US20220370588A1 - Application of pseudomonas aeruginosa vaccine in treating infection associated with burn or scald injury - Google Patents

Application of pseudomonas aeruginosa vaccine in treating infection associated with burn or scald injury Download PDF

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US20220370588A1
US20220370588A1 US17/637,057 US202017637057A US2022370588A1 US 20220370588 A1 US20220370588 A1 US 20220370588A1 US 202017637057 A US202017637057 A US 202017637057A US 2022370588 A1 US2022370588 A1 US 2022370588A1
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pseudomonas aeruginosa
cells
scald
burn
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Zhenling Wang
Yuquan Wei
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Sichuan University
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Sichuan University
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Priority claimed from CN201910777473.5A external-priority patent/CN112402601B/en
Priority claimed from CN201910777595.4A external-priority patent/CN112410212B/en
Priority claimed from CN201910777606.9A external-priority patent/CN112410240B/en
Priority claimed from CN201921369450.2U external-priority patent/CN211394490U/en
Priority claimed from CN201910777479.2A external-priority patent/CN112410239B/en
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Assigned to SICHUAN UNIVERSITY reassignment SICHUAN UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WANG, Zhenling, WEI, YUQUAN
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    • C12R2001/38—Pseudomonas
    • C12R2001/385—Pseudomonas aeruginosa

Definitions

  • the present invention belongs to the field of microbiology, and particularly relates to an application of a Pseudomonas aeruginosa vaccine in prevention and treatment of burn and scald complicated with bacterial infection.
  • burn and scald have become one of the most common accidental injuries in daily production and life. According to statistics, about 5000-10000 people per million people in China suffer from burn or scald every year.
  • the main parts of burn and scald include skin and/or mucosa, and in severe cases, subcutaneous and/or submucosal tissues, such as muscles, bones, joints and even internal organs, can be injured.
  • Scald is a type of thermal burns, and is tissue damage mainly caused by hot fluid, steam and high-temperature solid.
  • the depth of scald is judged according to the classification rule of “three degrees and four types” for burn severity, and according to the depth, pathological change and clinical manifestations of scald, it can be divided into: I degree, superficial II degree, deep II degree and III degree scalds.
  • Wound infection is the most common and serious complication after scald.
  • the bacteria which cause wound infection are mainly gram-negative bacilli, wherein Pseudomonas aeruginosa is one of the most common pathogens.
  • the detection rate of Pseudomonas aeruginosa in burn patients varies from 20% to more than 50% in different reports. Some studies have pointed out the probability that Pseudomonas aeruginosa could be isolated from burn wounds 7 days after burn was almost 100%. It is also reported that the mortality rate after Pseudomonas aeruginosa infection is 31%, especially in patients with burn. If the bacterial infection of burn wounds is not treated in time, it may cause serious consequences. In addition, if the bacteria in the burn wounds are not removed in time, delay of wound healing or healing with scar formation may impair the function of a wound site (joint).
  • the current treatment for scald complicated with Pseudomonas aeruginosa infection is generally surgical debridement combined with local and systemic antibiotics.
  • the most common antibacterial drugs include synthetic antibacterial drugs such as silver sulfadiazine and antibiotics such as ciprofloxacin, ceftazidime and cefoperazone/sulbactam.
  • synthetic antibacterial drugs such as silver sulfadiazine
  • antibiotics such as ciprofloxacin, ceftazidime and cefoperazone/sulbactam.
  • the therapeutic effect is unsatisfactory, such as progression into sepsis, which is life-threatening.
  • Pseudomonas aeruginosa sepsis is the main reason of death in scald patients, with an incidence rate from 8% to 42.5% and a mortality rate from 28% to 65%.
  • the objective of the present invention is to provide an application of a Pseudomonas aeruginosa vaccine in prevention and treatment of infection in burn and scald.
  • the present invention adopts the following technical solutions:
  • the burn and scald include burns and scalds, and degree of the scalds includes I degree, superficial II degree, deep II degree, or III degree scalds.
  • site of the scalds includes skin, mucosa or other tissues.
  • bacteria of the complicated with bacterial infection include one or more of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae and Mycobacterium tuberculosis.
  • the bacterial infection is Pseudomonas aeruginosa infection.
  • the burn and scald include burns and scalds, and degree of the scalds includes I degree, superficial II degree, deep II degree, or III degree scalds.
  • site of the scalds includes skin, mucosa or other tissues.
  • the Pseudomonas aeruginosa vaccine comprises inactivated Pseudomonas aeruginosa and/or Pseudomonas aeruginosa membrane vesicles.
  • immunization procedures of the Pseudomonas aeruginosa vaccine comprise: injection take places (i) 0, 3rd, and 7th days, and (ii) 0, 2nd and 4th weeks.
  • the inactivated Pseudomonas aeruginosa is inactivated by irradiation, and the Pseudomonas aeruginosa membrane vesicles are isolated from the Pseudomonas aeruginosa inactivated by irradiation.
  • the Pseudomonas aeruginosa vaccine prevents Pseudomonas aeruginosa infection, and reduces bacterial load in skin scald complicated with bacterial infection.
  • content of whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1 ⁇ 10 4 -1 ⁇ 10 10 /injection.
  • the content of whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1 ⁇ 10 4 /injection, 1 ⁇ 10 5 /injection, 1 ⁇ 10 6 /injection, 1 ⁇ 10 7 /injection, 1 ⁇ 10 8 /injection, 1 ⁇ 10 9 /injection and 1 ⁇ 10 10 /injection.
  • the Pseudomonas aeruginosa vaccine further contains an immunoadjuvant.
  • administration site of the Pseudomonas aeruginosa vaccine is subcutaneous, muscle and/or mucosa.
  • the medicament can also contain any pharmaceutically acceptable carrier and/or adjuvant.
  • the carrier is a liposome.
  • the experimental results of the present invention show that the Pseudomonas aeruginosa vaccine of the present invention can effectively prevent and treat burn and scald complicated with Pseudomonas aeruginosa infection caused by multidrug-resistant Pseudomonas aeruginosa by activating the specific immune response of the body.
  • the Pseudomonas aeruginosa vaccine of the present invention can reduce the bacterial load in the immunized subject through the established immunization procedures, thereby providing a technical solution that can effectively prevent burn and scald complicated with Pseudomonas aeruginosa infection, which avoids the technical problems caused by the use of antibiotics such as poor effectiveness, difficulty in curing and proneness to drug resistance in the prior art to a certain degree.
  • the experimental results of the present invention show that, the Pseudomonas aeruginosa vaccine of the present invention can be used to effectively inhibit the bacterial load in the site of burn and scald complicated with bacterial infection by activating the human immune response, prevent secondary infection caused by Pseudomonas aeruginosa and avoid the problem of drug resistance caused by antibiotics in the prior art, and has broad application scenarios in burn and scald complicated with bacterial infection.
  • FIG. 1 is a Transmission Electron Microscopy (TEM) image of irradiated Pseudomonas aeruginosa membrane vesicles (scale: 200 nm).
  • TEM Transmission Electron Microscopy
  • FIG. 2 shows the percentage of proliferation of CD4 + T cells after interacting with DC treated with different treatment methods.
  • FIG. 3 is a flow cytometry plot of proliferation of CD4 + T cells after interacting with DC treated with different treatment methods.
  • FIG. 4 shows irradiated membrane vesicles enhance the interaction between DC cells and T cells (GC: growth control, dendritic cell growth control group (unstimulated group); Cell+MVs (whole-cell bacteria+membrane vesicle treatment group); MVs (membrane vesicle treatment group)).
  • GC growth control, dendritic cell growth control group (unstimulated group); Cell+MVs (whole-cell bacteria+membrane vesicle treatment group); MVs (membrane vesicle treatment group)).
  • FIG. 5 shows the bacterial load of scalded sites of rabbits 24 h after Pseudomonas aeruginosa infection.
  • FIG. 6 shows the bacterial load of scalded sites of rabbits 24 h after Pseudomonas aeruginosa PA14 infection.
  • burn and scald in the present invention refers to the damage to tissues, mainly referring to skin and/or mucosa, caused by heat, including hot fluid (water, soup, oil, etc.), steam, high-temperature gas, flame, and red-hot metal liquid or solid (such as molten steel and steel ingots), which may damage subcutaneous and/or submucosal tissues in severe cases, such as muscles, bones, joints and even internal organs.
  • Scald is tissue damage caused by hot fluid, steam and high-temperature solid, and belongs to one of thermal burns.
  • the Pseudomonas aeruginosa vaccine of the present invention comprises (i) irradiation-inactivated Pseudomonas aeruginosa cells and/or (ii) Pseudomonas aeruginosa membrane vesicles.
  • FIG. 1 is a Transmission Electron Microscopy (TEM) photograph of purified membrane vesicles.
  • Embodiments 1-3 introduce some isolation methods for preparing vesicles.
  • the vesicles can be isolated from non-irradiated bacteria or isolated from irradiated bacteria or obtained by other methods.
  • a method for isolating membrane vesicles from Pseudomonas aeruginosa comprises the following steps: 1) isolating bacterial cells in bacterial solution for culture of bacteria from culture medium, and collecting supernatant 1; 2) centrifuging the supernatant 1 with a high-speed centrifuge, and collecting supernatant 2; and 3) centrifuging the supernatant 2 with an ultra-high-speed centrifuge to precipitate membrane vesicles.
  • isolation method in the step 1) comprises centrifugation, column chromatography, or dialysis bag concentration.
  • the supernatant 2 collected in the step 2) is subjected to dialysis bag concentration prior to the step 3).
  • the dialysis bag selected can concentrate substances greater than 100 KD.
  • the membrane vesicles are resuspended with a buffer solution
  • the buffer solution comprises 50 mM Tris, 5 mM NaCl and 1 mM MgSO 4 calculated as a volume unit of 1 L and has a pH of 7.4.
  • the bacterial cells and the membrane vesicles are prepared as a biological composition, and the preparation method comprises: collecting the bacterial cells isolated in the step 1) in the above isolation method for membrane vesicles, and mixing the bacterial cells with the membrane vesicles obtained in the step 3) to form the biological composition.
  • the supernatant 1 is filtered with a 0.3-0.5 ⁇ M filter to remove impurities.
  • the supernatant 1 is filtered with a 0.45 ⁇ M filter to remove impurities.
  • the isolation method in the step 1) is centrifugation, the centrifugation speed is 100-10000 g, and the centrifugation time is 10-60 min.
  • the centrifugation speed in the step 1) is 400-8000 g, and the centrifugation time is 10-30 min.
  • the high-speed centrifugation speed in the step 2) is 5000-25000 g, and the high-speed centrifugation time is 10-100 min.
  • the high-speed centrifugation speed in the step 2) is 10000-20000 g, and the high-speed centrifugation time is 30-60 min.
  • the ultra-high-speed centrifugation speed in the step 3) is 5000-150000 g, and the ultra-high-speed centrifugation time is 60-600 min.
  • the ultra-high-speed centrifugation speed in the step 3) is 15000-150000 g, and the ultra-high-speed centrifugation time is 60-180 min.
  • the method for preparing bacterial membrane vesicles further comprises the following steps: 1) Augmentation of membrane vesicles: culturing bacteria to logarithmic growth phase; collecting the bacterial cells, resuspending the bacterial cells and then irradiating them with ionizing irradiation to obtain irradiated bacteria; 2) Isolation and purification of membrane vesicles: isolating membrane vesicles produced by the irradiated bacteria from the irradiated bacteria to obtain the membrane vesicles using the method for isolating membrane vesicles described in Embodiment 1.
  • the ionizing irradiation is X-rays, and the irradiation dose is 500-3000 Gy.
  • the irradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gy, 900-1000 Gy, 1000-1100 Gy, 1100-1200 Gy, 1200-1300 Gy, 1300-1400 Gy, 1400-1500 Gy, 1500-1600 Gy, 1600-1700 Gy, 1700-1800 Gy, 1800-1900 Gy, 1900-2000 Gy, 2100-2200 Gy, 2200-2300 Gy, 2300-2400 Gy, 2400-2500 Gy, 2500-2600 Gy, 2600-2700 Gy, 2700-2800 Gy, 2800-2900 Gy and 2900-3000 Gy.
  • the irradiation dose is 500-1000 Gy.
  • the irradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gy and 900-1000 Gy.
  • OD 600 value of the bacteria in logarithmic growth phase in the step 1) is 0.3-0.8.
  • the OD 600 value of the bacteria in logarithmic growth phase in the step 1) is 0.5-0.7.
  • the bacterial cells are resuspended with phosphate buffer saline or sterile normal saline.
  • the bacterial cells are resuspended with phosphate buffer saline.
  • the bacterial cells are resuspended to an OD 600 value of 20-80.
  • the bacterial cells are resuspended to an OD 600 value of 40-60.
  • the content of nucleic acids and the content of proteins in the membrane vesicles prepared by the above method are increased by 10-20 times, compared with those prepared from bacteria not irradiated with ionizing irradiation.
  • the membrane vesicles prepared by the present invention have various application scenarios: for example, (i) the membrane vesicles can be used as an immunogen; (ii) the membrane vesicles can be used as an immune response enhancer; (iii) the membrane vesicles can be used as a vaccine for treating bacterial infectious diseases; (iv) the membrane vesicles can be used as a vaccine adjuvant (in some embodiments, the vaccine adjuvant non-specifically changes or enhances the antigen-specific immune response of the body); (v) the membrane vesicles can be used as an antigen-presenting cell function enhancer.
  • the above antigen-presenting cell includes dendritic cells (i.e., DC cells), macrophages and B cells.
  • dendritic cells i.e., DC cells
  • macrophages i.e., macrophages
  • B cells i.e., B cells.
  • the membrane vesicles obtained by irradiation, isolation and purification can be used as an enhancer for the maturation of the DC cells, and specifically, used as an enhancer for promoting the significant up-regulation of cell surface molecules CD80, CD86 and MHCII molecules of bone marrow-derived dendritic cells.
  • the membrane vesicles prepared by the present invention can be combined with DC cells in preparation of a proliferation agent for CD4 + T cells.
  • the method for promoting proliferation of CD4 + T cells comprises the following steps: co-culturing membrane vesicles-stimulated and OVA-antigen-phagocytosed DCs with CFSE-labeled CD4 + T lymphocytes in vitro, wherein the membrane vesicles are prepared by irradiation.
  • the method for isolating and preparing bacterial membrane vesicles comprises the following steps:
  • Culturing bacteria to logarithmic growth phase wherein OD 600 value of the bacteria in logarithmic growth phase is 0.3-0.8, and the OD 600 value of 0.5-0.8 is preferably selected (fermentation can also be performed here to further enrich bacterial cells); collecting bacterial cells, and resuspending the bacterial cells with an appropriate amount of phosphate buffer solution, wherein the ratio of the amount of the added phosphate buffer solution to the total amount of the bacterial cells is that the OD 600 value of the amount of the bacteria contained in every 1 ml of solution is 20-80, and the OD 600 value of 40-60 is preferably selected; after resuspension, irradiating the bacterial cells with ionizing irradiation to obtain irradiated bacteria; preferably, irradiating with X-rays, with an irradiation dose of 500-3000 Gy.
  • the irradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gy, 900-1000 Gy, 1000-1100 Gy, 1100-1200 Gy, 1200-1300 Gy, 1300-1400 Gy, 1400-1500 Gy, 1500-1600 Gy, 1600-1700 Gy, 1700-1800 Gy, 1800-1900 Gy, 1900-2000 Gy, 2100-2200 Gy, 2200-2300 Gy, 2300-2400 Gy, 2400-2500 Gy, 2500-2600 Gy, 2600-2700 Gy, 2700-2800 Gy, 2800-2900 Gy and 2900-3000 Gy.
  • the ionizing irradiation can stimulate Pseudomonas aeruginosa PAO1 to produce membrane vesicles.
  • the membrane vesicles are shown in FIG. 1 .
  • Dendritic cells are the main antigen-presenting cells of the body, and have the main function of phagocytosing and processing antigen molecules as well as presenting them to T cells.
  • the DCs are the known most powerful and the only professional antigen-presenting cell that can activate resting T cells in the body, and are a key link in initiating, regulating and maintaining immune responses.
  • the maturation of the DCs determines the immune response or immune tolerance of the body.
  • the main characteristics of mature DCs are changes in the expression of co-stimulatory molecules CD80 and CD86, reduced ability to phagocytose antigens and enhanced the ability to process and present antigens (increased MHCII molecules expression), and interaction with T lymphocytes.
  • BMDC mouse bone marrow-derived dendritic cells
  • BMDC stimulation taking the BMDC cells induced for 7 days, and repeatedly blowing the cells in a 6-well plate to detach adherent cells; collecting the cell suspension, centrifuging it at 1100 rpm for 5 min, removing supernatant, and adding 1 ml of medium to resuspend the cells, and adjusting the cell concentration to 1 ⁇ 10 6 /m1 after counting viable cells, and inoculating 2 ml of the cells into a new 6-well plate.
  • Each stimulator will be added respectively and uniformly mixed: whole-cell bacteria, whole-cell bacteria+vesicles, and vesicles at a final concentration of 15 ⁇ g/mL (based on protein). Continuing to culture them for 24 hours and adding an equal volume of PBS to the growth control group.
  • Maturation markers detection by flow cytometry after 24 h, taking out the 6-well plate, repeatedly blowing the cells to detach them, collecting the cell suspension into a Flow Cytometry Tube, centrifuging it at 1500 rpm for 3 min, removing supernatant, adding 1 ml of PBS to continue centrifugation at 1500 rpm for 3 min, then removing the supernatant and repeatedly washing for 3 times.
  • the vesicles of the experimental group (MVs) treated by X-rays can significantly up-regulate the surface costimulatory molecules CD80, CD86 and MHCII of DCs after stimulation, and these surface molecules are markers of dendritic cell maturation.
  • the vesicles can significantly promote the differentiation and maturation of DCs.
  • the phagocytic ability of DC cells is detected by detecting the fluorescence intensity of FITC-dextran: DC cells have strong antigen endocytosis and processing abilities. The DC cells have strong phagocytic ability in an immature state when not in contact with antigen. After in contact with antigen and activated, the DC cells become mature with low phagocytic ability and enhanced antigen-presenting ability. In the experiment, by detecting the fluorescence intensity of the FITC-dextran, the amount of the dextran phagocytosed by DC is determined to detect whether the phagocytic ability of DC is enhanced.
  • Stimulation collecting the cells on the 7th day, blowing down all the cells, then centrifuging and resuspending the cells for counting, then inoculating the cells into a 6-well plate with 1 ⁇ 10 6 cells per well, and respectively adding the stimulator: adding an equal volume of PBS to the GC group, adding the same concentration of membrane vesicles (by protein level) to the control group and the treatment group and then culturing them at 37° C. for 24 h.
  • Phagocytosis and detection adding the dextran (5 ⁇ g/ml), and after culture for 1 h, aspirating the cells into a Flow Cytometry Tube; washing the cells with PBS for 3 times; adding CD11c antibody and incubating at room temperature for 30 min in the dark; washing the cells with PBS for 3 times; and detecting the fluorescence of FITC by flow cytometry.
  • the present invention uses FITC-dextran as a model antigen for phagocytosis of DCs and detects the mean fluorescence intensity value of FITC of CD11c+DCs.
  • the experimental result shows that after the DCs are stimulated by membrane vesicles, the mean fluorescence intensity value of FITC is significantly reduced compared with that of the GC group (growth control group). This experimental result proves again that the vesicles can promote the maturation of DCs, thereby reducing their ability to take up antigen.
  • CFSE Fluorescent dye CFSE
  • CFDA-SE carboxyfluorescein diacetate, succinimidyl ester
  • the CFSE-labeled fluorescence can be equally distributed to two daughter cells, and the fluorescence intensity is half that of the parental cells. Therefore, the percentage of cells with weak CFSE fluorescence can be counted by flow cytometry to obtain the proportion of proliferating cells.
  • Antigen phagocytosis culturing DCs which are cultured for 7 days in a medium containing 10 ⁇ g/ml OVA for 24 h to serve as GC (growth control group); adding vesicles to the MVs group, then centrifuging and collecting antigen-phagocytosed DCs; resuspending the DCs in a normal medium; and applying the DCs in a 96-well plate at a density of 2 ⁇ 10 4 cells/well, with 100 ⁇ l per well, and 3 replicate wells per group.
  • T cell extraction on the second day, isolating and enriching OVA-specific CD4+T lymphocytes from the spleens of OT-II mice by a magnetic negative selection beads kit from Stem Cell Technologies company.
  • CFSE Fluorescent dye CFSE
  • CFDA-SE carboxyfluorescein diacetate, succinimidyl ester
  • the CFSE-labeled fluorescence can be equally distributed to two daughter cells, and the fluorescence intensity is half that of the parental cells. Therefore, the percentage of cells with weak CFSE fluorescence can be counted by flow cytometry to obtain the proportion of proliferating cells.
  • Antigen phagocytosis culturing DCs which are cultured for 7 days in a medium for 24 h to serve as GC (growth control group); adding vesicles to the MVs group, then centrifuging and collecting antigen-phagocytosed DCs; resuspending the DCs in a normal medium; and applying the DCs in a 96-well plate at a density of 4 ⁇ 10 4 cells/well, with 100 ⁇ l per well, and 3 replicate wells per group.
  • GC growth control group
  • T cell extraction on the second day, isolating and enriching the T cells of the mice from the spleens of mice one week after one MVs immunization using a magnetic negative selection beads kit from Stem Cell Technologies company.
  • mice are used in the experiment.
  • a scald model of rabbits is established by scalding rabbits with a hot-air gun one day before the rabbits are infected with Pseudomonas aeruginosa .
  • the scalded sites are subcutaneously infected with the homologous strain of Pseudomonas aeruginosa SKLBPA1 and Pseudomonas aeruginosa PA14 respectively (both purchased from ATCC).
  • the rabbits are sacrificed, and skin and subcutaneous muscle tissue of the scalded sites are taken, homogenized and counted for CFU.
  • the CFU of the model group and the vaccine group are compared to observe whether the vaccine of the present invention has a protective effect on scald complicated with Pseudomonas aeruginosa infection.
  • the results of the experiment will show that the Pseudomonas aeruginosa vaccine provided by the present invention can have preventive and therapeutic effects on burn and scald complicated with infection caused by different serotypes of Pseudomonas aeruginosa (two representative strains of SKLBPA1 and PA14 are used as examples in the experiment), and has wide application scenarios.
  • the content of the vaccine of the present invention includes: 10 8 CFU/ml
  • mice 6 rabbits, New Zealand White rabbits, weighing 2.210 kg-2.870 kg, female.
  • test vaccine (10 8 CFU/ml) is subcutaneously immunized with 1000 in the left and right groins of the rabbits for 3 times, with two immunization procedures of 0, 3, 7 d (injection take places at 0, 3rd, and 7th days) and 0, 2, 4 w (injection take places at 0, 2nd and 4th weeks).
  • the front and back hairs on the left and right sides of the rabbits are removed by scissors and depilatory cream.
  • the skin of the depilation sites is disinfected by 75% alcohol and applied with tetracaine hydrochloride mucilage for local anesthesia.
  • the temperature of the hot-air gun is set at 200° C.; the metal plate with square holes is disinfected by 75% alcohol and stuck onto the depilation sites.
  • the hot-air gun is turned on; and a hot air outlet is pointed towards the square holes to blow the hot air onto the rabbit skin for 5 s, causing a certain degree of scald model.
  • Pseudomonas aeruginosa SKLBPA1 and PA1 recovered from ⁇ 80° C. to TSA plates, and cultured overnight at 37° C.
  • Monoclonal colonies are picked from the plates respectively into TSB for shaking culture at 37° C. at 220 rpm overnight.
  • the overnight bacterial solution is diluted to measure OD 600 ; the original bacterial solution is inoculated into 100 ml of TSB (250 ml conical flask); and the bacteria are shaken at 37° C. at 220 rpm to logarithmic growth phase.
  • TSB 250 ml conical flask
  • the bacterial solution is collected into a 50 ml centrifuge tube, and centrifuged at 4100 rpm (3000 ⁇ g) at room temperature for 10 min; the supernatant is discarded and the precipitate is resuspended with 2 mL of 0.9% sodium chloride injection (about 5 OD/ml); and the bacterial solution is adjusted to 0.1 OD 600 (2 ⁇ 10 7 CFU/ml).
  • the adjusted bacterial solution is coated to a TSA plate, and cultured at 37° C. overnight, and CFU is counted.
  • Animals are sacrificed 24 hours after infection.
  • the skin is disinfected by spraying with 75% alcohol.
  • the skin and muscular tissue of the scalded sites are taken aseptically, and the tissue is homogenized, coated to the TSA plate, and cultured at 37° C. overnight.
  • the CFU is counted by a colony counter.
  • LOG 10 CFU scatter plots of skin and muscular tissue are drawn using Graphpad Prism software. The mean value of LOG 10 CFU is calculated, and between-group variation is analyzed by Analysis of Variance.
  • the body weights of the animals in each group are shown in Table 2.
  • the body weights of the rabbits in each immunization group and each model group are increased steadily, and there is no significant difference among the groups.
  • the hot-air gun is sleeved with an air nozzle with an outer diameter of 14 mm and an inner diameter of 10 mm; the temperature is set as 200° C.; and the air nozzle is kept close to the depilation sites on the backs of the rabbits for 5 seconds. After 2 hours, round blisters with a diameter of about 10 mm appear on the skins of the scalded sites, and after 24 hours, the skins of the scalded sites are scabbed.
  • the concentrations of Pseudomonas aeruginosa SKLBPA1 and PA14 bacterial suspensions used in the experiment are 0.1 OD/ml; the remaining bacterial suspensions after subcutaneous infection are diluted to 10 ⁇ 4 and 10 ⁇ 5 ; 50 ⁇ l of bacterial suspensions are evenly coated on the TSA plates, and incubated overnight in an incubator at 37° C.; and the CFU is counted.
  • Table 3 The results are shown in Table 3.
  • the animals are sacrificed 24 h after infection.
  • the skin and muscular tissue of the scalded and infected sites (infected with Pseudomonas aeruginosa SKLBPA1) are taken aseptically, homogenized, and coated to the TSA plates, and cultured overnight at 37° C.
  • the CFU is counted, and then the mean and the standard deviation of each group are compared based on the log values of the bacterial loads to the base of 10.
  • the results are shown in Table 4 and FIG. 5 .
  • Two immunization procedures of PA1 vaccine can significantly reduce the load of Pseudomonas aeruginosa SKLBPA1 (P ⁇ 0.01), which indicates that the vaccine has an obvious protective effect.
  • the skin and muscular tissue of the scalded and infected sites of Pseudomonas aeruginosa PA14 are taken aseptically, coated to the TSA plates by a homogenizer, and cultured overnight at 37° C.; the CFU is counted by a colony counter; and then the mean and the standard deviation of each group are compared based on the log values of the bacterial loads to the base of 10.
  • the results are shown in Table 5 and FIG. 6 .
  • the immunization procedure 0, 3, 7 d of the PA1 vaccine can significantly reduce the load of Pseudomonas aeruginosa PA14 (P ⁇ 0.05), with a reduction range of about 0.6-0.9 log. It indicates that the vaccine has a certain protective effect.
  • the immunization procedure 0, 3, 7 d can quickly generate an effective immune response, so the vaccine of the present invention can be used to prevent skin scald complicated with infection (that is, as a prophylactic vaccine), and may also possibly be used to inhibit and alleviate the infection after the onset of skin scald complicated with infection (that is, the possibility of serving as a therapeutic vaccine).

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Abstract

The present invention belongs to the field of microbiology, and particularly relates to an application of a Pseudomonas aeruginosa vaccine in prevention and treatment of burn and scald complicated with bacterial infection. The burn and scald of the present invention include burns and scalds, and degree of the scalds includes I degree, superficial II degree, deep II degree, or III degree scalds. Site of the scalds includes skin, mucosa or other tissues. The Pseudomonas aeruginosa vaccine of the present invention can effectively prevent and treat burn and scald complicated with Pseudomonas aeruginosa infection caused by multidrug-resistant Pseudomonas aeruginosa by activating the specific immune response of the body. The Pseudomonas aeruginosa vaccine of the present invention can reduce the bacterial load in the immunized subject through the established immunization procedures, thereby providing a technical solution that can effectively prevent burn and scald complicated with Pseudomonas aeruginosa infection, which avoids the technical problems caused by the use of antibiotics such as poor effectiveness, difficulty in curing and proneness to drug resistance in the prior art to a certain degree.

Description

    PRIORITY APPLICATIONS
  • The present application claims priority from Chinese invention patent applications 1) 201910777479.2 “BACTERIAL MEMBRANE VESICLE, AND PREPARATION METHOD AND APPLICATION THEREOF”, 2) 201910777473.5 “Staphylococcus aureus MEMBRANE VESICLE, AND PREPARATION METHOD AND APPLICATION THEREOF”, 3) 201910777606.9 “Pseudomonas aeruginosa MEMBRANE VESICLE, AND PREPARATION METHOD AND APPLICATION THEREOF”, 4) 201921369450.2 “A PRODUCTION SYSTEM, AND ISOLATION AND PURIFICATION SYSTEM FOR BACTERIAL MEMBRANE VESICLE”, 5) 201910777595.4 “A PRODUCTION SYSTEM, AND ISOLATION AND PURIFICATION SYSTEM AND METHOD FOR BACTERIAL MEMBRANE VESICLE” filed on Aug. 22, 2019, which are incorporated by reference in their entirety.
    • TECHNICAL FIELD
  • The present invention belongs to the field of microbiology, and particularly relates to an application of a Pseudomonas aeruginosa vaccine in prevention and treatment of burn and scald complicated with bacterial infection.
    • BACKGROUND
  • At present, burn and scald have become one of the most common accidental injuries in daily production and life. According to statistics, about 5000-10000 people per million people in China suffer from burn or scald every year. The main parts of burn and scald include skin and/or mucosa, and in severe cases, subcutaneous and/or submucosal tissues, such as muscles, bones, joints and even internal organs, can be injured. Scald is a type of thermal burns, and is tissue damage mainly caused by hot fluid, steam and high-temperature solid. The depth of scald is judged according to the classification rule of “three degrees and four types” for burn severity, and according to the depth, pathological change and clinical manifestations of scald, it can be divided into: I degree, superficial II degree, deep II degree and III degree scalds.
  • Wound infection is the most common and serious complication after scald. The bacteria which cause wound infection are mainly gram-negative bacilli, wherein Pseudomonas aeruginosa is one of the most common pathogens. The detection rate of Pseudomonas aeruginosa in burn patients varies from 20% to more than 50% in different reports. Some studies have pointed out the probability that Pseudomonas aeruginosa could be isolated from burn wounds 7 days after burn was almost 100%. It is also reported that the mortality rate after Pseudomonas aeruginosa infection is 31%, especially in patients with burn. If the bacterial infection of burn wounds is not treated in time, it may cause serious consequences. In addition, if the bacteria in the burn wounds are not removed in time, delay of wound healing or healing with scar formation may impair the function of a wound site (joint).
  • The current treatment for scald complicated with Pseudomonas aeruginosa infection is generally surgical debridement combined with local and systemic antibiotics. The most common antibacterial drugs include synthetic antibacterial drugs such as silver sulfadiazine and antibiotics such as ciprofloxacin, ceftazidime and cefoperazone/sulbactam. However, due to the irrational use of antibiotics and the emergence of multidrug-resistant bacteria, the therapeutic effect is unsatisfactory, such as progression into sepsis, which is life-threatening. According to statistics, in the process of scald complicated with Pseudomonas aeruginosa infection, Pseudomonas aeruginosa sepsis is the main reason of death in scald patients, with an incidence rate from 8% to 42.5% and a mortality rate from 28% to 65%.
    • SUMMARY
  • In view of this, the objective of the present invention is to provide an application of a Pseudomonas aeruginosa vaccine in prevention and treatment of infection in burn and scald.
  • To achieve the above objective, the present invention adopts the following technical solutions:
  • Use of a Pseudomonas aeruginosa vaccine in manufacture of a medicament for prevention and treatment of infection in burn and scald.
  • Further, the burn and scald include burns and scalds, and degree of the scalds includes I degree, superficial II degree, deep II degree, or III degree scalds.
  • Further, site of the scalds includes skin, mucosa or other tissues.
  • Further, the infection in burn and scald is burn and scald complicated with bacterial infection.
  • Further, bacteria of the complicated with bacterial infection include one or more of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae and Mycobacterium tuberculosis.
  • Further, the bacterial infection is Pseudomonas aeruginosa infection.
  • Further, the burn and scald include burns and scalds, and degree of the scalds includes I degree, superficial II degree, deep II degree, or III degree scalds.
  • Further, site of the scalds includes skin, mucosa or other tissues.
  • Further, the Pseudomonas aeruginosa vaccine comprises inactivated Pseudomonas aeruginosa and/or Pseudomonas aeruginosa membrane vesicles.
  • Further, immunization procedures of the Pseudomonas aeruginosa vaccine comprise: injection take places (i) 0, 3rd, and 7th days, and (ii) 0, 2nd and 4th weeks.
  • Further, the inactivated Pseudomonas aeruginosa is inactivated by irradiation, and the Pseudomonas aeruginosa membrane vesicles are isolated from the Pseudomonas aeruginosa inactivated by irradiation.
  • Further, the Pseudomonas aeruginosa vaccine prevents Pseudomonas aeruginosa infection, and reduces bacterial load in skin scald complicated with bacterial infection.
  • Further, content of whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1×104-1×1010/injection.
  • Further, the content of whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1×104/injection, 1×105/injection, 1×106/injection, 1×107/injection, 1×108/injection, 1×109/injection and 1×1010/injection.
  • Further, the Pseudomonas aeruginosa vaccine further contains an immunoadjuvant.
  • Further, administration site of the Pseudomonas aeruginosa vaccine is subcutaneous, muscle and/or mucosa.
  • Further, the medicament can also contain any pharmaceutically acceptable carrier and/or adjuvant.
  • Further, the carrier is a liposome.
  • The present invention has the following beneficial effects:
  • The experimental results of the present invention show that the Pseudomonas aeruginosa vaccine of the present invention can effectively prevent and treat burn and scald complicated with Pseudomonas aeruginosa infection caused by multidrug-resistant Pseudomonas aeruginosa by activating the specific immune response of the body. The Pseudomonas aeruginosa vaccine of the present invention can reduce the bacterial load in the immunized subject through the established immunization procedures, thereby providing a technical solution that can effectively prevent burn and scald complicated with Pseudomonas aeruginosa infection, which avoids the technical problems caused by the use of antibiotics such as poor effectiveness, difficulty in curing and proneness to drug resistance in the prior art to a certain degree.
  • The experimental results of the present invention also show that, the Pseudomonas aeruginosa vaccine of the present invention can be used to effectively inhibit the bacterial load in the site of burn and scald complicated with bacterial infection by activating the human immune response, prevent secondary infection caused by Pseudomonas aeruginosa and avoid the problem of drug resistance caused by antibiotics in the prior art, and has broad application scenarios in burn and scald complicated with bacterial infection.
    • DESCRIPTION OF DRAWINGS
  • To make the embodiments of the present invention or the technical solutions in the prior art clearer, the drawings required to be used in the description of the embodiments or the prior art will be briefly introduced below. It is obvious that the drawings described below are some embodiments of the present invention, and that other drawings can be obtained from these drawings for those of ordinary skill in the art without making inventive effort.
  • FIG. 1 is a Transmission Electron Microscopy (TEM) image of irradiated Pseudomonas aeruginosa membrane vesicles (scale: 200 nm).
  • FIG. 2 shows the percentage of proliferation of CD4+ T cells after interacting with DC treated with different treatment methods.
  • FIG. 3 is a flow cytometry plot of proliferation of CD4+ T cells after interacting with DC treated with different treatment methods.
  • FIG. 4 shows irradiated membrane vesicles enhance the interaction between DC cells and T cells (GC: growth control, dendritic cell growth control group (unstimulated group); Cell+MVs (whole-cell bacteria+membrane vesicle treatment group); MVs (membrane vesicle treatment group)).
  • FIG. 5 shows the bacterial load of scalded sites of rabbits 24 h after Pseudomonas aeruginosa infection.
  • FIG. 6 shows the bacterial load of scalded sites of rabbits 24 h after Pseudomonas aeruginosa PA14 infection.
    •  DETAILED DESCRIPTION
  • To make the objective, the technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in combination with drawings. It is obvious that the described embodiments are some of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making inventive effort shall belong to the protection scope of the present invention.
  • The term “burn and scald” in the present invention refers to the damage to tissues, mainly referring to skin and/or mucosa, caused by heat, including hot fluid (water, soup, oil, etc.), steam, high-temperature gas, flame, and red-hot metal liquid or solid (such as molten steel and steel ingots), which may damage subcutaneous and/or submucosal tissues in severe cases, such as muscles, bones, joints and even internal organs. Scald is tissue damage caused by hot fluid, steam and high-temperature solid, and belongs to one of thermal burns.
  • The Pseudomonas aeruginosa vaccine of the present invention comprises (i) irradiation-inactivated Pseudomonas aeruginosa cells and/or (ii) Pseudomonas aeruginosa membrane vesicles. FIG. 1 is a Transmission Electron Microscopy (TEM) photograph of purified membrane vesicles.
  • Embodiments 1-3 introduce some isolation methods for preparing vesicles. The vesicles can be isolated from non-irradiated bacteria or isolated from irradiated bacteria or obtained by other methods.
    • Embodiment 1 Isolation Method for Bacterial Membrane Vesicles
  • In some embodiments, a method for isolating membrane vesicles from Pseudomonas aeruginosa comprises the following steps: 1) isolating bacterial cells in bacterial solution for culture of bacteria from culture medium, and collecting supernatant 1; 2) centrifuging the supernatant 1 with a high-speed centrifuge, and collecting supernatant 2; and 3) centrifuging the supernatant 2 with an ultra-high-speed centrifuge to precipitate membrane vesicles.
  • Further, isolation method in the step 1) comprises centrifugation, column chromatography, or dialysis bag concentration.
  • Further, the supernatant 2 collected in the step 2) is subjected to dialysis bag concentration prior to the step 3). In some embodiments, the dialysis bag selected can concentrate substances greater than 100 KD.
  • Further, the membrane vesicles are resuspended with a buffer solution, the buffer solution comprises 50 mM Tris, 5 mM NaCl and 1 mM MgSO4 calculated as a volume unit of 1 L and has a pH of 7.4.
  • In some embodiments, the bacterial cells and the membrane vesicles are prepared as a biological composition, and the preparation method comprises: collecting the bacterial cells isolated in the step 1) in the above isolation method for membrane vesicles, and mixing the bacterial cells with the membrane vesicles obtained in the step 3) to form the biological composition.
  • Further, in the step 1), the supernatant 1 is filtered with a 0.3-0.5 μM filter to remove impurities.
  • Preferably, the supernatant 1 is filtered with a 0.45 μM filter to remove impurities.
  • Further, the isolation method in the step 1) is centrifugation, the centrifugation speed is 100-10000 g, and the centrifugation time is 10-60 min.
  • Preferably, the centrifugation speed in the step 1) is 400-8000 g, and the centrifugation time is 10-30 min.
  • Further, the high-speed centrifugation speed in the step 2) is 5000-25000 g, and the high-speed centrifugation time is 10-100 min.
  • Preferably, the high-speed centrifugation speed in the step 2) is 10000-20000 g, and the high-speed centrifugation time is 30-60 min.
  • Further, the ultra-high-speed centrifugation speed in the step 3) is 5000-150000 g, and the ultra-high-speed centrifugation time is 60-600 min.
  • Preferably, the ultra-high-speed centrifugation speed in the step 3) is 15000-150000 g, and the ultra-high-speed centrifugation time is 60-180 min.
    • Embodiment 2 Augmentation and Purification of Bacterial Membrane Vesicles
  • Further, in some embodiments, the method for preparing bacterial membrane vesicles further comprises the following steps: 1) Augmentation of membrane vesicles: culturing bacteria to logarithmic growth phase; collecting the bacterial cells, resuspending the bacterial cells and then irradiating them with ionizing irradiation to obtain irradiated bacteria; 2) Isolation and purification of membrane vesicles: isolating membrane vesicles produced by the irradiated bacteria from the irradiated bacteria to obtain the membrane vesicles using the method for isolating membrane vesicles described in Embodiment 1.
  • Further, the ionizing irradiation is X-rays, and the irradiation dose is 500-3000 Gy. The irradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gy, 900-1000 Gy, 1000-1100 Gy, 1100-1200 Gy, 1200-1300 Gy, 1300-1400 Gy, 1400-1500 Gy, 1500-1600 Gy, 1600-1700 Gy, 1700-1800 Gy, 1800-1900 Gy, 1900-2000 Gy, 2100-2200 Gy, 2200-2300 Gy, 2300-2400 Gy, 2400-2500 Gy, 2500-2600 Gy, 2600-2700 Gy, 2700-2800 Gy, 2800-2900 Gy and 2900-3000 Gy.
  • Preferably, the irradiation dose is 500-1000 Gy. The irradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gy and 900-1000 Gy.
  • Further, OD600 value of the bacteria in logarithmic growth phase in the step 1) is 0.3-0.8.
  • Preferably, the OD600 value of the bacteria in logarithmic growth phase in the step 1) is 0.5-0.7.
  • Further, in the step 1), the bacterial cells are resuspended with phosphate buffer saline or sterile normal saline.
  • Preferably, in the step 1), the bacterial cells are resuspended with phosphate buffer saline.
  • Further, in the step 1), the bacterial cells are resuspended to an OD600 value of 20-80.
  • Preferably, in the step 1), the bacterial cells are resuspended to an OD600 value of 40-60.
  • The content of nucleic acids and the content of proteins in the membrane vesicles prepared by the above method are increased by 10-20 times, compared with those prepared from bacteria not irradiated with ionizing irradiation.
  • The membrane vesicles prepared by the present invention have various application scenarios: for example, (i) the membrane vesicles can be used as an immunogen; (ii) the membrane vesicles can be used as an immune response enhancer; (iii) the membrane vesicles can be used as a vaccine for treating bacterial infectious diseases; (iv) the membrane vesicles can be used as a vaccine adjuvant (in some embodiments, the vaccine adjuvant non-specifically changes or enhances the antigen-specific immune response of the body); (v) the membrane vesicles can be used as an antigen-presenting cell function enhancer.
  • The above antigen-presenting cell includes dendritic cells (i.e., DC cells), macrophages and B cells. The membrane vesicles obtained by irradiation, isolation and purification can be used as an enhancer for the maturation of the DC cells, and specifically, used as an enhancer for promoting the significant up-regulation of cell surface molecules CD80, CD86 and MHCII molecules of bone marrow-derived dendritic cells.
  • In some embodiments, the membrane vesicles prepared by the present invention can be combined with DC cells in preparation of a proliferation agent for CD4+ T cells. Specifically, the method for promoting proliferation of CD4+ T cells comprises the following steps: co-culturing membrane vesicles-stimulated and OVA-antigen-phagocytosed DCs with CFSE-labeled CD4+ T lymphocytes in vitro, wherein the membrane vesicles are prepared by irradiation.
    • Embodiment 3 A Method for Isolating and Preparing Bacterial Membrane Vesicles
  • In some embodiments, the method for isolating and preparing bacterial membrane vesicles comprises the following steps:
  • 1. Culturing bacteria to logarithmic growth phase, wherein OD600 value of the bacteria in logarithmic growth phase is 0.3-0.8, and the OD600 value of 0.5-0.8 is preferably selected (fermentation can also be performed here to further enrich bacterial cells); collecting bacterial cells, and resuspending the bacterial cells with an appropriate amount of phosphate buffer solution, wherein the ratio of the amount of the added phosphate buffer solution to the total amount of the bacterial cells is that the OD600 value of the amount of the bacteria contained in every 1 ml of solution is 20-80, and the OD600 value of 40-60 is preferably selected; after resuspension, irradiating the bacterial cells with ionizing irradiation to obtain irradiated bacteria; preferably, irradiating with X-rays, with an irradiation dose of 500-3000 Gy. The irradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gy, 900-1000 Gy, 1000-1100 Gy, 1100-1200 Gy, 1200-1300 Gy, 1300-1400 Gy, 1400-1500 Gy, 1500-1600 Gy, 1600-1700 Gy, 1700-1800 Gy, 1800-1900 Gy, 1900-2000 Gy, 2100-2200 Gy, 2200-2300 Gy, 2300-2400 Gy, 2400-2500 Gy, 2500-2600 Gy, 2600-2700 Gy, 2700-2800 Gy, 2800-2900 Gy and 2900-3000 Gy.
  • 2. Collecting bacterial solution, centrifuging the bacterial solution and collecting supernatant, and filtering the supernatant with a 0.3-0.5 μM filter to remove the bacteria; wherein the centrifugation speed is 400-8000 g; and the centrifugation time is 10-30 min.
  • 3. Centrifuging the filtered supernatant with a high-speed centrifuge, collecting supernatant, and removing flagella; wherein the high-speed centrifugation speed is 10000-20000 g; and the high-speed centrifugation time is 30-60 min.
  • 4. Centrifuging the supernatant after removal of the flagella with an ultra-high-speed centrifuge to precipitate membrane vesicles; wherein the ultra-high-speed centrifugation speed is 15000-150000 g; and the ultra-high-speed centrifugation time is 60-180 min.
  • 5. Collecting the membrane vesicles to obtain purified membrane vesicles.
  • Preparation, isolation and purification of membrane vesicles by irradiating Pseudomonas aeruginosa PAO1 with ionizing irradiation:
  • 1. Streaking Pseudomonas aeruginosa PAO1 recovered from −80° C. onto LB plates, and culturing them in an incubator at 37° C. for 16-18 h.
  • 2. Picking monoclonal colonies from the LB plates, inoculating the monoclonal colonies in 20 mL of LB liquid medium, and culturing them at constant temperature of 37° C. at 250 rpm for 16-18 h.
  • 3. Inoculating overnight bacterial solution into 1 L of LB medium to an initial concentration of 0.05 OD600/mL and culturing the bacteria to logarithmic growth phase at 37° C. at 250 rpm, and measuring OD600 value of the bacterial solution.
  • 4. Transferring the above bacterial solution of the step 3 to a centrifugal barrel, centrifuging the bacterial solution at 5,000 g for 20 min, collecting the bacterial cells and resuspending the bacterial cells with normal saline, and adjusting the concentration of the bacterial cells to about 50 OD.
  • 5. Placing the above bacterial solution in an irradiator with an irradiation dose of 1000 Gy.
  • 6. Centrifuging the irradiated bacterial solution at 8,000×g for 20 min twice and collecting supernatant; filtering the supernatant with a 0.45 μM filter to remove the bacteria and collecting the supernatant again; at the same time, coating a small amount of the supernatant onto the LB plates and culturing them at 37° C. for 24-72 h to confirm that viable bacteria do not exist.
  • 7. Centrifuging the supernatant of the step 6 with a high-speed centrifuge to remove flagella in the supernatant.
  • 8. Centrifuging the supernatant of the step 7 with an ultra-high-speed centrifuge to precipitate membrane vesicles.
  • 9. Discarding the supernatant, resuspending the precipitate with MV buffer, and storing it at −80° C.
  • 10. Observing the extracted membrane vesicles of the normal group and the membrane vesicles of the experimental group of the present invention by transmission electron microscopy.
  • Experimental Results:
  • According to the results of transmission electron microscopy, the ionizing irradiation can stimulate Pseudomonas aeruginosa PAO1 to produce membrane vesicles. The membrane vesicles are shown in FIG. 1.
    • Embodiment 4 Immunomodulatory Effects of Irradiated Bacterial Membrane Vesicles—Promoting Maturation of Dendritic Cells
  • Dendritic cells (DCs) are the main antigen-presenting cells of the body, and have the main function of phagocytosing and processing antigen molecules as well as presenting them to T cells. The DCs are the known most powerful and the only professional antigen-presenting cell that can activate resting T cells in the body, and are a key link in initiating, regulating and maintaining immune responses. The maturation of the DCs determines the immune response or immune tolerance of the body. Co-stimulatory molecules B7 (B7-1=CD80 and B7-2=CD86) on the surfaces of the DCs can be bound to CD28 or CD152 molecules on the surfaces of T cells, to enhance or weaken the MHC-TCR signal transduction between DCs and T cells. The main characteristics of mature DCs are changes in the expression of co-stimulatory molecules CD80 and CD86, reduced ability to phagocytose antigens and enhanced the ability to process and present antigens (increased MHCII molecules expression), and interaction with T lymphocytes.
  • 1. Culture and induction of mouse bone marrow-derived dendritic cells (BMDC): taking 6-8 week old C57 female mice, aseptically separating mouse femurs, removing the muscles on the femurs, and cutting both ends of the femurs; rinsing the bone lumens with PBS until the bone lumens turn white; filtering PBS suspension and then centrifuging it at 1200 rpm for 5 min; removing supernatant; and adding 5 ml of red blood cell lysis buffer to resuspend the cells. After standing for 15 min, centrifuging the lysis product at 1200 rpm for 5 min, and removing the supernatant; adding 50 ml of 1640 complete medium (20 ng/ml GM-CSF, 10% FBS and 50 mM of 2-mercaptoethanol) to resuspend the cells. After uniform mixing, dividing the cells into 5 petri dishes and culturing them in an incubator. Changing the medium every 2 days and collecting the cells on the 7th day.
  • 2. BMDC stimulation: taking the BMDC cells induced for 7 days, and repeatedly blowing the cells in a 6-well plate to detach adherent cells; collecting the cell suspension, centrifuging it at 1100 rpm for 5 min, removing supernatant, and adding 1 ml of medium to resuspend the cells, and adjusting the cell concentration to 1×106/m1 after counting viable cells, and inoculating 2 ml of the cells into a new 6-well plate. Each stimulator will be added respectively and uniformly mixed: whole-cell bacteria, whole-cell bacteria+vesicles, and vesicles at a final concentration of 15 μg/mL (based on protein). Continuing to culture them for 24 hours and adding an equal volume of PBS to the growth control group.
  • 3. Maturation markers detection by flow cytometry: after 24 h, taking out the 6-well plate, repeatedly blowing the cells to detach them, collecting the cell suspension into a Flow Cytometry Tube, centrifuging it at 1500 rpm for 3 min, removing supernatant, adding 1 ml of PBS to continue centrifugation at 1500 rpm for 3 min, then removing the supernatant and repeatedly washing for 3 times. Adding CD11c/CD80/CD86/MHCII antibodies and incubating at room temperature for 30 min in the dark; at the same time, setting an isotype control group as the negative control group (adding isotype controls of CD11c/CD80/CD86/MHCII). After incubation, adding PBS to wash twice, then adding 200 μl of PBS to resuspend the cells, and detecting the cells by flow cytometry.
  • 4. Result processing: analyzing the ratio of CD80/CD86/MHCII in CD11c cells by flow cytometry software.
  • Experimental results: Compared with the whole-cell bacteria, the vesicles of the experimental group (MVs) treated by X-rays can significantly up-regulate the surface costimulatory molecules CD80, CD86 and MHCII of DCs after stimulation, and these surface molecules are markers of dendritic cell maturation. In conclusion, it is proved that the vesicles can significantly promote the differentiation and maturation of DCs.
  • The phagocytic ability of DC cells is detected by detecting the fluorescence intensity of FITC-dextran: DC cells have strong antigen endocytosis and processing abilities. The DC cells have strong phagocytic ability in an immature state when not in contact with antigen. After in contact with antigen and activated, the DC cells become mature with low phagocytic ability and enhanced antigen-presenting ability. In the experiment, by detecting the fluorescence intensity of the FITC-dextran, the amount of the dextran phagocytosed by DC is determined to detect whether the phagocytic ability of DC is enhanced.
  • 1. Culture and induction of BMDC cells (same as the above).
  • 2. Stimulation: collecting the cells on the 7th day, blowing down all the cells, then centrifuging and resuspending the cells for counting, then inoculating the cells into a 6-well plate with 1×106 cells per well, and respectively adding the stimulator: adding an equal volume of PBS to the GC group, adding the same concentration of membrane vesicles (by protein level) to the control group and the treatment group and then culturing them at 37° C. for 24 h.
  • 3. Phagocytosis and detection: adding the dextran (5 μg/ml), and after culture for 1 h, aspirating the cells into a Flow Cytometry Tube; washing the cells with PBS for 3 times; adding CD11c antibody and incubating at room temperature for 30 min in the dark; washing the cells with PBS for 3 times; and detecting the fluorescence of FITC by flow cytometry.
  • 4. Result processing: analyzing the ratio of FITC in CD11c cells by flow cytometry software.
  • Experimental results: in order to detect the phagocytic function of DCs, the present invention uses FITC-dextran as a model antigen for phagocytosis of DCs and detects the mean fluorescence intensity value of FITC of CD11c+DCs. The experimental result shows that after the DCs are stimulated by membrane vesicles, the mean fluorescence intensity value of FITC is significantly reduced compared with that of the GC group (growth control group). This experimental result proves again that the vesicles can promote the maturation of DCs, thereby reducing their ability to take up antigen.
    • Embodiment 5 Interaction Between Mature DCs and T Cells Stimulated by Bacterial Membrane Vesicles in X-Ray Treatment Group
  • A. Interaction Between Mature DCs and CD4+ T Cells:
  • The effective cross-antigen presentation of extracellular proteins by DCs plays an important role in the induction of specific cellular immune responses. Therefore, the cross-presentation effect of OVA antigen by DCs stimulated by membrane vesicles is detected. 72 h after co-culture of DCs-T cells, the proliferation of OT-II CD4+ T lymphocytes is detected by CFSE flow cytometry. Fluorescent dye CFSE (CFDA-SE), namely carboxyfluorescein diacetate, succinimidyl ester, is a cell staining reagent that can fluorescently label live cells. CFDA-SE can be irreversibly coupled to cellular proteins by binding to intracellular amines after entering cells. In the process of cell division and proliferation, the CFSE-labeled fluorescence can be equally distributed to two daughter cells, and the fluorescence intensity is half that of the parental cells. Therefore, the percentage of cells with weak CFSE fluorescence can be counted by flow cytometry to obtain the proportion of proliferating cells.
  • 1. Culture and induction of BMDC cells (same as the previous embodiment).
  • 2. Antigen phagocytosis: culturing DCs which are cultured for 7 days in a medium containing 10 μg/ml OVA for 24 h to serve as GC (growth control group); adding vesicles to the MVs group, then centrifuging and collecting antigen-phagocytosed DCs; resuspending the DCs in a normal medium; and applying the DCs in a 96-well plate at a density of 2×104 cells/well, with 100 μl per well, and 3 replicate wells per group.
  • 3. T cell extraction: on the second day, isolating and enriching OVA-specific CD4+T lymphocytes from the spleens of OT-II mice by a magnetic negative selection beads kit from Stem Cell Technologies company.
  • 4. Co-culture of DC and T cells: labeling the sorted CD4+ T cells with 1 μM CFSE according to the kit instructions. After labeling, washing the cells for 3 times with PBS and adding the cells to the 96-well plate at a density of 105 cells/well to a final culture volume of 200 μl (CD4:DC=5:1).
  • 5. On the 3rd day after co-culture, detecting the proliferation of CD4+ T cell population by CFSE decrement by flow cytometry.
  • Experimental results: co-culturing membrane vesicles-stimulated and OVA-antigen-phagocytosed DCs with CFSE-labeled OT-II mouse CD4+ T lymphocytes in vitro. The analysis results of flow cytometry for CFSE fluorescence intensity show that the proportion of proliferating CD4+ T cells is increased. The membrane vesicles (14.05%) can significantly increase the proliferation-promoting effect of OVA-antigen-phagocytosed DCs (6.80%) on specific CD4+ T cells. See FIG. 2 and FIG. 3 for details.
  • B. Promotion of T Cell Proliferation by DCs Treated by the Membrane Vesicles:
  • The effective cross-antigen presentation of extracellular proteins by DCs plays an important role in the induction of specific cellular immune responses. Therefore, the cross-presentation effect of OVA antigen by DCs stimulated by membrane vesicles is detected. 72 h after co-culture of DCs-T cells, the proliferation of T lymphocytes is detected by CFSE by flow cytometry. Fluorescent dye CFSE (CFDA-SE), namely carboxyfluorescein diacetate, succinimidyl ester, is a cell staining reagent that can fluorescently label live cells. CFDA-SE can be irreversibly coupled to cellular proteins by binding to intracellular amines after entering cells. In the process of cell division and proliferation, the CFSE-labeled fluorescence can be equally distributed to two daughter cells, and the fluorescence intensity is half that of the parental cells. Therefore, the percentage of cells with weak CFSE fluorescence can be counted by flow cytometry to obtain the proportion of proliferating cells.
  • 1. Culture and induction of BMDC cells (same as the previous embodiment).
  • 2. Antigen phagocytosis: culturing DCs which are cultured for 7 days in a medium for 24 h to serve as GC (growth control group); adding vesicles to the MVs group, then centrifuging and collecting antigen-phagocytosed DCs; resuspending the DCs in a normal medium; and applying the DCs in a 96-well plate at a density of 4×104 cells/well, with 100 μl per well, and 3 replicate wells per group.
  • 3. T cell extraction: on the second day, isolating and enriching the T cells of the mice from the spleens of mice one week after one MVs immunization using a magnetic negative selection beads kit from Stem Cell Technologies company.
  • 4. Co-culture of DC and T cells: labeling the sorted T cells with 1 μM CFSE according to the kit instructions. After labeling, washing the cells for 3 times with PBS and adding the cells to the 96-well plate at a density of 4×105 cells/well to a final culture volume of 200 μl (CD3:DC=10:1).
  • 5. On the 3rd day after co-culture, detecting the proliferation of CD3+, CD8+ and CD4+ T cell populations by CFSE decrement by flow cytometry.
  • Experimental results: co-culturing membrane vesicles-stimulated and OVA-antigen-phagocytosed DCs with CFSE-labeled OT-II mouse CD4+ T lymphocytes in vitro. The analysis results of flow cytometry for CFSE fluorescence intensity show that the proportion of proliferating CD4+ T cells is increased. As shown in the figure, the fluorescence intensity of the whole-cell bacteria plus vesicle stimulation group is 63.5%, and the fluorescence intensity of the vesicle stimulation group is 71%. It indicates that DCs after vesicle treatment can significantly stimulate the proliferation of CD4+ T cells. See FIG. 4.
    • Embodiment 6 Experiment of Pseudomonas aeruginosa Vaccine Against Scald Complicated with Pseudomonas aeruginosa Skin Infection
  • Experimental rabbits are used in the experiment. A scald model of rabbits is established by scalding rabbits with a hot-air gun one day before the rabbits are infected with Pseudomonas aeruginosa. After 24 h, the scalded sites are subcutaneously infected with the homologous strain of Pseudomonas aeruginosa SKLBPA1 and Pseudomonas aeruginosa PA14 respectively (both purchased from ATCC). 24 h after infection, the rabbits are sacrificed, and skin and subcutaneous muscle tissue of the scalded sites are taken, homogenized and counted for CFU. The CFU of the model group and the vaccine group are compared to observe whether the vaccine of the present invention has a protective effect on scald complicated with Pseudomonas aeruginosa infection. The results of the experiment will show that the Pseudomonas aeruginosa vaccine provided by the present invention can have preventive and therapeutic effects on burn and scald complicated with infection caused by different serotypes of Pseudomonas aeruginosa (two representative strains of SKLBPA1 and PA14 are used as examples in the experiment), and has wide application scenarios.
  • The content of the vaccine of the present invention includes: 108 CFU/ml
  • Experimental animals: 6 rabbits, New Zealand White rabbits, weighing 2.210 kg-2.870 kg, female.
  • 1. Experimental Groups
  • Three groups are set in the experiment with 2 animals in each group. The specific group information is shown in Table 1.
  • TABLE 1
    Group Information
    Group Immunization Bacteria for
    Number Group Name Vaccine Procedure Infection
    Group PA1 PA1 0, 3, 7 d (injection SKLBPA1
    1 immunization take places at 0,
    group 1 3rd, 7th days)
    Group PA1 0, 2, 4 w (injection
    2 immunization take places at 0,
    group 2 2nd, 4th weeks)
    Group PA1 model — —
    3 group
  • 2. Immunization
  • The test vaccine (108 CFU/ml) is subcutaneously immunized with 1000 in the left and right groins of the rabbits for 3 times, with two immunization procedures of 0, 3, 7 d (injection take places at 0, 3rd, and 7th days) and 0, 2, 4 w (injection take places at 0, 2nd and 4th weeks).
  • 3. Establishment of Scald Model
  • Two days before infection, the front and back hairs on the left and right sides of the rabbits are removed by scissors and depilatory cream. After 24 h, the skin of the depilation sites is disinfected by 75% alcohol and applied with tetracaine hydrochloride mucilage for local anesthesia. Then, the temperature of the hot-air gun is set at 200° C.; the metal plate with square holes is disinfected by 75% alcohol and stuck onto the depilation sites. The hot-air gun is turned on; and a hot air outlet is pointed towards the square holes to blow the hot air onto the rabbit skin for 5 s, causing a certain degree of scald model.
  • 4. Infection
  • 4.1 Recovery of Bacteria
  • Pseudomonas aeruginosa SKLBPA1 and PA1 recovered from −80° C. to TSA plates, and cultured overnight at 37° C.
  • 4.2 Overnight Bacteria Culture in a Shaker
  • Monoclonal colonies are picked from the plates respectively into TSB for shaking culture at 37° C. at 220 rpm overnight.
  • 4.3 Culture Expansion
  • The overnight bacterial solution is diluted to measure OD600; the original bacterial solution is inoculated into 100 ml of TSB (250 ml conical flask); and the bacteria are shaken at 37° C. at 220 rpm to logarithmic growth phase.
  • 4.4 Centrifugation and Washing
  • The bacterial solution is collected into a 50 ml centrifuge tube, and centrifuged at 4100 rpm (3000×g) at room temperature for 10 min; the supernatant is discarded and the precipitate is resuspended with 2 mL of 0.9% sodium chloride injection (about 5 OD/ml); and the bacterial solution is adjusted to 0.1 OD600 (2×107 CFU/ml).
  • 4.5 Infection
  • One week (the 7th day) after the last immunization, 50 μl of bacterial solution (1×106 CFU/site) is subcutaneously injected into the scalded skin of the rabbits.
  • 4.6 Plate Coating and Counting
  • The adjusted bacterial solution is coated to a TSA plate, and cultured at 37° C. overnight, and CFU is counted.
  • 5. Count of Bacteria of Skin and Muscular Tissue 24 Hours after Infection
  • Animals are sacrificed 24 hours after infection. The skin is disinfected by spraying with 75% alcohol. The skin and muscular tissue of the scalded sites are taken aseptically, and the tissue is homogenized, coated to the TSA plate, and cultured at 37° C. overnight. The CFU is counted by a colony counter.
  • 6. Data Processing
  • LOG10 CFU scatter plots of skin and muscular tissue are drawn using Graphpad Prism software. The mean value of LOG10 CFU is calculated, and between-group variation is analyzed by Analysis of Variance.
  • Experimental Results:
  • 1. Change in Body Weights of Rabbits
  • The body weights of the animals in each group are shown in Table 2. The body weights of the rabbits in each immunization group and each model group are increased steadily, and there is no significant difference among the groups.
  • TABLE 2
    Change in Body Weights of Rabbits
    Week
    1 Week 2 Week 3 Week 4 Week 5 Week 6
    Groups weight (kg) weight (kg) weight (kg) weight (kg) weight (kg) weight (kg)
    Group 1 2.460 ± 0.057 2.540 ± 0.057 2.590 ± 0.028 2.655 ± 0.035 2.740 ± 0.085 2.905 ± 0.205
    Group 2 2.540 ± 0.198 2.615 ± 0.177 2.700 ± 0.170 2.770 ± 0.184 2.925 ± 0.148 3.025 ± 0.021
    Group 3 2.555 ± 0.064 2.635 ± 0.064 2.735 ± 0.078 2.800 ± 0.071 2.845 ± 0.021 3.060 ± 0.156
  • 2. Modeling Results of Skin Scald of Rabbits
  • The hot-air gun is sleeved with an air nozzle with an outer diameter of 14 mm and an inner diameter of 10 mm; the temperature is set as 200° C.; and the air nozzle is kept close to the depilation sites on the backs of the rabbits for 5 seconds. After 2 hours, round blisters with a diameter of about 10 mm appear on the skins of the scalded sites, and after 24 hours, the skins of the scalded sites are scabbed.
  • 3. Infective Dose of Pseudomonas aeruginosa for Rabbit Skin
  • The concentrations of Pseudomonas aeruginosa SKLBPA1 and PA14 bacterial suspensions used in the experiment are 0.1 OD/ml; the remaining bacterial suspensions after subcutaneous infection are diluted to 10−4 and 10−5; 50 μl of bacterial suspensions are evenly coated on the TSA plates, and incubated overnight in an incubator at 37° C.; and the CFU is counted. The results are shown in Table 3.
  • TABLE 3
    Viable Count of Bacteria Results of Bacterial Solution for Infection
    Turbidity of Concentration of
    Bacterial Strain Bacterial Solution Bacterial Solution
    Name for Infection (OD/ml) for Infection (CFU/ml)
    SKLBPA1 0.1 8.00 × 107
    PA14 0.1 1.06 × 108
  • 4. Bacterial Load of Scalded Sites of Rabbits 24 h after Pseudomonas aeruginosa Infection
  • The animals are sacrificed 24 h after infection. The skin and muscular tissue of the scalded and infected sites (infected with Pseudomonas aeruginosa SKLBPA1) are taken aseptically, homogenized, and coated to the TSA plates, and cultured overnight at 37° C. The CFU is counted, and then the mean and the standard deviation of each group are compared based on the log values of the bacterial loads to the base of 10. The results are shown in Table 4 and FIG. 5. Two immunization procedures of PA1 vaccine can significantly reduce the load of Pseudomonas aeruginosa SKLBPA1 (P<0.01), which indicates that the vaccine has an obvious protective effect.
  • TABLE 4
    Bacterial Load of Scalded Sites of Rabbits 24 h
    After Pseudomonas Aeruginosa Infection
    Number
    Bacteria for of Skins LOG10 of
    Groups Vaccine Immunization Procedure Infection (blocks) Bacterial Load
    Group
    1 PA1 0, 3, 7 d (injection take places SKLBPA1 6 5.08 ± 0.61**
    at 0, 3rd, 7th days)
    Group 2 0, 2, 4 w (injection take places 6 4.68 ± 0.40**
    at 0, 2nd, 4th weeks)
    Group 3 — — 6 6.93 ± 0.57 
    (Note:
    **compared with each model group, P < 0.01 represents statistically highly significant differences.)
  • 5. Bacterial Load of Scalded Sites of Rabbits 24 h after Pseudomonas aeruginosa PA14 Infection
  • The skin and muscular tissue of the scalded and infected sites of Pseudomonas aeruginosa PA14 are taken aseptically, coated to the TSA plates by a homogenizer, and cultured overnight at 37° C.; the CFU is counted by a colony counter; and then the mean and the standard deviation of each group are compared based on the log values of the bacterial loads to the base of 10. The results are shown in Table 5 and FIG. 6. The immunization procedure 0, 3, 7 d of the PA1 vaccine can significantly reduce the load of Pseudomonas aeruginosa PA14 (P<0.05), with a reduction range of about 0.6-0.9 log. It indicates that the vaccine has a certain protective effect.
  • It is worth noting that in the experiments for SKLBPA1 and PA14, the immunization procedure 0, 3, 7 d can quickly generate an effective immune response, so the vaccine of the present invention can be used to prevent skin scald complicated with infection (that is, as a prophylactic vaccine), and may also possibly be used to inhibit and alleviate the infection after the onset of skin scald complicated with infection (that is, the possibility of serving as a therapeutic vaccine).
  • TABLE 5
    Bacterial Load of Scalded Sites of Rabbits 24 h
    after Pseudomonas Aeruginosa PA14 infection.
    Number
    Bacteria for of Skins LOG10 of
    Groups Vaccine Immunization Procedure Infection (blocks) Bacterial Load
    Group
    7 PA1 0, 3, 7 d (injection take places PA14 6  5.67 ± 0.54*
    at 0, 3rd, 7th days)
    Group 8 0, 2, 4 w (injection take places 6 6.22 ± 1.10
    at 0, 2nd, 4th weeks)
    Group 9 — — 18 6.30 ± 0.65
    (Note:
    *compared with each model group, P < 0.05 represents statistically significant differences.)
  • The embodiments of the present invention are described above in combination with drawings, but the present invention is not limited to the aforementioned specific embodiments. The aforementioned embodiments are merely illustrative and not limiting. For those of ordinary skill in the art, many forms can be made under the teaching of present invention without departing from the spirit of the present invention and the scope of the claims, all of which shall fall within the protection scope of the present invention.

Claims (19)

1. Use of a Pseudomonas aeruginosa vaccine in manufacture of a medicament for prevention and treatment of bacterial infection in burn and scald, wherein the burn and scald are complicated with the bacterial infection and the bacterial infection is Pseudomonas aeruginosa infection; the Pseudomonas aeruginosa vaccine comprises inactivated Pseudomonas aeruginosa and/or Pseudomonas aeruginosa membrane vesicles; wherein the inactivated Pseudomonas aeruginosa is inactivated by irradiation, and the Pseudomonas aeruginosa membrane vesicles are isolated from the Pseudomonas aeruginosa inactivated by irradiation.
2. The use according to claim 1, wherein the burn and scald include burns and scalds, and degree of the scalds includes I degree, superficial II degree, deep II degree, or III degree scalds.
3. The use according to claim 2, wherein site of the scalds includes skin, mucosa or other tissues.
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. The use according to claim 1, wherein site of the scalds includes skin, mucosa or other tissues.
9. (canceled)
10. The use according to claim 1, wherein immunization procedures of the Pseudomonas aeruginosa vaccine comprise: injection take places (i) 0, 3rd, and 7th days, and (ii) 0, 2nd and 4th weeks.
11. (canceled)
12. The use according to claim 1, wherein the Pseudomonas aeruginosa vaccine prevents Pseudomonas aeruginosa infection, and reduces bacterial load in skin scald complicated with bacterial infection.
13. The use according to claim 1, wherein content of whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1×104-1×1010/injection.
14. The use according to claim 13, wherein the content of whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1×104/injection, 1×105/injection, 1×106/injection, 1×107/injection, 1×108/injection, 1×109/injection and 1×1010/injection.
15. The use according to claim 1, wherein the Pseudomonas aeruginosa vaccine further contains an immunoadjuvant.
16. The use according to claim 15, wherein the immunoadjuvant is aluminum hydroxide.
17. The use according to claim 1, wherein administration site of the Pseudomonas aeruginosa vaccine is subcutaneous, muscle and/or mucosa.
18. The use according to claim 1, wherein the medicament can also contain any pharmaceutically acceptable carrier and/or adjuvant.
19. The use according to claim 18, wherein the carrier is a liposome.
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