substantially clearing the colonization of Pseudomonas aeruginosa in the lungs of the subject.

[0020] In some embodiments, a single dose of the drug includes approximately 10 8 to 10 11 of the Pseudomonas aeruginosa membrane vesicles.

[0021] In some embodiments, a single dose of the drug can be approximately 6.0×10 8 , 6.0×10 9 , 3.0×10 10individuals, 6.0×10 10 individuals, 1.2×10 11 individuals or 3.0×10 11 of the Pseudomonas aeruginosa membrane vesicles.

[0022] In some embodiments, the single dose of the drug can be about 6.0×10 8 of the Pseudomonas aeruginosa membrane vesicles.

[0023] In some embodiments, the drug comprises at least one dose.

[0024] Surprisingly, the Pseudomonas aeruginosa membrane vesicles (XMVs) prepared by the present invention have extremely strong immune protection. When the single dose is about 6.0×10 8 (about 0.1 μg) and immunized only once, it can effectively clear 99% of the Pseudomonas aeruginosa colonized in the lungs. When the single dose is increased, the Pseudomonas aeruginosa membrane vesicles prepared by the present invention can further effectively clear 99.99% of the Pseudomonas aeruginosa colonized in the lungs.

[0025] In some embodiments, the drug comprises 1 dose, 2 doses or 3 doses.

[0026] In some embodiments, the administration interval for each dose includes 7 - 28 days.

[0027] In some embodiments, the administration interval for each dose includes 14 days.

[0028] In some embodiments, the drug is an intramuscular or subcutaneous preparation.

[0029] In some embodiments, the drug is a vaccine.

[0030] In some embodiments, the drug is used for treating and / or preventing one or more of the following symptoms of acute pneumonia caused by Pseudomonas aeruginosa: alveolar rupture, vascular leakage, immune cell infiltration. The present invention discovers that the Pseudomonas aeruginosa membrane vesicles prepared by the present invention can effectively clear Pseudomonas aeruginosa, clear the inflammatory reaction caused by Pseudomonas aeruginosa, relieve the symptoms of acute pneumonia caused by Pseudomonas aeruginosa and protect the lung function of the subject.

[0031] In some embodiments, the drug is used to extend the survival time of the subject. The Pseudomonas aeruginosa membrane vesicles prepared in the present invention can effectively eliminate Pseudomonas aeruginosa that is challenged with a sub-lethal dose or a lethal dose and colonizes in the lungs at an extremely low dose (e.g., about 0.1 μg). In addition, it should be emphasized that all the experimental animals in the control group died 3 days after the challenge, while the survival rates of the experimental animals in each group immunized with the Pseudomonas aeruginosa membrane vesicles prepared in the present invention were almost able to remain at 100% 3 days after the challenge. Even as the observation time was extended (e.g., 10 days after the challenge), the survival rates of the experimental animals in each immunized group were still able to remain at 100%, and the experimental animals had bright hair and good mental state. The above experimental results indicate that the Pseudomonas aeruginosa membrane vesicles prepared in the present invention can substantially eliminate the Pseudomonas aeruginosa colonized in the lungs, reduce the risk of re-infection of Pseudomonas aeruginosa (including acute infection and chronic infection), and have good safety and low toxicity and side effects.

[0032] In some embodiments, the subject is a mammal.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects:

[0034] 1. The present invention selects the commonly used Pseudomonas aeruginosa strain PAO1, and centrifuges the Pseudomonas aeruginosa membrane vesicles produced by it by ultra-high speed centrifugation and purifies them by size exclusion chromatography to prepare XMVs with most of the protein components being intracellular proteins. The results show that when only a very low single dose (e.g., about 6.0×10 8 XMVs, 0.1 μg) is immunized once, the XMVs of the present invention can substantially eliminate 99% of the Pseudomonas aeruginosa colonized in the lungs. When the single dose is increased, the XMVs of the present invention can further substantially eliminate 99.99% of the Pseudomonas aeruginosa colonized in the lungs. The above experimental results also indicate that the Pseudomonas aeruginosa membrane vesicles prepared in the present invention have good safety, and even if the single dose is further increased, no obvious toxicity and side effects will be caused.

[0035] 2. The Pseudomonas aeruginosa membrane vesicles prepared in the present invention can effectively eliminate Pseudomonas aeruginosa that is challenged with a sub-lethal dose or a lethal dose and colonizes in the lungs at an extremely low dose. Compared with the control group with a mortality rate of 100%, the Pseudomonas aeruginosa membrane vesicles prepared in the present invention can significantly extend the survival time and survival rate of the subject (and even the survival rate can be maintained at 100% for a long time).

[0036] 3. It has been verified that the Pseudomonas aeruginosa membrane vesicles prepared in the present invention are also applicable to the treatment and / or prevention of acute infections caused by other serotypes of Pseudomonas aeruginosa, such as multi-drug resistant Pseudomonas aeruginosa W9 (serotype O20).

[0037] 4. Although the preparation process of the Pseudomonas aeruginosa membrane vesicles prepared by the present invention is relatively simple, in vivo experiments have proved that the Pseudomonas aeruginosa membrane vesicles provided by the present invention have extremely strong immunoprotective effects and are suitable for industrial production and actual clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0039] Figure 1 It is a result diagram of the preparation and characterization of XMVs;

[0040] Figure 2 It is a result diagram of the pulmonary bacterial load of each group of mice after immunization with different doses of XMVs (acute infection with sub-lethal dose of Pseudomonas aeruginosa);

[0041] Figure 3 It is a result diagram of the survival curve of each group of mice after immunization with different doses of XMVs (acute infection with lethal dose of Pseudomonas aeruginosa);

[0042] Figure 4 It is a result diagram of the body weight change curve of each group of mice after immunization with different doses of XMVs (acute infection with lethal dose of Pseudomonas aeruginosa);

[0043] Figure 5 It is a result diagram of the pulmonary bacterial load of each group of mice after immunization with different doses of XMVs (acute infection with sub-lethal dose of Pseudomonas aeruginosa);

[0044] Figure 6 It is a result diagram of the survival curve of each group of mice after immunization with different doses of XMVs (acute infection with lethal dose of Pseudomonas aeruginosa);

[0045] Figure 7 It is a result diagram of the evaluation of the pulmonary pathological changes in the acute pneumonia model of mice after immunization with XMVs. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0047] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0048] As used herein, "a plurality of" means two or more, i.e., it includes two, three, four, five, etc.

[0049] It should be noted that, as used herein, the term "comprises", "comprising", or any other variation thereof is intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a series of elements includes not only those elements but also other elements not expressly listed, or elements that are inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0050] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0051] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of the range 1 - 6 should be regarded as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0052] Example 1

[0053] Preparation of XMVs

[0054] 1.1 Culturing and Collecting Bacteria

[0055] (1) Take out the Pseudomonas aeruginosa PAO1 strain from the -80 °C ultra-low temperature freezer and streak it onto a TSA plate, and culture it overnight (16 - 18 h) at 37 °C.

[0056] (2) Pick a single colony and inoculate it into 100 mL of TSB, and culture it overnight (16 - 18 h) at 37 °C and 220 rpm.

[0057] (3) Take the overnight bacterial solution and measure its OD600 value, calculate the overnight bacterial liquid concentration, and inoculate it into 8 L of TSB medium to make the initial OD 600 = 0.05, and culture at 37 °C and 220 rpm until the logarithmic phase (OD 600 is 0.4 - 0.8).

[0058] (4) Centrifuge the above bacterial liquid (4000 rpm, 30 min), collect the bacterial cells and resuspend them with 0.9% sodium chloride injection, and adjust OD 600 , the specific method is as follows: zero adjustment, take two test tubes and add 3 ml of TSB liquid medium to each, and zero adjust OD 600 ; measurement, add an appropriate amount of bacterial suspension in the logarithmic phase to the test tube, measure OD 600 value, multiply the measured OD 600 value by the dilution factor, which is the actual OD 600 value of the bacterial suspension. Take 100 μL of the bacterial suspension, serially dilute it 10-fold with 0.9% sodium chloride injection and calculate the viable bacteria count.

[0059] 1.2 X-ray irradiation

[0060] Take 30 mL of the bacterial suspension in the above (4) (OD 600 is approximately 50) into a 50 mL sterile centrifuge tube and place it in an irradiator. The parameters of the irradiator include: 160 kV / 25 mA, the dose rate is 7.068 Gy / min, and the total irradiation dose is 1 kGy.

[0061] 1.3 Isolation of Pseudomonas aeruginosa membrane vesicles

[0062] Centrifuge the irradiated bacterial suspension (in this example, specifically 8,000 g, 30 min, 4 °C), and collect the first supernatant (to remove the bacterial cells). Filter the first supernatant through a 0.45 μm filter (Millipore) and then centrifuge it at high speed (in this example, specifically 16,000 g, 30 min, 4 °C), and collect the second supernatant (to remove flagella and most cell debris). Then, ultracentrifuge the second supernatant (in this example, specifically 100,000 g, 2 h, 4 °C) to obtain the precipitate of Pseudomonas aeruginosa membrane vesicles. Resuspend the precipitate of Pseudomonas aeruginosa membrane vesicles with 1 mL of sterile PBS (pH 7.2) and filter it through a 0.45 μm filter (Millipore) again to obtain an enriched suspension of Pseudomonas aeruginosa membrane vesicles.

[0063] 1.4 Preparation of XMVs

[0064] In this example, size exclusion chromatography (specifically, using a qEVoriginal Column (IZON, New Zealand) with a separation range of 35 nm) was used to further purify the enriched Pseudomonas aeruginosa membrane vesicle suspension.

[0065] (1) Place the qEV chromatography column on a support and make it horizontal (ensure the qEV chromatography column is vertical), and prepare a 50 mL centrifuge tube to collect the void volume.

[0066] (2) After the qEV chromatography column is equilibrated at room temperature, leave the Luer slip cap at the bottom in place, carefully remove the top cap, and replace the electrolyte in the column top with PBS.

[0067] (3) Remove the bottom Luer slip cap and rinse the qEV chromatography column with at least 10 mL of PBS.

[0068] (4) Pipette 500 μL of the enriched Pseudomonas aeruginosa membrane vesicle suspension and slowly add it above the chromatography column, while collecting the eluate with a 5 mL centrifuge tube below the chromatography column. When the Pseudomonas aeruginosa membrane vesicle suspension completely enters the chromatography column, promptly supplement PBS (to avoid drying of the chromatography column). After collecting 3 mL of eluate (take 50 μL as sample 1), use a centrifuge tube to collect 500 μL of the eluted Pseudomonas aeruginosa membrane vesicle fraction (take 50 μL as sample 2), continue to collect 1 mL of the Pseudomonas aeruginosa membrane vesicle fraction (take 50 μL as sample 3), and continue to collect the subsequent eluted fractions (take 50 μL each as sample 4, sample 5, and sample 6).

[0069] 1.5 Detection of components collected during the purification process of Pseudomonas aeruginosa membrane vesicles by silver staining method

[0070] (1) Preparation of 12.5% polyacrylamide gel SDS-PAGE: Prepare the separating gel according to the instructions of the SDS-PAGE gel preparation kit and let it stand at room temperature for 30 min. After the separating gel has completely solidified, prepare the stacking gel. Insert the well-forming comb into the stacking gel, being careful to avoid air bubbles. After standing at room temperature for 30 min, gently and evenly remove the comb, fix the gel plate on the electrophoresis apparatus, and add 1× SDS-PAGE electrophoresis buffer to the electrophoresis tank.

[0071] (2) According to the loading volume and the required protein amount, respectively take 20 μL of the collected Pseudomonas aeruginosa membrane vesicle fraction samples 1 - 6 and add 5 μL of 5× protein loading buffer, and perform a 10 min boiling water bath at 100 °C to fully lyse the samples, and place them on ice.

[0072] (3) Protein electrophoresis: Add 1 μL of protein Marker and 10 μL of the sample into the PAGE gel wells in sequence, adjust the initial voltage to 80 V, and start electrophoresis. After the protein Marker bands are separated (about 20 min), increase the voltage to 120 V, and stop electrophoresis when the bromophenol blue runs to the bottom of the gel.

[0073] (4) Silver staining of PAGE gel: Refer to the instruction manual of the PAGE gel silver staining kit. After electrophoresis, transfer the PAGE gel to a clean brown glass petri dish, rinse it once with ddH 2 O, and fix it with the fixing solution for 30 min. Transfer the PAGE gel to the sensitizing solution, immerse the gel with the dye solution, and place it on a shaker to shake slowly for 30 min. Discard the sensitizing solution, and rinse it 3 times with ddH 2 O, 10 min each time. Transfer the PAGE gel to the silver staining solution, immerse the gel with the dye solution, and shake it at room temperature for 40 min. Transfer the PAGE gel to the developing solution, immerse the gel with the developing solution, shake it at room temperature for development for 10 min, discard the developing solution, add the terminating solution to terminate the development, image, and save the photo.

[0074] 1.6 Determination of protein concentration of each sample collected during the purification process of Pseudomonas aeruginosa membrane vesicles by BCA (bicinchoninic acid) method

[0075] (1) Prepare protein standard: Add 1.2 mL of protein standard preparation solution to a tube of protein standard (30 mg BSA), fully dissolve it to prepare a 25 mg / mL protein standard solution. Take an appropriate amount of the 25 mg / mL protein standard solution and dilute it with 0.9% NaCl or PBS to a final concentration of 0.5 mg / mL.

[0076] (2) Prepare BCA working solution: According to the number of samples, prepare an appropriate amount of BCA working solution by mixing 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and mix well.

[0077] (3) Protein concentration detection: Add the protein standard to the standard wells of a 96-well plate at 0, 1, 2, 4, 8, 12, 16, 20 μL, add the standard diluent to make up to 20 μL, corresponding to protein standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL respectively.

[0078] (4) Add an appropriate volume of the sample to the sample wells of the 96-well plate. If the sample is less than 20 μL, add the standard diluent to make up to 20 μL.

[0079] (5) Add 200 μL of BCA working solution to each well and incubate at 37 °C for 20 - 30 minutes.

[0080] (6) Using an ELISA reader to measure the absorbance at A562 nm or wavelengths between 540-595 nm. The protein concentration of the sample (i.e., the total protein concentration of the XMVs prepared in this example after cleavage) was calculated based on the standard curve and the sample volume used. The results are shown in Table 1.

[0081] Table 1

[0082] Samples 3-5 with higher protein concentrations were collected for subsequent testing (in the present invention, the purified Pseudomonas aeruginosa membrane vesicles are referred to as XMVs).

[0083] 1.7 Scanning electron microscopy observation of XMVs morphology

[0084] (1) Cover glass coating: A circular cover glass with a diameter of 8 mm (from Jiangsu Shitai) was used. It was first cleaned with anhydrous ethanol, ultrasonically cleaned and air-dried, then immersed in a 2.5% chitosan solution for about 5 seconds and dried at room temperature.

[0085] (2) Fixation: Prepare a 24-well plate and place a coverslip coated with chitosan in the well. Use a pipette to draw 20 μL of purified XMVs and drop them on the center of the coverslip. Let it stand for 30 min to allow the bacteria to fully adhere to the coverslip. Add 1 mL of 2.5% glutaraldehyde to completely soak the coverslip and let it stand at 4°C for fixation overnight.

[0086] (3) Gradient dehydration: discard glutaraldehyde and wash three times with PBS, 5 min each time. Then use different concentrations of ethanol for gradient dehydration: 30% (once), 50% (once), 70% (once), 90% (once), 100% (3 times), 5 min / time. Finally, soak the sample in a small amount of 100% anhydrous ethanol.

[0087] (4) Drying: The sample was dried using the critical point drying method. The dryer was first precooled for about 30 minutes, and the cover glass with the sample was carefully placed into the dryer with tweezers. Liquid CO was injected. 2 , then CO 2 Replace, heat and vaporize, and finally discharge, open the dryer and take out the sample.

[0088] (5) Coating: Before metal coating, the sample is coated with a Leit C Plast of appropriate size. TM The sample was pasted with carbon conductive glue and placed on the metal sample stage of the ion sputtering instrument. Then, the sample was sprayed with gold for 120 seconds using the vacuum sputtering method to make the sample conductive.

[0089] (6) Sampling: After the sample was prepared, the surface morphology of XMVs was observed by SEM.

[0090] 1.8 Observation of the morphological structure of XMVs by transmission electron microscopy (TEM)

[0091] Take 10 μL of the purified XMVs suspension and drop it on a copper grid, and let it stand for 10 min. Then, add 10 μL of 1% phosphotungstic acid solution for negative staining for 5 min. Use filter paper to absorb the excess liquid, dry it at room temperature for 5 min, let the grid air dry, and then perform transmission electron microscopy observation and image acquisition.

[0092] 1.9 Detection of the particle size distribution and concentration of XMVs by nanoparticle tracking analyzer (NTA)

[0093] The particle size and concentration of XMVs were measured using NTA and analyzed using Zetaview analysis software. Briefly, take the purified XMVs sample, thaw it on ice and dilute it in ultrapure water (1:200 - 1:8000). Inject 1 mL of the diluted sample using Zetaview software for quantifying the particle size distribution and concentration of XMVs. The specific analysis parameters are: maximum particle size: 2000, minimum particle size 0. All measurements were carried out at room temperature (24.5 °C ± 0.1).

[0094] Figure 1 In Figure 1 A shows the purification of XMVs using a qEV column and the detection of each component by SDS-PAGE silver staining. Figure 1 B shows the morphological structure of XMVs observed by scanning electron microscopy (scale bar: 1 μm). Figure 1 C shows the morphological structure of XMVs observed by transmission electron microscopy (scale bar: 100 nm). The results of scanning electron microscopy and transmission electron microscopy show that the XMVs prepared in the present invention are spherical structures. Figure 1 D shows the particle size distribution of XMVs measured by nanoparticle tracking analyzer, and its average particle size is about 150 nm.

[0095] 1.10 Proteomic analysis of the protein localization in XMVs

[0096] (1) Lyse XMVs using 1% SDS, and measure the protein concentration using a BCA kit. Trypsin digestion: Add dithiothreitol to the protein solution to a final concentration of 5 mM, and reduce it at 56 °C for 30 min. Then add iodoacetamide to a final concentration of 11 mM, and incubate in the dark at room temperature for 15 min. Finally, dilute the urea concentration of the sample to less than 2 M. Add trypsin at a mass ratio of 1:50 (trypsin:protein), and digest overnight at 37 °C. Then add trypsin at a mass ratio of 1:100 (trypsin:protein) and continue digestion for 4 h.

[0097] (2) Liquid chromatography - mass spectrometry analysis: The peptide segments were dissolved in mobile phase A (0.1% (v / v) formic acid aqueous solution) of liquid chromatography and separated using a NanoElute ultra - high - performance liquid system. Mobile phase A is an aqueous solution containing 0.1% formic acid; mobile phase B is an acetonitrile solution containing 0.1% formic acid. Liquid phase gradient settings: 0 - 70 min, 6% - 22% B; 70 - 84 min, 22% - 32% B; 84 - 87 min, 32% - 80% B; 87 - 90 min, 80% B, and the flow rate was maintained at 300 nL / min. After separation by the ultra - high - performance liquid system, the peptide segments were injected into a Capillary ion source for ionization and then analyzed by a tims - TOF Pro mass spectrometer. The ion source voltage was set to 1.4 kV, and both the peptide segment parent ions and their secondary fragments were detected and analyzed using TOF. The secondary mass spectrometry scan range was set to 100 - 1700 m / z. The data acquisition mode used parallel accumulation serial fragmentation (PASEF) mode. After one acquisition of the first - level mass spectrum, 10 PASEF mode acquisitions were performed to obtain secondary spectra with the parent ion charge number in the range of 0 - 5. The dynamic exclusion time for tandem mass spectrometry scanning was set to 24 seconds to avoid repeated scanning of parent ions.

[0098] (3) Database search: The secondary mass spectrometry data was retrieved using Maxquant (v1.6.6.0). Retrieval parameter settings: The database was Pseudomonas_aeruginosa_strain_ATCC_15692_208964_UP_20190712 (5563 sequences). A reverse library was added to calculate the false discovery rate (FDR) caused by random matching, and a common contaminant library was added to the database to eliminate the influence of contaminant proteins in the identification results. The digestion method was set to Trypsin / P; the number of missed cleavage sites was set to 2; the mass error tolerances for the first - level parent ions in the First search and Main search were set to 50 ppm and 50 ppm respectively, and the mass error tolerance for the secondary fragment ions was 0.02 Da. Cysteine alkylation was set as a fixed modification, and the variable modifications were oxidation of methionine, acetylation of the protein N - terminus, and deamidation. The FDR for protein identification and PSM identification was both set to 1%.

[0099] (4) Mass spectrometry quality control detection: Most peptide segments were distributed among 7 - 20 amino acids, which conforms to the general rule based on trypsin digestion and HCD fragmentation method. Among them, peptide segments with less than 5 amino acids cannot produce effective sequence identification due to too few fragment ions generated. Peptide segments with more than 20 amino acids are not suitable for the HCD fragmentation method due to their high mass and charge number. The distribution of the lengths of the peptide segments identified by mass spectrometry meets the quality control requirements.

[0100] (5) Subcellular localization: Using Wolfpsort v.0.2 (http: / / www.genscript.com / psort / wolf_psort.html).

[0101] Through proteomic analysis of the protein components of the lysed XMVs, a total of 2320 proteins were identified. In this example, the localization of these proteins was carried out, and the results are shown in Table 2. Most of the proteins contained in the XMVs prepared in this example (i.e., non-free proteins) were localized in the cytoplasm.

[0102] Table 2 Protein localization Quantity Cytoplasm 1678 Periplasm 292 Inner membrane 196 Outer membrane 122 Others 32

[0103] In this example, proteomic analysis was performed on the protein components of the outer membrane vesicles (OMVs) produced by Pseudomonas aeruginosa PAO1 under normal growth conditions in comparison with XMVs. It was found that 425 proteins were relatively increased (up-regulated), and 423 proteins were relatively decreased (down-regulated). The localization of these proteins was carried out, and the results are shown in Table 3. Most of the up-regulated proteins in XMVs were localized in the cytoplasm, and most of the down-regulated proteins were localized in the non-cytoplasmic part. In other words, the XMVs prepared in the present invention are enriched with a large number of cytoplasmic proteins.

[0104] Table 3 Protein localization Up-regulated Down-regulated Cytoplasm 384 116 Periplasm 31 104 Inner membrane - 99 Outer membrane - 89 Extracellular - 10 Others 10 -

[0105] Example 2

[0106] Establishment of a mouse model of acute Pseudomonas aeruginosa pneumonia

[0107] 2.1 Preparation of Pseudomonas aeruginosa PAO1 bacterial solution

[0108] (1) Take out the preserved strain PAO1 from the -80 °C refrigerator, streak it on a TSA plate, and incubate it in a 37 °C incubator overnight for 14 - 16 h. Pick a single colony and inoculate it into 3 ml of TSB liquid medium, and incubate it overnight at 37 °C and 220 rpm for 14 - 16 h. Measure the concentration of the overnight bacterial solution with a spectrophotometer and inoculate it into 20 ml of TSB liquid medium (initial OD 600 = 0.05), and incubate it at 37 °C and 220 rpm until the logarithmic phase (2.5 - 3 h). Take 1 ml of the bacterial solution in the logarithmic phase and measure its absorbance value at 600 nm (OD 600 value).

[0109] (2) Transfer the remaining bacterial solution into a 50 ml centrifuge tube, centrifuge it at 3900 g for 10 min at 4 °C. Discard the supernatant, and resuspend the bacterial cells with 10 ml of 0.9% sodium chloride injection, and measure the OD 600 value.

[0110] 2.2 Establishment of Acute Infected Sublethal and Lethal Dose Pseudomonas aeruginosa Pneumonia Models in Mice

[0111] (1) Adjust the bacterial suspension concentration: Adjust the PAO1 bacterial suspension concentration to 1×10 7 CFU / mL (0.01 OD), 2×10 7 CFU / mL (0.02 OD), 5×10 7 CFU / mL (0.05 OD), 1×10 8 CFU / mL (0.1 OD), 2×10 8 CFU / mL (0.2 OD).

[0112] (2) Inoculation: After anesthetizing the mice by intraperitoneal injection of 10% chloral hydrate (40 μL / 10 g calculated by body weight), make an airway incision. Bend an insulin needle at a 90-degree angle to form a right-angled needle, aspirate the prepared PAO1 bacterial suspension (1×10 7 CFU / mL, 2×10 7 CFU / mL, 5×10 7 CFU / mL, 1×10 8 CFU / mL, 2×10 8 CFU / mL), and perfuse 50 μL into the airway. Suture the wound of the mice with suture thread, and take heat preservation measures for the mice and wait for them to wake up. The mice after waking up are fed normally.

[0113] (3) Observation and recording of mice: Continuously observe for 10 days, and record the death situation of the mice and the overall score of the surviving mice every 12 h. Through comprehensive scoring of piloerection, posture, movement, respiration, and nasal secretions, it is divided into: non-infected (0 - 1), slightly infected (2 - 4), moderately infected (5 - 7), severely infected (8 - 10). After the observation period, the surviving mice are euthanized by CO 2 inhalation method.

[0114] (4) Analysis of pulmonary bacterial colonization: The mice are inoculated with PAO1 bacterial suspension (1×10 7 CFU / mL, 2×10 7 CFU / mL, 5×10 7 CFU / mL, 1×10 8 CFU / mL, 2×10 8Instill 50 μL into the airway (CFU / mL), and the control group was instilled with an equal amount of 0.9% sodium chloride injection into the trachea, with 6 mice in each group. At 2 h and 24 h after infection, the surviving mice were sacrificed by cervical dislocation, the lung tissues were aseptically removed, weighed, rinsed with 0.9% sodium chloride injection, placed in a tissue homogenizer, added with 0.9% sodium chloride injection to a final volume of 1 mL, ground with a homogenizer to obtain the original tissue homogenate. The original solution was serially diluted 10-fold with 0.9% sodium chloride injection. 50 μL of each dilution was spread on TSA plates, with three replicates for each dilution. After culturing overnight at 37 °C in an incubator, the culture results were observed. The countable TSA plates (30 - 300 CFU) of each dilution were counted, and the average of the counting results was divided by the weight of the corresponding organ to obtain the number of CFU of bacteria colonized in each lung. According to the experimental results, the present invention selected 2×10 7 CFU / mL as the sublethal dose for the mouse acute Pseudomonas aeruginosa pneumonia model, and 1×10 8 CFU / mL as the lethal dose for the mouse acute Pseudomonas aeruginosa pneumonia model.

[0115] 2.3 Evaluation of the immunoprotective efficacy of different doses of XMVs against acute sublethal-dose Pseudomonas aeruginosa pneumonia in immunized mice

[0116] (1) Immunization: Prepare 7 groups of C57BL / 6 mice, 6 - 8 weeks old, with 6 mice in each group. One group was used as the control group, and the others were used as the immunized groups. Immunize three times at 0, 2, and 4 weeks respectively, and the immunization site was the left inguinal subcutaneous of the mice. The immunization doses were 0.1, 1, 5, 10, 20, 50 μg XMVs (the corresponding numbers were approximately 6.0×10 8 、6.0×10 9 、3.0×10 10 、6.0×10 10 、1.2×10 11 、3.0×10 11 XMVs), and the volume was 100 μL.

[0117] (2) Challenge: The immunized mice and the control mice were challenged with PAO1 bacterial solution (2×10 7Airway instillation of 50 μL of CFU / mL). All mice were sacrificed by cervical dislocation 24 h after infection. The lung tissues were aseptically removed, weighed, rinsed with 0.9% sodium chloride injection, placed in a tissue homogenizer, and made up to 1 mL with 0.9% sodium chloride injection. The homogenizer was used for grinding to obtain the original tissue homogenate. The original solution was serially diluted 10-fold with 0.9% sodium chloride injection. 50 μL of each dilution was spread on TSA plates, with three replicates for each dilution. After overnight incubation at 37 °C in an incubator, the TSA plates (30 - 300 CFU) that could be counted at each dilution were counted. The average of the counting results was divided by the weight of the corresponding organ to obtain the number of CFU of bacteria colonized in the lungs of each mouse. The experimental results are as Figure 2 shown. An immunization dose of 0.1 μg (approximately 6.0×10 8 XMVs) could clear more than 99% of the bacteria colonized in the lungs; when a dose of 5 μg (approximately 3.0×10 10 XMVs) was given, more than 99.99% of the bacteria colonized in the lungs could be cleared.

[0118] 2.4 Acute infectious lethal dose of Pseudomonas aeruginosa pneumonia in mice immunized with different doses of XMVs

[0119] (1) Immunization: A total of 7 groups of 6 - 8-week-old C57BL / 6 mice, with 10 mice in each group, one group as the control group and the others as the immunization groups. Immunization was carried out three times at 0, 2, and 4 weeks respectively, and the immunization site was the subcutaneous area of the left groin of the mice. The immunization doses were 0.1, 1, 5, 10, 20, 50 μg XMVs (the corresponding quantities were approximately 6.0×10 8 , 6.0×10 9 , 3.0×10 10 , 6.0×10 10 , 1.2×10 11 , 3.0×10 11 XMVs) with a volume of 100 μL.

[0120] (2) Challenge: The immunized mice and the control mice were instilled with 50 μL of PAO1 bacterial solution (1×10 8 CFU / mL) through the airway. The mice were continuously observed for 10 days, and the death situation and weight change of the mice were recorded every 12 h. The surviving mice were euthanized by CO 2 inhalation at the end of the observation period.

[0121] The experimental results are as Figure 3 shown. An immunization dose of 0.1 μg (approximately 6.0×10 8 XMVs) of XMVs could protect 70% of the mice from infection with a lethal dose of Pseudomonas aeruginosa; when 1 μg (approximately 6.0×10 9When the dose of XMVs is at or above [specific value], it can protect all mice from lethal-dose Pseudomonas aeruginosa infection.

[0122] Figure 4 Figure [specific number] shows the weight change curves of control group and immunized group mice after challenge test after being immunized with different doses of XMVs. In the figure, the original weight of the mice is taken as 100%, and the percentage change is calculated based on this weight. The results show that the weights of all mice showed a downward trend in the first three days after infection. Except for the control group mice and the immunized group (6.0×10 8 XMVs) two groups of mice, the weight changes of the remaining immunized group mice did not exceed 20%, and the mental state of the mice was good and the hair was shiny. The weight of the surviving mice in the immunized group (6.0×10 8 XMVs) also gradually increased three days after infection. While the weight of the control group mice decreased rapidly after infection and all died on the third day. Therefore, the present invention speculates that Figure 3 in the 6.0×10 8 XMVs group, the death of mice may be natural death. The above results indicate that the XMVs provided by the present invention have good immune protection effects. Lower doses of XMVs can already substantially clear the pulmonary colonization of Pseudomonas aeruginosa, and increasing the dose of XMVs can further substantially clear the pulmonary colonization of Pseudomonas aeruginosa, and the survival rates of each immunized group are almost all 100% (the mortality rate of the control group is 100%). In addition, it should be emphasized that the mice in the immunized group still maintained a 100% survival rate 10 days after challenge, and had shiny hair and good mental state. This shows that the XMVs provided by the present invention are highly safe and have low toxic and side effects.

[0123] Example 3

[0124] Determination of pulmonary bacterial colonization in mice with acute infection of sub-lethal dose Pseudomonas aeruginosa pneumonia after immunization with different doses of XMVs

[0125] (1) Immunization: Prepare 4 groups of C57BL / 6 mice, 6 - 8 weeks old, 6 mice in each group. One group is used as the control group, and the others are used as immunized groups. The immunized groups are given 1 dose, 2 doses, and 3 doses of immunization respectively, with an interval of 2 weeks. The immunization site is the subcutaneous area of the left groin of the mice, and the immunization dose is 3.0×10 10 XMVs, with a volume of 100 μL.

[0126] (2) Challenge: The immunized group mice and the control group mice are respectively challenged with PAO1 bacterial solution (2×10 7Airway instillation of 50 μL of (CFU / mL). All mice were sacrificed by cervical dislocation 24 h after infection. The lung tissues were aseptically removed, weighed, rinsed with 0.9% sodium chloride injection, placed in a tissue homogenizer, and fixed to 1 mL with 0.9% sodium chloride injection. The homogenizer was used for grinding to obtain the original tissue homogenate. The original solution was serially diluted 10-fold with 0.9% sodium chloride injection. 50 μL of each dilution was spread on TSA plates, with three replicates for each dilution. After culturing overnight at 37 °C in an incubator, the countable TSA plates (30 - 300 CFU) of each dilution were counted. The average of the counting results was divided by the weight of the corresponding organ to obtain the number of CFU of bacteria colonized in the lungs of each mouse. The experimental results are as Figure 5 shown. Administering only one immunization dose of XMVs (3.0×10 10 XMVs) can clear more than 99.99% of the bacteria colonized in the lungs.

[0127] Determination of the survival of mice with acute infectious lethal dose of Pseudomonas aeruginosa pneumonia after immunization with different doses of XMVs

[0128] (1) Immunization: Prepare 4 groups of C57BL / 6 mice, 6 - 8 weeks old, with 10 mice in each group. One group was used as the control group, and the others were used as the immunization groups. The immunization groups were immunized with 1 dose, 2 doses, and 3 doses respectively, with an interval of 2 weeks. The immunization site was the left inguinal subcutaneous area of the mice, and the immunization dose was 5 μg XMVs (about 3.0×10 10 XMVs), with a volume of 100 μL.

[0129] (2) Challenging: The immunized mice and the control mice were instilled with 50 μL of PAO1 bacterial solution (1×10 8 CFU / mL) into the airway. The mice were continuously observed for 10 days, and the death situation of the mice was recorded every 12 h. After the observation period, the surviving mice were euthanized by CO 2 inhalation.

[0130] The experimental results are as Figure 6 shown. Administering only one immunization dose of XMVs (about 3.0×10 10 XMVs) can protect all mice from infection with a lethal dose of Pseudomonas aeruginosa. In addition, it should be emphasized that the mice in the immunization group still maintained a 100% survival rate 10 days after challenging, and had bright hair and good mental state. This shows that the XMVs provided by the present invention have a very high immunoprotective effect and low toxicity and side effects.

[0131] Evaluation of pulmonary pathological changes

[0132] (1) Immunization: Prepare 2 groups of C57BL / 6 mice, 6 - 8 weeks old, with 18 mice in each group. The immunization site was the left inguinal subcutaneous area of the mice, and the immunization dose was 3.0×1010 XMVs were given a single immunization with a volume of 100 μL.

[0133] (2) Challenge: The immunized mice and the control mice were respectively instilled with 50 μL of PAO1 bacterial solution (2×10 7 CFU / mL) into the airway. All the mice were decapitated and sacrificed at 0 h (before infection), 24 h, and 72 h after infection. The intact lung tissues were aseptically removed and placed in a 50 ml centrifuge tube, and 40 ml of 4% paraformaldehyde was added to fix them for 3 - 4 days.

[0134] (3) Dehydration and clearing of specimens: The specimens were successively immersed in ethanol with concentrations of 80%, 90%, 95%, and 100% for 2 h each, and finally immersed in xylene for 30 min to make the specimens transparent.

[0135] (4) Impregnation and embedding of specimens: Paraffin was infiltrated into the lung tissue specimens to replace the xylene contained in the tissues, and then they were placed in molten solid paraffin until the paraffin solidified.

[0136] (5) Sectioning and mounting: After removing the excess paraffin along the tissue edge by 0.1 - 0.2 cm according to the tissue size, they were mounted on a wax holder, and the section thickness was adjusted to 5 μm for sectioning. The cut wax ribbons were spread flat on the water surface at 45 °C to make the wax ribbons flatten naturally.

[0137] (6) Mounting and baking of sections: The wax sections were fished onto glass slides and baked in an oven of a 60 °C section floating and baking temperature controller for 20 min.

[0138] (7) Staining and coverslipping: The H.E staining kit was used to operate according to the following procedure. Dewaxing to water: Xylene I for 5 - 10 min; Xylene II for 5 - 10 min; absolute ethanol for 5 min; 90% alcohol for 2 min; 70% alcohol for 2 min; distilled water for 2 min; washing with tap water. Staining: Hematoxylin staining solution for 5 - 10 min; washing with tap water; 1% hydrochloric acid solution for 30 s; washing with tap water for 5 min; 1% ammonia water for blueing for 10 s; washing with tap water for 20 min; 95% alcohol for 5 s; eosin staining solution for 30 s - 2 min; dehydration, clearing and coverslipping: 95% alcohol I for 2 min; 95% alcohol II for 2 min; xylene I for 5 min; xylene II for 5 min; mounting with neutral gum.

[0139] All the sections were observed and photographed under an optical microscope (magnification of 200×), and pathological scores were made according to the degrees of tissue congestion, edema, hemorrhage, and neutrophil infiltration. The results are as Figure 7As shown, severe pathological damage occurred in the lungs of the control group mice 24 hours after infection: alveolar rupture, vascular leakage, and immune cell infiltration; the lung damage was more severe at 72 hours. After immunization with XMVs, the pathological damage, bleeding, and immune cell infiltration in the lungs of mice were significantly reduced.

[0140] The above results indicate that the XMVs prepared by the present invention can significantly reduce the organ load of Pseudomonas aeruginosa in the acute pneumonia model of mice and significantly improve the survival rate of mice, and will not cause other toxic side effects to mice. In addition, the XMVs prepared by the present invention only require a low dose and immunization times to enable mice to reduce or even avoid the colonization and / or infection of Pseudomonas aeruginosa under the threat of sub-lethal or lethal doses of Pseudomonas aeruginosa, and are suitable for preparing drugs (such as vaccines) for treating and / or preventing acute pneumonia caused by Pseudomonas aeruginosa.

[0141] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. All of these are within the protection scope of the present invention.

Claims

1. Use of Pseudomonas aeruginosa membrane vesicles in the preparation of a drug for treating and / or preventing acute pneumonia caused by Pseudomonas aeruginosa in a subject, wherein, the drug is used for treating and / or preventing colonization and / or infection of the Pseudomonas aeruginosa in the lungs of the subject, and the Pseudomonas aeruginosa membrane vesicles are obtained by subjecting a Pseudomonas aeruginosa suspension to X-ray irradiation treatment, followed by centrifugation using an ultra-high speed centrifugation method and elution using a size exclusion chromatography method.

2. The use according to claim 1, wherein, the Pseudomonas aeruginosa strain includes Pseudomonas aeruginosa PAO1.

3. The use according to claim 1, wherein, the drug is used for treating and / or preventing colonization of the Pseudomonas aeruginosa in the lungs of the subject.

4. The use according to claim 3, wherein, the drug is used to substantially eliminate the colonization of the Pseudomonas aeruginosa in the lungs of the subject.

5. The use according to claim 1, wherein, The single dose of the drug includes 10 8 to 10 11 of the Pseudomonas aeruginosa membrane vesicles.

6. The use according to claim 5, wherein, the drug comprises at least one dose.

7. The use according to claim 1, wherein, the drug is an intramuscular or subcutaneous preparation.

8. The use according to claim 1, wherein, the drug is a vaccine.

9. The use according to claim 1, wherein, the drug is used for treating and / or preventing one or more of the following symptoms of acute pneumonia caused by the Pseudomonas aeruginosa: alveolar rupture, vascular leakage, immune cell infiltration.

10. The use according to claim 1, wherein, the drug is used to prolong the survival time of the subject.

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