Establishment of a guinea pig surrogate efficacy test model for swine contagious pleuropneumonia vaccine

Abstract

The present application relates to the technical field of biological medicine, more specifically, it relates to a guinea pig substitute efficacy test model for porcine contagious pleuropneumonia vaccine. The virulence of porcine contagious pleuropneumonia bacteria to host animals (pigs) is determined; a substitute animal is screened; vaccine I is prepared; the host animals are immunized with vaccine I; the substitute animals are immunized with vaccine I; the screened substitute animals are immunized with different doses of vaccine I; and the host animals and the screened substitute animals are immunized with vaccine II. The present application establishes a high-efficiency and low-cost guinea pig substitute efficacy test model method for porcine contagious pleuropneumonia vaccine, which can effectively replace the host animals (pigs) to evaluate the efficacy of the vaccine, greatly saves resources under the premise of ensuring reliable testing, and is suitable for popularization and application in vaccine research and development and quality testing.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the establishment of a guinea pig substitution efficacy testing model for porcine infectious pleuropneumonia vaccine. Background Technology

[0002] Porcine contagious pleuropneumonia (PCP) is an important bacterial respiratory disease in pigs caused by Actinobacillus pleuropneumoniae (APP). The typical pathological features of the disease are hemorrhagic fibrinous pneumonia in the acute phase, with affected pigs often exhibiting bleeding from the mouth and nose and even sudden death; in the chronic phase, it manifests as fibrinous necrotizing pleuropneumonia, with infected pigs becoming asymptomatic sources of infection, easily leading to secondary infections with other pathogens, resulting in long-term sub-health in the herd and causing serious economic losses to the pig industry. Vaccination is a key means of controlling this disease. Currently, the commonly used vaccines in China are mainly inactivated vaccines and subunit vaccines, while novel vaccines such as attenuated live vaccines, oral vaccines, bacterial shadow vaccines, DNA vaccines, and nano-vaccines are also under active development.

[0003] Vaccine production must meet the basic requirements of safety and efficacy; therefore, safety and efficacy testing are particularly important. Animal experiments remain irreplaceable in efficacy assessment. By comparing antibody levels and protective effects against challenge in non-host animals (mice, guinea pigs, etc.) and host animals (pigs) after vaccination, establishing a correlation between the two, it is hoped that non-host animals can be used to replace host animals for efficacy testing, simplifying operations and reducing costs and manpower. Currently, research based on animal substitution methods has been conducted for various animal diseases, such as using BALB / c mice to replace cattle in efficacy evaluation of foot-and-mouth disease inactivated vaccines. However, for porcine infectious pleuropneumonia vaccines, a reliable non-host animal efficacy substitution testing method is currently lacking. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a model for testing the guinea pig alternative efficacy of a porcine contagious pleuropneumonia vaccine, the specific steps of which are as follows: S1: Determine the virulence of porcine infectious pleuropneumonia bacteria to host animals, pigs: The porcine infectious pleuropneumonia bacteria strain was propagated, cultured, diluted, and injected intratracheally into pigs. The time of death was recorded and the pigs were dissected. All pigs that did not die were dissected 7 days later. S2: Screening alternative animals and challenge methods: Porcine infectious pleuropneumonia bacteria were propagated, cultured, diluted, and injected intraperitoneally and intranasally into mice and guinea pigs, respectively. The time of death was recorded and the animals were dissected. All surviving animals were dissected 7 days later. S3: Prepare vaccine I, administer 2.0 mL / animal via intramuscular injection in the neck of the host animal (pig). Administer a second immunization 21 days after the first immunization, and challenge the virus 14 days after the second immunization. Observe the immune protection effect and pathological changes. S4: Immunize alternative animals with vaccine I, using the same immunization and challenge times as the host animals (pigs), and observe the immune protection effect and pathological changes; S5: Immunize selected alternative animals with different doses of vaccine I and observe the protective effect of the immunization. S6: Simultaneously immunize the host animal (pig) and selected alternative animals with vaccine II, and observe the immunoprotective effect and pathological changes.

[0005] Preferably, the porcine infectious pleuropneumonia bacteria is APP1 type.

[0006] Preferably, the APP1 type is obtained by isolating, purifying, and identifying lung tissue from diseased pigs in a pig farm, and is freeze-dried and preserved by our company, with the preservation number CGMCC No.47172.

[0007] Preferably, the alternative animals are mice and guinea pigs.

[0008] Preferably, the vaccine I is an APP subunit vaccine.

[0009] Preferably, the APP subunit vaccine is JY20250711, and the APP subunit vaccine is formulated from ApXⅠ, ApXⅡ, ApXⅢ and OmpA proteins with aluminum gel.

[0010] Preferably, the protein ratio of ApXⅠ, ApXⅡ, ApXⅢ and OmpA is 1:1:1:1; the ratio of antigen to aluminum gel is 4:1; the concentration of ApXⅠ antigen is 500 μg / mL, the concentration of ApXⅡ antigen is 1288 μg / mL, the concentration of ApXⅢ antigen is 2925 μg / mL, and the concentration of OmpA antigen is 860 μg / mL.

[0011] Preferably, the vaccine II is JY20240927, and the vaccine II is formulated with ApXⅠ, ApXⅡ, OmpA and OmpD proteins and aluminum glue, wherein the ratio of ApXⅠ, ApXⅡ, OmpA and OmpD proteins is 1:1:1:1; the ratio of antigen to aluminum glue is 4:1; the concentration of ApXⅠ antigen is 1055 μg / mL, the concentration of ApXⅡ antigen is 1860 μg / mL, the concentration of OmpA antigen is 1250 μg / mL, and the concentration of OmpD antigen is 680 μg / mL.

[0012] The beneficial effects of this invention are as follows: This invention, through a systematic comparison of the infection effects of alternative animals and challenge methods, has for the first time discovered that intranasal challenge in guinea pigs can induce the most significant consolidation lesions in the lungs of guinea pigs, and these lesions are basically consistent with those in the host animal, pig. This method is simple to operate, has good reproducibility, and can effectively avoid guinea pig damage caused by mechanical manipulation or non-specific infection. It has successfully established a stable and reliable animal model of infection, which not only solves the technical difficulties in constructing alternative animal models in the field of disease research, but also meets the requirements of laboratory animal welfare ethics.

[0013] This invention effectively overcomes the key technical bottlenecks that have long existed in the study of the pathogenic biological characteristics of APP and vaccine development, such as the scarcity of experimental pigs, high animal husbandry costs, and stringent experimental conditions. The constructed guinea pig model significantly reduces the difficulty of experimental operations, saves manpower, financial resources, and time costs, and strictly follows the principles of "reduction, replacement, and optimization" in experimental animal welfare. This method provides a stable, reliable, and economical experimental platform for in-depth exploration of the pathogenic characteristics of APP and accelerating vaccine development, and has significant scientific research value and practical application benefits.

[0014] This invention establishes a highly efficient and low-cost guinea pig alternative efficacy testing model for porcine infectious pleuropneumonia vaccine. It can effectively replace pigs in vaccine efficacy evaluation, significantly saving resources while ensuring testing reliability. It is suitable for widespread application in vaccine research and development and quality inspection.

[0015] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. Preservation information: Classification and name: Actinobacillus pleuropneumoniae, depository institution: China General Microbiological Culture Collection Center, deposit date: July 2, 2026, accession number: CGMCC NO. 47172. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is an anatomical diagram of the host animal (pig) for APP1 virus detection; Figure 2 This is an anatomical diagram of the host animal (pig) for APP1 virus detection; Figure 3 This is an anatomical diagram of the host animal (pig) for APP1 virus detection; Figure 4 This is an anatomical diagram of the host animal (pig) for APP1 virus detection; Figure 5 This is an anatomical diagram of the host animal (pig) for APP1 virus detection; Figure 6 This is an anatomical diagram of the host animal (pig) for APP1 virus detection; Figure 7 This is an anatomical diagram of APP1 type mouse for toxicity testing; Figure 8 This is an anatomical diagram of APP1 type guinea pig nasal droplet toxicity test; Figure 9 This is an anatomical diagram of APP1 type guinea pig nasal droplet toxicity test; Figure 10 This is an anatomical diagram of the peritoneal toxicity test in guinea pigs using the APP1 method; Figure 11 This is an anatomical diagram of the peritoneal toxicity test in guinea pigs using the APP1 method; Figure 12 These are anatomical images of pigs challenged with the vaccine group; Figure 13 These are anatomical diagrams of guinea pigs challenged with the vaccine group; Figure 14 This is an anatomical diagram of the control group guinea pigs challenged with the virus. Detailed Implementation

[0017] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0018] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0019] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0020] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments. Example

[0021] APP Animal Virulence Determination in Pigs The APP1 type freeze-dried bacterial culture was reconstituted with BD TSB. 1 mL of the reconstituted culture was added to a test tube containing 9 mL of TSB liquid medium (containing 4% newborn calf serum and 2‰ NAD). The culture was incubated at 37°C and 120 rpm for 8 hours, then incubated at 4°C overnight. 4 mL of the culture was then added to 200 / 500 mL of TSB liquid medium (containing 4% newborn calf serum and 2‰ NAD). The culture was incubated at 37°C and 120 rpm for 8 hours, then diluted with TSB medium to a bacterial count of 1.0-2.0 × 10⁻¹⁰. 4 1.0-2.0*10 3 1.0-2.0*10 2 To detect the toxicity of pigs at CFU / mL, 8-week-old pigs were selected. Two pigs were injected with each titer via tracheal injection, 10 mL per pig. After testing, any pigs that died were promptly recorded and necropsies were performed. Seven days later, all surviving pigs were necropsies to observe pathological changes. Results are shown in Tables 1 and 2. Figure 1-6 As shown, the APP1 type tracheal injection in pigs has a 1 MLD of 1-2 * 10. 4 CFU, and the main lesions of the dead pigs were adhesions and hemorrhage.

[0022] Table 1. Results of APP1 virus detection in pigs

[0023] Table 2. Results of APP1 type toxicity detection in pigs via autopsy.

[0024] Example 2 Screening alternative animals and methods of challenge The APP1 type lyophilized bacterial strain was reconstituted with BD TSB. 1 mL of the reconstituted bacterial solution was added to a test tube containing 9 mL of TSB liquid medium (containing 4% newborn calf serum and 2‰ NAD). The culture was incubated at 37°C and 120 rpm for 8 hours, then incubated at 4°C overnight. 4 mL of the bacterial solution was added to 200 / 500 mL of TSB liquid medium (containing 4% newborn calf serum and 2‰ NAD), and incubated at 37°C and 120 rpm for 8 hours. The culture was then diluted to the injection gradient shown in Table 3 and administered intraperitoneally and intranasally to mice and guinea pigs (mice weighing 16–20 g, guinea pigs weighing 280–320 g). The injection volume for mice was 0.2 mL, and for guinea pigs, it was 0.5 mL. The time of death was recorded. Dead animals were dissected promptly, and all surviving animals were dissected 7 days later. The toxicity test results are shown in Table 3. The groups with consistent and effective toxicity testing after APP1 bacterial culture challenge were the mouse intraperitoneal group, the guinea pig intraperitoneal group, and the guinea pig nasal drop group. Therefore, these three groups were selected for subsequent experiments. The MLD of APP1 intraperitoneally injected into mice was 5.0-10.0*10. 7 CFU, guinea pig MLD is 5.0-10.0*108 CFU, administered intranasally to guinea pigs, has a MLD of 1.5-2.0*10. 9 CFU. Meanwhile, the autopsy results (Table 4) revealed that mice that died from intraperitoneal toxicity testing showed no obvious lesions, while the lesions in guinea pigs that died from intraperitoneal and nasal toxicity testing were almost identical to those in pigs that died from the same disease. Figure 7-11 As shown.

[0025] Table 3. Results of APP1 Alternative Animal Virus Testing

[0026] Table 4. Dissection results of APP1-type guinea pigs.

[0027] Example 3 Verification of the protective effect of this animal vaccine The purified ApXⅠ, ApXⅡ, ApXⅢ, and OmpA proteins were formulated into a vaccine using aluminum gel at the dosages shown in Table 5. The vaccine formulation is illustrated in Table 5, and the vaccine batch number is JY20250711. Five 8-week-old pigs were immunized via intramuscular injection in the neck at a dose of 2.0 mL / pig. A second immunization was administered 21 days after the first immunization, using the same vaccine at the same dose and method. Fourteen days after the second immunization, the pigs were challenged with a dose of 1 MLD (10 mL / pig) via tracheal injection. Five control pigs were also included. The vaccine efficacy is shown in Table 6. After the second immunization and challenge, no pigs survived in the control group, while five pigs survived in the immunized group, indicating a 100% protective effect. Figure 12 As shown.

[0028] Table 5. Vaccine Preparation Protocol

[0029] Table 6. Results of protection against this animal vaccine

[0030] Example 4 Verification of the protective effect of alternative animal vaccines Twenty guinea pigs were immunized with the same batch of vaccine JY20250711 via intramuscular injection, with a tentative dose of 0.5 mL / pig. Simultaneously, ten mice were immunized subcutaneously, with a tentative dose of 0.2 mL / mouse. Twenty-one days after the first immunization, a second immunization was administered with the same vaccine at the same dose and method. Fourteen days after the second immunization, mice were challenged with 1 MLD (0.2 mL / mouse) and guinea pigs with 1 MLD (0.5 mL / mouse). The challenge methods were intraperitoneal injection for mice, intranasal injection for 10 mice, and intraperitoneal injection for 10 guinea pigs. Ten mice and twenty guinea pigs served as control groups. The vaccine efficacy is shown in Table 7. After the second immunization, none of the control group mice survived. In the immunized group, 7 mice survived in the intraperitoneal challenge group (immunization efficacy 70%), 10 mice survived in the guinea pig intranasal challenge group (immunization efficacy 100%), and 5 mice survived in the guinea pig intraperitoneal challenge group (immunization efficacy 50%). Figure 13 As shown.

[0031] Table 7. Protective Results of Alternative Animal Vaccines

[0032] Example 5 Alternative animal immunization dose screening According to Example 4, the guinea pig nasal drop group was the optimal choice for alternative animals and challenge methods in establishing a model for testing the efficacy of porcine infectious pleuropneumonia vaccine. Thirty guinea pigs were immunized with the same batch of vaccine JY20250711 and divided into three groups of 10 each. The immunization method was intramuscular injection, with doses of 0.2 mL / guinea, 0.5 mL / guinea, and 1.0 mL / guinea, respectively. Twenty-one days after the first immunization, a second immunization was administered using the same vaccine at the same dose and method. Fourteen days after the second immunization, challenge was performed with a challenge dose of 1 MLD (0.5 mL) of guinea pig nasal drop injection. Ten guinea pigs were also used as a control group. The vaccine efficacy is shown in Table 8. After the second immunization, none of the control group survived. In the 0.2 mL group, 6 guinea pigs survived (60% immunization efficacy); in the 0.5 mL group, 10 guinea pigs survived (100% immunization efficacy); and in the 1.0 mL group, 10 guinea pigs survived (100% immunization efficacy). Figure 14 As shown.

[0033] Table 8. Guinea pig vaccine protection results

[0034] Example 6 Repeated verification of substitution effect Five pigs and ten guinea pigs were simultaneously immunized using another batch of vaccine JY20240927 prepared by our company (vaccine preparation protocol shown in Table 9). Pigs were immunized via intramuscular injection in the neck, with a dose of 2.0 mL / pig. Twenty-one days after the first immunization, a second immunization was administered using the same vaccine at the same dose and method. Fourteen days after the second immunization, pigs were challenged via tracheal injection at a dose of 1 MLD (10 mL / pig). Five control pigs were also included. Guinea pigs were immunized via intramuscular injection, with a dose of 0.5 mL / guinea pig. Twenty-one days after the first immunization, pigs were immunized via intramuscular injection, with a dose of 0.5 mL / guinea pig. Fourteen days after the second immunization, guinea pigs were challenged via nasal drops at a dose of 1 MLD (0.5 mL / guinea pig). Ten guinea pigs served as a control group. The vaccine efficacy is shown in Table 10. After the second immunization challenge, none of the control group survived, while 4 pigs survived in the immunized group, with an immunization protection rate of 80%, and 7 guinea pigs survived, with an immunization protection rate of 70%. The results show that the protection results of pigs and guinea pigs after immunization challenge were basically consistent, the replacement effect was well replicated, and the protection effect of guinea pigs was slightly lower than that of pigs, which is helpful for judging the vaccine protection effect.

[0035] Table 9. Vaccine Formulation Protocol

[0036] Table 10. Results of repeated validation of substitution effects

[0037] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. Establishment of a guinea pig substitution efficacy testing model for porcine contagious pleuropneumonia vaccine, characterized in that: The specific steps for establishing this are as follows: S1: Determine the virulence of porcine infectious pleuropneumonia bacteria to host animals, pigs: The porcine infectious pleuropneumonia bacteria strain was propagated, cultured, diluted, and injected intratracheally into pigs. The time of death was recorded and the pigs were dissected. All pigs that did not die were dissected 7 days later. S2: Screening alternative animals and challenge methods: Porcine infectious pleuropneumonia bacteria were propagated, cultured, diluted, and injected intraperitoneally and intranasally into mice and guinea pigs, respectively. The time of death was recorded and the animals were dissected. All surviving animals were dissected 7 days later. S3: Prepare vaccine I, administer 2.0 mL / animal via intramuscular injection in the neck of the host animal (pig). Administer a second immunization 21 days after the first immunization, and challenge the virus 14 days after the second immunization. Observe the immune protection effect and pathological changes. S4: Immunize alternative animals with vaccine I, using the same immunization and challenge times as the host animals (pigs), and observe the immune protection effect and pathological changes; S5: Immunize selected alternative animals with different doses of vaccine I and observe the protective effect of the immunization. S6: Simultaneously immunize the host animal (pig) and selected alternative animals with vaccine II, and observe the immunoprotective effect and pathological changes.

2. The establishment of a guinea pig substitution efficacy testing model for porcine infectious pleuropneumonia vaccine according to claim 1, characterized in that: The bacteria causing the porcine infectious pleuropneumonia were APP1 type.

3. The establishment of a guinea pig substitution efficacy testing model for porcine infectious pleuropneumonia vaccine according to claim 2, characterized in that: The APP1 type was isolated, purified, and identified from the lung tissue of diseased pigs in a certain pig farm, and was freeze-dried and preserved by our company, with the accession number CGMCC No.4717.

4. The establishment of a guinea pig substitution efficacy testing model for porcine infectious pleuropneumonia vaccine according to claim 1, characterized in that: The alternative animals selected were mice and guinea pigs.

5. The establishment of a guinea pig substitution efficacy testing model for porcine infectious pleuropneumonia vaccine according to claim 1, characterized in that: The vaccine I mentioned is an APP subunit vaccine.

6. The establishment of a guinea pig substitution efficacy testing model for porcine infectious pleuropneumonia vaccine according to claim 5, characterized in that: The APP subunit vaccine is JY20250711, which is formulated from ApXⅠ, ApXⅡ, ApXⅢ and OmpA proteins with aluminum gel.

7. The establishment of a guinea pig substitution efficacy testing model for porcine infectious pleuropneumonia vaccine according to claim 6, characterized in that: The ratio of ApXⅠ, ApXⅡ, ApXⅢ, and OmpA proteins is 1:1:1:1; the ratio of antigen to aluminum gel is 4:1; the concentration of ApXⅠ antigen is 500 μg / mL, the concentration of ApXⅡ antigen is 1288 μg / mL, the concentration of ApXⅢ antigen is 2925 μg / mL, and the concentration of OmpA antigen is 860 μg / mL.

8. The establishment of a guinea pig substitution efficacy testing model for porcine infectious pleuropneumonia vaccine according to claim 1, characterized in that: The vaccine II is JY20240927. Vaccine II is formulated with ApXⅠ, ApXⅡ, OmpA and OmpD proteins and aluminum glue. The ratio of ApXⅠ, ApXⅡ, OmpA and OmpD proteins is 1:1:1:1; the ratio of antigen to aluminum glue is 4:1; the concentration of ApXⅠ antigen is 1055 μg / mL, the concentration of ApXⅡ antigen is 1860 μg / mL, the concentration of OmpA antigen is 1250 μg / mL, and the concentration of OmpD antigen is 680 μg / mL.

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