CTLA-4 / B7-based targeted APC immune-enhanced echinococcosis multi-epitope vaccine

Abstract

In order to seek an echinococcosis antigen which has good immunogenicity and can activate an organism to generate an effective immune response against echinococcosis granulosa infection, the invention provides an echinococcosis multi-epitope antigen and application thereof in preparation of a vaccine for preventing echinococcosis. The multi-epitope antigen is formed by sequentially connecting CTLA-4-lgV, a T cell epitope of EgA31, a T cell epitope of EgG1Y162, a B cell epitope of EgA31 and a B cell epitope of EgG1Y162 in series, the multi-epitope antigen can be specifically combined with serum of a cystic echinococcosis patient, a mouse immunized by a vaccine can generate specific B cell response and generate specific cellular immunity and an antibody, and the multi-epitope antigen has good immunogenicity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a CTLA-4 / B7-targeted APC immune-enhanced echinococcosis multi-epitope vaccine. Background Art

[0002] Hydatid disease (Hydatidosis, Hydatid Disease) is scientifically known as echinococcosis (Echinococcosis). The pathogen is Echinococcus, whose larvae parasitize in humans and livestock. It is a serious zoonosis, mainly including cystic echinococcosis (CE) caused by Echinococcus granulosus (Eg) in dogs and alveolar echinococcosis (AE) caused by Echinococcus multilocularis (Em) in foxes. Other tapeworms that cause echinococcosis include Echinococcus oligarthtus (Eo) and Echinococcus vogeli (Ev).

[0003] The final hosts of Echinococcus granulosus are canine meat animals such as dogs, jackals, and wolves, and the intermediate hosts are various herbivorous animals such as sheep, cattle, camels, and humans. The adult parasitizes in the upper part of the small intestine of the final host, and fixes to the crypt at the base of the intestinal villi with the small hooks and suckers on the apex. The gravid segments or eggs are excreted with the host's feces. The intermediate hosts, including humans, swallow the eggs or gravid segments, and the six hook larvae hatch in the intestine, drill into the intestinal wall, and circulate through the blood to organs such as the liver and lungs. After 3 to 5 months, they develop into echinococcosis with a diameter of 1 to 3 cm. Generally, the diameter of the cyst reaches 0.5 to 1.0 cm six months after infection, and then increases by 1 to 5 cm each year, and can grow to a maximum of 30 to 40 cm. Echinococcus can survive in the human body for 40 years or even longer, but in case of secondary infection or trauma, it may undergo degeneration and die. The cyst fluid is turbid and is eventually absorbed and calcified. After the echinococcosis is ingested by the definitive hosts such as dogs and wolves, each protoscolex it contains can develop into a worm. Since the echinococcosis contains a large number of protoscolex, the number of parasitic worms in the intestines of dogs and wolves can reach thousands to tens of thousands. It takes about 8 weeks from infection to development and maturity, excretion of eggs and gestation, and the lifespan of the adult worm is about 5-6 months.

[0004] The harm of echinococcosis to humans is mainly manifested in the formation of space-occupying cysts in organs such as the liver, lungs, brain or kidneys, which cause serious damage to these important organs in the form of mechanical compression and nutrient consumption, and even lead to individual death. Children and young people are the high-risk population, and those under 40 years old account for about 80%. The severity depends on the number of echinococcosis, parasitic time and location. After the six hook larvae invade the host tissue, inflammatory reactions and cell infiltration occur around it, and a slender outer capsule is gradually formed. Because echinococcosis grows slowly, symptoms often appear 5-20 years after infection. Primary echinococcosis infection is mostly single, and secondary infection is often multiple, accounting for more than 20% of patients, and can affect several organs at the same time. For livestock, echinococcosis will cause major economic problems such as reduced production and quality of livestock, and as an important link in the life history of parasitic infection, it poses a huge hidden danger to people's lives and property safety.

[0005] The disease is distributed worldwide and is a global public health problem. According to recent surveys, cases of the disease have been reported in 25 provinces, municipalities and autonomous regions in my country, among which Xinjiang, Ningxia, Gansu, Qinghai, Tibet, Sichuan and Inner Mongolia are the most serious. The high-incidence areas account for about 44% of the country's area and the threatened population is about 50 million. The disease has been listed as one of the parasitic diseases that my country focuses on preventing and controlling.

[0006] Since the infection is not easy to detect in the early stage, most patients are mainly treated with drugs, and antiparasitic drugs such as albendazole are widely used to treat echinococcosis. Although these drugs can play a certain role, they need to be taken for a long time and cause many side effects. Therefore, exploring new treatment methods is an ideal measure for the prevention and treatment of echinococcosis. The preparation of echinococcosis vaccines can be used as an effective way of immune prevention, but there is currently no commercially available vaccine. Therefore, for those skilled in the art, seeking candidate molecules for prevention is a technical problem that needs to be solved in this field. Summary of the invention

[0007] In order to overcome the above technical problems, a hydatid disease antigen with good immunogenicity that can activate the body to produce an effective immune response against Echinococcus granulosus infection is sought. The present invention provides a hydatid disease multi-epitope antigen and its use in the preparation of a vaccine for the prevention of hydatid disease.

[0008] To achieve the above-mentioned purpose of the present invention, specifically, the echinococcosis multi-epitope antigen CVE31-162 provided by the present invention is composed of CTLA-4-lgV, the T cell epitope of EgA31, the B cell epitope of EgA31, the T cell epitope of EgG1Y162, and the B cell epitope of EgG1Y162 connected in series in sequence.

[0009] Wherein, the CTLA-4-lgV is the immunoglobulin variable region of CTLA-4.

[0010] The amino acid sequence of the immunoglobulin variable region of CTLA-4 is shown in SEQ ID No.1.

[0011] In an optional embodiment, the T cell epitope of EgA31 is composed of the following amino acid sequences connected in series:

[0012] SEQ ID No.2LRRALITQESRNLELQNDLERLQ;

[0013] SEQ ID No.3LERDLNDMKGLVGFYKAFAK;

[0014] SEQ ID No.4IQVQIQLLRGGYLDLFHDKIGHY;

[0015] SEQ ID No.5LQESETKLL.

[0016] In an optional embodiment, the T cell epitope of EgG1Y162 is composed of the following amino acid sequences connected in series:

[0017] SEQ ID No. 6FRWIHVGSRSL;

[0018] SEQ ID No. 7IKLTANLYTTY;

[0019] SEQ ID No. 8EVVVQAF.

[0020] In an optional embodiment, the B cell epitope of EgA31 is composed of the following amino acid sequences connected in series:

[0021] SEQ ID No. 9AQNNIRTQES;

[0022] SEQ ID No. 10AEKQAMDDSTASSD;

[0023] SEQ ID No. 11RETRDELDEQKAE;

[0024] SEQ ID No.12SKLTESGE

[0025] SEQ ID No. 13KKEKDA.

[0026] In an optional embodiment, the B cell epitope of EgG1Y162 is composed of the following amino acid sequences connected in series:

[0027] SEQ ID No. 14 IRV;

[0028] SEQ ID No. 15TT;

[0029] SEQ ID No. 16LKP;

[0030] SEQ ID No. 17KG;

[0031] SEQ ID No. 18APGEDGA.

[0032] Furthermore, the echinococcosis multi-epitope antigen provided by the present invention also comprises a connecting peptide sequence.

[0033] In an optional embodiment, the connecting peptide is used to connect T cell epitopes in series, and optionally, the sequence of the connecting peptide is AAT.

[0034] In an optional embodiment, the connecting peptide is used to connect B cell epitopes in series, and optionally, the sequence of the connecting peptide is KK.

[0035] In an optional embodiment, the connecting peptide is used to connect to CTLA-4-lgV. Optionally, the sequence of the connecting peptide is GGGGSGGGGSGGGG.

[0036] In an optional embodiment, the connecting peptide is used to connect the T cell epitope of EgA31 and the B cell epitope of EgA31, the B cell epitope of EgA31 and the T cell epitope of EgG1Y162, and the T cell epitope of EgG1Y162 and the B cell epitope of EgG1Y162, and the sequence of the connecting peptide is GPSL.

[0037] In an optional embodiment, the amino acid sequence of the echinococcosis multi-epitope antigen CVE31-162 is shown as SEQ ID No.19.

[0038] The present invention further provides the nucleotide sequence of the echinococcosis multi-epitope antigen CVE31-162, as shown in SEQ ID No.20.

[0039] The present invention further provides a method for preparing the echinococcosis multi-epitope antigen CVE31-162, which is characterized by comprising the following steps:

[0040] The present invention also provides the use of the echinococcosis multi-epitope antigen CVE31-162 in the preparation of a drug for preventing or treating echinococcosis.

[0041] Furthermore, the drug for preventing or treating echinococcosis is a vaccine.

[0042] The present invention also provides a vaccine comprising the echinococcosis multi-epitope antigen CVE31-162.

[0043] In an optional embodiment, the vaccine further comprises a pharmaceutically acceptable carrier or agent.

[0044] The EgA31 described in the present invention is hydatid fibrin, one of the main antigens of echinococcosis, and mainly exists in the microhairs of the early adult stage.

[0045] The EgG1Y162 protein described in the present invention is a new antigen gene amplified from Echinococcus granulosus. The antigen has T cell epitopes and B cell epitopes, but its immunogenicity as a single epitope vaccine is low.

[0046] In this study, Echinococcus granulosus was used as the target antigen, and the amino acid sequences of EgA31 and EgG1Y62 proteins were obtained from the National Center for Biotechnology Information (NCBI) database.

[0047] The accession number of protein EgA31 is AAC21558.1, and the amino acid sequence is shown in SEQ ID No.21.

[0048] The accession number of protein EgG1Y162 is BAH28839.1, and the amino acid sequence is shown in SEQ ID No.22.

[0049] The accession number of the protein CTLA-4 is AAD00698.1, wherein amino acids 2 to 115 are CTLA-4lgV, and the amino acid sequence is shown in SEQ ID No.23.

[0050] Of course, the present invention is not limited to the above-mentioned specific EgA31, EgG1Y62 protein and CTLA-4 sequences. Any antigen epitope sequence obtained by the same or similar method based on the concept of the present invention is within the scope of the concept of the present invention.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The present invention is the first to use the EgA31 protein and EgG1Y162 protein of Echinococcus granulosus as target antigens, combined with dominant epitope sequences, to design a multi-epitope antigen sequence. In vitro experiments have found that it can specifically bind to the serum of patients with cystic echinococcosis, and mice immunized with the vaccine can produce specific B cell responses, specific cellular immunity and antibodies, and have good immunogenicity.

[0053] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is a connection sequence diagram of each sequence in Example 1;

[0055] Figure 2 Detailed connection diagram of each sequence in Example 1;

[0056] Figure 3 3A-B are schematic diagrams of the optimal docking posture of CVE31-162 and B7-1, and 3C-D are schematic diagrams of the optimal docking posture of CVE31-162 and B7-2;

[0057] Figure 4 The molecular dynamics simulation results in Example 2 are shown in Figure 4A, where 4B is the RMSF fluctuation diagram of B7-1, 4C is the RMSF fluctuation diagram of B7-2, and 4A is the RMSD fluctuation diagram of B7-2;

[0058] Figure 5 5A is a diagram showing the results of the principal component analysis in Example 2, wherein 5A is a diagram showing the distribution of PC1 and PC2 of B7-1, and 5B is a diagram showing the distribution of PC1 and PC2 of B7-2;

[0059] Figure 6 Figures 6A and 6B show the levels of IgM, IgG1+IgG2 and IgG1 after immune injection, 6C shows the levels of active T cells, and 6D shows the levels of cytotoxic T cells (CTL).

[0060] Figure 7 The Western blot result in Example 2, wherein lanes 1-4 are serum from the normal control group, and lanes 5-9 are serum from infected patients;

[0061] Figure 8 The results of the CD4+ and CD8+ t cell levels and ELISA antibody levels in Example 2 are shown in Figure 8A, wherein 8A is a graph showing the levels of specific CD4+ T cells and CD8+ T cells, 8B is a statistical analysis graph showing the percentages of CD4+ T cells and CD8+ T cells, and 8C is a graph showing the results of specific antibody levels after mouse immunization;

[0062] Fig. 9 9A is a flow cytometry result diagram of macrophages in Example 2, wherein 9A is a flow cytometry result diagram of spleen macrophages, and 9B is a flow cytometry result diagram of liver macrophages;

[0063] Fig.1010A is a graph showing the macrophage level in the liver, and 10B is a graph showing the macrophage level in the spleen;

[0064] Fig.11 This is the result diagram of macrophages capturing vaccine protein in Example 2;

[0065] Fig.12 This is a graph showing the weight results of mouse hydatid cysts in Example 2.

[0066] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION

[0067] If no specific conditions are specified in the examples of the present invention, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0068] Example 1 Preparation of CVE31-162

[0069] The T cell and B cell antigen epitope nucleotide sequences of the synthesized echinococcosis proteins EgA31 and EgG1Y162 were connected with the synthesized CTLA-4lgV nucleotide sequence in sequence with different connecting peptides to obtain the CVE31-162 protein sequence of the present invention (sequence synthesis was completed by Xinjiang Youkang Biological Co., Ltd.); the specific preparation process is: after the synthesis of each nucleotide, the gene sequence is determined, and after the sequencing results are correct, it is seamlessly cloned into the plasmid vector; after the construction of the multi-epitope vaccine plasmid is completed, it is transfected into Escherichia coli, and then cultured in a selective culture medium containing appropriate antibiotics to screen out successfully transfected Escherichia coli. When Escherichia coli grows to an appropriate density, the expression of the target gene is initiated by adding the inducer IPTG, so that Escherichia coli produces the multi-epitope protein required for the vaccine, and then the protein expression is further enhanced by shaking culture, and finally the desired vaccine candidate protein is obtained through collection and purification steps. ,

[0070] The amino acid sequence of the immunoglobulin variable region of CTLA-4 and the amino acid sequence of each antigen epitope in the above sequence are shown in Table 1, and the connection sequence diagram and detailed connection diagram of each sequence are shown in Figure 1 , 2 shown.

[0071] The amino acid sequence of the hydatid disease multi-epitope antigen CVE31-162 obtained by connection is shown in SEQ ID No.19; and the nucleotide sequence thereof is shown in SEQ ID No.20.

[0072] Example 2

[0073]

[0074] 1. Molecular docking

[0075] 1.1 Obtaining the B7 molecule

[0076] The human B7-1 / CTLA-4 co-stimulatory complex and B7-2 / CTLA-4 co-stimulatory complex were obtained from the PDB database. Their IDs are 1I8L and 1I85, respectively. Then, the obtained dimeric co-stimulatory complexes were edited using DS software to obtain the monomeric structural models of B7-1 and B7-2.

[0077] 1.2Interfacing CVE31-162 with B7

[0078] The molecular docking of CVE31-162 and B7 was simulated using the LZerD server. In the molecular docking, B7 was used as the receptor and CVE31-162 was used as the ligand. Finally, we obtained CVE31-162 with B7-1 and CVE31-162 with B7-2. According to the output results of the LZerD server, the most reasonable docking pose was selected from the top ten lists. The docking scores and rankings are shown in Table 1. The best docking poses of CVE31-162 and B7-1 are shown in Table 1. Figure 3 As shown in A and 3B, the optimal docking posture of CVE31-162 and B7-2 is as follows Figure 3 C and 3D.

[0079] Since the CTLA-4lgV sequence in CVE31-162 can enhance the antigen presentation ability of APC, and the CTLA-4lgV structure has a strong affinity for B7 molecules, the simulation docking results show that its fusion with the antigen epitope can significantly enhance the antigen presentation ability of APC, thereby enhancing the immune response.

[0080] Table 1 Final model scores

[0081]

[0082] 2. Molecular dynamics simulation and principal component analysis

[0083] 2.1 Molecular dynamics simulation

[0084] RMSD and RMSF were obtained using the gmx rmsd and gmx rmsf programs in GROMAC. The trajectory analysis time for the vaccine-B7 docking complex was 40 ns. The RMSF of B7-1 fluctuated between 0.1 and 0.8 ( Figure 4 A), the RMSD fluctuates between 0-0.65 ( Figure 4B). The RMSF of B7-2 fluctuates between 0.05-0.5 ( Figure 4 C), the RMSD fluctuates between 0.1-0.65 ( Figure 4 D).

[0085] The RMSD (root mean square deviation) value shows that the deviation of the complex structure from the initial conformation is low and the fluctuation is small, indicating that the complex has good overall stability; the trajectory analysis of the CVE31-162-B7 docking complex was performed at a speed of 40ns. The RMSD value indicates the degree to which the structure deviates from its initial conformation, reflecting the overall stability of the complex. For B7-1, RMSD fluctuations between 0 and 0.65 indicate medium stability ( Figure 4 B), while RMSF values ​​between 0.1 and 0.8 indicate that there are elastic regions inside the protein ( Figure 4 A). Similarly, for B7-2, the RMSD fluctuation between 0.1 and 0.65 also indicates moderate stability ( Figure 4 D), RMSF values ​​between 0.05 and 0.5 indicate relatively lower flexibility compared with B7-1 ( Figure 4 C) These analyses indicate that both B7-1 and B7-2 have stable and flexible action regions that can interact well with CVE31-162.

[0086] 2.2 Principal Component Analysis

[0087] Molecular dynamics simulations (MDS) were performed using Gromacs2022.3, and small molecule pretreatment was performed using AmberTools22 and Gaussian16W. The simulations were performed at a temperature of 300K and a pressure of 1 bar, using TIP3P water and Na+ ions to ensure neutrality. For energy minimization, the steepest descent methods were the NVT and NPT equilibrium stages, each lasting 100ps. One execution lasted 100ns, with a running time step of 2fs. In order to explore the conformational state of the vaccine, principal component analysis (PCA) was performed on the atoms in the composite system with a total duration of 0 to 40ns to study the conformational state of the protein in the PC phase space. PCA is a statistical method. The trajectories of MDS will be projected to the principal modes by Gromacs, and the most important key principal components 1 (PC1) and 2 (PC2) will be visualized ( Figure 5 ).

[0088] From the results, for B7-1, PC1 ranges from -5 to 8 nm, and PC2 ranges from -6 to 6 nm, indicating that B7-1 has a wider distribution and variability in the conformational landscape ( Figure 5A). For B7-2, PC1 ranges from -9 to 4 nm, and PC2 ranges from -3 to 4 nm, indicating that the conformational space is more constrained compared to B7-1 ( Figure 5 B). These results suggest that B7-1 has greater structural flexibility, which may enhance its ability to interact with other molecules and regulate immune responses; B7-2 has a narrower conformational range and higher stability than B7-1, and both are able to have a better adaptive interaction with CVE31-162.

[0089] In summary, the results of molecular dynamics simulation and principal component analysis showed that the designed multi-epitope vaccine CVE31-162 had a strong binding affinity and reasonable stability with the B7 molecule, which is an important indicator for vaccine design and efficacy, indicating that the antigen in the present invention is an effective and stable candidate molecule for echinococcosis vaccine.

[0090] 3. Immune simulation

[0091] The constructed CVE31-162 protein sequence was input, and HLA-A (HLA-A*11:01, HLA-A*02:01), HLA-B (HLA-B*15:01, HLA-B*35:01) and HLA-DR (HLA-DRB1*07:01, HLA-DRB1*15:01) were selected. The three specific injection time intervals used were 1, 84 and 168, respectively. Finally, the software settings were restored to the default configuration, and the random seed parameters of the simulation were established as 12345, the volume of the simulation was set to 50, and the total simulation steps were configured to 1050. The C-ImmSim server was used to perform immune simulations to simulate the immune response produced in the body when exposed to the vaccine designed in Example 1, and the results are shown in Figure 1. Figure 6 shown.

[0092] As for the results of the primary immune response, the first antibody produced was mainly IgM. After the first injection of the vaccine structure, the amount of IgM increased. After the second and third injections, the level of IgM+IgG increased significantly and the antigen level decreased. At the same time, the levels of IgM, IgG1+IgG2 and IgG1 all increased significantly ( Figure 6 A). After stimulating the body, B lymphocytes also change in different stages, especially stimulating the proliferation of memory B cells to produce different subtypes of antibodies ( Figure 6 B). Two booster injections lead to a rapid increase in TH cells and an increase in active T cells ( Figure 6 C) can also stimulate the changes of cytotoxic T cells (CTL) at different stages ( Figure 6 D).

[0093] 2. In vitro experiments

[0094] A total of 5 patients (adults aged 18-60 years) diagnosed with cystic echinococcosis in the Department of Infectious Diseases of the First Affiliated Hospital of Xinjiang Medical University were included in this study. In addition, we also included 4 healthy individuals (adults aged 18-60 years) as a control group. All enrolled patients complied with the Chinese guidelines for the diagnosis and treatment of cystic echinococcosis (CE), and any other conditions that might confound the experimental results were excluded. Peripheral blood (4 ml per person) of patients and healthy participants was collected, and serum was separated from it for Western blot (WB) detection to analyze the specific antibodies against CVE31-162 in the serum of infected patients. The results were as follows: Figure 7 As shown, antibodies that can specifically bind to the antigen of the present invention were found in the CE group, while no specific antibodies were found in the uninfected control group, indicating that the antigen of the present invention can effectively bind to the echinococcosis antibody specifically, help neutralize or eliminate the pathogen, and provide immune protection against echinococcosis infection.

[0095] The specific steps of Western Blot assay were as follows: the buffer containing CVE31-162 (1 lg / lL) was separated by SDS-PAGE and the protein was transferred to a PVDF membrane. After blocking with a 5% skim milk powder solution, the PVDF membrane was immersed in human serum (1:200 dilution) from CE patients (n=10) and healthy subjects (n=10) and incubated at 4°C overnight. Then, the PVDF membrane was incubated with a rabbit anti-human IgG secondary antibody labeled with HRP (1:5000 dilution) at room temperature for 1 hour. Finally, the target protein bands were observed using an ECL detection kit (BL523B, Biosharp, Beijing, China) and exposed on Amersham Hyperfilm (GE Healthcare).

[0096] 3. In vivo experiments

[0097] In this study, mice were injected with CVE31-162 vaccine protein to establish an in vivo animal experimental model, and serum was collected to determine the level of specific antibodies. FCM was used to evaluate the levels of specific CD4+T cells and CD8+T cells, thus confirming that CVE31-162 can activate specific cellular immunity in mice. ELISA technology was used to detect the production of specific antibodies in the supernatant to verify the antigenicity of the vaccine.

[0098] The specific experimental process is:

[0099] 8-week-old SPF BALB / C mice (from the Animal Experiment Center of Xinjiang Medical University) were randomly divided into two groups: CVE31-162 group (n=6), receiving intraperitoneal injection of CVE31-162 mixed with an equal amount of adjuvant (50μg / mouse), and control group (n=6), receiving intraperitoneal injection of PBS mixed with an equal amount of adjuvant (50μg / mouse); immunization was performed once every two weeks for a total of three times, the first immunization used complete Freund's adjuvant (CFA), and the second and third booster immunizations used incomplete Freund's adjuvant (IFA). Two weeks after the final immunization, 6 mice in each group were euthanized, and serum and spleen cells were collected for the following tests.

[0100] 1. Detection of specific CD4+ and CD8+ T cell levels by flow cytometry FCM

[0101] Two weeks after the final immunization, spleen cells were removed from the mice. Lymphocytes were counted using a hemocytometer, and spleen cell suspension was prepared with the cell concentration adjusted to 2×10^6 cells / mL. The spleen cell suspension was then incubated with anti-CD3 (APC) and anti-CD4 (FITC) monoclonal antibodies for 20 minutes. Subsequently, CD3+CD4+CD8+ cells were gated using FlowJo software for flow cytometric analysis. The flow cytometer used in this study was BeamCyte-1026-0097.

[0102] FCM was used to assess the levels of specific CD4+T cells and CD8+T cells ( Figure 8 A), the results showed that the levels of specific CD4+T cells and CD8+T cells in the CVE31-162 group were significantly higher than those in the control group (P<0.001, P<0.01) ( Figure 8 B).

[0103] 2. Enzyme-linked immunosorbent assay (ELISA) to detect antibody levels

[0104] CVE31-162 mice were immunized with CVE31-162 vaccine, and serum was collected to determine the level of specific antibodies. Figure 8 As shown in C, compared with the control group, the level of specific antibodies in the CVE31-162 group increased significantly 2 weeks after the first immunization (P<0.01), reaching a peak 2 weeks after the third immunization (week 6). In addition, the serum specific antibody level of the CVE31-162 group at week 4 was also increased compared with the specific antibody level at week 2 before CVE31-162 immunization (week 0, P<0.01) and 2 weeks after the first immunization (P<0.01). These results indicate that CVE31-162 can increase the serum specific antibody level of this animal model and activate humoral immunity.

[0105] 2. Detection of macrophage levels in spleen and liver by flow cytometry (FCM)

[0106] Mouse liver leukocytes were separated by 40% percoll solution. The suspension of spleen cells and liver leukocytes was incubated with monoclonal antibodies against CD45 (APC), F4 / 80 (FITC), and CD11b (APC-Cy7) for 20 minutes. Subsequently, CD45+ cells were gated using FlowJo software, and finally, F4 / 80+CD11b+ was gated to identify macrophages. The flow cytometer used in this study was BD FACSLyric.

[0107] The results of FCM detection method to evaluate the level of macrophages are as follows Fig. 9 A and B show that the levels of macrophages in the liver and spleen of the CVE31-162 group were significantly higher than those of the control group (P<0.001) ( Fig.10 A, B), indicating that CVE31-162 can significantly activate and increase the number of macrophages and has good immunogenicity.

[0108] 3. Evaluation of CVE31-162 Uptake by Macrophages

[0109] Evaluation of macrophage uptake of CVE31-162 Splenocytes were cultured in a medium containing 1% glutamine, 1% diabody, 10% fetal bovine serum, and 88% DMEM at 37°C and 5% carbon dioxide overnight. Subsequently, His-tagged CVE31-162 recombinant protein was added to the cultured cells (final concentration of 10 μg / mL) and incubated at 37°C for 4 hours. The cell suspension was then collected in a 15 mL centrifuge tube and centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in pre-cooled PBS. The cells were then incubated with anti-CD45 (APC), F4 / 80 (FITC), CD11b (APC-Cy7), and anti-His tag (PE) at 4°C in the dark for 20 minutes to evaluate the uptake of CVE31-162 protein by macrophages.

[0110] Given the strong interaction between CTLA-4 and B7 molecules, we used FCM to detect the uptake of the vaccine by macrophages expressing B7 molecules. The results showed that the proportion of His-tag positive cells was 4.54%, indicating that the vaccine protein had been successfully captured by macrophages ( Fig.11 ), which can effectively activate multiple immune responses including phagocytosis, antibody production, and cellular immune response, thereby enhancing the body's immune defense against Echinococcus granulosus infection.

[0111] IV. Evaluation of the protective effect of CVE31-162

[0112] To evaluate the potential protective effect of CVE31-162, thereby conferring resistance to infection with the protoscolecus of Echinococcus granulosus, we subjected two groups of mice to in vivo experiments (animal experiments) and subjected them to protoscolecus infection for 22 weeks. We dissected the mice and measured the weight of the Echinococcus granulosus cysts to evaluate the protective effect of the multi-epitope vaccine.

[0113] The results showed that there was a statistically significant difference in the weight of hydatid cysts between the immunized group and the control group (P<0.001). Fig.12 Specifically, the average weight of hydatid cysts in immunized mice was significantly lower than that in non-immunized mice, indicating that the vaccine exhibited a strong protective effect and could effectively reduce the infection caused by the protoscoleces of Echinococcus granulosus.

[0114] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A multi-epitope antigen for echinococcosis CVE31-162, characterized in that It is composed of CTLA-4-lgV, the T cell epitope of EgA31, the B cell epitope of EgA31, the T cell epitope of EgG1Y162, and the B cell epitope of EgG1Y162 in series; Wherein, the CTLA-4-lgV is the immunoglobulin variable region of CTLA-4.

2. The echinococcosis multi-epitope antigen CVE31-162 according to claim 1, characterized in that The amino acid sequence of the CTLA-4-lgV is shown in SEQ ID No.

1.

3. The echinococcosis multi-epitope antigen CVE31-162 according to claim 1, characterized in that The T cell epitope of EgA31 is composed of the following amino acid sequences connected in series: SEQ ID No.2LRRALITQESRNLELQNDLERLQ; SEQ ID No.3LERDLNDMKGLVGFYKAFAK; SEQ ID No.4IQVQIQLLRGGYLDLFHDKIGHY; SEQ ID No.5LQESETKLL.

4. The echinococcosis multi-epitope antigen CVE31-162 according to claim 1, characterized in that The T cell epitope of EgG1Y162 is composed of the following amino acid sequences connected in series: SEQ ID No. 6FRWIHVGSRSL; SEQ ID No. 7IKLTANLYTTY; SEQ ID No. 8EVVVQAF.

5. The echinococcosis multi-epitope antigen CVE31-162 according to claim 1, characterized in that The B cell epitope of EgA31 is composed of the following amino acid sequences connected in series: SEQ ID No. 9AQNNIRTQES; SEQ ID No. 10AEKQAMDDSTASSD; SEQ ID No. 11RETRDELDEQKAE; SEQ ID No.12SKLTESGE SEQ ID No. 13KKEKDA.

6. The echinococcosis multi-epitope antigen CVE31-162 according to claim 1, characterized in that The B cell epitope of EgG1Y162 is composed of the following amino acid sequences connected in series: SEQ ID No. 14 IRV; SEQ ID No. 15TT; SEQ ID No. 16LKP; SEQ ID No. 17KG; SEQ ID No. 18APGEDGA.

7. The echinococcosis multi-epitope antigen CVE31-162 according to claim 1, characterized in that The echinococcosis multi-epitope antigen further comprises a connecting peptide sequence; preferably, the connecting peptide is used to connect T cell epitopes in series, and the sequence of the connecting peptide is AAT; Preferably, the connecting peptide is used for tandem B cell epitopes, and the sequence of the connecting peptide is KK; Preferably, the connecting peptide is used to connect to CTLA-4-lgV, and the sequence of the connecting peptide is GGGGSGGGGSGGGG. Preferably, the connecting peptide is used to connect the T cell epitope of EgA31 and the B cell epitope of EgA31, the B cell epitope of EgA31 and the T cell epitope of EgG1Y162, the T cell epitope of EgG1Y162 and the B cell epitope of EgG1Y162, and the sequence of the connecting peptide is GPSL.

8. The echinococcosis multi-epitope antigen CVE31-162 according to claim 1, characterized in that The amino acid sequence of the echinococcosis multi-epitope antigen CVE31-162 is shown in SEQ ID No.

19.

9. The nucleotide sequence of the echinococcosis multi-epitope antigen CVE31-162 according to claim 1, characterized in that The sequence is shown as SEQ ID No.

20.

10. Use of the echinococcosis multi-epitope antigen CVE31-162 of any one of claims 1 to 8 of the present invention or the nucleotide of claim 9 in the preparation of a drug for preventing or treating echinococcosis; Preferably, the drug for preventing or treating echinococcosis is a vaccine; Preferably, the vaccine further comprises a pharmaceutically acceptable carrier or agent.

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