CN114364396A - Application of pseudomonas aeruginosa vaccine in resisting burn and scald infection - Google Patents
Application of pseudomonas aeruginosa vaccine in resisting burn and scald infection Download PDFInfo
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Abstract
The application of the pseudomonas aeruginosa vaccine in preventing and treating burn and scald combined with bacterial infection is provided, wherein the burn and scald comprise burn and scald, and the degree of the burn comprises the following steps: scald of degree I, superficial degree II, deep degree II and degree III, the scald part comprises skin, mucous membrane or other tissues.
Description
The application requires Chinese invention patent 1) 201910777479.2' bacterial membrane vesicle and preparation method and application thereof, which are submitted in 2019, 8, month and 22 days; 2)201910777473.5 'Staphylococcus aureus membrane vesicle, its preparation method and application'; 3)201910777606.9 'Pseudomonas aeruginosa membrane vesicle, preparation method and application thereof'; 4)201921369450.2 'production system and separation and purification system of bacterial membrane vesicles'; 5)201910777595.4 priority of a bacterial membrane vesicle production system and separation and purification system and method, the five priority patent applications are incorporated by reference in their entirety.
The invention belongs to the field of microbiology, and particularly relates to application of a pseudomonas aeruginosa vaccine in preventing and treating burn and scald complicated bacterial infection.
At present, burns and scalds become one of the most common accidental injuries in daily production and life. According to statistics, about 5000-. The main sites of burns and scalds include the skin and/or mucous membranes, and even severe cases may injure subcutaneous and/or submucosal tissues, such as muscles, bones, joints and even internal organs. Scalding is one of the thermal burns, mainly the tissue damage caused by hot liquid, steam and high temperature solid. The judgment of the scald depth is divided according to a three-degree quartering method, and the scald depth, pathological changes and clinical manifestations can be divided into: scald of I degree, superficial II degree, deep II degree and III degree.
Wound infection is the most common and serious complication after scald. The bacteria causing wound infection are mainly gram-negative bacilli, wherein pseudomonas aeruginosa is one of the most common pathogenic bacteria. The detection rate of pseudomonas aeruginosa in burn patients varies from 20% to more than 50% in different reports, and researches indicate that the pseudomonas aeruginosa can be separated from burn wounds of 100% almost 7 days after burns. In addition, the death rate of pseudomonas aeruginosa after infection reaches 31 percent, and particularly in burn patients, if the bacterial infection of the wound surface after burn cannot be cured in time, serious consequences can be caused. In addition, if infectious bacteria are not removed in a timely manner, wound healing is delayed, or scars heal, which can impair wound site (joint) function.
At present, the treatment mode adopted for scald combined with pseudomonas aeruginosa infection is generally surgical debridement operation, and antibiotics are used locally and systemically in a combined manner. The most common antibacterial drugs include the synthetic antibacterial drugs sulfadiazine silver and the antibiotics ciprofloxacin, ceftazidime and cefoperazone/sulbactam, etc. However, due to the inappropriate use of antibiotics and the emergence of multidrug-resistant bacteria, the therapeutic effect is unsatisfactory, such as developing sepsis, life-threatening. Statistically, pseudomonas aeruginosa sepsis is the main cause of death in scald patients during scald combined with pseudomonas aeruginosa infection, and the incidence rate is between 8% and 42.5%, and the death rate is between 28% and 65%.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of pseudomonas aeruginosa vaccine in preventing and treating burn and scald infection.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the pseudomonas aeruginosa vaccine is applied to the preparation of the medicine for preventing and treating burn and scald infection.
Further, the burns and scalds include burns and scalds, and the degree of the scalds includes I-degree, superficial II-degree, deep II-degree or III-degree scalds.
Further, the scald site includes skin, mucous membranes, or other tissue.
Further, the burn and scald infection is burn and scald complicated bacterial infection.
Further, the bacteria with combined bacterial infection comprise one or more of pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, staphylococcus aureus, streptococcus pneumoniae and mycobacterium tuberculosis.
Further, the bacterial infection is a pseudomonas aeruginosa infection.
Further, the burns and scalds include burns and scalds, and the degree of the scalds includes I-degree, superficial II-degree, deep II-degree or III-degree scalds.
Further, the scald site includes skin, mucous membranes, or other tissue.
Further, the pseudomonas aeruginosa vaccine comprises inactivated pseudomonas aeruginosa and/or pseudomonas aeruginosa membrane vesicles.
Further, the immunization program of the pseudomonas aeruginosa vaccine comprises the following steps: the injection time interval was (i)0, 3, 7 days, (ii)0, 2, 4 weeks.
Further, the inactivated pseudomonas aeruginosa is subjected to irradiation inactivation, and the pseudomonas aeruginosa membrane vesicles are separated from the pseudomonas aeruginosa subjected to irradiation inactivation.
Further, the pseudomonas aeruginosa vaccine prevents pseudomonas aeruginosa infection, and reduces the bacterial load in the skin scald combined bacterial infection.
Further, the pseudomonas aeruginosa vaccine contains the whole pseudomonas aeruginosa thallus in the content of: 1X 104-1×10 10A needle.
Further, the pseudomonas aeruginosa vaccine contains the whole pseudomonas aeruginosa thallus in the content of: 1X 104Needle, 1X 105Needle, 1X 106Needle, 1X 107Needle, 1X 108Needle, 1X 10 9Needle and 1X 1010A needle.
Further, the pseudomonas aeruginosa vaccine also contains an immunologic adjuvant.
Further, the administration site of the pseudomonas aeruginosa vaccine is subcutaneous, muscle and/or mucosa.
Further, the medicament may further comprise any pharmaceutically acceptable carrier and/or auxiliary agent.
Further, the carrier is a liposome.
The invention has the beneficial effects
The experimental result shows that the pseudomonas aeruginosa vaccine can effectively prevent and treat burn and scald complicated pseudomonas aeruginosa infection caused by multidrug resistant pseudomonas aeruginosa by activating the atopic immunoreaction of the organism. Through a given immunization program, the bacterial load of the body infected by the immune organism is reduced, so that a technical scheme which can effectively prevent and treat burns and scalds combined with pseudomonas aeruginosa infection is provided, and the technical problems of poor antibiotics, difficult radical cure, easy generation of drug resistance and the like in the prior art are avoided to a certain extent.
The experimental result also shows that the pseudomonas aeruginosa vaccine adopted by the invention can effectively inhibit the bacterial load in the burn and scald complicated bacterial infection part by activating the human immune response, prevent the superinfection caused by the pseudomonas aeruginosa, avoid the problems of drug resistance and the like caused by adopting antibiotics in the prior art, and has wide application scenes in the burn and scald complicated bacterial infection.
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.
FIG. 1 Transmission Electron microscopy of irradiated Pseudomonas aeruginosa membrane vesicles (scale: 200 nm).
Figure 2 percentage proliferation of CD4+ T cells after interaction with DC after different treatment modalities.
FIG. 3 flow chart of proliferation of CD4+ T cells after interaction with DCs after different treatment modalities.
FIG. 4 irradiation-treated membrane vesicles enhance interaction of DC cells with T cells (GC: growth control, dendritic Cell growth control (unstimulated group); Cell + MVs (whole Cell + vesicle-treated group); MVs (vesicle-treated group)).
FIG. 5 bacterial load in rabbit scald sites 24h after Pseudomonas aeruginosa infection.
FIG. 6 bacterial load of the scald part of the rabbit 24h after Pseudomonas aeruginosa PA14 infection.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a burn and scald treatment, which is a tissue damage caused by heat, including hot liquid (water, soup, oil and the like), steam, high-temperature gas, flame, hot metal liquid or solid (such as molten steel and steel ingot) and the like, mainly refers to skin and/or mucous membrane, and can also injure subcutaneous and/or submucosal tissues such as muscle, bone, joint and even viscera in severe cases. Scald is a tissue injury caused by hot liquid, steam, high-temperature solid and the like, and belongs to one of thermal burns.
The pseudomonas aeruginosa vaccine of the invention comprises (i) inactivated pseudomonas aeruginosa thallus by irradiation and/or (ii) pseudomonas aeruginosa membrane vesicles. FIG. 1 is a transmission electron micrograph of purified membrane vesicles.
Examples 1-3 describe some isolation methods for preparing vesicles, either from non-irradiated bacteria or irradiated bacteria, or by other means.
Example 1 bacterial membrane vesicle separation method
In some embodiments, the method of isolating membrane vesicles from pseudomonas aeruginosa comprises the steps of: 1) separating thallus in a bacterial liquid for culturing bacteria from a culture solution, and collecting to obtain a supernatant 1; 2) centrifuging the supernatant 1 by using a high-speed centrifuge, and collecting a supernatant 2; 3) and centrifuging the supernatant 2 by using an ultra-high speed centrifuge, and precipitating into membrane vesicles.
Further, the separation method in the step 1) comprises centrifugation, column chromatography or membrane-packed concentration.
Further, the supernatant 2 obtained in the step 2) is concentrated by membrane packing before entering the step 3). In some embodiments, the selected membrane package can concentrate materials greater than 100 KD.
Further, the membrane vesicles were resuspended in a buffer comprising 50mM Tris,5mM NaCl,1mM MgSO 1L volume4And a pH of 7.4.
In some embodiments, the bacterial cells and membrane vesicles are prepared as a biological composition by a method comprising: collecting the bacteria separated in step 1) in the membrane vesicle separation method, and mixing the bacteria and the membrane vesicles obtained in step 3) to form the biological composition.
Further, in the step 1), the supernatant 1 is filtered by using a 0.3-0.5 μ M filter to remove impurities.
Preferably, the supernatant 1 is filtered to remove impurities using a 045 μ M filter.
Further, the separation method in the step 1) is centrifugation, and the centrifugation rate of the centrifugation is 10000 g; the centrifugation time is 10-60 min.
Preferably, the rate of centrifugation in step 1) is 400-8000 g; the centrifugation time is 10-30 min.
Further, the high-speed centrifugation rate in the step 2) is 5000-25000 g; the centrifugation time is 10-100 min.
Preferably, the high speed centrifugation rate in step 2) is 10000-; the centrifugation time is 30-60 min.
Further, the ultra-high speed centrifugation rate in the step 3) is 5000-; the centrifugation time is 60-600 min.
Preferably, the ultra-high speed centrifugation rate in the step 3) is 15000-150000 g; the centrifugation time is 60-180 min.
Example 2 bacterial membrane vesicle bulking and purification
Further, in some embodiments, the method of preparing bacterial membrane vesicles further comprises the steps of: 1) membrane vesicle increment: culturing the bacteria to logarithmic growth phase; collecting thalli, and performing irradiation treatment on the thalli by using ionizing rays after resuspension to obtain irradiated bacteria; 2) separating and purifying membrane vesicles: the irradiated cells and the membrane vesicles produced by the irradiated cells were separated by the method for separating membrane vesicles described in example 1, to obtain membrane vesicles.
Further, the ionizing radiation irradiation treatment mode is X-ray, and the irradiation dose is 500-3000 Gy. The irradiation dose specifically includes: 600Gy, 600-700Gy, 700-800Gy, 800-900Gy, 900-1000Gy, 1000-1100Gy, 1100-1200Gy, 1200-1300Gy, 1300-1400Gy, 1400-1500Gy, 1500-1600Gy, 1600-1700Gy, 1700-1800-1900 Gy, 1900-2000Gy,2100-2200Gy,2200-2300Gy,2300-2400Gy,2400-2500Gy, 2500-2600-Gy, 2700-2800Gy,2800-2900Gy, 2900-3000-Gy.
Preferably, the irradiation dose is 500-1000 Gy. The irradiation dose specifically includes: 600Gy at 500, 700Gy at 600, 800Gy at 700, 900Gy at 800, and 1000Gy at 900.
Further, OD of the bacteria in the logarithmic growth phase in the step 1)600The value is 0.3-0.8.
Preferably, the OD of the bacteria in the logarithmic growth phase in the step 1)600The value is 0.5-0.7.
Further, the step 1) is to resuspend the thalli by using phosphate buffer solution or sterile physiological saline.
Preferably, the cells are resuspended in a phosphate buffer in step 1).
Further, the thallus is resuspended to OD in the step 1)600The value is 20-80.
Preferably, the bacterial cells are resuspended to OD in the step 1)600The value is 40-60.
Compared with the bacteria which are not irradiated by ionizing rays, the membrane vesicle prepared by the method has the advantages that the content of nucleic acid and the content of protein in the membrane vesicle are improved by 10-20 times. .
The membrane vesicle prepared by the invention has various application scenes: for example, (i) can be used as an immunogen; (ii) can be used as immune response promoter; (iii) can be used as vaccine for treating bacterial infection diseases; (iv) can be used as a vaccine adjuvant (in some embodiments, the vaccine adjuvant is non-specifically altering or enhancing the body's specific immune response to an antigen); (v) can be used as antigen presenting cell function promoter.
Such antigen presenting cells include dendritic cells (i.e., DC cells), macrophages, and B cells. The membrane vesicle obtained by radiation and separation and purification can be used as a DC cell maturation promoter, and specifically can be used as a promoter for promoting the significant up-regulation of marrow-derived dendritic cell surface molecules CD80, CD86 and MHCII molecules.
In some embodiments, the membrane vesicles prepared by the invention can be combined with DC cells for preparing CD4+T cell proliferators. In particular, CD4 is promoted+The method for T cell proliferation comprises the following steps: the membrane vesicles prepared by irradiation treatment, DCs phagocytizing OVA antigen and CFSE labeled CD4+T lymphocytes were co-cultured in vitro.
Example 3A method for the isolation and preparation of bacterial membrane vesicles
In some embodiments, the method for preparing bacterial membrane vesicles by isolation comprises the steps of:
1. OD of bacteria grown to logarithmic growth phase600The value is 0.3-0.8; preferably, OD is selected600The value is 0.5-0.8 (hair can also be sent here)Fermenting to further enrich the thallus); collecting thallus, re-suspending the thallus with appropriate amount of phosphate buffer solution, wherein the ratio of the amount of phosphate buffer solution to the total amount of thallus is OD600A value of 20-80; preferably, OD is selected600A value of 40-60; after resuspension, using ionizing rays to perform irradiation treatment to obtain irradiated bacteria; the X-ray radiation treatment is preferably carried out, the radiation dose is 500-3000Gy, and the method specifically comprises the following steps: 600Gy, 600-700Gy, 700-800Gy, 800-900Gy, 900-1000Gy, 1000-1100Gy, 1100-1200Gy, 1200-1300Gy, 1300-1400Gy, 1400-1500Gy, 1500-1600Gy, 1600-1700Gy, 1700-1800-1900 Gy, 1900-2000Gy,2100-2200Gy,2200-2300Gy,2300-2400Gy,2400-2500Gy, 2500-2600-Gy, 2700-2800Gy,2800-2900Gy, 2900-3000-Gy.
2. Collecting bacterial liquid, centrifuging the bacterial liquid and collecting supernatant, and filtering and sterilizing the supernatant by using a 03-0.5 mu M filter; the centrifugal rate is 400-8000 g; the centrifugation time is 10-30 min.
3. Centrifuging the filtered supernatant by using a high-speed centrifuge, collecting the supernatant, and removing flagella; the high-speed centrifugation rate is 10000-; the centrifugation time is 30-60 min.
4. Centrifuging the supernatant without flagella by an ultra-high speed centrifuge, and precipitating membrane vesicles; the ultra-high speed centrifugation speed is 15000-; the centrifugation time is 60-180 min.
5. And collecting the membrane vesicles to obtain the purified membrane vesicles.
Ionizing ray irradiation pseudomonas aeruginosa PAO1 preparation, separation and purification of membrane vesicles:
1. recovering pseudomonas aeruginosa PAO1 from-80 ℃ and streaking to an LB plate, and culturing for 16-18h in a culture box at 37 ℃.
2. The single colony is picked from the LB plate and inoculated in 20mL LB liquid culture medium, and cultivated for 16-18h at 37 ℃ and 250rpm constant temperature.
3. Inoculating overnight bacterial liquid into 1L LB culture medium to initial concentration of 0.05OD600In mL, cultured at 37 ℃ and 250rpm until logarithmic growth phase, and measured for OD600The value is obtained.
4. Transferring the bacterial liquid of the step 3) to a centrifugal barrel, centrifuging for 20min at 5,000g, collecting the thallus, re-suspending the thallus with physiological saline, and adjusting the thallus concentration to about 50 OD.
5. And (3) placing the bacterial liquid in an irradiator, wherein the irradiation dose is 1000 Gy.
6. Centrifuging the irradiated bacterial liquid for 20min at 8,000 Xg twice, and collecting the supernatant; the supernatant was collected again after filtration sterilization using a 0.45 μ M filter, and a small amount of the supernatant was applied to an LB plate and cultured at 37 ℃ for 24 to 72 hours to confirm the absence of viable bacteria.
7. Centrifuging the supernatant obtained in the step 6) by a high-speed centrifuge, and removing flagella in the supernatant.
8. Centrifuging the supernatant obtained in the step 7) by an ultra-high speed centrifuge, and precipitating the membrane vesicles.
9. The supernatant was discarded and the pellet resuspended in MV buffer and stored at-80 ℃.
10. And (3) carrying out transmission electron microscope observation on the extracted normal and experimental membrane vesicles.
The experimental results are as follows:
the electron microscope results show that the ionizing radiation can stimulate pseudomonas aeruginosa PAO1 to produce membrane vesicles. The membrane vesicles are shown in FIG. 1.
Example 4 immunomodulation of irradiated bacterial Membrane vesicles-promotion of dendritic cell maturation
Dendritic Cells (DCs) are the main antigen presenting Cells of the body, whose main function is to phagocytose, process and process antigen molecules, and present them to T Cells. Is a known professional antigen presenting cell with the strongest function and the only ability to activate resting T cells in vivo, and is a central link for starting, regulating and maintaining immune response. The maturation of dendritic cells determines the body's development of an immune response or tolerance. Costimulatory molecules B7(B7-1 ═ CD80 and B7-2 ═ CD86) on DCs surfaces can bind to CD28 or CD152 molecules on T cell surfaces, either enhancing or attenuating MHC-TCR signaling between DCs and T cells. The expression of costimulatory molecules CD80 and CD86 is changed, the antigen phagocytosis capacity is weakened, the antigen presentation capacity is improved (the expression of MHCII molecules is increased), and the interaction with T lymphocytes is mainly shown on mature DCs.
1. Induction culture of mouse bone marrow-derived dendritic cell (BMDC) cells: taking a C57 female mouse with 6-8 weeks, aseptically separating the femur of the mouse, cleaning the muscle on the femur, cutting the two ends of the femur, washing the bone lumen with PBS until the femur is whitish, filtering the PBS suspension, separating at 1200rpm for 5min, removing the supernatant, and adding 5ml of erythrocyte lysate for resuspending the cells. After standing for 15min, centrifugation is carried out at 1200rpm for 5min, the supernatant is removed, and 50ml of 1640 complete medium (20ng/ml GM-CSF, 10% FBS, 50mM 2-mercaptoethanol) is added to resuspend the cells. After mixing uniformly, the mixture is divided into 5 culture dishes to be cultured in an incubator, the culture solution is changed every 2 days, and cells are collected on the 7 th day.
BMDC stimulation: repeatedly blowing BMDC cells for inducing for 7 days into a 6-hole plate to make adherent cells fall off, collecting cell suspension, centrifuging at 1100rpm for 5min, removing supernatant, adding 1ml culture medium to resuspend cells, counting viable cells, and adjusting cell concentration to 1 × 106Perml, inoculate 2ml into a new 6-well plate. Adding each stimulant and mixing evenly, respectively: whole bacteria, whole bacteria + vesicles, vesicles to a final concentration of 15 μ g/mL (protein standard), incubation was continued for 24 hours and an equal volume of PBS was added to the growth control.
3. Flow detection of mature marker: and taking out the 6-hole plate after 24h, repeatedly blowing cells to enable the cells to fall off, collecting cell suspension to a flow tube, centrifuging at 1500rpm for 3min, removing supernatant, adding 1ml of PBS, continuing to centrifuge at 1500rpm for 3min, removing supernatant, and repeatedly cleaning for 3 times. Adding CD11c/CD80/CD86/MHCII antibody, incubating at room temperature in dark for 30min, and setting up a isotype control group and a coliform control group of CD11c/CD80/CD 86/MHCII. After incubation, PBS was added to wash the cells for 2 times, and 200. mu.l PBS was added to resuspend the cells for detection on an up-flow cytometer.
4. And (4) processing a result: flow cytometry software analyzed the CD80/CD86/MHCII ratio in CD11c cells.
The experimental results are as follows: compared with whole thallus, the X-ray treated experimental group (MVs) vesicles can obviously up-regulate DCs surface co-stimulatory molecules CD80, CD86, MHCII and the like after stimulation, and the surface molecules are markers of dendritic cell maturation. Taken together, vesicles were shown to significantly promote differentiation and maturation of DCs.
Phagocytic capacity of DC cells was detected by measuring the fluorescence intensity of FITC-labeled dextran: the DC cells have extremely strong antigen endocytosis and processing capacity. When not contacted with antigen substance, the product is in an immature state, has strong phagocytic ability, and becomes mature after being activated by contacting with antigen, so that the phagocytic ability is weakened, and the antigen presenting ability is enhanced. The experiment determines the amount of phagocytic glucan of the DC by detecting the fluorescence intensity of FITC-labeled glucan so as to detect whether the phagocytic capacity of the DC is enhanced.
BMDC cell induction culture (same as the previous example).
2. Stimulation: cells were collected on day 7, blown down, centrifuged, resuspended, counted, and plated into 6-well plates at 1X 10 cells/well6Adding stimulators into each cell: GC (Growth Control) groups were incubated at 37 ℃ for 24h with equal volumes of PBS, Control and Treatment groups added with membrane vesicles (at protein level) at the same concentrations.
3. Phagocytosis and detection: dextran (5. mu.g/ml) was added, the cells were further cultured for 1 hour, aspirated into a flow tube, washed with PBS 3 times, added with CD11c antibody, incubated at room temperature in the dark for 30min, washed with PBS 3 times, and then flow-assayed for FITC fluorescence.
4. And (4) processing a result: flow cytometry software analyzed the proportion of FITC in CD11c cells.
Experimental data: in order to detect the phagocytosis function of DCs, the invention adopts FITC-glucan as a model antigen for phagocytosis of DCs, and detects the FITC average fluorescence intensity value of CD11c + DCs. The experimental results show that the mean fluorescence intensity value of FITC is obviously reduced compared with that of a GC group (growth control) after DCs receive membrane vesicle stimulation. This experimental result again demonstrates that vesicles can promote maturation of DCs, thereby reducing their uptake capacity for antigen.
Example 5 mature DCs after stimulation of X-ray treated bacterial membrane vesicles interact with T cells:
A. mature DC and CD4+T cell interaction:
efficient cross-antigen presentation of extracellular proteins by DCs plays an important role in inducing specific cellular immune responses. Thus, cross-presentation of OVA antigen by DCs following membrane vesicle stimulation was examined. Proliferation of OT-II CD4+ T lymphocytes was detected by CFSE flow cytometry at 72h after DCs-T cell co-culture. The fluorescent dye CFSE (CFDA-SE), namely hydroxy fluorescein diacetate succinimide ester, is a cell staining reagent which can carry out fluorescent labeling on living cells. It can be coupled to a cell protein irreversibly by binding to an amino group in the cell after entering the cell. During cell division and proliferation, CFSE marker fluorescence can be equally distributed to two daughter cells, with half the intensity of the parent cell. Therefore, the percentage of cells with weak CFSE fluorescence can be counted by flow cytometry to obtain the proportion of proliferating cells.
BMDC cell induction culture (same as above example).
2. Antigen phagocytosis: DCs cultured for 7 days were cultured in OVA medium containing 10. mu.g/ml for 24 hours as GC (growth control), and membrane vesicles were additionally added to the MVs group, followed by centrifugation to collect antigen-phagocytized DCs and resuspension in normal medium at 2X 104The density of cells/well was plated in 96-well plates, 100. mu.l per well, 3 replicates per group.
T cell extraction: on the next day, OT-II mice splenic OVA-specific CD4+ T lymphocytes were isolated and enriched using the Stem Cell Technologies negative magnetic bead screening kit.
Co-culture of DCs with T cells: selected CD4+ T cells were labeled with 1 μ M CFSE according to the kit instructions. After labeling, wash 3 times with PBS at 105The density of cells/well was added to a 96-well plate to give a final culture volume of 200. mu.l (CD4: DC ═ 5: 1).
5. On day 3 post co-culture, CD4 was detected by CFSE depletion using flow cytometry+Proliferation of T cell populations.
The experimental results are as follows: DCs stimulated by membrane vesicles and phagocytosing OVA antigen and CFSE-labeled OT-II mouse CD4+T lymphocytes were co-cultured in vitro. The result of flow analysis on CFSE fluorescence intensity shows that the proliferation CD4+The proportion of T cells increases. Membrane vesicles (14.05%) were able to significantly increase phagocytosis of OVA antigen by DCs (6.80%) for specific CD4+T cellsThe proliferation promoting effect of (1). See fig. 2 and 3 for details.
B. Membrane vesicle-treated DCs promote T cell proliferation:
efficient cross-antigen presentation of extracellular proteins by DCs plays an important role in inducing specific cellular immune responses. Thus, cross-presentation of OVA antigen by DCs following vesicle stimulation was examined. Proliferation of T lymphocytes was examined by CFSE flow cytometry at 72h after DCs-T cell co-culture. The fluorescent dye CFSE (CFDA-SE), namely hydroxy fluorescein diacetate succinimide ester, is a cell staining reagent which can carry out fluorescent labeling on living cells. It can be coupled to a cell protein irreversibly by binding to an amino group in the cell after entering the cell. During cell division and proliferation, CFSE marker fluorescence can be equally distributed to two daughter cells, with half the intensity of the parent cell. Therefore, the percentage of cells with weak CFSE fluorescence can be counted by flow cytometry to obtain the proportion of proliferating cells.
BMDC cell induction culture (same as above example).
2. Antigen phagocytosis: DCs cultured for 7 days were cultured in a medium for 24 hours as GC (growth control), and in MVs group, vesicles were additionally added, followed by centrifugation to collect antigen-phagocytized DCs and resuspension in a normal medium at 4X 104The density of cells/well was plated in 96-well plates, 100. mu.l per well, 3 replicates per group.
T cell extraction: on the following day, T cells enriched in mice were isolated using a negative magnetic bead screening kit from mouse spleen Stem Cell Technologies one week after one immunization with MVs.
Co-culture of DCs with T cells: selected T cells were labeled with 1 μ M CFSE according to kit instructions. After labeling, wash 3 times with PBS at 4X 105The density of cells/well was added to a 96-well plate to make the final culture volume 200. mu.l (CD3: DC ═ 10: 1).
5. On day 3 post co-culture, CD3 was detected by CFSE depletion using flow cytometry+，CD8 +，CD4 +Of T cell populationsProliferation status.
The experimental results are as follows: DCs that vesicle-stimulated and phagocytized OVA antigen were co-cultured in vitro with CFSE-labeled OT-II mouse CD4+ T lymphocytes. Flow analysis CFSE fluorescence intensity results indicated an increased proportion of proliferating CD4+ T cells. As shown in the figure, the fluorescence intensity of the cell and vesicle stimulation group was 63.5%, and the fluorescence intensity of the vesicle stimulation group was 71%. Indicating that DCs after vesicle processing can remarkably stimulate the proliferation of CD4+ T cells. See fig. 4.
Example 6 experiment of Pseudomonas aeruginosa vaccine against scald complicated by Pseudomonas aeruginosa skin infection
According to the experiment, experimental rabbits are adopted, a scald model is formed on the rabbits by using a hot air electric baking gun one day before the rabbits are infected with pseudomonas aeruginosa, the subcutaneous parts of the scald parts are respectively infected with the same pseudomonas aeruginosa SKLBPA1 and pseudomonas aeruginosa PA14 (both purchased from ATCC) after 24 hours, the rabbits are killed after 24 hours after infection, the skin and subcutaneous muscle tissues of the scald parts are taken, CFU counting is carried out after homogenate, CFU of a model group and CFU of a vaccine group are compared, and whether the vaccine has a protection effect on scald and pseudomonas aeruginosa infection is observed. The result of the experiment shows that the pseudomonas aeruginosa vaccine provided by the invention can have prevention and treatment effects on burn and scald complicated infection caused by pseudomonas aeruginosa with different serum types (two representative strains of SKLBPA1 and PA14 are taken as examples in the experiment), and has wide application scenes.
The vaccine content of the invention comprises: 108CFU/ml
Experimental animals: rabbits, white big ears in New Zealand, 2.210-2.870 kg body weight, female, 6.
1. Experiment grouping
3 groups of 2 animals were used in this experiment, and the specific grouping is shown in Table 3.
TABLE 2 Experimental group Table
2. Immunization
Test vaccine (10)8CFU/ml) was immunized 3 times subcutaneously in the left and right groins of rabbits at intervals of 3-4 days (i.e., immunization schedule of 0, 3, 7 days) or 2 weeks (i.e., immunization schedule of 0, 2, 4 weeks).
3. Scald model establishment
Removing front and back hair on the left side and the right side of a rabbit by using scissors and depilatory cream 2 days before infection, disinfecting the skin of a depilated part by using 75% alcohol after 24 hours, smearing tetracaine hydrochloride mucilage for local anesthesia, setting the temperature of a hot air electric baking gun to be 200 ℃, disinfecting a metal plate with square holes by using 75% alcohol, pasting the metal plate on the depilated part, starting the electric baking gun, aligning a hot air port with the square holes to blow hot air to the skin of the rabbit for 5 seconds, and causing a certain degree of scald to the model.
4. Infection with viral infection
4.1 Strain recovery
Pseudomonas aeruginosa SKLBPA1 and PA14 were recovered from-80 ℃ to TSA plates and incubated overnight at 37 ℃.
4.2 shake the fungus overnight
Single clones were picked from the plates separately in 3ml TSB and shaken overnight at 37 ℃ and 220 rpm.
4.3 expansion culture
Diluting overnight bacteria liquid to measure OD600The original strain liquid is inoculated into 100ml TSB (250ml conical flask), and the strain is shaken at 220rpm at 37 ℃ until the logarithmic phase.
4.4 centrifugal washing
Collecting the bacterial liquid into a 50mL centrifuge tube, centrifuging at 4100rpm (3000 Xg) for 10min at room temperature, discarding the supernatant, suspending 2mL of 0.9% sodium chloride injection (about 5OD/mL), adjusting the bacterial liquid to 0.1OD600(2×10 7CFU/ml)。
4.5 infection
After 1 week (day 7) of the last immunization, 50. mu.l of the bacterial solution (1X 10) was injected subcutaneously into the scalded skin of the rabbit6CFU/site).
4.6 plate count
The adjusted bacterial solution was applied to a TSA plate, cultured overnight at 37 ℃ and the CFU was counted.
5. Bacterial counts in skin and muscle tissue 24 hours post infection
Animals were sacrificed 24 hours after infection, sterilized by spraying 75% ethanol to the skin, aseptically removed the skin and muscle tissue of the scalded area, homogenized, plated on TSA plates, incubated overnight at 37 ℃, and counted for CFU using a colony counter.
6. Data processing
LOG for skin and muscle tissue is manufactured by Graphpad Prism mapping software10CFU scatter diagram, calculating LOG10CFU means, and analysis of group differences using analysis of variance.
Results of the experiment
1. Change in rabbit body weight
The weights of animals in each group are shown in table 3, the weights of rabbits in each immune group and each model group are steadily increased, and no significant difference exists between the groups.
TABLE 3 animal weight Change
2. Modeling result for rabbit skin scald
Setting the temperature of a tuyere with the outer diameter of 14mm and the inner diameter of 10mm on a hot air electric baking gun sleeve at 200 ℃, and sticking the tuyere to the hair removal part on the back of the rabbit for 5 seconds. After 2 hours, circular blisters with the diameter of about 10mm appear on the skin of the scald part, and after 24 hours, the skin of the scald part is scabbed.
3. Pseudomonas aeruginosa infection amount of rabbit skin
The concentrations of the pseudomonas aeruginosa SKLBPA1 and PA14 bacterial suspensions used in the experiment are both 0.1OD/ml, and the bacterial suspension left after subcutaneous infection is diluted to 10-4、10 -550 μ l of the suspension was spread evenly on a TSA plate, incubated overnight in an incubator at 37 ℃ and the number of colonies was counted, as shown in Table 5.
TABLE 4 viable bacteria count results of infected bacteria
| Strain name | Turbidity (OD/ml) of infectious bacteria liquid | Concentration of infectious bacterium solution (CFU/ml) |
| SKLBPA1 | 0.1 | 8.00×10 7 |
| PA14 | 0.1 | 1.06×10 8 |
4. Bacterial load capacity of rabbit scald part 24h after pseudomonas aeruginosa infection
The animals were sacrificed 24h after infection, skin and muscle tissues at the site of the strain were aseptically scalded and infected, TSA plates were applied to a homogenizer, cultured overnight at 37 ℃, CFU was counted, and then the amount of the bacteria was counted as 10 as a base log, and the average and standard deviation were compared for each group, and the results are shown in Table 5 and FIG. 10. Both immunization programs of the PA1 vaccine can obviously reduce the load of the vaccine (P <0.01), and the vaccine has obvious protective effect.
TABLE 5 bacterial load of rabbit scald sites 24h after Pseudomonas aeruginosa infection
(Note: ratio of star to each model group, p <0.01, significant differences)
5. Bacterial load of rabbit scald part 24h after pseudomonas aeruginosa PA14 infection
Skin and muscle tissues of the scalded and infected pseudomonas aeruginosa PA14 part are aseptically taken, a TSA plate is coated on a homogenizer, the mixture is cultured overnight at 37 ℃, a colony counter is used for counting CFU, the bacterial load is counted by taking 10 as a base counter value, the average and standard deviation of each group are compared, and the results are shown in Table 6 and figure 11. The PA1 vaccine is immunized in 0, 3 and 7 days, and can obviously reduce the load of the P14 of the pseudomonas aeruginosa (P is less than 0.05), and the reduction range is about 0.6-0.9 LOG. The vaccine is proved to have a certain protective effect.
It is noted that in the experiments against SKLBPA1 and PA14, 0, 3 and 7d of immunization programs can rapidly generate effective immune responses, so that the vaccine of the present invention can be used not only for preventing skin scald complicated infection (i.e. as a preventive vaccine), but also for inhibiting infection and alleviating infection after skin scald complicated infection and hair dyeing (i.e. as a therapeutic vaccine).
TABLE 6 bacterial load of rabbit scald sites 24h after Pseudomonas aeruginosa PA14 infection
(Note: compared to each model group, p <0.05, significant differences)
While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (19)
- The pseudomonas aeruginosa vaccine is applied to the preparation of the medicine for preventing and treating burn and scald infection.
- The use of claim 1, wherein the burns and scalds include burns and scalds, and the extent of the burns and scalds includes: scald of degree I, superficial degree II, deep degree II and degree III.
- The use of claim 2, wherein the burn site comprises skin, mucous membranes, or other tissue.
- The use of claim 1, wherein the burn infection is a burn and scald complicated with bacterial infection.
- The use of claim 4, wherein the bacterial infection is caused by one or more of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis.
- The use of claim 4, wherein the bacterial infection is a Pseudomonas aeruginosa infection.
- The use of claim 4, wherein the burns and scalds comprise burns and scalds, and the extent of the burns and scalds comprise burns of degree I, superficial degree II, deep degree II and degree III.
- The use of claim 7, wherein the burn site comprises skin, mucous membranes, or other tissue.
- The use of any one of claims 1-8, wherein the Pseudomonas aeruginosa vaccine comprises inactivated Pseudomonas aeruginosa and/or Pseudomonas aeruginosa membrane vesicles.
- The use of claim 9, wherein the pseudomonas aeruginosa vaccine immunization program comprises: the injection time interval was (i)0, 3, 7 days, (ii)0, 2, 4 weeks.
- The use of claim 9, wherein said inactivated pseudomonas aeruginosa is inactivated by irradiation, and said pseudomonas aeruginosa membrane vesicles are isolated from said pseudomonas aeruginosa inactivated by irradiation.
- The use of claim 9, wherein the pseudomonas aeruginosa vaccine prevents pseudomonas aeruginosa infection and reduces the bacterial load in the scald complicated bacterial infection.
- The use of claim 9, wherein the pseudomonas aeruginosa vaccine comprises pseudomonas aeruginosa whole cells in an amount comprising: 1X 104-1×10 10A needle.
- The use of claim 13, wherein the pseudomonas aeruginosa vaccine comprises pseudomonas aeruginosa whole cells in an amount comprising: 1X 104Needle, 1X 105Needle, 1X 106Needle, 1X 107Needle, 1X 108Needle, 1X 109Needle and 1X 1010A needle.
- The use of claim 1, wherein the pseudomonas aeruginosa vaccine further comprises an immunological adjuvant.
- The use of claim 15, wherein the immunoadjuvant is aluminum hydroxide.
- The use of claim 1, wherein the pseudomonas aeruginosa vaccine is administered at a site that is subcutaneous, intramuscular and/or mucosal.
- The use of claim 1, wherein the medicament may further comprise any pharmaceutically acceptable carrier and/or adjuvant.
- The use of claim 18, wherein the carrier is a liposome.
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| CN201910777595.4A CN112410212B (en) | 2019-08-22 | 2019-08-22 | Production system, separation and purification system and method of bacterial membrane vesicles |
| CN201910777606.9A CN112410240B (en) | 2019-08-22 | 2019-08-22 | Pseudomonas aeruginosa membrane vesicle and preparation method and application thereof |
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