CN114364396B - Application of pseudomonas aeruginosa vaccine in burn and scald infection resistance - Google Patents
Application of pseudomonas aeruginosa vaccine in burn and scald infection resistance Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application of the pseudomonas aeruginosa vaccine in preventing and treating burns and scalds combined with bacterial infection is provided, wherein the burns and scalds comprise burns and scalds, and the degree of the scalds comprises: degree I, shallow degree II, deep degree II, and degree III scalds, the site of the scald comprising skin, mucous membrane, or other tissue.
Description
The application requires that Chinese patent 1) 201910777479.2 'bacterial membrane vesicle submitted in 2019, 8 and 22 days, and preparation method and application' thereof; 2) 201910777473.5 staphylococcus aureus membrane vesicles, and preparation method and application thereof; 3) 201910777606.9 'Pseudomonas aeruginosa membrane vesicle, and preparation method and application thereof'; 4) 201921369450.2 "a bacterial membrane vesicle production system and separation and purification system"; 5) 201910777595.4 "production System and separation purification System and method for bacterial Membrane vesicles", which are incorporated by reference in their entirety.
Technical Field
The invention belongs to the field of microbiology, and particularly relates to application of a pseudomonas aeruginosa vaccine in preventing and treating burns and scalds combined bacterial infection.
Background
At present, burns and scalds become one of the most common accidental injuries in daily production and life. It is counted that about 5000-10000 people in every million people in our country suffer from burns or scalds every year. The main sites of burns and scalds include skin and/or mucous membranes, and severe cases can even injure subcutaneous and/or submucosal tissues such as muscles, bones, joints and even viscera. The scald belongs to one of thermal burns, and is mainly tissue injury caused by hot liquid, steam and high-temperature solids. The judgment of the depth of the scald is divided according to a three-degree quartering method, and the judgment can be divided into the following steps according to the depth, pathological changes and clinical manifestations of the scald: degree I, shallow degree II, deep degree II and degree III scalds.
Wound infection is the most common and serious complication after scalding. Bacteria responsible for wound infection are mainly gram-negative bacilli, of which 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 pseudomonas aeruginosa can be separated from nearly 100% of burn wounds after 7 days of burn. It is also reported that mortality rates of up to 31% after pseudomonas aeruginosa infection, especially in burn patients, can cause serious consequences if bacterial infection of the wound surface after burn is not cured in time. In addition, if the infectious bacteria are not cleared in time, wound healing is delayed, or scars heal, the wound site (joint) function is impaired.
At present, the treatment mode adopted for scalding and combining pseudomonas aeruginosa infection is generally surgical debridement operation, and simultaneously, antibiotics are used in a combined local mode and a systemic mode. The most common antibacterial agents include the artificially synthesized antibacterial agents silver sulfadiazine and the antibiotics ciprofloxacin, ceftazidime, cefoperazone/sulbactam, etc. However, due to the unreasonable use of antibiotics and the emergence of multi-drug resistant bacteria, the therapeutic effect is unsatisfactory, for example, the development of sepsis is life threatening. It is counted that pseudomonas aeruginosa sepsis is the main cause of death in scalded patients during the course of scalding and pseudomonas aeruginosa infection, the incidence rate is between 8% and 42.5%, and the mortality rate is between 28% and 65%.
Disclosure of Invention
Therefore, the invention aims to provide the application of the pseudomonas aeruginosa vaccine in preventing and treating burn and scald infection.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the application of the pseudomonas aeruginosa vaccine in preparing medicines for preventing and treating burns and scalds infection.
Further, the burns and scalds include burns and scalds, and the degree of the scalds includes I degree, shallow II degree, deep II degree, or III degree scalds.
Further, the site of the burn includes skin, mucous membrane, or other tissue.
Further, the burn and scald infection is a burn and scald combined bacterial infection.
Further, the bacteria infected by the combined bacteria comprise one or more of pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, staphylococcus aureus, streptococcus pneumoniae and tubercle bacillus.
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, shallow II degree, deep II degree, or III degree scalds.
Further, the site of the burn includes skin, mucous membrane, or other tissue.
Further, the pseudomonas aeruginosa vaccine includes inactivated pseudomonas aeruginosa and/or pseudomonas aeruginosa membrane vesicles.
Further, the immunization program of the pseudomonas aeruginosa vaccine comprises: the injection time intervals were (i) 0, 3, 7 days, (ii) 0, 2, 4 weeks.
Further, the inactivated pseudomonas aeruginosa is inactivated by irradiation, and the pseudomonas aeruginosa membrane vesicles are separated from the pseudomonas aeruginosa inactivated by irradiation.
Further, the pseudomonas aeruginosa vaccine prevents pseudomonas aeruginosa infection and reduces bacterial load in the skin scalding combined bacterial infection.
Further, the pseudomonas aeruginosa vaccine contains the whole pseudomonas aeruginosa thallus with the contents that: 1X 10 4 -1×10 10 Needle.
Further, the pseudomonas aeruginosa vaccine contains the whole pseudomonas aeruginosa thallus with the contents that: 1X 10 4 Needle, 1X 10 5 Needle, 1X 10 6 Needle, 1X 10 7 Needle, 1X 10 8 Needle, 1X 10 9 Needle and 1X 10 10 Needle.
Further, the pseudomonas aeruginosa vaccine also contains an immunoadjuvant.
Further, the administration site of the pseudomonas aeruginosa vaccine is subcutaneous, intramuscular and/or mucosal.
Further, the medicament may further comprise any pharmaceutically acceptable carrier and/or adjuvant.
Further, the carrier is a liposome.
The invention has the beneficial effects that
The experimental result shows that the pseudomonas aeruginosa vaccine can effectively prevent and treat burn and scald caused by multidrug-resistant pseudomonas aeruginosa and pseudomonas aeruginosa infection by activating organism atopic immune reaction. The bacterial load of the immune organism infection is reduced through the established immunization program, so that the technical scheme which can effectively prevent and treat burns and scalds combined with pseudomonas aeruginosa infection is provided, and the technical problems that antibiotics are not good, are difficult to radically cure, and are easy to generate drug resistance in the prior art are avoided to a certain extent.
The experimental result also shows that the pseudomonas aeruginosa vaccine can effectively inhibit bacterial load in the sites of the burn and scald complicated bacterial infection by activating human immune reaction, prevent double infection caused by the pseudomonas aeruginosa, avoid the problems of drug resistance and the like caused by antibiotics in the prior art, and have wide application prospect in the burn and scald complicated bacterial infection.
Drawings
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 will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.
FIG. 1 transmission electron microscopy of irradiated P.aeruginosa membrane vesicles (scale bar: 200 nm).
FIG. 2CD4+T cells proliferation percentage after DC interaction with different treatments.
FIG. 3CD4+T cell proliferation flow chart after interaction with DC from different treatments.
FIG. 4 bacterial load at the site of scalding of rabbits 24h after infection with Pseudomonas aeruginosa.
FIG. 5 bacterial load at the site of scalding of rabbits 24h after infection with P.aeruginosa PA 14.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The term "burn and scald" as used herein refers to the damage to tissues caused by heat, including hot liquid (water, soup, oil, etc.), steam, hot gas, flame, hot metal liquid or solid (e.g., molten steel, steel ingot), etc., mainly skin and/or mucous membrane, and subcutaneous and/or submucosal tissues such as muscles, bones, joints, and even viscera may be injured by severe cases. The 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 present invention comprises (i) radiation inactivated pseudomonas aeruginosa cells and/or (ii) pseudomonas aeruginosa membrane vesicles. FIG. 1 is a transmission electron micrograph of the purified membrane vesicles.
Examples 1-3 describe some isolation methods for preparing vesicles, either from non-irradiated bacteria or from 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 thalli in a bacterial liquid for culturing bacteria from the bacterial liquid, and collecting to obtain a supernatant 1; 2) Centrifuging the supernatant 1 by using a high-speed centrifuge, and collecting a supernatant 2; 3) Centrifuging the supernatant 2 by using an ultra-high speed centrifuge, and precipitating to form membrane vesicles.
Further, the separation method in the step 1) comprises centrifugation, column passing or membrane package concentration.
Further, the supernatant 2 collected in the step 2) is concentrated by a membrane package before entering the step 3). In some embodiments, the selected membrane package may concentrate substances greater than 100 KD.
Further, the membrane vesicles are resuspended in buffer, 1L of bufferProduct calculation, including 50mM Tris,5mM NaCl,1mM MgSO 4 And the pH was 7.4.
In some embodiments, the thallus and the membrane vesicle are prepared as a biological composition, and the preparation method comprises: collecting the bacterial cells separated in the step 1) in the method for separating 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 off with a 045. Mu.M filter.
Further, the separation method in the step 1) is centrifugation, and the centrifugation speed of the centrifugation is 100-10000g; the centrifugation time is 10-60min.
Preferably, the rate of centrifugation in step 1) is from 400 to 8000g; the centrifugation time is 10-30min.
Further, the high-speed centrifugation rate in the step 2) is 5000-25000g; the centrifugation time is 10-100min.
Preferably, the high speed centrifugation rate in step 2) is 10000-20000g; the centrifugation time is 30-60min.
Further, the ultra-high speed centrifugation rate in the step 3) is 5000 to 150000g; the centrifugation time is 60-600min.
Preferably, the ultra-high speed centrifugation rate in step 3) is 15000-150000g; the centrifugation time is 60-180min.
Example 2 bacterial Membrane vesicle enhancement and purification
Further, in some embodiments, the method of preparing a bacterial membrane vesicle further comprises the steps of: 1) Membrane vesicle increment: culturing the bacteria to a logarithmic growth phase; collecting thalli, re-suspending the thalli, and then carrying out irradiation treatment on the thalli by using ionizing rays to obtain irradiated bacteria; 2) Membrane vesicle separation and purification: the membrane vesicles produced by the irradiation cells and the irradiation 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 radiation, and the irradiation dose is 500-3000Gy. The irradiation dose specifically comprises: 500-600Gy,600-700Gy,700-800Gy,800-900Gy,900-1000Gy,1000-1100Gy,1100-1200Gy,1200-1300Gy,1300-1400Gy,1400-1500Gy, 1500-1100 Gy,1600-1700Gy, 1700-2500 Gy, 1800-2500 Gy,1900-2000Gy,2100-2200Gy,2200-2300Gy, 2300-240Gy, 2400-2500Gy,2500-2600Gy,2600-2700Gy,2700-2800Gy, 2900-3000Gy.
Preferably, the irradiation dose is 500-1000Gy. The irradiation dose specifically comprises: 500-600Gy,600-700Gy,700-800Gy,800-900Gy and 900-1000Gy.
Further, the OD of the bacteria in the logarithmic growth phase in the step 1) 600 The value is 0.3-0.8.
Preferably, the OD of the bacteria in the logarithmic growth phase in step 1) 600 The value is 0.5-0.7.
Further, the cells are resuspended in the step 1) with a phosphate buffer or sterile physiological saline.
Preferably, in the step 1), the cells are resuspended with a phosphate buffer.
Further, the step 1) of re-suspending the bacteria to OD 600 The value is 20-80.
Preferably, in said step 1), said bacteria are resuspended to OD 600 The value is 40-60.
Compared with bacteria which are not irradiated by ionizing radiation, the content of nucleic acid and protein in the membrane vesicle prepared by the method is improved by 10-20 times. .
The membrane vesicle prepared by the invention has various application scenes: for example, (i) can act as an immunogen; (ii) can be used as an immune response enhancer; (iii) can be used as a vaccine for the treatment of bacterial infection diseases; (iv) May be used as a vaccine adjuvant (in some embodiments, the vaccine adjuvant is a non-specific agent that alters or enhances the specific immune response of the body to an antigen); (v) can act as an antigen presenting cell function promoter.
Such antigen presenting cells include dendritic cells (i.e., DC cells), macrophages and B cells. The membrane vesicles obtained through radiation, separation and purification can be used as an accelerator for DC cell maturation, in particular, can be used as an accelerator for promoting the remarkable up-regulation of bone marrow-derived dendritic cell fine surface molecules CD80, CD86 and MHCII.
In some embodiments, the membrane vesicles made according to the invention can be used in conjunction with DC cells in the preparation of CD4 + T cell proliferating agents. Specifically, promotion of CD4 + The method for T cell proliferation comprises the following steps: DCs and CFSE labeled CD4 of membrane vesicles prepared by irradiation treatment and phagocytizing OVA antigen + T lymphocytes were co-cultured in vitro.
Example 3A method for the isolated preparation of bacterial membrane vesicles
In some embodiments, the method of separately preparing a bacterial membrane vesicle comprises the steps of:
1. culturing the bacteria to logarithmic growth phase, and OD of the bacteria in logarithmic growth phase 600 The value is 0.3-0.8; preferably, the OD is selected 600 A value of 0.5 to 0.8 (fermentation may also be performed here to further enrich the cells); collecting thallus, re-suspending the thallus with phosphate buffer solution in the ratio of 1ml to OD 600 Values 20-80; preferably, the OD is selected 600 A value of 40-60; after re-suspension, the bacteria are irradiated by ionizing radiation; preferably, the method uses X-ray radiation to irradiate, the irradiation dose is 500-3000Gy, and the method specifically comprises the following steps: 500-600Gy,600-700Gy,700-800Gy,800-900Gy,900-1000Gy,1000-1100Gy,1100-1200Gy,1200-1300Gy,1300-1400Gy,1400-1500Gy, 1500-1100 Gy,1600-1700Gy, 1700-2500 Gy, 1800-2500 Gy,1900-2000Gy,2100-2200Gy,2200-2300Gy, 2300-240Gy, 2400-2500Gy,2500-2600Gy,2600-2700Gy,2700-2800Gy, 2900-3000Gy.
2. Collecting bacterial liquid, centrifuging the bacterial liquid, collecting supernatant, and filtering and sterilizing the supernatant by using a 03-0.5 mu M filter; the centrifugation speed is 400-8000g; the centrifugation time is 10-30min.
3. Centrifuging the filtered supernatant with a high-speed centrifuge, collecting supernatant, and removing flagella; the high-speed centrifugation rate is 10000-20000g; the centrifugation time is 30-60min.
4. Centrifuging the supernatant after flagellum removal by using an ultra-high speed centrifuge, and precipitating membrane vesicles; the ultra-high speed centrifugation rate is 15000-150000g; the centrifugation time is 60-180min.
5. Collecting the membrane vesicles to obtain purified membrane vesicles.
Preparation, separation and purification of membrane vesicles by ionizing radiation irradiation of pseudomonas aeruginosa PAO 1:
1. the pseudomonas aeruginosa PAO1 streaks are resuscitated from-80 ℃ to LB plates and cultured in a 37 ℃ incubator for 16-18 hours.
2. The monoclonal colony is picked from the LB plate and inoculated into 20mL of LB liquid medium, and the culture is carried out for 16-18h at a constant temperature of 37 ℃ and a constant speed of 250 rpm.
3. Inoculating overnight bacterial liquid into 1L LB culture medium to initial concentration of 0.05OD 600 Culture at 37℃and 250rpm to logarithmic growth phase and OD measurement 600 Values.
4. Transferring the bacterial liquid in the step 3) into a centrifugal barrel, centrifuging for 20min at 5,000g, collecting bacterial cells, re-suspending the bacterial cells by using physiological saline, and adjusting the bacterial cell concentration to be about 50 OD.
5. The bacterial liquid is taken and placed in an irradiation instrument, and the irradiation dose is 1000Gy.
6. Centrifuging the irradiated bacterial liquid for 8,000Xg and 20min twice, and collecting supernatant; the supernatant was filtered with a 0.45 μm filter to sterilize and the resultant supernatant was collected again while a small amount of the supernatant was smeared on an LB plate and cultured at 37 ℃ for 24-72 hours to confirm the presence of a sterile.
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 a super-high speed centrifuge to precipitate membrane vesicles.
9. The supernatant was discarded and the pellet was resuspended in MV buffer and stored at-80 ℃.
10. And performing transmission electron microscope observation on the extracted normal group membrane vesicles and the extracted experimental group membrane vesicles.
Experimental results:
from the electron microscopy results, the ionizing radiation was able to stimulate the production of membrane vesicles by pseudomonas aeruginosa PAO 1. Membrane vesicles are shown in figure 1.
EXAMPLE 4 immunomodulatory effects of irradiation-treated bacterial Membrane vesicles-promotion of dendritic cell maturation
Dendritic Cells (DCs) are the primary antigen presenting Cells of the body, whose primary functions are phagocytosis, processing of antigen molecules, and presentation to T Cells. Is the only professional antigen presenting cell capable of activating resting T cells with the strongest in vivo function and is the central link for starting, regulating and maintaining immune response. The maturation of dendritic cells determines the immune response or tolerance of the body. Costimulatory molecules B7 (B7-1 = CD80 and B7-2 = CD 86) on the surface of DCs can bind to CD28 or CD152 molecules on the surface of T cells, enhancing or attenuating MHC-TCR signaling between DCs and T cells. The expression of the co-stimulatory molecules CD80 and CD86 is mainly shown on mature DCs, and the expression of the co-stimulatory molecules CD80 and CD86 is changed, the phagocytic antigen capacity is reduced, the antigen presenting capacity is enhanced (the MHCII molecule expression is increased), the interaction with T lymphocytes is realized, and the like.
1. Mouse bone marrow derived dendritic cell (BMDC) cells induction culture: taking a C57 female mouse of 6-8 weeks, aseptically separating the femur of the mouse, removing the muscles on the femur cleanly, cutting the two ends of the femur, flushing the bone lumen by PBS until the bone lumen turns white, filtering the PBS suspension, separating for 5min at 1200rpm, removing the supernatant, and adding 5ml of erythrocyte lysate to resuspend the cells. After 15min of standing, centrifugation was carried out at 1200rpm for 5min, the supernatant was removed, and 50ml of 1640 complete medium (20 ng/ml GM-CSF, 10% FBS, 50mM 2-mercaptoethanol) was added to resuspend the cells. After mixing, the mixture is placed into a culture box in 5 culture dishes for culture, the liquid is changed every 2 days, and the cells are collected on the 7 th day.
Bmdc stimulation: collecting BMDC cells induced for 7 days, repeatedly blowing in 6-well plate to remove adherent cells, collecting cell suspension, centrifuging at 1100rpm for 5min, removing supernatant, adding 1ml culture medium to resuspend cells, counting living cells, and adjusting cell concentration to 1×10 6 Per ml, 2ml was inoculated into a new 6-well plate. Adding each stimulus and uniformly mixing, wherein the steps are as follows: whole bacteria, whole bacteria+vesicles, vesicles to a final concentration of 15. Mu.g/mL (protein standard), and incubation was continued for 24 hours, with equal volumes of PBS added to the growth control.
3. Stream detection mature marker: taking out the 6-hole plate after 24 hours, repeatedly blowing the cells to fall off, collecting cell suspension to a flow tube, centrifuging at 1500rpm for 3 minutes, removing supernatant, adding 1ml PBS, centrifuging at 1500rpm for 3 minutes, removing supernatant, and repeatedly cleaning for 3 times. The CD11c/CD80/CD86/MHCII antibody was added and incubated at room temperature for 30min in the absence of light, while the negative control group should be established with a isotype control group added with the same control of CD11c/CD80/CD 86/MHCII. After incubation was completed, PBS was added for 2 washes, 200. Mu.l PBS was added to resuspend the cells, and detection was performed by an upflow cytometer.
4. And (3) result processing: flow cytometry software analyzed the CD80/CD86/MHCII ratio in CD11c cells.
Experimental results: x-ray treated panel (MVs) vesicles significantly up-regulate the surface costimulatory molecules CD80, CD86, MHCII, etc. of DCs after stimulation, as compared to whole cells, which are markers of dendritic cell maturation. Taken together, vesicles have been shown to significantly promote differentiation and maturation of DCs.
Phagocytic capacity of DC cells was tested by detecting FITC-labeled dextran fluorescence intensity: DC cells have extremely strong antigen endocytosis and processing capacity. When not contacted with antigen, the antigen is in a non-mature state, has strong phagocytic capacity, becomes mature after being activated by contacting with antigen, has weak phagocytic capacity and has enhanced antigen presenting capacity. The experiment determines how much of the phagocytic glucan of the DC is by detecting the fluorescence intensity of the FITC-labeled glucan so as to detect whether the phagocytic capacity of the DC is enhanced.
Bmdc cells were induced (as in the previous example).
2. Stimulation: cells were collected on day 7, and after all cells were blown down, they were counted by centrifugation and resuspended and then plated in 6-well plates, each well plated with 1X 10 6 Individual cells, each with the addition of a stimulus: the GC (Growth Control) group was added with equal volume of PBS, and the Control group and the Treatment group of Treatment were incubated at 37℃for 24 hours after adding membrane vesicles (at protein level) at the same concentration.
3. Phagocytosis and detection: dextran (5 μg/ml) was added, after further incubation for 1h, cells were aspirated into flow tubes, washed 3 times with PBS, then incubated with CD11c antibody at room temperature in the dark for 30min, and washed 3 times with PBS to detect FITC fluorescence in a flow-through manner.
4. And (3) result processing: flow cytometry software analyzed the FITC proportion in CD11c cells.
Experimental data: in order to detect phagocytic function of DCs, FITC-dextran is used as a mode antigen for phagocytosis of DCs, and the average fluorescence intensity value of FITC of CD11c+ DCs is detected. Experimental results showed that the FITC mean fluorescence intensity values were significantly reduced for DCs after receiving membrane vesicle stimulation compared to GC group (growth control). Again, this experimental result demonstrates that vesicles can promote maturation of DCs, thereby reducing their ability to uptake antigen.
Example 5 interaction of mature DCs with T cells after stimulation of bacterial membrane vesicles in X-ray treatment groups:
A. mature DC and CD4 + T cell interaction:
DCs have an important role in inducing specific cellular immune responses for the cross-antigen presentation of extracellular proteins. Thus, cross-presentation of DCs to OVA antigen following membrane vesicle stimulation was examined. Proliferation of OT-II CD4+ T lymphocytes was detected by CFSE flow cytometry 72h after DCs-T cell co-culture. The fluorescent dye CFSE (CFDA-SE), namely hydroxy fluorescein diacetate succinimidyl ester, is a cell staining reagent which can carry out fluorescent marking on living cells. It can be coupled to cellular proteins irreversibly by binding to intracellular amino groups after entry into the cell. During cell division and proliferation, CFSE-labeled fluorescence can be equally divided into two daughter cells, with half the fluorescence intensity of the parent cell. Thus, flow cytometry can be used to count the percentage of cells that have weaker CFSE fluorescence, and thus the proportion of proliferating cells.
Bmdc cells were induced (as in example above).
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), MVs groups were additionally added with membrane vesicles, and antigen-phagocytized DCs were collected by centrifugation and resuspended in normal medium at 2X 10 4 The density of cells/well was plated in 96-well plates at 100 μl per well, 3 multiplex wells per group.
T cell extraction: on the next day, spleen OVA-specific cd4+ T lymphocytes from OT-II mice were isolated and enriched using a Stem Cell Technologies company negative magnetic bead screening kit.
Co-culture of DCs with T cells: the sorted CD4+ T cells were labeled with 1. Mu.M CFSE according to the kit instructions. After labeling, washing 3 times with PBS, 10 5 The cell/well density was added to a 96-well plate to give a final culture volume of 200 μl (CD 4: dc=5:1).
5. On day 3 after co-cultivation, CD4 was detected by CFSE decrease using flow cytometry + Proliferation of T cell populations.
Experimental results: DCs stimulated by membrane vesicles and phagocytosed to OVA antigen were combined with CFSE-labeled OT-II mouse CD4 + T lymphocytes were co-cultured in vitro. The flow analysis of CFSE fluorescence intensity results show that proliferation of CD4 + The proportion of T cells increases. Membrane vesicles (14.05%) significantly increased the number of DCs phagocytosed to OVA antigen (6.80%) for specific CD4 + Proliferation promoting effect of T cells. See fig. 2 and 3 for details.
B. The membrane vesicle treated DCs promote T cell proliferation:
DCs have an important role in inducing specific cellular immune responses for the cross-antigen presentation of extracellular proteins. Thus, cross-presentation of DCs to OVA antigen following vesicle stimulation was examined. Proliferation of T lymphocytes was examined by CFSE flow cytometry 72h after DCs-T cell co-culture. The fluorescent dye CFSE (CFDA-SE), namely hydroxy fluorescein diacetate succinimidyl ester, is a cell staining reagent which can carry out fluorescent marking on living cells. It can be coupled to cellular proteins irreversibly by binding to intracellular amino groups after entry into the cell. During cell division and proliferation, CFSE-labeled fluorescence can be equally divided into two daughter cells, with half the fluorescence intensity of the parent cell. Thus, flow cytometry can be used to count the percentage of cells that have weaker CFSE fluorescence, and thus the proportion of proliferating cells.
Bmdc cells were induced (as in example above).
2. Antigen phagocytosis: DCs cultured for 7 days were cultured in medium for 24 hours as GC (growth control), MVs were additionally added with vesicles, and antigen-phagocytized DCs were collected by centrifugation and resuspended in normal medium at 4X 10 4 The density of cells/well was plated in 96-well plates at 100 μl per well, 3 multiplex wells per group.
T cell extraction: on the next day, T cells of the enriched mice were isolated using a negative magnetic bead screening kit from mouse spleen Stem Cell Technologies company one week after MVs immunization.
Co-culture of DCs with T cells: the T cells selected by the sorting were labeled with 1 μm CFSE according to the kit instructions. After labeling, the sample was washed 3 times with PBS to 4X 10 5 The cell/well density was added to a 96-well plate to give a final culture volume of 200 μl (CD 3: dc=10:1).
5. On day 3 after co-cultivation, CD3 was detected by CFSE decrease using flow cytometry + ，CD8 + ，CD4 + Proliferation of T cell populations.
Experimental results: DCs that were vesicle stimulated and phagocytosed to OVA antigen were co-cultured in vitro with CFSE-labeled OT-II mouse CD4+ T lymphocytes. Flow analysis of CFSE fluorescence intensity results indicated an increase in the proportion of proliferating cd4+ T cells. As shown in the figure, the fluorescence intensity of the cell-plus-vesicle stimulated group was 63.5%, and the vesicle stimulated group was 71%. It is shown that DCs after vesicle treatment significantly stimulated the proliferation of CD4+ T cells.
EXAMPLE 6 experiment of Pseudomonas aeruginosa vaccine against scalding and Pseudomonas aeruginosa skin infection
The experiment adopts experimental rabbits, a hot air electric roasting gun is used for causing a scalding model to the rabbits the day before the rabbits are infected with pseudomonas aeruginosa, the same strain of pseudomonas aeruginosa SKLBPA1 and pseudomonas aeruginosa PA14 (both purchased from ATCC) are respectively infected subcutaneously at the scalding part after 24 hours, the rabbits are sacrificed 24 hours after infection, skin and subcutaneous muscle tissues at the scalding part are taken, the homogenate is used as CFU count, the CFU of the model group and the vaccine group are compared, and whether the vaccine has a protection effect on the scalding combined pseudomonas aeruginosa infection is observed. The results of the experiment show that the pseudomonas aeruginosa vaccine provided by the invention can have prevention and treatment effects on burn and scald combined infection caused by pseudomonas aeruginosa of different serum types (in the experiment, two representative strains of SKLBPA1 and PA14 are adopted as examples), and has wide application scenes.
The vaccine content of the invention comprises: 10 8 CFU/ml
Experimental animals: rabbits, new Zealand white, weight 2.210kg-2.870kg, female, 6.
1. Experimental grouping
The experiment was carried out in 3 groups of 2 animals, and the specific grouping is shown in Table 3.
Table 2 experimental grouping table
2. Immunization
Subject vaccine (10) 8 CFU/ml) was immunized 100 μl subcutaneously in the groin of rabbits, 3 times, 3-4 days apart (i.e., immunization schedule 0, 3, 7 days) or 2 weeks (i.e., immunization schedule 0, 2, 4 weeks).
3. Scald model establishment
Removing hair on the front and back of the left and right sides of a rabbit with scissors and depilatory cream 2 days before infection, sterilizing the skin of the depilatory part with 75% alcohol after 24 hours, smearing the butyl caine hydrochloride mucilage for local anesthesia, setting the temperature of a hot wind power baking gun to 200 ℃, sterilizing a metal plate with square holes with 75% alcohol, sticking the metal plate on the depilatory part, starting the electric baking gun, and blowing hot wind towards the skin of the rabbit with a hot wind port aligned to the square holes for 5 seconds to cause a model of a certain degree of scald.
4. Infection with
4.1 Strain resuscitation
Pseudomonas aeruginosa SKLBPA1 and PA14 were recovered from-80℃to TSA plates and incubated overnight at 37 ℃.
4.2 shaking overnight
The individual colonies were picked from the plates in 3ml TSB and shaken overnight at 37℃at 220 rpm.
4.3 expanded culture
Diluting the fungus liquid overnight to measure OD 600 The stock solution was inoculated into 100ml TSB (250 ml Erlenmeyer flask), and shaken at 37℃and 220rpm to logarithmic growth phase.
4.4 centrifugal washing
Collecting bacterial liquid into 50ml centrifuge tube, centrifuging at 4100rpm (3000 Xg) at room temperature for 10min, discarding supernatant, re-suspending 2mL of 0.9% sodium chloride injection (about 5 OD/ml), and adjusting bacterial liquid to 0.1OD 600 (2×10 7 CFU/ml)。
4.5 infection
1 week after the last immunization (day 7), 50. Mu.l of bacterial liquid (1X 10) was subcutaneously injected into the skin of the rabbit scalded 6 CFU/site).
4.6 plating count
The adjusted bacterial liquid is taken, coated on a TSA plate, cultured overnight at 37 ℃, and CFU is counted.
5. Bacterial count of skin and muscle tissue 24 hours post infection
Animals were sacrificed 24 hours after infection, 75% ethanol was sprayed to disinfect the skin, skin and muscle tissue at the site of scalding was removed by aseptic manipulation, tissue homogenated, TSA plates were applied, incubated overnight at 37 ℃, and CFU was counted with a colony counter.
6. Data processing
Skin and muscle tissue LOG was made using Graphpad Prism mapping software 10 CFU scatter diagram, calculate LOG 10 CFU mean, and variance analysis was used to analyze group-to-group differences.
Experimental results
1. Rabbit weight change
The body weights of animals in each group are shown in Table 3, and the body weights of rabbits in each immune group and each model group are steadily increased, so that no significant difference exists between the groups.
TABLE 3 animal weight variation
2. Modeling result for skin scald of rabbit
And (3) a tuyere with the outer diameter of 14mm and the inner diameter of 10mm is arranged on the hot wind power baking gun sleeve, the temperature is set to be 200 ℃, and the tuyere is attached to the dehairing 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 at the scalded part, and after 24 hours, the skin at the scalded part forms crusts.
3. Pseudomonas aeruginosa infection amount of rabbit skin
The concentrations of the bacterial suspensions of the pseudomonas aeruginosa SKLBPA1 and the PA14 used in the experiment are 0.1OD/ml, and the bacterial suspension remained after the subcutaneous infection is diluted to 10 -4 、10 -5 Mu.l of the colonies were evenly spread on TSA plates and incubated overnight in a 37℃incubator, and the number of colonies was counted, and the results are shown in Table 5.
TABLE 4 viable count results of infectious microbe liquid
[ Table 0001]
| Strain name | Turbidity of infectious microbe liquid (OD/ml) | Concentration of infectious microbe liquid (CFU/ml) |
Table 0002
| SKLBPA1 | 0.1 | 8.00×10 7 |
| PA14 | 0.1 | 1.06×10 8 |
4. Bacterial load of scalded part of rabbit after 24h infection of pseudomonas aeruginosa
Animals were sacrificed 24h after infection, scalded and infected skin and muscle tissues at the bacteria site were aseptically removed, a TSA plate was coated on a homogenizer, incubated overnight at 37℃and CFU was counted, bacterial load was counted again based on 10, and the average and standard deviation were compared for each group, and the results are shown in Table 5 and FIG. 4. Both immunization programs of the PA1 vaccine can obviously reduce the load capacity (P < 0.01) of the strain, which shows that the vaccine has obvious protective effect.
TABLE 5 bacterial load at the site of scalding in rabbits 24h after Pseudomonas aeruginosa infection
(note: x vs. model groups, p <0.01, significant differences
5. Bacterial load of scalding part of rabbit after 24h infection of pseudomonas aeruginosa PA14
The skin and muscle tissues of the PA14 part of the pseudomonas aeruginosa are aseptically scalded and infected, a TSA plate is coated on a homogenizer, the bacteria are cultured overnight at 37 ℃, CFU is counted by a colony counter, bacterial load is counted by taking 10 as a base, and the average value and the standard deviation of each group are compared, so that the results are shown in Table 6 and figure 5. The PA1 vaccine is immunized in 0, 3 and 7 days, and can obviously reduce the load capacity (P < 0.05) of the pseudomonas aeruginosa PA14 by about 0.6-0.9 LOG. The vaccine has a certain protection effect.
It is noted that in experiments with SKLBPA1 and PA14, the immunization program of 0, 3, and 7d can generate an effective immune response rapidly, so that the vaccine of the present invention can be used not only for preventing skin scald complicated infection (i.e. as a prophylactic vaccine), but also for inhibiting infection and alleviating infection after skin scald complicated infection occurs (i.e. as a therapeutic vaccine).
Table 6 bacterial load at scalding sites of rabbits 24h after infection with pseudomonas aeruginosa PA14
(note: p <0.05, significant differences from each model group ratio
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.
Claims (11)
1. The application of the pseudomonas aeruginosa vaccine in preparing the medicine for preventing and treating burns and scalds and combining pseudomonas aeruginosa infection is characterized in that the pseudomonas aeruginosa vaccine is used for reducing the bacterial load in the burns and scalds; the pseudomonas aeruginosa vaccine comprises pseudomonas aeruginosa inactivated by irradiation and membrane vesicles separated from the pseudomonas aeruginosa inactivated by irradiation; the irradiation inactivation is performed by X-ray irradiation, and the irradiation dose is 900-1000Gy.
2. The use of claim 1, wherein the burns and scalds include burns and scalds, and the degree of the scalds includes: degree I, shallow degree II, deep degree II and degree III scalds.
3. The use according to claim 2, wherein the site of scalding comprises skin, mucous membrane.
4. The use according to claim 1, wherein the pseudomonas aeruginosa vaccine is for the prevention of pseudomonas aeruginosa infection.
5. The use according to claim 1, wherein the pseudomonas aeruginosa vaccine contains pseudomonas aeruginosa whole cell content comprising: 1X 10 4 -1×10 10 Needle.
6. The use according to claim 5, wherein the pseudomonas aeruginosa vaccine contains pseudomonas aeruginosa whole cell content comprising: 1X 10 4 Needle, 1X 10 5 Needle, 1X 10 6 Needle, 1X 10 7 Needle, 1X 10 8 Needle, 1X 10 9 Needle and 1X 10 10 Needle.
7. The use according to claim 1, wherein the pseudomonas aeruginosa vaccine further comprises an immunoadjuvant.
8. The use according to claim 7, wherein the immunoadjuvant is aluminium hydroxide.
9. The use according to claim 1, wherein the site of administration of the pseudomonas aeruginosa vaccine is subcutaneous, intramuscular and/or mucosal.
10. The use according to claim 1, wherein the medicament further comprises any pharmaceutically acceptable carrier and/or adjuvant.
11. The use according to claim 10, 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 |
| CN201910777479.2A CN112410239B (en) | 2019-08-22 | 2019-08-22 | Bacterial membrane vesicle and preparation method and application thereof |
| CN201910777606.9A CN112410240B (en) | 2019-08-22 | 2019-08-22 | Pseudomonas aeruginosa membrane vesicle and preparation method and application thereof |
| CN2019107774792 | 2019-08-22 | ||
| PCT/CN2020/110383 WO2021032179A1 (en) | 2019-08-22 | 2020-08-21 | Application of pseudomonas aeruginosa vaccine in treating infection associated with burn or scald injury |
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