Multi-epitope vaccine method for preventing alveolar echinococcosis

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses a multi-epitope vaccine method for preventing alveolar echinococcosis, which is technically characterized in that the method adopts an immunoinformatics high-throughput prediction technology to realize deep excavation and analysis of antigen epitopes. Through the advanced technology, the selection number of HLA-class I and HLA-class II alleles is remarkably increased, so that a wider and richer basis is provided for subsequent research. In the research process, CD8 + T cell epitopes, CD4 + T cell epitopes and B cell epitopes are particularly screened, and the stability of the epitopes is strictly verified, so that the effectiveness and reliability of the selected epitopes in vaccine development are ensured. The screened high-frequency antigen epitopes play a positive role in improving the coverage rate of the vaccine in different human species in the world, so that the protection group of the vaccine is effectively expanded, and the public health level is enhanced.

Description

Technical Field

[0001] The patent of this invention relates to the field of molecular biology technology, specifically, to a multi-epitope vaccine method for preventing alveolar echinococcosis. Background Art

[0002] Hydatid disease, also known as echinococcosis, is a disease caused by the larvae of Echinococcus granulosus and Echinococcus multilocularis in humans. The disease is prevalent in many livestock breeding areas in the world, generally occurring in plateau areas. The final hosts of Echinococcus granulosus are dogs, wolves, foxes, sheep, mice, horses, etc. as intermediate hosts. The clinical manifestations of echinococcosis are highly diverse and reflect the location and size of the larvae. Most of the symptoms are due to the mechanical pressure of the larvae or the outflow of blister fluid causing foreign antibodies. The diagnosis can be made by detecting the worms during cyst biopsy.

[0003] Current diagnostic methods for alveolar echinococcosis are limited, and available drugs are toxic and ineffective, posing challenges to surgical intervention and overall disease management. Therefore, there is an urgent need to improve diagnostic tools, discover new drug and vaccine targets, and develop effective prevention strategies, of which vaccine development is a key approach.

[0004] To this end, the present invention aims to provide a multi-epitope vaccine method for preventing alveolar echinococcosis to solve the above-mentioned problems. Summary of the invention

[0005] The purpose of the present invention is to solve the technical problems raised in the above-mentioned background technology and to provide a multi-epitope vaccine method for preventing alveolar echinococcosis.

[0006] The above-mentioned purpose of the present invention is achieved like this:

[0007] A multi-epitope vaccine method for preventing alveolar echinococcosis comprises the following steps:

[0008] S1. Candidate antigens for vaccine design: Using E. multilocularis protein as the target antigen, the amino acid sequences of Em-TSP3 and Em-TIP proteins were obtained from the National Center for Bioinformation database;

[0009] S2. Overall evaluation of candidate protein prediction: The transmembrane domains of proteins EmTSP3 and EmTIP were analyzed by the online server TMHMM; the single signal peptides of EmTSP3 and EmTIP were predicted by the online prediction server SignalP-5.0; the main phosphorylation sites of proteins EmTSP3 and EmTIP were predicted by the NetPhos 3.1 server; the secondary structures of proteins EmTSP3 and EmTIP were determined by the DNAstar software online server; the tertiary structures of proteins EmTSP3 and EmTIP were constructed by SWISS-MODEL;

[0010] S3. Prediction of antigen epitopes: The online servers IEDB, NetMHCIIpan-4.0 and NetCTLpan-4.0 were used to predict the helper T lymphocyte epitopes and cytotoxic T lymphocyte epitopes of Em-TSP3 and Em-TIP, and HLA-a*1101, HLA-A*0201, HLA-A*0301, HLA-DRB1*0701, HLA-DRB1*1501 and HLA-DRB1*0301 were selected as prediction objects; linear B lymphocyte epitopes and B cell conformational epitopes were predicted using DNASTAR and IEDB servers;

[0011] Antigenicity was assessed using the VaxiJen v2.0 platform, allergenic potential was determined using the AllergenFP v1.0 platform, and epitope toxicity was assessed using the ToxinPred platform; the results showed that highly antigenic, non-toxic, and non-allergenic TEs were screened from the top ten epitopes, and highly antigenic, non-toxic, and non-allergenic epitopes were screened from the top ten scoring BEs for vaccine construction;

[0012] S4. Construction of vaccine proteins: covalently combining the above selected epitopes using specific linkages to form a complex vaccine sequence;

[0013] S5. Prediction of secondary and tertiary structures of vaccine proteins: SOMPA predicts the secondary structure of MEV, uses the AlphaFold2 server to build a prediction model for the tertiary structure of MEV, and selects the model with the highest score; uses the GalaxyWEB server to refine the model structure, and finally selects the model with the highest GDT-HA score;

[0014] S6. Molecular docking: Use energy minimization and spatial structural complementarity to predict ligand-receptor interactions at active sites; generate multiple models and select the top model for analysis; use PyMOL to visualize the selected docked structure in 3D and use Discovery Studio to create 2D interaction plots.

[0015] Furthermore, the amino acid sequences of Em-TSP3 and Em-TIP proteins are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0016] Furthermore, in S4, the constructed vaccine contained dominant epitopes, specifically 7 CTL epitopes, 7 Th epitopes, 5 LB epitopes and 2 CB epitopes, with a total length of 436 amino acids.

[0017] Furthermore, the molecular weight of the vaccine is 48.37 kDa and the molecular formula is: 2131 H 3389 N635 O 624 S 15 . The isoelectric point is 27.61.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The method of the present invention designs and develops a multi-epitope recombinant vaccine MEV with these two proteins as target antigens, which can stimulate the body to produce an immune response against E. multilocularis and block the transmission between livestock and humans;

[0020] 2. The method of the present invention adopts high-throughput prediction technology of immunoinformatics to achieve in-depth mining and analysis of antigenic epitopes; significantly increases the number of HLA-I and HLA-II alleles to be selected, thereby providing a broader and richer basis for subsequent research; in the present invention, CD8+T cell epitopes, CD4+T cell epitopes and B cell epitopes are specially screened, and their stability is strictly verified to ensure the effectiveness and reliability of the selected epitopes in vaccine development; these screened high-frequency antigenic epitopes will play a positive role in improving the coverage of vaccines among different ethnic groups around the world, thereby effectively expanding the protected population of vaccines and enhancing the level of public health;

[0021] 3. The present invention connects the screened high-frequency antigenic epitopes with linker proteins, and pays attention to the adaptability and pertinence of linker selection. The EAAAK linker peptide helps maintain the stability of the protein and enhance its antigenicity; β-defensin-3 has significant immunomodulatory properties; PADRE is a multifunctional HLA-DR binding epitope; the specific connection strategy ensures the optimal presentation and function of each epitope in the vaccine; and the His tag facilitates the purification and detection of the vaccine. The use of these advantageous antigenic epitopes and the optimized design of linker proteins can theoretically enhance the immunogenicity of vaccine proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the transmembrane domain of the proteins EmTSP3 and EmTIP analyzed by the online server TMHMM in the embodiment of the present invention;

[0023] Figure 2 is the signal peptide of EmTSP3 and EmTIP proteins in the embodiments of the present invention;

[0024] Figure 3 It is the NetPhos 3.1 analysis in the embodiment of the present invention;

[0025] Figure 4 In the embodiment of the present invention, DNASTAR is used to predict the secondary structure of proteins EmTSP3 and EmTIP;

[0026] Figure 5 The tertiary structures of proteins EmTSP3 and EmTIP are constructed using SWISS-MODEL in the embodiments of the present invention;

[0027] Figure 6 is the epitope sequence in the MEV construction in the embodiment of the present invention;

[0028] Figure 7 is the length of the query sequence for constructing the vaccine in the embodiment of the present invention;

[0029] Figure 8 :(A) The secondary structure prediction results are: α-helix 47.94%, random coil 37.61%, extended chain 14.45%. (B) In the MEV structure displayed using Discovery Studio. (C) The pink area is the hydrogen bond donor and the green area is the hydrogen bond acceptor;

[0030] Fig. 9 :(A) Statistics of non-bonded interactions between different atom types. (B) The red area is the allowed area, the yellow area is the maximum allowed area, and the blank area is the unallowed area. (C) Z-score plot obtained from ProSA-web, the light blue area represents the structure group analyzed using X-ray, and the dark blue area represents the group analyzed using nuclear magnetic resonance (NMR);

[0031] Fig.10 :(A) The interaction analysis of the MEV-TLR4 complex was performed using the visualization tool PyMol, and its three-dimensional image was taken. (B) The interaction of the complex was analyzed using the visualization tool Discovery Studio, and its two-dimensional image was taken. ;

[0032] Fig.11 :(A) Molecular dynamics simulation of MEV-TLR4 complex. (B, C) RMSD of TLR4 and MEV. (D) Radius of gyration analysis;

[0033] Fig.12 It is the protein conformation at the lowest point of the free energy graph;

[0034] Fig.13 It is to predict and optimize the immune response of MEV vaccine ((A) B cell subtypes. (B) Helper T cell (Th cell) subtypes. (C) Cytotoxic T cell (TC cell) subtypes. (D) Natural killer cells (NK cells). (E) Dendritic cells (DC cells). (F) Epithelial cells (EP cells). (G) Immunoglobulins. (H) Interleukins and cytokines);

[0035] Fig.14is the multiple cloning site integrated into the plasmid in the embodiment of the present invention;

[0036] Fig.15 The embodiment of the present invention comprehensively uses computational simulation and molecular biology techniques to improve vaccine immunogenicity and expression efficiency ((A) Optimize the adaptability of codons. (B) GC content. (C) MEV (1320 bp) after amplification);

[0037] Fig.16 This is an in vitro experiment in the embodiment of the present invention;

[0038] Fig.17 It is a representative band of Western Blot in the embodiment of the present invention;

[0039] Fig.18 This is an in vivo experiment in the embodiments of the present invention; Fig.19 This is the in vivo experimental result in the embodiment of the present invention. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0042] The same or similar numbers in the drawings of this embodiment correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right" and the like indicate directions or positional relationships based on the directions or positional relationships shown in the drawings, it is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limitations on this patent. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0043] As shown in the drawings, a preferred embodiment of the present invention is provided.

[0044] The embodiment of the present invention provides a multi-epitope vaccine method for preventing alveolar echinococcosis, comprising:

[0045] 1. Vaccine design candidate antigens:

[0046] In this study, the amino acid sequences of Em-TSP3 and Em-TIP proteins were obtained from the database of the National Center for Biotechnology Information (NCBI). The physicochemical parameters of proteins EmTSP3 and EmTIP were analyzed by ProtParam. EmTSP3 is composed of 148 amino acids, a total of 2304 atoms, and its chemical formula is C 724 H 1176 N 184 O 206 S 14 , molecular weight is 16kda. The theoretical isoelectric point PI is 6.70. It contains 15 acidic amino acids and 15 basic amino acids. The calculated instability index (II) is 31.84, the fat index is 111.96, and the overall average hydrophilicity (GRAVY) is 0.389. The protein EmTIP is composed of 592 amino acids, a total of 8992 atoms, and the chemical formula is:

[0047] C 2886 H 4474 N 748 O 861 S 23 , the molecular weight is 64kda. PI is 5.11, it contains 57 acidic amino acids and 44 basic amino acids, the fat index is 91.10, and the GRAVY is 0.089.

[0048] The amino acid sequence of Em-TSP3 protein is:

[0049] MLKTFAVIVAILLVAEIVCGIVLLVYRHEFVGLVGKEMQREIKDLTAHGRNASD

[0050] PLLKSIYKLQEELECCGGVGPTDWSKPYPASCCKSGKENCTQPYQQGCAVAM YEQIKDSSLAFGLIILIVCLIQIGAVICACCLAKKVNEYEKV;

[0051] The amino acid sequence of Em-TIP is:

[0052] MQAYAYILTVWFLGVQCVAITGPGGSSFSLVASWSDADLAAFADVNTDRRTDA

[0053] IVLSSSESALYALLAPNAKQTDGKPERKKLFSLTHSLPLRSIAVADFDGDSVADF

[0054] LLVFRDTSKYSVNVWYGGKNVTRSVGSFATQPLVCDVNADMIADVYGEMEN

[0055] HRVVALGEPNAFSLQNFTHSATSLSELSSAGFVSLAFNPNPSLVTLAVDTIEVFN

[0056] DLTPKGDGSPSSYPLPIELKGANRPIGKLVFGDFDMSGRIQLLIAGCSDNTCRHS

[0057] YIFMHSLTGNAVWEAIAVEWNPPEMEGSCSLAPASVDQFSSAAIIGLSLGDADL

[0058] DGYPDLAVGLKCTRSSGLRPIILPAILRNLAGAGRKVRFQAYLLPGVESQETLK

[0059] QIAFYDYNEDGILDLYMSYEGRGGVSTSLYMQKLTKEAYFLKVMLTTGRCGSP

[0060] SQCPDGVLPYGLPGYGFRASYETQGADGGRIRSSAAFVTSSSCCGALQLPFTTF

[0061] GFGDFATYIENVPVSVPAPTQQARKHKLTFIVPNAQVVVVPYPPDNPSNWQAK

[0062] LFLQPLYDMKVIYVAITLLVTCIVLLVVVGILQYLEVRSDQKERMQESQRFHFD

[0063] AM

[0064] 2. Overall evaluation of candidate protein prediction:

[0065] The transmembrane domains of proteins EmTSP3 and EmTIP were analyzed by online server TMHMM ( Figure 1 ). The online prediction server SignalP-5.0 predicted that the EmTSP3 protein and the EmTIP protein each have a single signal peptide. The signal peptide is located at residues 1-21 of EmTSP3 and residues 1-20 of EmTIP, and the probability of occurrence is very high ( Figure 2 ). Using NetPhos 3.1 server analysis, the main phosphorylation sites of protein EmTSP3 and protein EmTIP were predicted ( Figure 3 The secondary structures of proteins EmTSP3 and EmTIP were determined using DNAstar software online server ( Figure 4 ). The tertiary structures of proteins EmTSP3 and EmTIP were constructed using SWISS-MODEL, and the sequence identities with protein models B6VFH5.1 A and A0A068WNA4.1 were 100% and 98.77%, respectively ( Figure 5 ).

[0066] like Figure 2 As shown, it is the signal peptide of EmTSP3 and EmTIP proteins

[0067] like Figure 3 As shown in the figure, NetPhos 3.1 analysis showed that the EmTSP3 protein had 6 serine, 3 threonine and 9 tyrosine phosphorylation sites, and was mainly targeted by PKC, PKA and CKI. The EmTIP protein had 39 serine phosphorylation sites, 21 threonine phosphorylation sites and 5 tyrosine phosphorylation sites, and was mainly targeted by PKC, PKA, DNA-PK and cDC2.

[0068] like Figure 4 As shown, the Gramier-Robson method is represented by lines A, B, T, and C, with colors representing different structural elements: α-helix (red), β-fold (green), β-turn (blue), and random coil (yellow). In addition, the Chou-Fasman method uses lines A, E, and T to depict the same structural elements without including the random coil structure.

[0069] like Figure 5 As shown in (A), it is the tertiary structure of EmTSP3 protein. Figure 5 (B) shows the tertiary structure of the EmTIP protein. Next to each structure is a Ramachandran diagram showing the allowed and disallowed peptide bond rotation angles. The dark green circle is the fully allowed region, the light green circle is the allowed region, and the outside of the green circle is the disallowed region. A good model has more than 90% of the scattering within these circles.

[0070] 3. Prediction of antigenic epitopes:

[0071] The online servers IEDB, NetMHCIIpan-4.0 and NetCTLpan-4.0 were used to predict the helper T lymphocyte epitopes (HTLE) and cytotoxic T lymphocyte epitopes (CTL) of Em-TSP3 and Em-TIP, and HLA-a*1101 (13.46%), HLA-A*0201 (12.50%), HLA-A*0301 (10.10%), HLA-DRB1*0701 (16.35%), HLA-DRB1*1501 (8.65%) and HLA-DRB1*0301 (7.69%) were selected as prediction objects. Linear B lymphocyte epitopes (BLEs) and B cell conformational epitopes (CBEs) were predicted using DNASTAR and IEDB servers.

[0072] Antigenicity was evaluated using the VaxiJenv2.0 platform, allergen potential was determined using the AllergenFPv1.0 platform, and epitope toxicity was evaluated using the ToxinPred platform. The results showed that highly antigenic, non-toxic, and non-allergenic TEs were screened from the top ten epitopes (Tables 1, 2), and highly antigenic, non-toxic, and allergenic epitopes were screened from the top ten scoring BEs for vaccine construction (Tables 3, 4).

[0073] Table 1 List of finally selected Th epitopes

[0074]

[0075]

[0076] Table 2 List of finally selected CTL epitopes

[0077]

[0078] Table 3 List of finally selected linear epitopes

[0079]

[0080]

[0081] Table 4 List of final selected conformational epitopes

[0082]

[0083] 4. Construction of vaccine protein

[0084] These carefully selected epitopes were covalently linked using specific linkages to form a complex vaccine sequence. The constructed vaccine contained dominant epitopes, specifically 7 CTL epitopes, 7 Th epitopes, 5 LB epitopes and 2 CB epitopes, with a total length of 436 amino acids ( Figure 6). Use tools such as ProtParam, VaxiJen, and AllergenFP to predict the physicochemical properties, antigenicity, solubility, and allergenicity of vaccine proteins. The molecular weight of the MEV vaccine is 48.37 kDa, and the molecular formula is C 2131 H 3389 N 635 O 624 S 15 . The isoelectric point is 27.61, indicating that it is a stable protein. The GRAVY value is -0.662, indicating its hydrophilicity. The antigenicity is 0.7621, which exceeds the threshold and is non-allergenic. The solubility is 0.92, which is a soluble protein antigen. The homology between the vaccine and humans is only 10%, which is not enough to trigger an autoimmune response ( Figure 7 ).

[0085] like Figure 7 As shown, the diagram above illustrates the length of the query sequence, which is approximately 430 amino acids or nucleotides. The initial matching region (positions 1-80) is highlighted with a pink bar. This shows that it is highly similar to the known β-defensin 3 protein, which only accounts for about 10% of the entire sequence.

[0086] 5. Prediction of secondary and tertiary structures of vaccine proteins:

[0087] The prediction results of SOMPA for the secondary structure of MEV are as follows: Figure 8 We used the AlphaFold2 server to build a prediction model for the MEV tertiary structure and selected the model with the highest score ranking. Subsequently, we used the GalaxyWEB server to refine the model structure and finally selected the model with the highest GDT-HA score, indicating the best quality assessment of the model ( Fig. 9 ).exist Figure 8 In C, the donor and acceptor contribute to the stability of the vaccine structure and may bind to antibodies or T-cell receptors during immunization.

[0088] 6. Molecular docking:

[0089] Molecular docking uses energy minimization and spatial structural complementarity to predict ligand-receptor interactions at the active site. Multiple models were generated and the top model was selected for analysis. The best docking result score was -280.32 and the ligand RMSD was Use PyMOL to visualize the selected docked structures in 3D ( Fig.10 A) and create a 2D interactive diagram using Discovery Studio.

[0090] 7. Molecular dynamics simulation evaluation of MEV

[0091] The molecular dynamics simulation results showed that the MEV-TLR4 complex had good structural stability throughout the simulation. Subsequent analysis included examination of RMSD, RMSF, RoG, and potential energy to assess conformational and structural integrity. The RMSD curve showed stability throughout the simulation, with slight fluctuations at 65ns, indicating that the overall structure was stable ( Fig.11 ). RMSF values ​​showed little fluctuation for TLR4 and vaccine proteins, indicating sustained structural integrity. RoG maintained a consistent trend with no significant deviation, indicating that the compactness and structural integrity of the complex were maintained during the simulation. Free energy landscape analysis showed a clear low energy region at 100 ns, indicating a stable conformation of the protein ( Fig.12 ). Binding free energy calculations showed that there was a strong interaction between the vaccine and TLR4, showing a strong affinity (Table 5).

[0092] Table 5 △G binding free energy

[0093]

[0094] 8. Immune simulation and recombinant plasmid construction:

[0095] This study used multiple computational approaches to predict and optimize the immune response to MEV vaccines ( Fig.12 ). First, the immune response after MEV vaccination was simulated using the C-ImmSim server. Second, the MEV sequence was codon optimized to increase the codon adaptation index (CAI) and GC content, and BamHI and XhoI sites were introduced to facilitate its integration into the multiple cloning site of the plasmid ( Fig.14 ). Finally, the construction of the recombinant plasmid was verified by simulated PCR and agarose gel electrophoresis. These methods combine computational simulation and molecular biology techniques to optimize the design of MEV vaccines and improve their immunogenicity and expression efficiency ( Fig.15 ).

[0096] 9. In vitro experiments:

[0097] This study included 46 patients (aged 18-60 years) diagnosed with AE in the Department of Infectious Diseases of the First Affiliated Hospital of Xinjiang Medical University. In addition, we recruited 12 healthy individuals (adults aged 18-60 years). The selected patients met the Chinese AE diagnosis and treatment guidelines and excluded other conditions that may affect the experimental results. Peripheral blood (4 ml per person) was collected from patients and healthy controls, and serum was separated for Western blot analysis. The Ficoll Paque method was used to separate the patient's peripheral blood mononuclear cells (PBMCs) for in vitro cell experiments; the codon optimization and protein expression of MEV protein and V-C4MEV protein were completed by Shanghai Shenggong Biotechnology Co., Ltd.

[0098] 10. In vivo experiments:

[0099] In this study, 8-week-old SPF-grade BALB / C mice were obtained from the Animal Experimental Center of Xinjiang Medical University and randomly divided into two groups: MEV group (n=12) and control group (n=12). MEV group mice received intraperitoneal injection of MEV and an equal volume of Freund's adjuvant (50 μg / mouse), and control group mice received intraperitoneal injection of PBS and an equal volume of Freund's adjuvant (50 μg / mouse). Immunization was performed every two weeks for a total of three times, with complete Freund's adjuvant (CFA) used for the first immunization and incomplete Freund's adjuvant (IFA) used for the second and third booster immunizations. Before each immunization, blood samples were collected through the inner canthus of the eye to monitor the dynamic changes of antibody levels. Two weeks after the last immunization, six mice in each group were killed, and their serum and spleen cells were collected for flow cytometry (FCM), ELISA, and ELISPOT analysis. In addition, the remaining six mice in each group were subjected to potential protection tests to evaluate whether MEV could induce host resistance to original scolex infection.

[0100] The problems solved by the embodiments of the present invention are:

[0101] 1. Currently, the diagnostic methods for alveolar echinococcosis are limited, and the available drugs are toxic and ineffective, which poses challenges to surgical intervention and overall disease management. Therefore, there is an urgent need to improve diagnostic tools, discover new drug and vaccine targets, and develop effective prevention strategies, among which vaccine development is a key method. A multi-epitope recombinant vaccine MEV with these two proteins as target antigens was designed and developed. In theory, it can stimulate the body to produce an immune response against E. multilocularis and block the transmission between livestock and humans;

[0102] 2. This study used high-throughput prediction technology from immunoinformatics to achieve in-depth mining and analysis of antigenic epitopes. Through this advanced technology, we have significantly increased the number of HLA-I and HLA-II alleles to choose from, thus providing a broader and richer foundation for subsequent research. During the research process, we specifically screened CD8+T cell epitopes, CD4+T cell epitopes, and B cell epitopes, and rigorously verified their stability to ensure the effectiveness and reliability of the selected epitopes in vaccine development. These screened high-frequency antigenic epitopes will play a positive role in increasing vaccine coverage among different ethnic groups around the world, thereby effectively expanding the vaccine's protected population and enhancing public health levels;

[0103] 3. This study will use linker proteins to connect the high-frequency antigenic epitopes screened, while focusing on the adaptability and pertinence of linker selection. The EAAAK linker peptide helps maintain the stability of the protein and enhance its antigenicity; β-defensin-3 has significant immunomodulatory properties; PADRE is a multifunctional HLA-DR binding epitope; specific connection strategies ensure the optimal presentation and function of each epitope in the vaccine; His tags facilitate vaccine purification and detection. The use of these advantageous antigenic epitopes and the optimized design of linker proteins can theoretically enhance the immunogenicity of vaccine proteins;

[0104] 4. This study comprehensively examined the physicochemical properties of MEV, analyzed its secondary and tertiary structures in detail, and studied the interaction between MEV and Toll-like receptor 4 (TLR4) through molecular docking simulation, thereby verifying the basic physicochemical properties and structural characteristics

[0105] 5. The study also performed molecular dynamics simulations to further evaluate the stability and dynamic properties of the docking results. C-ImmSim simulations provide valuable predictions about the dynamics of MEV vaccine-induced immunity, highlighting the role of specific HLA alleles in shaping immune responses. These findings can provide information for future vaccine design and optimization for specific populations, ensuring improved vaccine efficacy and protection.

[0106] 6. The study performed codon optimization and expression verification by simulated PCR and agarose gel electrophoresis. Through reasonable codon optimization and precise molecular cloning strategies, the recombinant plasmid of multi-epitope vaccine can be effectively constructed, laying a solid foundation for subsequent vaccine expression and functional verification. This process not only improves the expression efficiency of the vaccine, but also provides strong technical support for the development of multi-epitope vaccines.

[0107] 7. This study successfully established an animal experimental model of MEV vaccination in mice, which provides a basis for the subsequent evaluation of vaccine effects. After three vaccinations of mice, we used ELISA to detect the cytokines IFN-γ and IL-4 in the mouse spleen cell culture fluid, and the results showed that MEV can effectively activate specific cellular immune responses. This finding further confirms the potential of vaccines in activating the body's immune system. In addition, we detected the production of specific antibodies by ELISA technology, and used flow cytometry (FCM) to analyze the ratio of CD4+T cells and CD8+T cells to verify the antigenicity of the vaccine. The results showed that the vaccine has good immunogenicity and can effectively activate the immune response against E. multilocularis infection. In summary, MEV shows broad prospects in the prevention and control of alveolar echinococcosis, and is expected to become an important means to block transmission between livestock and humans.

[0108] In summary, in the above embodiments of the present invention:

[0109] 1. The scheme of the present invention identified two protein antigen epitopes by immunoinformatics methods, with TSP3 and TIP proteins of E. multilocularis as target antigens. Using key technologies such as molecular docking, a multi-epitope vaccine was designed, which can theoretically enhance the body's immune response and produce specific antibodies against E. multilocularis, thereby achieving effective prevention of the parasite. Therefore, MEV vaccination may become an important means of preventing alveolar echinococcosis in the future.

[0110] 2. In the in vitro experiment, the secretion of IFN-γ and IL-4 was detected after PBMCs were stimulated by MEV and PBS. The average secretion of IFN-γ in the control group was 50.86pg / mL, and that in the MEV group was 389.70pg / mL, and the difference was statistically significant (p<0.05). The average secretion of IL-4 in the control group was 25.96pg / mL, and that in the MEV group was 246.4pg / mL. Two independent sample t-tests were used to compare the differences in IFN-γ and IL-4 secretion levels between the control group and the MEV group. The secretion of IFN-γ in the MEV group was significantly higher than that in the Control group (P<0.001)( Fig.16 Similarly, the IL-4 secretion in the MEV group was significantly higher than that in the Control group (P<0.001) ( Fig.16 B in the figure). Western Blot was used to detect MEV-specific antibodies in the serum of infected patients. Therefore, when MEV protein was incubated with patient serum, a target band of about 48 kDa was generated. The results are shown in Fig.17 shown.

[0111] Fig.16 The results of in vitro experiments, among which (A) is the statistical analysis of IFN-γ concentration levels between the control group and the MEV group, and the statistical method is two independent sample t test (***P<0.01). (B) is the statistical analysis of IL-4 concentration levels between the control group and the MEV group, and the statistical method is two independent sample t test (***P<0.01).

[0112] Fig.17 The figure shows representative bands of Western Blot. The letter "M" indicates the position of the marker. Bands 1-4 are from healthy individuals, while bands 5-9 are from patients with alveolar echinococcosis. The black bands represent the blot of MEV protein.

[0113] 3. In the in vivo experiment, the flowchart of the mouse vaccination strategy is as follows Fig.18As shown in A. Flow cytometry (FCM) was used to assess the levels of specific CD4+T cells and CD8+T cells ( Fig.18 The results showed that the levels of specific CD4+T cells and CD8+T cells in the MEV group were significantly higher than those in the control group (P<0.001, P<0.01) ( Fig.18 C in the study). The ability of the vaccine to induce CD4+ T cell immune response was evaluated by ELISPOT detection of IFN-γ and IL-4 released by mouse spleen cells. Results ( Fig.19 Figures A, C) show that the levels of IFN-γ and IL-4 produced by the MEV group were significantly higher than those of the control group, and the differences were statistically significant (both P < 0.05). Significant differences were also observed between the MEV group and the PBS and SAG4 groups (both P < 0.001). The number of IFN-γ and IL-4 producing cells visualized by spots further illustrates the differences in the results ( Fig.19 Our studies have shown that MEV can effectively induce a strong cellular immune response in animal models.

[0114] 3.1 To evaluate the antigenicity of MEV, mice were immunized with MEV protein and serum was collected to measure the levels of specific antibodies. Fig.19 As shown in Figure E, compared with the control group, the level of specific antibodies in the MEV group was significantly increased two weeks after the first immunization (week 2) (P<0.01), and reached a peak two weeks after the third immunization (week 6). In addition, the serum specific antibody level of the MEV group at week 4 was significantly increased compared with the baseline level before MEV immunization (week 0, P<0.01) and the level two weeks after the first immunization (week 2, P<0.01). These results show that MEV can increase serum specific antibody levels and activate humoral immunity in animal models.

[0115] 3.2 To verify the potential protective effect of the vaccine developed in this study, we established an animal infection model using mice infected with Echinococcus multilocularis protoscolex. The results showed that there was a significant difference in the vesicle weight between the control group and the MEV group (P < 0.001), as shown in Fig.13 As shown in F. The average vesicle weight of the MEV group was lower than that of the control group, indicating that the vaccine exhibited a strong protective effect and effectively prevented infection with Echinococcus multilocularis protoscolex.

[0116] 3.3 In summary, our study used multiple immunoinformatics analyses to identify beneficial epitopes of EmTSP3 and EmTIP proteins. After comprehensive analysis of allergenicity, toxicity, antigenicity, and molecular binding properties, the selected antigenic epitopes were linked to adjuvants using appropriate linkers to obtain the MEV structure. The vaccine efficacy was subsequently evaluated by molecular docking and molecular dynamics simulations, and its interaction with TLR4 was assessed. MEV can effectively activate cellular and humoral immune responses, resulting in significant protection against parasitic infection. Taken together, these results indicate that MEV has the potential to become an efficient candidate vaccine for the prevention of Echinococcus multilocularis infection.

[0117] Fig.18 The results of the in vivo experiment are shown in Figure 1. (A) The mouse vaccination strategy flow chart shows the steps of the mouse vaccination experiment. (B) This is a representative flow cytometry strategy diagram for CD4+T cells and CD8+T cells. (C) This figure shows the statistical analysis of the percentage of CD4+T cells and CD8+T cells in the control group and the MEV group. The statistical method is the two independent sample T test (***P<0.001, **P<0.01).

[0118] like Fig.19 As shown, it is the result of in vivo experiment. Among them, (A) The number of T cells that can produce IFN-γ in spleen cell specimens after antigen stimulation. (B) Representative ELISPOT spot diagram of IFN-γ. (C) The number of T cells that can produce IL-4 in spleen cell specimens after antigen stimulation. (D) Representative ELISPOT spot diagram of IL-4. Statistical analysis was performed using one-way analysis of variance. * indicates P<0.01 compared with the control group. # indicates P<0.01 in the MEV group compared with the PBS group. & indicates P<0.01 in the MEV group compared with the SAG4 group. ns indicates P>0.05. (E) Dynamic detection of specific antibodies. * indicates P<0.01 compared with the control group. # indicates P<0.01 in the MEV group compared with the 2nd week. & indicates P<0.01 in the MEV group compared with the 4th week. (F) Cyst weight. The difference in cyst weight between the control group and the MEV group was statistically significant. (***P<0.001).

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A multi-epitope vaccine method for preventing alveolar echinococcosis, characterized in that: The following steps are involved: S1. Candidate antigens for vaccine design: Using E. multilocularis protein as the target antigen, the amino acid sequences of Em-TSP3 and Em-TIP proteins were obtained from the National Center for Bioinformation database; S2. Overall evaluation of candidate protein prediction: The transmembrane domains of proteins EmTSP3 and EmTIP were analyzed using the online server TMHMM; the single signal peptides of EmTSP3 and EmTIP proteins were predicted using the online prediction server SignalP-5.0; the main phosphorylation sites of proteins EmTSP3 and EmTIP were predicted using the NetPhos 3.1 server; The secondary structures of proteins EmTSP3 and EmTIP were determined using DNAstar software online server; the tertiary structures of proteins EmTSP3 and EmTIP were constructed using SWISS-MODEL; S3. Prediction of antigen epitopes: The online servers IEDB, NetMHCIIpan-4.0 and NetCTLpan-4.0 were used to predict the helper T lymphocyte epitopes and cytotoxic T lymphocyte epitopes of Em-TSP3 and Em-TIP, and HLA-a*1101, HLA-A*0201, HLA-A*0301, HLA-DRB1*0701, HLA-DRB1*1501 and HLA-DRB1*0301 were selected as prediction objects; linear B lymphocyte epitopes and B cell conformational epitopes were predicted using DNASTAR and IEDB servers; Antigenicity was assessed using the VaxiJen v2.0 platform, allergenic potential was determined using the AllergenFP v1.0 platform, and epitope toxicity was assessed using the ToxinPred platform; the results showed that highly antigenic, non-toxic, and non-allergenic TEs were screened from the top ten epitopes, and highly antigenic, non-toxic, and non-allergenic epitopes were screened from the top ten scoring BEs for vaccine construction; S4. Construction of vaccine proteins: covalently combining the above selected epitopes using specific linkages to form a complex vaccine sequence; S5. Prediction of secondary and tertiary structures of vaccine proteins: SOMPA predicts the secondary structure of MEV, uses the AlphaFold2 server to build a prediction model for the tertiary structure of MEV, and selects the model with the highest score; uses the GalaxyWEB server to refine the model structure, and finally selects the model with the highest GDT-HA score; S6. Molecular docking: Use energy minimization and spatial structural complementarity to predict ligand-receptor interactions at active sites; generate multiple models and select the top model for analysis; use PyMOL to visualize the selected docked structure in 3D and use Discovery Studio to create 2D interaction plots.

2. A multi-epitope vaccine method for preventing alveolar echinococcosis according to claim 1, characterized in that: The amino acid sequences of Em-TSP3 and Em-TIP proteins are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

3. A multi-epitope vaccine method for preventing alveolar echinococcosis according to claim 1, characterized in that: In S4, the constructed vaccine contained dominant epitopes, specifically 7 CTL epitopes, 7 Th epitopes, 5 LB epitopes, and 2 CB epitopes, with a total length of 436 amino acids.

4. A multi-epitope vaccine method for preventing alveolar echinococcosis according to claim 3, characterized in that: The molecular weight of the vaccine is 48.37 kDa and the molecular formula is: C 2131 H 3389 N 635 O 624 S 15 . The isoelectric point is 27.61.

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