A method for constructing a piglet live attenuated vaccine aerosol immunization rabbit animal model and application thereof

Abstract

The application discloses a kind of hog cholera live vaccine aerosol immunization domestic rabbit animal model construction method and its product and application, the method includes: (1) first immunization: domestic rabbit is atomized with vaccine, so that domestic rabbit inhales vaccine aerosol in closed space 15-45 minutes;Rabbit infection amount 750 RID / head share;(2) secondary immunization: in the 28-42 days after first immunization, according to the method of step (2) is immunized for the second time.The animal model established in the application can comprehensively and objectively evaluate the efficacy and safety of hog cholera live vaccine aerosol immunization, and overcomes the shortcomings of traditional muscle injection model, such as high stress and complicated operation.The model provides a standardized experimental tool and scientific basis for the research and development, effect evaluation and immunization program optimization of the new immunization route (aerosol immunization) of hog cholera vaccine.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, and relates to a method for constructing and applying a rabbit animal model immunized with a live swine fever vaccine via aerosol. Background Technology

[0002] Classical swinefever (CSF) is a highly contagious viral disease caused by the classical swinefever virus. It is widespread, with extremely high morbidity and mortality rates, posing a significant threat to the pig industry. The World Organisation for Animal Health (OIE) classifies it as a Category A infectious disease. Vaccination is the most effective strategy for controlling CSF. Currently, CSF vaccines (such as the rabbit-attenuated live attenuated CSF vaccine) are mainly administered via intramuscular injection. While effective, this method has significant drawbacks: 1) it causes considerable stress to animals during capture, restraint, and injection, affecting animal welfare and production performance; 2) in large-scale farms, immunization is time-consuming and labor-intensive, requiring substantial manpower and resources; 3) there is a risk of cross-infection or immunization failure due to needle contamination or injection technique problems.

[0003] Aerosol immunization, as a novel immunization route, has many potential advantages: 1) needle-free injection, which can greatly reduce animal stress and the labor intensity of operators; 2) it can mimic the natural infection route of classical swine fever virus (respiratory tract), and is expected to induce local mucosal immunity in the respiratory and digestive tract mucosa, establishing the first line of defense; 3) it can achieve rapid herd immunity. However, the application of aerosol immunization in classical swine fever vaccines is still in the research stage, and its immunogenicity, safety, and optimal immunization regimen lack systematic evaluation and standardized models.

[0004] Rabbit cross-immunization assays are a classic method for evaluating the efficacy of live swine fever vaccines, primarily by observing the fever response of rabbits after immunization to determine vaccine virulence and immunogenicity. However, traditional rabbit assays are mainly used to evaluate intramuscularly administered vaccines and lack standardized animal models and comprehensive evaluation index systems for systematically evaluating aerosol immunization.

[0005] Therefore, developing a rabbit animal model that can systematically and comprehensively evaluate the aerosol immunization effect of swine fever live vaccine is crucial for promoting the research and application of aerosol immunization technology. Summary of the Invention

[0006] Purpose of the Invention: The purpose of this invention is to provide a standardized and reproducible rabbit animal model for aerosol immunization with a live classical swine fever vaccine and its construction method. This model can comprehensively and objectively evaluate the immunogenicity, safety, and immune response kinetics of aerosol immunization, and can be effectively compared with the traditional intramuscular injection route, providing key technical support for the research and optimization of novel immunization strategies for classical swine fever vaccines.

[0007] Technical solution: This invention provides a method for constructing a rabbit animal model for evaluating the aerosol immunization effect of a live swine fever vaccine, comprising the following steps: (1) First immunization: administer vaccine aerosol to rabbits, allowing them to inhale the vaccine aerosol in a closed space for 15-45 minutes; (2) Second immunization: On days 28-42 after the first immunization, the second immunization is performed according to the method in step (2).

[0008] The equipment used for vaccine atomization in step (1) is the equipment disclosed in utility model patent CN215231175U.

[0009] The immunization dose mentioned in step (1) is 2 doses per rabbit; the rabbit infection dose is 750 RID per dose.

[0010] The present invention also provides a rabbit animal model constructed by the method described above.

[0011] The present invention also provides an application of the rabbit animal model described above in evaluating the aerosol immunization effect of a live swine fever vaccine.

[0012] Specifically, the applications include: evaluating the immunogenicity, safety, and ability to induce cellular and humoral immunity of the aerosol immunization route of the swine fever live vaccine, or comparing the advantages and disadvantages of aerosol immunization with traditional intramuscular injection immunization.

[0013] This invention also provides a method for evaluating the aerosol immunization effect of a live swine fever vaccine, comprising the following steps: (1) Preparation and grouping of experimental animals: Healthy rabbits of a specific age and weight were selected and randomly and evenly divided into five groups: blank control group, intramuscular injection group of swine fever heat-resistant live vaccine, aerosol immunization group of swine fever heat-resistant live vaccine, intramuscular injection group of swine fever passaged cell live vaccine, and aerosol immunization group of swine fever passaged cell live vaccine.

[0014] (2) Initial immunization procedure: Intramuscular injection group: The corresponding vaccine was injected intramuscularly into the hind limbs of rabbits, with the preferred dosage being 2 doses per rabbit.

[0015] Aerosol Immunization Group: Using a dedicated aerosol immunization device, the vaccine is atomized in a closed immunization chamber, allowing rabbits to naturally inhale the vaccine aerosol. The preferred exposure time is 30 minutes.

[0016] Blank control group: Injected with an equal volume of sterile vaccine diluent.

[0017] (3) Second booster immunization: On the 35th day after the first immunization, all immunized groups were given a second immunization in strict accordance with the method and dosage of the first immunization.

[0018] (4) Multi-dimensional indicator monitoring and data collection: This is the core of model building and requires systematic monitoring. Temperature pattern response: After each immunization, measure rectal temperature every 6 hours and monitor continuously for at least 4-5 days, recording the fever pattern (fixed fever, mild fever, no response).

[0019] Clinical symptoms: Observe the rabbit's mental state, appetite, and feces daily, and score them according to the standard scoring system; weigh the rabbit regularly and record the amount of food consumed.

[0020] Systemic immunological evaluation: a. Humoral immunity: Blood was collected at different time points after immunization, serum was separated, and the titers of swine fever virus-specific IgG antibodies and E2 protein antibodies were detected by ELISA.

[0021] b. Cellular immunity and cytokines: The dynamic changes in the concentration of Th1 (such as IL-2, IFN-γ, etc.) and Th2 (such as IL-4, IL-10, etc.) cytokines in serum were detected by ELISA.

[0022] c. Lymphocyte function: Peripheral blood lymphocytes were isolated, and the lymphocyte transformation rate under stimulation by mitogen PHA (T cell stimulant) and LPS (B cell stimulant) was detected by MTT assay to assess the status of cellular and humoral immune function.

[0023] (5) Model Validation and Data Analysis: Statistical methods were applied to compare and analyze the differences in all the above indicators between the aerosol immunization group, the intramuscular injection group, and the blank control group. Data demonstrated that: ① Aerosol immunization effectively stimulates specific immune responses in rabbits (e.g., antibody seroconversion, increased cytokines, and increased lymphocyte conversion rate); ② Aerosol immunization exhibits immune response characteristics different from intramuscular injection (e.g., milder thermogenic response, potentially superior mucosal antibodies); ③ Aerosol immunization has good safety (mild clinical symptoms, no impact on growth). When the aerosol immunization group significantly outperforms the blank control group in key immunological indicators, and has its own characteristics or comparable effects to the intramuscular injection group, while also exhibiting better safety indicators, the animal model is considered successfully constructed and can be used to evaluate aerosol immunization.

[0024] The present invention also provides a method for improving the immunization efficiency of a swine fever vaccine, comprising the following steps: (1) First immunization: administer vaccine aerosol to rabbits, allowing them to inhale the vaccine aerosol in a closed space for 15-45 minutes; (2) Second immunization: On days 28-42 after the first immunization, the second immunization is performed according to the method in step (2).

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Comprehensive evaluation system: For the first time in a rabbit model, the classic "rabbit thermotype response" and the modern "serological-cellular immunology" multidimensional evaluation indicators are integrated, which can comprehensively evaluate aerosol immunity from multiple perspectives such as immunogenicity, safety, and immune response type.

[0026] 2. Strong model comparability: The parallel intramuscular injection control group is set up in the same experiment, which can directly and scientifically compare the advantages and disadvantages of aerosol immunization and traditional methods, and the conclusions are reliable.

[0027] 3. High standardization and reproducibility: Key parameters such as animal selection, grouping, immunization dosage, time points, and detection methods have been clearly defined, and standardized operating procedures have been established, which is conducive to the comparison of results between different studies and the promotion of the model.

[0028] 4. Clear application value: This model can not only be used to evaluate the efficacy of aerosol immunization, but also to screen suitable vaccine strains for aerosol immunization, optimize aerosol particle size, immunization dosage and procedure, etc., thus accelerating the industrialization of aerosol immunization technology.

[0029] 5. Ethical and practical significance: This model reduces the need for extensive exploratory experiments on target animals (pigs), conforms to the "3R" principle of animal experiments, and rabbits are low-cost and easy to manage, which can significantly reduce the initial research and development costs. Attached Figure Description

[0030] Figure 1 The graph shows the body temperature change curves of the group receiving intramuscular injection of classical swine fever heat-resistant live vaccine (rabbit-derived) after secondary immunization; Figure 2 A graph showing the body temperature change after secondary immunization of the group immunized with the heat-resistant live swine fever vaccine (rabbit-derived) via aerosol. Figure 3 A graph showing the body temperature change after secondary immunization of the classical swine fever cell live vaccine administered via intramuscular injection. Figure 4 A graph showing the body temperature change after secondary immunization of the classical swine fever cell live vaccine aerosol immunization group. Figure 5 Clinical symptom scores of experimental animals in each group were recorded 1 day after the first immunization (a) and 1 week after the second immunization (b). Figure 6 The changes in body weight of animals in each group after the first immunization (a) and the second immunization (b); Figure 7 Blood pathogen quantification for the heat-resistant intramuscular injection group, the passage intramuscular injection group (a), the heat-resistant aerosol group, and the passage aerosol group (b) of classical swine fever; Figure 8 Analysis of E2 antibody levels in classical swine fever four weeks after the first immunization; Figure 9 Serum IL-2 concentration; Figure 10 Serum IL-6 concentration; Figure 11 Serum IL-10 concentration; Figure 12 Serum IFN-γ concentration; Figure 13 Results of the lymphocyte transfer experiment stimulated by PHA (day 18 after secondary immunization); Figure 14 The results of the LPS-stimulated lymphocyte transfer experiment (day 18 after secondary immunization). Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0032] 1. Main reagents: Swine fever heat-resistant protective live vaccine (rabbit source, 750 RID / dose, Shandong Lvdou Biotechnology Co., Ltd., Veterinary Drug Approval No. (2013) 151821050), swine fever passaged cell live vaccine (7500 RID / dose, Shandong Lvdou Biotechnology Co., Ltd., Veterinary Drug Approval No. (2013) 151821084), swine fever virus nucleic acid fluorescent RT-PCR detection kit, etc. (Shandong Lvdou Biotechnology Co., Ltd.); GeneJET virus DNA and RNA purification kit (#K0821, ThermoScientific).

[0033] 2. Experimental animals: Healthy New Zealand White rabbits (20 females and 20 males, aged 14 weeks and weighing 1.5-2.0 kg) were provided by our institute's experimental animal farm. Their body temperature was initially measured and basal body temperature was measured one day before the experiment.

[0034] Example 1

[0035] Forty 14-week-old New Zealand White rabbits were divided into five groups of eight (four males and four females) each: a blank control group, a classical swine fever (CSF) heat-resistant live vaccine intramuscular injection group (ST), a CSF heat-resistant live vaccine aerosol immunization group, a CSF passaged cell live vaccine intramuscular injection group, and a CSF passaged cell live vaccine aerosol immunization group. First immunization: For the CSF heat-resistant live vaccine aerosol immunization group and the CSF passaged cell live vaccine aerosol immunization group, the rabbits were immunized with CSF heat-resistant live vaccine (rabbit-derived, 750 RID / dose) and CSF passaged cell live vaccine (7500 RID / dose) aerosol immunizations, respectively. The average dose per rabbit was two doses. Aerosol immunization was performed using an aerosol immunization device (patent number CN215231175U, purchased from Shandong Lvdou Biotechnology Co., Ltd.). After spraying, the rabbits were sealed in a closed room for 30 minutes to ensure they fully inhaled the vaccine. For the swine fever heat-resistant live vaccine intramuscular injection group and the swine fever passaged cell live vaccine intramuscular injection group, rabbits were injected intramuscularly into their legs with the swine fever heat-resistant live vaccine (rabbit-derived) and the swine fever passaged cell live vaccine, respectively. The immunization dose was 2 doses (8 rabbits / group), 2 mL per rabbit. The blank control group (8 rabbits / group) received an equal volume of vaccine diluent intramuscularly, 2 mL per rabbit. A second immunization was administered 35 days later, with the same method and dosage as the first immunization. After the first immunization of all experimental animals, body temperature was measured and recorded every 6 hours for 5 consecutive days. After the second immunization of all experimental animals, body temperature was measured and recorded every 6 hours for 5 consecutive days.

[0036] Rabbit cross-immunization test after secondary immunization, such as Figure 1 As shown, the results were analyzed according to the methods described in the literature (Ge Ye, Zhang Xingjuan, Zhu Qinghu, Han Qiuying, Wang Mouping, Li Weijie, Sun Jianhong, Qiu Huaji. Parallel relationship between quantitative RT-PCR and rabbit typing heat test for detecting classical swine fever rabbit-attenuated live vaccine [J]. Chinese Journal of Preventive Veterinary Medicine, 2011, 33(09):699-703; Wen Hong, Fu Anjing, Li Hongyan. Detection effect of classical swine fever vaccine by rabbit cross-immunization test [J]. Heilongjiang Journal of Animal Husbandry and Veterinary Medicine, 2009(24):81). Figures 1-4As shown, after the second immunization, all four groups exhibited bimodal curves, occurring between 18-24 hours and 36-48 hours post-immunization. The overall trend of body temperature change was a return to normal body temperature from post-immunization high temperature. For the second immunization, in the heat-resistant live vaccine intramuscular immunization group, 4 rabbits developed mild fever, and the remaining rabbits died. In the heat-resistant live vaccine aerosol immunization group, 5 rabbits developed mild fever, 2 showed no significant change, and 1 died. In the passaged cell live vaccine intramuscular injection group, 1 rabbit developed type fever, 3 rabbits developed mild fever, 2 showed no significant change, and 2 died. In the passaged cell live vaccine aerosol immunization group, 2 rabbits developed type fever, 5 rabbits developed mild fever, and 1 died. Monitoring of body temperature changes showed that both intramuscular injection and aerosol immunization resulted in mild fever. The number of rabbits with type fever in the aerosol groups was higher than in the corresponding intramuscular injection groups, and the curve appearance was similar.

[0037] A comparison of aerosol immunization and intramuscular injection methods revealed that both methods resulted in fever patterns in the body temperature of the experimental rabbits. Therefore, this indicates that the aerosol immunization method described in this invention is effective for experimental animals. After the second immunization, the number of patterned or mild fevers observed in the aerosol-immunized group was higher than that in the intramuscular injection group, suggesting that aerosol immunization is more effective.

[0038] Example 2

[0039] Forty 14-week-old New Zealand White rabbits were randomly divided into five groups of eight each (four males and four females): a blank control group, a group receiving intramuscular injection of classical swine fever (CSF) heat-resistant live vaccine, a group receiving aerosol immunization with CSF heat-resistant live vaccine, a group receiving intramuscular injection of CSF passaged cell live vaccine, and a group receiving aerosol immunization with CSF passaged cell live vaccine. For the first immunization, the rabbits in the CSF heat-resistant live vaccine aerosol immunization group and the CSF passaged cell live vaccine aerosol immunization group were immunized with CSF heat-resistant live vaccine (rabbit-derived, 750 RID / dose) and CSF passaged cell live vaccine (7500 RID / dose), respectively, via aerosol immunization. The average dose per rabbit was two doses. Aerosol immunization was performed using an aerosol immunization device (patent number CN215231175U, purchased from Shandong Lvdou Biotechnology Co., Ltd.). After spraying, the rabbits were sealed in a closed room for 30 minutes to ensure they fully inhaled the vaccine. For the swine fever heat-resistant live vaccine intramuscular injection group and the swine fever passaged cell live vaccine intramuscular injection group, rabbits were injected intramuscularly into their legs with the swine fever heat-resistant live vaccine (rabbit-derived) and the swine fever passaged cell live vaccine, respectively. The immunization dose for both groups was 2 doses (8 rabbits / group), 2 mL per rabbit. The blank control group (8 rabbits / group) was injected intramuscularly with an equal volume of vaccine diluent, 2 mL per rabbit. A second immunization was performed 35 days later, with the immunization method and dose for each group being the same as the first immunization.

[0040] Starting from day 0, blood samples were collected every 7 days until 30 days after the second immunization. Nasal and rectal swabs were collected simultaneously and placed in PBS (containing 0.1% BSA, pH 7.2-7.4). Blood samples were incubated overnight at 4°C, and serum was collected for testing. Nasal and rectal swabs were incubated overnight at 4°C, centrifuged at 10,000 rpm for 1 min, and the supernatant was collected for testing. All samples were stored at -20°C for later use.

[0041] Record any injection site injuries and animal behavior. After vaccination, observe the animal's safety daily. Monitor rabbit weight weekly. For the first 7 days after vaccination, monitor rabbit clinical symptoms weekly thereafter (record rectal temperature, and observe appetite, feces, mental state, and any lesions on the eyelids, conjunctiva, and skin, etc., and strictly score according to the clinical symptom scoring system in Table 1).

[0042] Table 1 Clinical symptom scoring system for experimental animals

[0043] Blood samples were collected from rabbits in each group one day or one week after the first immunization. Nucleic acid was extracted from the blood according to the instructions of the GeneJET Viral DNA and RNA Purification Kit (#K0821, purchased from Thermo Scientific). Using the extracted RNA as a template, Real-time PCR was performed according to the instructions of the Classical Swine Fever Virus Nucleic Acid Fluorescent RT-PCR Detection Kit (purchased from Shandong Lvdou Biotechnology Co., Ltd.).

[0044] Operation procedure: FastMode, HoldingStage; reps: 1; 95℃ 20s, cyclingstage, numberofcycles: 45, 95℃ 15s, 59℃ 30s, 68℃ 30s.

[0045] Descriptive statistics were used to test the normality and homogeneity of variance of all data. Parametric analysis was only performed if the data met the requirements of normality and homogeneity of variance. One-way ANOVA in SPSS 16.0 (SPSS Inc., Chicago, IL, USA) was used to analyze all data. Rabbit weight was statistically analyzed using repeated measures ANOVA in the general linear model of SPSS 16.0. This model included the main effect of treatment, the main effect of time, and the interaction between treatment and time. Significance level. P A value <0.05 is considered statistically significant. All data are expressed as "mean ± SEM".

[0046] After immunization, the rabbits' body temperature was recorded, and their appetite, feces, mental state, and any signs of lesions on the eyelids, conjunctiva, and skin were observed. Scoring was strictly performed according to a clinical symptom scoring system. Results showed that, for example... Figure 5 As shown in Figure a, 1 day after immunization, there was no significant difference in clinical symptom scores between the heat-resistant aerosol group, the heat-resistant intramuscular injection group, the passaged aerosol group, and the passaged intramuscular injection group and the control group. p >0.05), the clinical symptom score of the passaged intramuscular injection group was significantly higher than that of the control group ( p <0.01). There was no significant difference in clinical symptom scores between the heat-resistant intramuscular injection group and the heat-resistant aerosol group. p >0.05), there was no significant difference in clinical symptom scores between the passaged aerosol group and the passaged intramuscular injection group. p >0.05). This indicates that after that immunization, the passaged muscle group produced a certain degree of stress response, while the stress responses of the other groups were not significantly different.

[0047] like Figure 5 As shown in b, one week after the second immunization, there was no significant difference in clinical symptom scores between the heat-resistant aerosol group, the heat-resistant intramuscular injection group, the passaged aerosol group, and the passaged intramuscular injection group and the control group compared with the control group. p >0.05), there was no significant difference in clinical symptom scores between the heat-resistant intramuscular injection group and the heat-resistant aerosol group. p >0.05), there was no significant difference in clinical symptom scores between the passaged aerosol group and the passaged intramuscular injection group. p >0.05). This indicates that after the second immunization, the stress response among the groups was not significantly different.

[0048] like Figure 6 As shown in Figure a, analysis of rabbit body weight using Repeated Measures analysis revealed that within 5 weeks after immunization: the within-group factorial test results showed that the time factor had an effect ( P <0.01), the body weight of rabbits in all groups increased significantly. The average body weight of all rabbits at week 0 was 1.07±0.254 kg, and the average body weight increase of all rabbits at week 5 after the first immunization was 1.67±0.479 kg; however, there was no interaction between time and different immunization groups. P >0.05); the results of the intergroup factor test showed that the grouping factor had no effect ( P >0.05), and none of the treatment groups had a significant effect on the body weight of rabbits. Figure 6 As shown in b, the within-group factor test results within 5 weeks after the second immunization (6-10 weeks after the first immunization) indicate that the time factor has an effect. P<0.01), with the experimental time, the weight of rabbits in each group increased significantly. The average weight of all rabbits in the first week after the second immunization was 1.85±0.072 kg, and the average weight of all rabbits in the fifth week after the first immunization was 2.65±0.068 kg; however, there was no interaction between time and different immunization groups. P >0.05); the results of the intergroup factor test showed that the grouping factor had no effect ( P >0.05), and the second immunization had no significant effect on the weight of rabbits in any of the treatment groups. This indicates that the vaccine immunization did not affect the growth of rabbits.

[0049] Blood samples were collected one week after the first immunization for classical swine fever (CSF) pathogen fluorescence quantitative PCR detection. The procedure was performed according to the instructions of the CSF virus nucleic acid fluorescence RT-PCR detection kit (purchased from Shandong Lvdou Biotechnology Co., Ltd.). For both the CSF heat-resistant live vaccine (rabbit-derived) and the CSF passaged cell live vaccine (ST), no vaccine strain was detected in either the intramuscular injection group or the aerosol immunization group (see [link to relevant documentation]). Figure 7 During the first week of immunization, ear vein blood was collected from rabbits in each group, and real-time quantitative PCR was used to detect classical swine fever virus A as a negative control. Figure 7 In a, H is the positive control. Figure 7 In group b, F is the positive control, and the other groups are the experimental groups.

[0050] For both the classical swine fever heat-resistant live vaccine (rabbit-derived) and the classical swine fever passaged cell live vaccine (ST), whether in the intramuscular injection group or the aerosol immunization group, the rabbits had normal weight and no pathogens were detected in their blood.

[0051] Example 3 Forty 14-week-old New Zealand White rabbits were randomly divided into five groups of eight each (four males and four females): a blank control group, a group receiving intramuscular injection of classical swine fever (CSF) heat-resistant live vaccine, a group receiving aerosol immunization with CSF heat-resistant live vaccine, a group receiving intramuscular injection of CSF passaged cell live vaccine, and a group receiving aerosol immunization with CSF passaged cell live vaccine. For the first immunization, the rabbits in the CSF heat-resistant live vaccine aerosol immunization group and the CSF passaged cell live vaccine aerosol immunization group were immunized with CSF heat-resistant live vaccine (rabbit-derived, 750 RID / dose) and CSF passaged cell live vaccine (7500 RID / dose), respectively, via aerosol immunization. The average dose per rabbit was two doses. Aerosol immunization was performed using an aerosol immunization device (patent number CN215231175U, purchased from Shandong Lvdou Biotechnology Co., Ltd.). After spraying, the rabbits were sealed in a closed room for 30 minutes to ensure they fully inhaled the vaccine. For the heat-resistant intramuscular injection group and the passaged intramuscular injection group, rabbits were injected intramuscularly into their legs with the heat-resistant live swine fever vaccine (rabbit-derived) and the passaged live swine fever vaccine, respectively. The immunization dose was 2 doses (8 rabbits / group), 2 mL per rabbit. The blank control group (8 rabbits / group) received an equal volume of vaccine dilution via intramuscular injection, 2 mL per rabbit. A second immunization was administered 35 days later, with the same immunization method and dose for each group as the first immunization. Blood was collected from the marginal ear vein on days 10, 20, and 30 after the first immunization.

[0052] 1. E2 antibody level measurement Whole blood was collected from the experimental animals. After coagulation, the blood was centrifuged (4000 r / min) for 10 min. Serum IgA antibody levels were detected using ElISA. Washing buffer was prepared (10 ml of washing buffer mixed with 190 ml of distilled water). The serum to be tested and the positive and negative controls were placed in a dilution plate and diluted 2-fold (60 μL of diluent mixed with 60 μL of the sample to be tested). 300 μL of the diluted washing buffer was added to each well of the antigen plate. After washing twice, the plate was patted dry on absorbent material. 100 μL of the diluted serum was added to each well, shaken well, covered with sealing film, and incubated in a 37 ℃ water bath for 2 h. After discarding the serum from the plate, the diluent was added to wash each well. 100 μL of enzyme-labeled protein was added, and the plate was incubated in a 37 ℃ water bath for 30 min. The enzyme-labeled protein was discarded, and the plate was washed again with washing buffer. 100 μL of substrate chromogenic solution was added to each well (20-25 ℃, protected from light for 10 min). Add 50 μL of stop solution to each well. Place in an ELISA reader (ODL). 630 The test is conducted to determine the positive or negative result: a blocking rate ≥40% is considered positive, a blocking rate ≤30% is considered negative, and a rate between 30% and 40% is considered questionable.

[0053] The concentrations of IL-2, IL-4, IL-6, and IL-10 in the serum of rabbits in each treatment group were detected using an ELISA kit (see the instruction manual for Jiangsu Baolai (Enzyme Immunoassay) Biological Cytokine ELISA Kit).

[0054] like Figure 8 As shown, 4 weeks after the first immunization, the E2 antibody blocking rate of the swine fever heat-resistant live vaccine intramuscular injection group, the swine fever heat-resistant live vaccine aerosol immunization group, the swine fever passaged cell live vaccine intramuscular injection group, and the swine fever passaged cell live vaccine aerosol immunization group was extremely significantly or significantly higher than that of the control group. p <0.01 or p <0.05), the E2 antibody blocking rate in the serum of the classical swine fever heat-resistant live vaccine intramuscular injection group (heat-resistant intramuscular injection) was significantly higher than that of the classical swine fever heat-resistant live vaccine aerosol immunization group (heat-resistant aerosol group). p <0.01), but there was no significant difference between the classical swine fever cell live vaccine intramuscular injection group (cell live injection) and the classical swine fever cell live vaccine aerosol immunization group (cell live aerosol). p >0.05).

[0055] like Figure 9 As shown, after the first immunization, the serum IL-2 levels in both the passaged intramuscular injection group and the passaged aerosol group showed strong and similar responses, while the heat-resistant vaccine did not elicit a response; similar changes were observed after the second immunization. This indicates that after the first and second immunizations, the passaged cell vaccine effectively stimulated cellular immunity in rabbits, and the effects of aerosol immunization and intramuscular injection were similar, while the heat-resistant vaccine did not elicit a response.

[0056] Th2 cells primarily secrete cytokines such as IL-4, IL-6, and IL-10 to regulate humoral immune responses. For example... Figure 10 As shown, the changes in IL-6 concentration in each group were not significant at weeks 2, 4, 6, 7, and 8 after the initial immunization. This indicates that humoral immunity was not activated by IL-6 in any group.

[0057] like Figure 11 As shown, for IL-10, both the passaged intramuscular injection group and the passaged aerosol group showed strong and similar responses after the first immunization, while the heat-resistant vaccine did not elicit a response; the changes were similar after the second immunization. This indicates that only the passaged cell vaccine could effectively stimulate humoral immunity in rabbits after the first immunization, and the effects of aerosol immunization and intramuscular injection were similar, while the heat-resistant vaccine did not elicit a response; after the second immunization, the passaged cell vaccine could still effectively stimulate humoral immunity in rabbits, while the heat-resistant intramuscular injection group and the heat-resistant aerosol group also produced some humoral immune responses.

[0058] like Figure 12 As shown, there were no significant changes in IFN-γ concentrations in any group at weeks 2, 4, 6, and 8 after the initial immunization. At week 7 after the initial immunization, there were no significant differences between the heat-resistant intramuscular injection group and the heat-resistant aerosol group, or between the passaged intramuscular injection group and the passaged aerosol group.

[0059] 2. Experiment on the transformation of peripheral blood lymphocytes in rabbits immunized with swine fever vaccine Mix 5 mL of blood with 50 IU of heparin. Add 4 mL of fresh anticoagulated whole blood to a centrifuge tube containing 4 mL of physiological saline and mix well. Then add the diluted whole blood to a centrifuge tube containing 4 mL of lymphocyte separation medium. Centrifuge at 900 g for 20 min and aspirate the lymphocytes in the middle layer. Wash three times with PBS; the bottom layer contains lymphocytes. Add RPMI 1640 complete culture medium to each tube to a final volume of 1.5 mL. After resuspending the cells, take 100 μL of the cell suspension and count the cells using a cell counter.

[0060] Add 80 μL of single-cell suspension to a 96-well plate, and add 20 μL of PHA or LPS to a final concentration of 20 μg / mL PHA and 5 μg / mL LPS. Include a negative control. Perform triplicate for each sample. Incubate at 37°C with 5% CO2 for 44 h, then add 20 μL of MTT per well and continue incubation for another 4 h. Centrifuge at 1000g for 10 min at room temperature, carefully remove the supernatant, and finally add 100 μL of DMSO to each well. Gently vortex on a small micro-shaker for approximately 15 min to dissolve the precipitate in each well.

[0061] OD measured on an ELISA reader 570nm The obtained data were statistically processed, and their average value was calculated. The Stimulation Index (SI) was used as a parameter to determine the degree of lymphocyte transformation. Lymphocyte transformation rate = OD of test wells 570nm Average value / control well OD 570nm Average value * 100%.

[0062] The efficiency of lymphocytes transforming into lymphoblasts under the stimulation of antigens and mitogens PHA and LPS is one of the important indicators for assessing the body's immune function. PHA induces T lymphocyte proliferation, while LPS induces B lymphocyte proliferation. Analysis of the lymphocyte transformation assay shows that ( Figure 13 , Figure 14 In the lymphocyte transformation experiment, all treatment groups showed strong and similar responses after the first immunization with either PHA or LPS stimulation. This indicates that both vaccines, regardless of aerosol or intramuscular injection, can induce good cellular and humoral immunity in rabbits after the first immunization. However, after the second immunization, only the passaged intramuscular injection group and the passaged aerosol group showed strong and similar responses, while the heat-resistant vaccine did not elicit a response. This indicates that after the second immunization, only the passaged cell vaccine produced good cellular and humoral immunity, while the heat-resistant vaccine did not elicit a response. In summary, all experimental results show that aerosol immunization with the classical swine fever vaccine has shown initial success in rabbit animal models.

Claims

1. A method for constructing a rabbit animal model for evaluating the aerosol immunization effect of a live swine fever vaccine, characterized in that, Includes the following steps: (1) First immunization: The rabbits are given vaccine aerosol and inhaled in a closed space for 15-45 minutes; the rabbit infection dose is 750 RID / dose. (2) Second immunization: On days 28-42 after the first immunization, the second immunization is performed according to the method in step (2).

2. The construction method according to claim 1, characterized in that, The equipment used for vaccine nebulization in step (1) is the equipment disclosed in CN215231175U.

3. The construction method according to claim 1, characterized in that, The immunization dose mentioned in step (1) is 2 doses per animal.

4. A rabbit animal model constructed by the method described in any one of claims 1 to 3.

5. The application of the rabbit animal model of claim 4 in evaluating the aerosol immunization effect of a live swine fever vaccine.

6. The application according to claim 5, characterized in that, The specific applications include: evaluating the immunogenicity, safety, and ability to induce cellular and humoral immunity of the swine fever live vaccine via aerosol immunization, or comparing the advantages and disadvantages of aerosol immunization with traditional intramuscular injection immunization.

7. A method for evaluating the aerosol immunization efficacy of a live swine fever vaccine, characterized in that, Includes the following steps: (1) Preparation and grouping of experimental animals: Healthy rabbits of specific ages and weights were selected and divided into five groups: blank control group, intramuscular injection group of swine fever heat-resistant live vaccine, aerosol immunization group of swine fever heat-resistant live vaccine, intramuscular injection group of swine fever passaged cell live vaccine, and aerosol immunization group of swine fever passaged cell live vaccine. (2) Initial immunization procedure: Intramuscular injection group: The corresponding vaccine was injected intramuscularly into the hind limbs of rabbits, with the preferred dosage being 2 doses per rabbit; Aerosol Immunization Group: Using a dedicated aerosol immunization device, the vaccine is atomized in a closed immunization chamber, allowing rabbits to naturally inhale the vaccine aerosol. The preferred exposure time is 30 minutes. Blank control group: injected with an equal volume of sterile vaccine diluent; (3) Second booster immunization: On the 35th day after the first immunization, all immunized groups were given a second immunization in strict accordance with the method and dosage of the first immunization; (4) Multi-dimensional indicator monitoring and data collection: This is the core of model building and requires systematic monitoring. Temperature pattern response: After each immunization, measure rectal temperature every 6 hours and monitor continuously for at least 4-5 days, recording the fever pattern (fixed fever, mild fever, no response). Clinical symptoms: Observe the rabbit's mental state, appetite, and feces daily, and score them according to the standard scoring system; weigh the rabbit regularly and record its feed intake; Systemic immunological evaluation: a. Humoral immunity: Blood was collected at different time points after immunization, serum was separated, and the titers of swine fever virus-specific IgG antibodies and E2 protein antibodies were detected by ELISA. b. Cellular immunity and cytokines: The dynamic changes in the concentrations of Th1 and Th2 cytokines in serum were detected by ELISA. c. Lymphocyte function: Peripheral blood lymphocytes were isolated, and the lymphocyte transformation rate under mitogen PHA and LPS stimulation was detected by MTT assay to assess the status of cellular and humoral immune function. (5) Model validation and data analysis: When the aerosol immunization group is superior to the blank control group in terms of immunological indicators, and the immunization effect is comparable to that of the intramuscular injection group, and the safety indicators are better, it indicates that the animal model has been successfully constructed and can be used to evaluate aerosol immunization.

8. A method for improving the immunization efficiency of a classical swine fever vaccine, characterized in that, Includes the following steps: (1) First immunization: administer vaccine aerosol to rabbits, allowing them to inhale the vaccine aerosol in a closed space for 15-45 minutes; (2) Second immunization: On days 28-42 after the first immunization, the second immunization is performed according to the method in step (1).

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