Establishment and application method of acute pneumonia animal model for acinetobacter baumannii vaccine immune effect test
By establishing an acute pneumonia animal model in BALB/c mice and administering different doses of live Acinetobacter baumannii in groups, the problem of poor model controllability in existing technologies was solved, and a highly reliable and reproducible vaccine effect assessment was achieved.
Patent Information
- Application Number
- CN202511473828.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-15
- Publication Date
- 2026-01-02
AI Technical Summary
Current technologies lack animal models that can effectively simulate the common clinical infection routes, infection sites, pathogenic mechanisms and pathological changes of Acinetobacter baumannii. Furthermore, existing models are cumbersome to operate, have poor controllability, and cannot accurately assess the immunoprotective effect of vaccines.
An acute pneumonia animal model was established using BALB/c mice. Different doses of Acinetobacter baumannii live bacterial solution were administered to different groups of mice. Combined with strict feeding and environmental control, statistical analysis was conducted to ensure the reliability and reproducibility of the model.
This provides a highly reliable and reproducible animal model that can accurately simulate clinical infection with Acinetobacter baumannii, improving the accuracy and controllability of vaccine efficacy assessment. It is applicable to different strains and animal species, facilitating cross-sectional comparison of different experimental results.
Smart Images
Figure CN121241981A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine. Background Technology
[0002] Acinetobacter baumannii is a Gram-negative opportunistic pathogen that has recently become a significant cause of hospital-acquired infections worldwide. Multidrug-resistant (MDR) Acinetobacter baumannii infections often occur in debilitated patients, particularly those in intensive care units (ICUs) and / or those with severe chronic illnesses, those undergoing invasive procedures (e.g., mechanical ventilation), or those with long-term hospitalizations or receiving broad-spectrum antibiotics. The most common clinical manifestations of Acinetobacter baumannii are ICU infections such as ventilator-associated pneumonia (VAP) and bacteremia, with morbidity and mortality rates as high as 52%. Other hospital-acquired Acinetobacter baumannii infections include urinary tract infections, wound infections, and meningitis. Due to the lack of new antibiotics for treating MDR-resistant Acinetobacter baumannii infections, the Infectious Diseases Society of America (IDSA) describes Acinetobacter baumannii as “a classic example of an unmet medical need that is mismatched with the current pipeline of antimicrobial development.”
[0003] Current technology lacks an animal model that can best simulate the common clinical infection routes, infection sites, pathogenic mechanisms, pathological changes, and clinical manifestations of Acinetobacter baumannii. For example, the basic principle for establishing conventional animal models of Acinetobacter baumannii infection is to suppress the immune function of mice with immunosuppressants, and then construct localized infectious lesions in the lungs using microtracheal injection or ultrasonic nebulization. Although this method partially simulates the infection situation in clinical immunodeficient patients, it is cumbersome to operate in animal experiments, has poor experimental controllability, low success rate, poor reproducibility, and the use of immunosuppression has an antagonistic effect on the immunoprotective effect of vaccines, which is not conducive to the assessment of vaccine immune effects. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides a method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine.
[0005] This invention is achieved using the following technical solution: A method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine, comprising the following steps: S1: Select and raise laboratory animals; S2: Experimental grouping and labeling; S3: Constructing an animal model; S4: Perform statistical analysis of the data.
[0006] Furthermore, the experimental animals were BALB / c mice; grade: SPF; weight: 18-21g; age: 10-12w; number and sex: 160 mice, female.
[0007] Furthermore, the rearing conditions include: cage type: IVC or negative pressure isolator; stocking density: 2-5 animals / cage; rearing environment standard: GB14925-2010; rearing environment control system: Honeywell; temperature: 20-26℃; humidity: 40-70%; lighting: animal lighting: 10-20 Lux, 12h light / 12h darkness; working lighting: 100-200 Lux; air exchange rate: ≥ 15 times / hour, 100% fresh air; the air exchange rate can be reduced during non-working hours, but should not be less than 10 times / hour.
[0008] Furthermore, the experimental groups were: normal control group, low-dose model group, medium-dose model group and high-dose model group; wherein, low-dose model group: 0.2×10⁹ CFU / animal; medium-dose model group: 0.8×10⁹ CFU / animal; high-dose model group: 1.6×10⁹ CFU / animal.
[0009] Furthermore, the identification includes animal identification and cage tag identification.
[0010] Further, step S3 specifically involves administering 25 μL of sodium chloride injection to the lungs of the experimental animals in the normal control group; and administering 25 μL of Acinetobacter baumannii live bacterial solution of different concentrations to the lungs of the experimental animals in the low-dose model group, medium-dose model group, and high-dose model group.
[0011] Furthermore, step S4 specifically involves using IBM SPSS Statistics 22.0 for statistical analysis of the data.
[0012] The beneficial effects of this invention are as follows: This invention is a method for preparing an animal model of Acinetobacter baumannii acute pneumonia with high reliability, repeatability, applicability and controllability. The method is simple to operate and applicable to different sources of challenge strains, animal breeds and strains, and facilitates horizontal comparison of experimental results from different batches and groups.
[0013] This invention can simulate animal models of Acinetobacter baumannii, including common clinical infection routes, infection sites, pathogenic mechanisms, pathological changes, and clinical manifestations, to the greatest extent possible, thereby improving the pertinence and accuracy of judging and predicting the efficacy of candidate vaccines.
[0014] This invention is applicable to acute infection animal models of Acinetobacter baumannii strains from various sources (such as ATCC standard strains, clinical isolates, highly virulent strains, and low-virulence strains). The animal models exhibit high short-term lethality and allow for accurate LD50 (or LD90) determination, ensuring stable and controllable morbidity and mortality, making them suitable for challenge trials of the protective effect against the virus in candidate vaccine-treated animals. Attached Figure Description
[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.
[0016] Figure 1 This is a flowchart of the present invention. Detailed Implementation
[0017] To make the objectives, 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 with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.
[0018] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.
[0019] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.
[0020] See Figure 1 The establishment and application of an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine includes the following steps: S1: Select and raise laboratory animals; S2: Experimental grouping and labeling; S3: Constructing an animal model; S4: Perform statistical analysis of the data.
[0021] The selection criteria for experimental animals: BALB / c mice have a relatively clear genetic and biological background (including normal ranges for various data such as anatomy, physiology, and clinicopathology) and similar clinical pathogenesis, and have been reported in similar drug development both domestically and internationally. The rationale for animal number selection: In accordance with the 3R principle, the fewest possible number of animals were used while meeting research objectives, scientific standards, and regulatory requirements. In this experiment, BALB / c mice were divided into 4 groups, totaling 160 mice, with 40 mice in each group. Weight: 18-21g; Age: 10-12 weeks.
[0022] Specifically, the housing conditions for laboratory animals are as follows: Cage type: IVC or negative pressure isolator; Stocking density: 2-5 animals / cage; Cage space displacement frequency: Bedding and cage bottom should be changed weekly. Environmental standards: GB14925-2010; Environmental control system: Honeywell; Temperature: 20-26℃; Humidity: 40-70%; Lighting: Animal lighting: 10-20 Lux, 12h light / 12h dark; Working lighting: 100-200 Lux; Air exchange rate: ≥ 15 times / hour, 100% fresh air; Air exchange rate can be reduced during non-working hours, but should not be lower than 10 times / hour.
[0023] Feed type: Breeding feed for rats and mice; Feeding method: Ad libitum; Nutritional composition testing: Test reports provided by the feed supplier for each batch; Standard and semi-standard nutritional components: crude protein, crude fat, crude fiber, crude ash, moisture, calcium, and phosphorus; Amino acid components: cystine + methionine, lysine, in accordance with the People's Republic of China National Standard GB14924.3-2010. Testing is conducted quarterly; Confirmation of feed contaminant content: Test reports provided by the feed supplier for each batch; Chemical contaminant components: arsenic, lead, mercury, cadmium, hexachlorocyclohexane (HCH), DDT, aflatoxin B1, total bacterial count, coliform bacteria, mold and yeast count, pathogenic bacteria (Salmonella), in accordance with the People's Republic of China National Standard GB / T 14924.2-2001. Testing is conducted quarterly; Feed shelf life: 90 days; Sterilization method: Irradiation sterilization; Feed usage period: After opening the bag within the barrier, store in a sealed container and use within one week; monthly sterility testing is required. Drinking water: Type: Ultrafiltration / reverse osmosis + sodium hypochlorite (3-10ppm residual chlorine), pH 6.5-7.5, Water supply method: GB5749-2022, Automatic drinking water system (Avidity); Total bacterial count test: Monthly sterility test; Routine water quality indicators test: Residual chlorine test, pH test, once a month.
[0024] Group design: 40 nippons were assigned to the normal control group, 40 nippons to the low-dose model group (0.2 × 10⁹ CFU / nippon), 40 nippons to the medium-dose model group (0.8 × 10⁹ CFU / nippon), and 40 nippons to the high-dose model group (1.6 × 10⁹ CFU / nippon). The model design is shown in Table 1. Table 1. Model Design
[0025] See Table 2 for specific group information. Table 2 Grouping
[0026] Note 1: The first digit of the animal number represents the group. 1, 2, 3, and 4 represent the normal control group, low-dose model group, medium-dose model group, and high-dose model group, respectively. F represents female. The last 3 digits represent the animal serial number.
[0027] Note 2: The day of model making is defined as D1.
[0028] Animal identification, adaptation / experimentation period: Cage tags and ear tags (identification numbers) are used as animal identification marks, and tail markings are used as auxiliary identification marks.
[0029] Cage labeling: Before grouping: Use uniform white cage cards, clearly indicating the experiment number, animal strain, cage number, animal number, ear tag (identification number), number of animals, entry time, and any additional information. After grouping: Use different cage cards to distinguish between groups: normal control group, low-dose model group, medium-dose model group, and high-dose model group. The cage card should indicate the experiment number, animal strain, cage number, animal number, identification number (as shown in the table), treatment factor, number of animals, and expected start and end dates of the experiment. See Table 3 for cage label colors after grouping. Table 3 Cage Identification Colors
[0030] Animal model selection criteria: Acinetobacter baumannii can cause hospital-acquired pneumonia, bloodstream infections, abdominal infections, central nervous system infections, urinary tract infections, and skin and soft tissue infections. The most common site of nosocomial infection with Acinetobacter baumannii is the lungs, and it is a significant pathogen in hospital-acquired pneumonia (HAP), especially ventilator-associated pneumonia (VAP). Animal models of Acinetobacter baumannii pneumonia often use immunosuppressants combined with strains, which cannot meet the requirements for pharmacodynamic evaluation of vaccine-related biological products. To meet the evaluation requirements of vaccine-related biological products, an acute Acinetobacter baumannii pneumonia animal model was established using immunocompetent animals combined with a standard Acinetobacter baumannii strain.
[0031] Modeling method Modeling time: D1.
[0032] Normal control group: 25 μL of sodium chloride injection was administered to the lungs.
[0033] For the remaining groups: 25 μL of Acinetobacter baumannii live bacterial solution of different concentrations was administered into the lungs.
[0034] Environmental requirements: Conducted in an ABSL-2 laboratory biosafety cabinet.
[0035] The main operations are as follows: 1) Anesthesia: Mice were injected intramuscularly with a compound anesthetic (containing Sutacetin-50: 10 mg / mL and celazine hydrochloride: 2 mg / mL). Sutacetin-50 50 mg / kg and celazine hydrochloride 10 mg / kg were administered to confirm the depth of anesthesia.
[0036] 2) After anesthesia, fix the mouse in a supine position on the endotracheal intubation table (15°~20°). Use a rubber band to pull the mouse's upper incisors backward to fix them, which is conducive to exposing the glottis.
[0037] 3) Illuminate the skin of the mouse's neck with an LED flexible tube spotlight, pull out the mouse's tongue with tweezers, and insert another curved ophthalmic tweezer into the mouse's mouth, pressing one end against the mouse's palate and the other end against the base of the tongue, until the mouse's airway opening and closing with the mouse's breathing is clearly visible.
[0038] 4) At this point, insert the gel pipette tip, which has been pre-absorbed with 25 μL of bacterial solution, into the airway. After insertion, the level of bacterial solution in the pipette can be observed to decrease with the frequency of the mouse's breathing. After the liquid in the pipette tip is completely aspirated, remove the pipette tip.
[0039] 5) After inhaling the bacterial solution, the mouse was kept in a supine position on the intubation table for about 10 seconds, then removed. After observing the mouse's condition, it was put back into the cage.
[0040] Model evaluation indicators and basis 1) Selection criteria for modeling indicators: According to similar literature reports, mice with different degrees of modeling will exhibit arched backs, piloerection, or even death, as well as colonization of Acinetobacter baumannii in the lungs.
[0041] 2) Model evaluation indicators; HE staining of lung tissue pathological sections should show varying degrees of alveolar epithelial cell degeneration and necrosis or accumulation of alkaline substances, and some samples may have hemorrhage or fibrous tissue hyperplasia; bronchoalveolar lavage fluid smears should show Acinetobacter baumannii.
[0042] The detection indicators included animal survival, peripheral blood collection, bronchoalveolar lavage fluid collection, and histopathology. The final experimental data were statistically analyzed using IBM SPSS Statistics 22.0. All quantitative data are expressed as Mean ± SEM s. Graph Pad Prism 8 software was used to plot the parameters before and after drug administration in different groups of animals. Data were analyzed using SPSS 22.0 statistical software. The Levene test was used to test the homogeneity of variance. When the variances were homogeneous (P≥0.05), the Dunnett's & LSD method in one-way ANOVA was used to compare differences between groups. When the variances were unequal (P<0.05), the Mann-Whitney U test (MW method) in the Kruskal-Wallis H rank-sum test (KW method) was used to compare differences between groups.
[0043] Through the above experimental procedures and related tests, we have demonstrated that all animals treated with the medium dose showed clinical and pathologically confirmed inflammatory pathological damage to the lungs. The mortality rate of untreated model animals reached 100% within one week of model formation, indicating successful model replication.
[0044] For the foregoing embodiments, in order to simplify the description, they are all described as a series of actions. However, those skilled in the art should understand that this application is not limited to the described order of actions, because according to this application, some steps can be performed in other orders or simultaneously. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions involved are not necessarily essential to this application.
[0045] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Modifications and variations made by those skilled in the art without departing from the spirit and scope of the invention should be within the protection scope of the appended claims.
Claims
1. A method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine, characterized in that... Includes the following steps: S1: Select and raise laboratory animals; S2: Experimental grouping and labeling; S3: Constructing an animal model; S4: Perform statistical analysis of the data.
2. The method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine as described in claim 1, characterized in that, The experimental animals were BALB / c mice; Rating: SPF; Weight: 18-21g; Age: 10-12 weeks; Number and sex: 160, female.
3. The method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine as described in claim 1, characterized in that, The conditions for rearing include: cage type: IVC or negative pressure isolator; stocking density: 2-5 animals / cage; environmental conditions standard: GB14925-2010; environmental control system: Honeywell; temperature: 20-26℃; humidity: 40-70%; lighting: animal lighting: 10-20 Lux, 12h light / 12h dark; working lighting: 100-200 Lux; air exchange rate: ≥ 15 times / hour, 100% fresh air; the air exchange rate can be reduced during non-working hours, but should not be lower than 10 times / hour.
4. The method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine as described in claim 1, characterized in that, The experimental groups were: normal control group, low-dose model group, medium-dose model group and high-dose model group; among them, the low-dose model group: 0.2×10⁹ CFU / animal; the medium-dose model group: 0.8×10⁹ CFU / animal; and the high-dose model group: 1.6×10⁹ CFU / animal.
5. The method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine as described in claim 1, characterized in that, The identification includes animal identification and cage tag identification.
6. The method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine as described in claim 1, characterized in that, Step S3 specifically involves administering 25 μL of sodium chloride injection to the lungs of the experimental animals in the normal control group; and administering 25 μL of Acinetobacter baumannii live bacterial solution of different concentrations to the lungs of the experimental animals in the low-dose model group, medium-dose model group, and high-dose model group.
7. The method for establishing and applying an acute pneumonia animal model for testing the immune effect of Acinetobacter baumannii vaccine as described in claim 1, characterized in that, Step S4 specifically involves using IBM SPSS Statistics 22.0 for statistical analysis of the data.