Immune effect evaluation method of cysticercus cellulosae recombinant TPx protein vaccine

Abstract

The invention discloses an immune effect evaluation method of a cysticercus cellulosae recombinant TPx protein vaccine, and belongs to the technical field of biological medicines. The vaccine takes recombinant thioredoxin peroxidase (TPx) from cysticercus cellulosae as an immunogen, and an adjuvant (such as a Freund's adjuvant) can be optionally supplemented to enhance the immune effect. A mouse is immunized through subcutaneous injection of the vaccine, it is proved that the vaccine can effectively induce an organism to generate humoral immune response and cellular immune response, specifically, the levels of serum IgG, IgG2a and IgG1 antibodies are remarkably increased, the proliferation capacity of splenic lymphocytes is enhanced, and the secretion levels of Th1 type cell factors (TNF-alpha and IL-12) and Th2 type cell factors (IL-13 and IL-10) are remarkably increased. The invention provides a new candidate vaccine for prevention of cysticercosis, and has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a porcine cysticercus recombinant TPx protein vaccine-induced immune response and potential for preventing cysticercosis. BACKGROUND

[0002] Cysticercosis is a serious parasitic disease that harms human health and livestock production caused by the larva of Taenia solium, i.e., porcine cysticercus. The cysticercus can parasitize in the brain, eye, subcutaneous tissue and muscle of the human body, etc. Among them, brain cysticercosis can cause epilepsy, intracranial hypertension and even death, etc. The treatment is difficult and the prognosis is poor.

[0003] At present, the prevention and treatment of cysticercosis mainly depends on chemical drugs such as praziquantel, albendazole and surgical treatment. However, long-term use of anthelmintics has the risk of toxic side effects and drug residues, and surgical treatment has high risk and trauma. Therefore, developing a safe and effective preventive vaccine is an ideal strategy to control the disease from the source. Although the development of cysticercosis vaccine has become a research hotspot at home and abroad, there is still a lack of candidate vaccine molecules that can efficiently induce comprehensive immune protection in the prior art, especially vaccine schemes based on porcine cysticercus-specific antigens and evaluated by systematic immunology still need to be developed.

[0004] Thioredoxin peroxidase (TPx) is a member of the superfamily of peroxiredoxin, which is widely expressed in different developmental stages of various parasites and their excreted-secreted antigens. Existing studies have shown that TPx protein in other parasites such as Toxoplasma gondii and Schistosoma japonicum can be used as a potential diagnostic antigen and vaccine candidate molecule, and can induce certain protective immune responses in the host. However, whether the recombinant TPx protein derived from porcine cysticercus has the potential to be used as a cysticercosis vaccine candidate molecule, and whether it can effectively induce humoral and cellular immune responses in the host, have not been reported and studied before the present application. SUMMARY

[0005] The main purpose of the present application is to provide a porcine cysticercus recombinant TPx protein vaccine to solve the technical problem in the prior art that there is a lack of cysticercosis candidate vaccine that can effectively induce humoral and cellular immunity and achieve complete protection. In addition, the present application also provides the immune response induced by the above vaccine in mice and the potential for preventing cysticercosis, and provides a system and method for evaluating the immune effect of porcine cysticercus vaccine, which provides an effective tool for screening and verifying cysticercosis vaccine candidate molecules.

[0006] To achieve the above purpose, the technical scheme adopted is: A porcine cysticercus recombinant TPx protein vaccine, comprising a recombinant thioredoxin peroxidase TPx protein derived from porcine cysticercus as an immunogen.

[0007] Furthermore, the recombinant TPx protein is derived from the TPx protein expressed in the excretory secretion antigen ESA of porcine cysticercosis.

[0008] Furthermore, the vaccine also includes a pharmaceutically acceptable adjuvant, which is Freund's complete adjuvant (CFA) and / or Freund's incomplete adjuvant (IFA).

[0009] And a method for inducing an immune response in mice, comprising the following steps: 1) Immunize mice with the above-mentioned vaccine; 2) The immunization method is multiple subcutaneous injections; 3) The immunization program includes the initial immunization and at least one booster immunization.

[0010] Furthermore, in the method for inducing an immune response in mice, the immunization procedure involves first performing an initial immunization, followed by booster immunizations in the second and fourth weeks, for a total of three immunizations.

[0011] And a system for evaluating the immunization efficacy of a porcine cysticercosis vaccine, comprising: 1) A module for detecting serum levels of IgG, IgG2a, IgG1, and IgE; 2) Module for detecting the proliferation level of splenic lymphocytes; 3) A module for detecting the levels of TNF-α, IL-12, IL-13, and IL-10 in the supernatant of spleen lymphocyte culture.

[0012] Furthermore, in the system, the detection methods include indirect ELISA, CCK-8 assay, and double-antibody sandwich ELISA.

[0013] And the application of the system in screening or evaluating candidate molecules for anti-porcine cysticercosis vaccines.

[0014] And a method for evaluating the immunization efficacy of a porcine cysticercosis vaccine, including: 1) Immunize mice with the vaccine described above; 2) Serum and spleen lymphocytes were collected at different time points after immunization; 3) Detect immune indicators through the above-mentioned system; 4) The efficacy of vaccine-induced humoral and cellular immune responses was comprehensively evaluated based on serum IgG, IgG2a, IgG1, IgE levels, spleen lymphocyte proliferation levels, and TNF-α, IL-12, IL-13, and IL-10 levels in spleen lymphocyte culture supernatant.

[0015] Compared with the prior art, the porcine cysticercosis recombinant TPx protein vaccine provided by the present invention has the following significant beneficial effects: 1. Strong immunogenicity, capable of inducing a highly efficient humoral immune response: The vaccine of this invention can significantly increase the levels of specific IgG and its subclasses (IgG2a, IgG1) in the serum of immunized mice, indicating that it can effectively activate B cells and plasma cells and produce high levels of protective antibodies.

[0016] 2. Stimulates a significant cellular immune response: The vaccine of this invention can effectively promote the proliferation of splenic lymphocytes and significantly increase the secretion levels of Th1 cytokines (such as TNF-α and IL-12) and Th2 cytokines (such as IL-13 and IL-10), indicating that it can induce the body to produce a balanced Th1 / Th2 mixed immune response, which is crucial for effectively combating intracellular parasitic infections.

[0017] 3. Significant synergistic effect of adjuvant: When the vaccine of this invention is used in combination with Freund's adjuvant, all detected immune indicators (antibody level, lymphocyte proliferation, cytokine secretion) are significantly higher than those of the group using vaccine protein alone, proving that the adjuvant can greatly enhance the immune effect of the vaccine.

[0018] 4. Provides new candidate vaccine molecules for the prevention and control of cysticercosis: This invention is the first to systematically verify the effectiveness and feasibility of recombinant TPx protein from porcine cysticercosis as a vaccine candidate molecule, laying a solid experimental foundation for the development of safe and effective cysticercosis prevention vaccines, and has important theoretical value and clinical application prospects. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0020] Cysticercosis is a serious zoonotic parasitic disease caused by the larvae of the porcine tapeworm—the cysticercus of the pig—parasitizing humans or pigs. It poses a significant threat to human health and livestock production. [1] Cysticerci can parasitize the human brain, eyes, subcutaneous tissue, and muscles. Neurocysticercosis is particularly serious, causing central nervous system symptoms such as seizures, increased intracranial pressure, and neuropsychiatric disorders, and in severe cases, death. [4] Currently, treatment for this disease mainly relies on chemotherapy and surgery. However, long-term use of anthelmintics (such as praziquantel and albendazole) carries serious toxic side effects, and drug residues in meat products may harm the human body. Surgical treatment, on the other hand, is quite invasive. The development of a cysticercosis vaccine has become a hot research topic both domestically and internationally.

[0021] Thioredoxin peroxidase (TPx) is a member of the peroxiredoxin superfamily. This protein is widely expressed in various growth and development stages of parasites and their excretory-secretory antigens (ESA). It can not only participate in host immune regulation but also be used as a diagnostic antigen for the immunodiagnosis of parasitic diseases and a candidate vaccine molecule for the immunoprevention of parasitic diseases. [5] However, there have been no reports on the recombinant TPx protein of Taenia solium cysticerci as a candidate vaccine molecule for cysticercosis. Therefore, based on the previous preparation of the recombinant TPx protein of Taenia solium cysticerci in our research group [6] , we immunized mice with it to observe the immune response effect induced in mice, providing a new candidate vaccine molecule for the prevention and treatment of cysticercosis.

[0022] Example 1 1 Materials and Methods 1.1 Materials 1.1.1 Recombinant TPx protein vaccine of Taenia solium cysticerci: Derived from the previous preparation in our research group [6] .

[0023] Experimental animals: 60 SPF-grade Kunming mice, 8 weeks old, half male and half female, purchased from the Experimental Animal Center of Zunyi Medical University [Animal License Number: SCXK (Qian) 2021-0002].

[0024] Main instruments and reagents: Mouse serum immunoglobulin (Ig) G, IgG2a, IgG, IgE detection kits, as well as tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-13, IL-10 detection kits were purchased from Shanghai Youningwei Biotechnology Co., Ltd. (Brand name: elabscience); RPMI 1640 medium; Complete Freund's adjuvant (CFA) and Incomplete Freund's adjuvant (IFA) were purchased from Shanghai Yuchun Biotechnology Co., Ltd. (Brand name: Ron); CCK-8 cell proliferation detection kit was purchased from Biosharp Biotechnology Co., Ltd., China. Mouse spleen lymphocyte separation kit was purchased from Shanghai Yuchun Biotechnology Co., Ltd. (Brand name: Solarbio). The microplate reader was purchased from Thermo Company, USA.

[0025] Methods 1.2.1 Grouping of experimental animals: 60 8-week-old SPF-grade Kunming mice were randomly divided into 3 groups, namely normal saline control group (control group), recombinant TPx protein vaccine group (vaccine group), and recombinant TPx protein vaccine + adjuvant group (vaccine + adjuvant group), with 20 mice in each group.

[0026] 1.2.2 Animal Immunization: A multi-point subcutaneous injection method was used. Two booster immunizations were administered after the initial immunization at week 0, for a total of three immunizations, with each injection spaced two weeks apart. Control group: injected with 100 μl of physiological saline; Vaccine group: injected with 50 μl of recombinant TPx protein vaccine (1 μg / μl) + 50 μl of physiological saline; Vaccine + Adjuvant group: injected with 50 μl of recombinant TPx protein vaccine (1 μg / μl) + 50 μl of adjuvant (CFA for the first dose, IFA for the second and third doses). Mice were allowed free access to food during the experiment.

[0027] 1.2.3 Detection of mouse serum antibody levels: Four mice were selected from each of the three groups at 0, 2, 4, 6, and 8 weeks after the initial immunization. Blood was collected by enucleation, incubated at 4 ℃ for 12 h, centrifuged at 3000 r / min for 10 min, and the serum was separated and stored at -20 ℃ for later use. The levels of mouse serum IgG, IgG2a, IgG1, and IgE were detected using an indirect ELISA method. The specific procedures were performed according to the instructions of the mouse IgG, IgG2a, IgG1, and IgE detection kits.

[0028] 1.2.4 Detection of Splenic Lymphocyte Proliferation Level in Mice: At 0, 2, 4, 6, and 8 weeks after the initial immunization, spleens were aseptically harvested from four mice that had undergone blood collection. Splenic lymphocyte suspensions were prepared according to the instructions of the mouse spleen lymphocyte isolation kit, and the cell concentration was adjusted to 5 × 10⁶ cells / mL using RPMI 1640 medium. 6 / ml (stock solution). In a 96-well plate, add 200 μl of stock solution, or 200 μl of stock solution + 2 μl of recombinant TPx protein vaccine (1 μg / μl), with 3 wells per sample. Incubate at 37℃ and 5% CO2 for 48 h. Two h before the end of incubation, add 20 μl of CCK-8 solution to each well and measure the absorbance (A) using a microplate reader. 450nm value. 1.2.5 Detection of TNF-α, IL-12, IL-13, and IL-10 levels in mouse spleen lymphocyte culture supernatant: In 24-well culture plates, 500 μl of the stock solution and 500 μl of the stock solution + 5 μl of recombinant TPx protein vaccine (1 μg / μl) were added, with three wells for each sample. The plates were incubated at 37℃ and 5% CO2 for 48 h, then centrifuged at 4000 r / min for 5 min. The supernatant was collected and stored at -20℃ for later use. The levels of TNF-α, IL-12, IL-13, and IL-10 in the spleen lymphocyte culture supernatant were detected using a double-antibody sandwich ELISA method. The specific procedures were performed according to the instructions of the TNF-α, IL-12, IL-13, and IL-10 detection kits. 1.3 Statistical Analysis: Statistical analysis was performed using IBM SPSS Statistics 27 software. All measurement data were expressed as mean ± standard deviation. (S) indicates the group. One-way ANOVA was used for comparisons among multiple groups, and LSD test was used for further pairwise comparisons between groups; independent samples t-test was used for comparisons between two groups, and P < 0.05 was considered statistically significant.

[0029] result 2.1 Results of serum antibody level detection in mice: 2–8 weeks post-immunization, the serum levels of IgG, IgG2a, and IgG1 in the vaccine group and the vaccine + adjuvant group were higher than those in the control group, and the levels in the vaccine + adjuvant group were also higher than those in the vaccine group. P All values ​​were <0.05; there were no statistically significant differences in serum IgE levels among the three groups of mice. P (All > 0.05). See Tables 1-4.

[0030] Table 1. Comparison of serum IgG levels in the three groups of mice at different immunization times ( )

[0031] Note: The control group was the saline control group; the vaccine group was the recombinant TPx protein vaccine group; the vaccine + adjuvant group was the recombinant TPx protein vaccine + adjuvant group; compared with the control group... Compared with the vaccine group, .

[0032] Table 2 Comparison of serum IgG2a levels in the three groups of mice at different immunization times ( )

[0033] Note: The control group was the saline control group; the vaccine group was the recombinant TPx protein vaccine group; the vaccine + adjuvant group was the recombinant TPx protein vaccine + adjuvant group; compared with the control group... Compared with the vaccine group, .

[0034] Table 3. Comparison of serum IgG1 levels in the three groups of mice at different immunization times ( )

[0035] Note: The control group was the saline control group; the vaccine group was the recombinant TPx protein vaccine group; the vaccine + adjuvant group was the recombinant TPx protein vaccine + adjuvant group; compared with the control group... Compared with the vaccine group, .

[0036] Table 4. Comparison of serum IgE levels in the three groups of mice at different immunization times ( )

[0037] Note: The control group was the saline control group; the vaccine group was the recombinant TPx protein vaccine group; the vaccine + adjuvant group was the recombinant TPx protein vaccine + adjuvant group.

[0038] 2.2 Results of mouse spleen lymphocyte proliferation level detection: 2–8 weeks post-immunization, the spleen lymphocyte proliferation levels in the vaccine group and the vaccine + adjuvant group were higher than those in the control group, and the vaccine + adjuvant group was higher than that in the vaccine group. P All <0.05); Under different treatment factors within the group, the proliferation level of spleen lymphocytes during antigen stimulation at 0–8 weeks of immunization was higher than that of the original solution ( P (All values ​​< 0.05). See Table 5.

[0039] Table 5. Comparison of splenic lymphocyte proliferation levels at different immunization times in the three groups of mice.

[0040]

[0041] Note: The control group was the saline control group; the vaccine group was the recombinant TPx protein vaccine group; the vaccine + adjuvant group was the recombinant TPx protein vaccine + adjuvant group.

[0042] 2.3 Results of TNF-α, IL-12, IL-13, and IL-10 levels in mouse spleen lymphocyte culture supernatant: From 2 to 8 weeks post-immunization, the levels of TNF-α, IL-12, IL-13, and IL-10 in the spleen lymphocyte culture supernatant of the vaccine group and the vaccine + adjuvant group were all higher than those in the control group, and the levels in the vaccine + adjuvant group were all higher than those in the vaccine group. P All <0.05); Under different treatment factors within the group, the levels of TNF-α, IL-12, IL-13, and IL-10 in the supernatant of spleen lymphocyte culture 0–8 weeks after antigen stimulation were higher than those in the original solution ( P All values ​​are <0.05. See Tables 6-9.

[0043] Table 6. TNF-α levels in the supernatant of spleen lymphocyte culture from three groups of mice at different immunization times.

[0044]

[0045] Note: The control group was the saline control group; the vaccine group was the recombinant TPx protein vaccine group; the vaccine + adjuvant group was the recombinant TPx protein vaccine + adjuvant group.

[0046] Table 7. Comparison of IL-12 levels in spleen lymphocyte culture supernatant from three groups of mice at different immunization times.

[0047]

[0048] Note: The control group was the saline control group; the vaccine group was the recombinant TPx protein vaccine group; the vaccine + adjuvant group was the recombinant TPx protein vaccine + adjuvant group.

[0049] Table 8. Comparison of IL-13 levels in spleen lymphocyte culture supernatant from three groups of mice at different immunization times.

[0050]

[0051] Note: The control group was the saline control group; the vaccine group was the recombinant TPx protein vaccine group; the vaccine + adjuvant group was the recombinant TPx protein vaccine + adjuvant group.

[0052] Table 9. Comparison of IL-10 levels in spleen lymphocyte culture supernatant from three groups of mice at different immunization times.

[0053]

[0054] Note: The control group was the saline control group; the vaccine group was the recombinant TPx protein vaccine group; the vaccine + adjuvant group was the recombinant TPx protein vaccine + adjuvant group.

[0055] discuss Vaccination is a safe and effective tool for preventing parasitic infections. Research has found that TPx protein is an important candidate vaccine molecule for parasitic diseases. Currently, recombinant TPx protein is being studied extensively in the fields of Toxoplasma gondii, Schistosoma japonicum, and Fasciola hepatica. Research results suggest that TPx protein can induce an effective protective immune response in the host in corresponding disease models, thereby resisting parasitic infections. [6-8] This study aims to observe the induced immune response in mice by immunizing them with a previously prepared recombinant TPx protein vaccine from porcine cysticercosis, providing an important candidate vaccine for the immunoprophylaxis of cysticercosis.

[0056] In the development of cysticercosis vaccines, humoral immunity, cellular immunity, and CD4+ in T lymphocytes are all important factors. + CD8 +Content is an important indicator for judging vaccine efficacy. Antigens stimulate B lymphocytes to differentiate into plasma cells, which produce protective proteins (antibodies). These antibodies specifically bind to the corresponding antigens and are the main immune molecules in the body's humoral immune response. The main antibody molecules in the body can be divided into five classes: IgG, IgM, IgA, IgD, and IgE. IgG is the most widely distributed antibody component in serum and is also the main antibody molecule in the body's immune response. Human IgG is divided into four subclasses: IgG1, IgG2, IgG3, and IgG4, while mouse IgG is divided into four subclasses: IgG1, IgG2a, IgG2b, and IgG3. [9] In this study, serum IgG, IgG2a, and IgG1 levels in mice in the vaccine group and the vaccine + adjuvant group all increased to varying degrees after the first immunization, reaching higher levels at week 6 post-immunization, while serum IgE levels showed no statistically significant difference. This indicates that the recombinant TPx protein vaccine can induce a humoral immune response in mice.

[0057] Lymphocytes are the body's main immune cells. Lymphocyte proliferation is a crucial step in the immune response, and their proliferation and differentiation levels are important indicators of cellular immune response capacity, often used to evaluate lymphocyte activity. When cultured T lymphocytes are stimulated by specific antigens in vitro, they undergo mitosis, resulting in increased protein and nucleic acid synthesis, a phenomenon known as lymphocyte transformation.

[10] This study used the simple and highly sensitive CCK-8 assay to detect the proliferation level of splenic lymphocytes in mice. The results showed that the proliferation level of splenic lymphocytes in both the vaccine and vaccine + adjuvant groups increased after the first immunization, reaching a relatively high level at week 6 post-immunization. These results preliminarily demonstrate that immunization with recombinant TPx protein can induce a cellular immune response in mice.

[0058] T lymphocytes can be classified into helper T cells (Th cells), cytotoxic T cells, and regulatory T cells based on their functions. Among them, Th cells play an important role in cell-mediated immunity. Based on the different cytokines they secrete, they can be divided into Th1 and Th2 helper cells. Cytokines participate in antibody and cell-mediated immunity, playing a crucial role in the immune response. CD4 + T cells and CD8 + T cells are the two major subsets of T lymphocytes, among which CD4 + T cells can differentiate into Th1 cells and Th2 cells. The expression levels of IgG and its subsets are related to the regulation of cytokines produced by Th1 and Th2 cells. Th1 cells mediate cellular immune responses by secreting TNF-α and IL-12. TNF-α is a pro-inflammatory cytokine produced by lymphocytes and monocytes / macrophages, which can promote macrophage activation, enhance its phagocytic effect, and activate other cytokines.[11,12] TNF-α also has immunoprotective effects, activating macrophages to produce large amounts of reactive oxygen species, H2O2, and NO, thereby combating parasitic infections. IL-12, produced by dendritic cells, macrophages, B lymphocytes, and other antigen-presenting cells, promotes Th1 cell proliferation and is a multifunctional cytokine.

[13] In parasitic immunity, IL-12 can directly induce the proliferation of T lymphocytes and natural killer (NK) cells, and the differentiation of Th0 cells into Th1 cells, promoting the production of IgG and IgG2a, thereby exerting an anti-parasitic effect. Furthermore, Th2 cells mediate humoral immune responses by secreting IL-13 and IL-10. IL-13, mainly produced by activated T lymphocytes and mast cells, can induce the activation, proliferation, and differentiation of B lymphocytes, promoting the production of IgG1 and IgE.

[14] Furthermore, IL-10 has an inhibitory effect on Th1 cells and can promote B lymphocyte proliferation, differentiation, and IgG production. Extensive data indicate that both Th1 and Th2 immune responses play crucial roles in the occurrence and development of cysticercosis.

[15] This study used a recombinant TPx protein vaccine to immunize mice. The results showed that after immunization with the recombinant TPx protein, the levels of TNF-α, IL-12, IL-13, and IL-10 in the supernatant of mouse spleen lymphocyte culture increased from 2 to 8 weeks after immunization, and reached a relatively high level in the 4th week, indicating that the vaccine can induce a mixed Th1 and Th2 immune response in the body.

[0059] In summary, this study used a recombinant TPx protein vaccine from porcine cysticercosis to immunize mice, and dynamically observed and analyzed the serum levels of IgG, IgG2a, IgG1, and IgE, the proliferation level of spleen lymphocytes, and the levels of TNF-α, IL-12, IL-13, and IL-10 in the spleen lymphocyte culture supernatant to explore the potential of the recombinant TPx protein from porcine cysticercosis as a candidate molecule for a vaccine. The results showed that the serum levels of IgG, IgG2a, and IgG1, the proliferation level of spleen lymphocytes, and the levels of TNF-α, IL-12, IL-13, and IL-10 in the spleen lymphocyte culture supernatant all showed varying degrees of increase. These indicators preliminarily demonstrate that the vaccine can induce both cellular and humoral immune responses in mice. The use of adjuvants can further promote the induction of immune responses by the vaccine, laying an important foundation for subsequent research on the immunoprotective efficacy of the recombinant TPx protein vaccine from porcine cysticercosis.

[0060] Example 2 This invention provides a recombinant TPx protein vaccine against cysticercosis (Taenia solium), comprising recombinant thioredoxin peroxidase (TPx) protein derived from cysticercosis as an immunogen. The recombinant TPx protein is derived from the TPx protein expressed in the excretory secretory antigen (ESA) of cysticercosis. The vaccine also includes a pharmaceutically acceptable adjuvant, namely Freund's complete adjuvant (CFA) and / or Freund's incomplete adjuvant (IFA). This recombinant TPx protein vaccine against cysticercosis solves the technical problem of the prior art lacking candidate vaccines for cysticercosis that can effectively induce humoral and cellular immunity to achieve complete protection.

[0061] This invention provides a method for inducing an immune response in mice, comprising the following steps: 1) Immunize mice with the vaccine described above; 2) The immunization method is multiple subcutaneous injections; 3) The immunization program includes an initial immunization and at least one booster immunization. In the method for inducing an immune response in mice, the immunization program involves an initial immunization, followed by booster immunizations in weeks 2 and 4, for a total of three immunizations.

[0062] This invention provides a system for evaluating the immunization efficacy of a porcine cysticercosis vaccine, comprising: 1) a module for detecting serum levels of IgG, IgG2a, IgG1, and IgE; 2) a module for detecting splenic lymphocyte proliferation levels; and 3) a module for detecting TNF-α, IL-12, IL-13, and IL-10 levels in splenic lymphocyte culture supernatant. The detection methods in this system include indirect ELISA, CCK-8 assay, and double-antibody sandwich ELISA. The invention also describes the application of this system in screening or evaluating candidate molecules for anti-porcine cysticercosis vaccines.

[0063] This invention provides a method for evaluating the immunization efficacy of a porcine cysticercosis vaccine, comprising: 1) Immunize mice with the vaccine described above; 2) Collect serum and spleen lymphocytes at different time points after immunization; 3) Detect immune indicators using the above-described system; 4) Evaluate the vaccine-induced humoral and cellular immune responses based on serum IgG, IgG2a, IgG1, and IgE levels, spleen lymphocyte proliferation levels, and TNF-α, IL-12, IL-13, and IL-10 levels in spleen lymphocyte culture supernatant. This provides an effective tool for screening and validating candidate molecules for cysticercosis vaccines.

[0064] References [1]Hossain MS, Shabir S, Toye P, et al. Insights into the diagnosis, vaccines, and control of Taenia solium, a zoonotic, neglected parasite. Parasit Vectors. 2023 , 16(1):380. [2]Gulelat Y, Eguale T, Kebede N, et al. Epidemiology of porcinecysticercosis in eastern and southern Africa: Systematic review and meta-analysis[J]. Front Public Health, 2022, 10: 836177. [3]Rahantamalala A, Rakotoarison RL, Rakotomalala E, et al. Prevalence and factors associated with human Taenia solium taeniosis and cysticercosis in twelve remote villages of Ranomafana rainforest, Madagascar[J]. PLoS Negl Trop Dis, 2022, 16(4): e0010265. [4] He Wei, Luo Bo, Zhou Biying. Research progress on the role of recombinant TPx, an important human parasite, in immune regulation, immune diagnosis and immune prevention [J]. Chinese Journal of Endemic Diseases, 2022, 41(10): 856-860. [5] Wang Qianfei, Yuan Fengling, Wang Shimin, et al. Expression, purification and polyclonal antibody preparation of TPx protein excreted and secreted by cysticercosis of pig[J]. Chinese Journal of Pathogenic Biology, 2023, 18(3): 310-314+318. [6] Madhumathi J, Anugraha G, Prince PR, et al. Proliferative responses of Brugia malayi TPX-1 and its epitopic peptide(29-43) in an endemicpopulation of human lymphatic filariasis[J]. Micro-bes and infection vol,2011, 13(6): 602-606. [7]Dalton JP, Robinson MW, Mulcahy G, et al. Immunomodulatorymolecules of Fasciola hepatica : candidates for both vaccine and immunotherapeutic development[J]. Veterinary parasitology, 2013, 195(3-4):272-285. [8]Robinson MW, Hutchinson AT, Dalton JP, et al. Peroxiredoxin: acentral player in immune modulation[J]. Parasite Immunol, 2010, 32(5): 305-313. [9] Wang Liangliang, Chen Minyan, Zheng Weiqiong, et al. Immune response induced in mice by recombinant Mycobacterium smegmatis vaccine containing Taenia solium TSOL18[J]. Chinese Journal of Endemic Diseases, 2019, 38 (10): 793-799.

[10] Shao Anliang, Mu Yufeng, Qu Shuxin, et al. Optimization and application of human peripheral blood lymphocyte proliferation assay [J]. Journal of Pharmaceutical Analysis, 2019, 39(8): 1354-1361.

[11] Liu Y, Zhou LJ, Wang J, et al. TNF-α differentially regulates synaptic plasticity in the hippocampus and spinal cord by microglia-dependent mechanisms after peripheral nerve injury[J]. J Neurosci, 2017, 37(4): 871-881.

[12] Dalvi PS, Chalmers JA, Luo V, et al. High fat induces acuteandchronic inflammation in the hypothalamus: effect of high-fat diet, palmitate and TNF-α on appetite-regulating NPY neurons[J]. Int J Obes (Lond), 2017, 41(1): 149-158.

[13] Nikitina EY, Desai SA, Zhao X, et al. Versatile prostatecancertreatment with inducible caspase and interleukin-12[J]. Cancer Res, 2005, 65(10): 4309-4319.

[14] Liu Xiuxing, Huang Ping, Xiong Junping, et al. The role of IL-13 in allergic diseases [J]. Journal of Cellular and Molecular Immunology, 2017, 33(7): 996-1000.

[15] Zhang Yue, Liu Meichen, Zhou Biying. Research progress on the immunopathogenesis of cysticercosis [J]. Chinese Journal of Endemic Diseases, 2019, 38(3): 253-258.

Claims

1. A recombinant TPx protein vaccine of Cysticercus cellulosae of swine characterized in that, The vaccine comprises a recombinant thioredoxin peroxidase (TPx) protein derived from a pork cysticercus as an immunogen.

2. The vaccine of claim 1, wherein The recombinant TPx protein is derived from a TPx protein expressed in an excretory-secretory antigen (ESA) of a pork cysticercus.

3. The vaccine as described in claim 1, characterized in that, The vaccine further comprises a pharmaceutically acceptable adjuvant, which is a Freund's complete adjuvant (CFA) and / or a Freund's incomplete adjuvant (IFA).

4. A method for inducing an immune response in mice, characterized in that, The method comprises the following steps: 1) immunizing a mouse with the vaccine according to any one of claims 1-3; 2) the immunization mode is multi-point subcutaneous injection; 3) the immunization procedure comprises a primary immunization and at least one booster immunization.

5. The method of claim 4, wherein, The immunization procedure comprises a primary immunization, and then a booster immunization at the 2nd week and the 4th week, for a total of 3 immunizations.

6. A system for evaluating the immunization effect of a porcine cysticercus vaccine, characterized by, The method comprises: 1) a module for detecting IgG, IgG2a, IgG1 and IgE levels in serum; 2) a module for detecting spleen lymphocyte proliferation level; 3) a module for detecting TNF-α, IL-12, IL-13 and IL-10 levels in spleen lymphocyte culture supernatant.

7. The system of claim 6, wherein, The detection method comprises indirect ELISA, CCK-8 method and double-antibody sandwich ELISA method.

8. The system of claim 6 is used for screening or evaluating anti-pork cysticercus vaccine candidate molecules.

9. A method for evaluating the immunization effect of a porcine cysticercus vaccine, characterized by, The method comprises: 1) immunizing a mouse with the vaccine according to any one of claims 1-3; 2) collecting serum and spleen lymphocytes at different time points after immunization; 3) detecting immune indexes by the system according to any one of claims 6-8; 4) comprehensively evaluating the effects of vaccine-induced humoral immune response and cellular immune response according to serum IgG, IgG2a, IgG1 and IgE levels, spleen lymphocyte proliferation level, and TNF-α, IL-12, IL-13 and IL-10 levels in spleen lymphocyte culture supernatant.

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