Preferred epitope peptide of CCHFV Gc, tandem multi-epitope vaccine, expression vector and application

Abstract

The invention belongs to the technical field of microbial immunity and DNA vaccines, and particularly relates to a preferred epitope peptide of a Crimean-Congo hemorrhagic fever virus envelope glycoprotein C terminal (CCHFV Gc), a tandem multi-epitope vaccine, an expression vector and application. The preferable epitope peptide is composed of any one amino acid sequence as shown in SEQ ID NO.1-SEQ ID NO.21, and the preferable epitope peptide has high affinity, strong immunogenicity and interspecific amp; and an intraspecific conservative preferred epitope. Preferably epitopes are connected in series through a linker to synthesize a series multi-epitope vaccine, the series multi-epitope vaccine enters cells to be expressed to form recombinant protein, then T lymphocytes are activated, and cellular immune response is induced. The in-vivo vaccine evaluation of animals is carried out by using the tandem multi-epitope vaccine, and it is proved that the vaccine can effectively induce anti-CCHFV specific cellular immune response and the application safety in BALB / c mice. The invention is helpful for understanding the immunobiology of CCHFV Gc and perfecting the epitope vaccine design for the virus in the future.

Description

Technical Field

[0001] This invention belongs to the field of microbial immunology technology, specifically relating to a preferred epitope peptide of the C-terminus of the envelope glycoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV Gc), a tandem multi-epitope vaccine, an expression vector, and its applications. Background Technology

[0002] Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread pathogen, increasingly prevalent due to tick-borne transmission. CCHFV can cause Crimean-Congo hemorrhagic fever (CCHF) with a case fatality rate of up to 40%, and there is currently no specific vaccine. CCHFV is classified as a Biosafety Level 4 (BSL-4) pathogen and is listed as a high-priority pathogen by the US National Institutes of Health (NIH / NIAID) and the World Health Organization (WHO).

[0003] CCHFV is a member of the genus Orthovirus in the family Neloviridae, possessing a three-sense negative-strand RNA genome. The M segment encodes a glycoprotein precursor (GPC), which cleaves to form Gn, Gc, etc. Current data indicate that both Gn and Gc can be recognized by immune cells and provide immune protection, with an immune response against Gc leading to prolonged survival. Furthermore, Gc-mediated immune responses have been confirmed in infected individuals. Therefore, Gc is considered the most valuable immune target against CCHFV.

[0004] Cytotoxic T lymphocytes (CTLs) restricted by major histocompatibility complex (MHC) class I molecules are important effector cells against viral infections. T cell recognition of epitopes and induction of immune responses play a crucial role in the body's immune response. In virus-host symbiosis, viruses can interfere with the killing activity of CTLs in various ways, such as modifying or reducing protein sequences or immune epitopes that T cells can recognize, thereby evading host clearance. These interference patterns impair the host's immune homeostasis and immunity. Only high-affinity peptides that can bind to MHC class I molecules can activate cells and trigger an immune response. Therefore, high-affinity epitopes are essential for T cell immune responses against CCHFV. Furthermore, conserved epitopes are a key factor in "herd immunity" against widely mutated viral strains; therefore, in virus-host symbiosis, conserved epitopes can provide long-term immune protection for the host.

[0005] Although a series of general methods have been developed for predicting and screening viral antigenic epitopes, such as a combination of computational tools based on MHC binding affinity, immunogenicity, and conservation, the sporadic nature and lack of systematic research on the Crimean-Congo hemorrhagic fever outbreak have limited the exploration of its pathogenesis, host-virus interactions, and immune response mechanisms. The effectiveness of existing general epitope screening methods when directly applied to Crimean-Congo hemorrhagic fever virus is also significantly limited. It is currently difficult to obtain preferred epitopes that possess high affinity, strong immunogenicity, interspecies conservation, and the ability to effectively stimulate cellular immune responses, thus restricting the treatment and containment of CCHFV.

[0006] Therefore, it is urgent to analyze the host's immune protection mechanism against CCHFV by examining its virological and immunological characteristics, and to identify key immune-optimal epitope peptides, so as to lay the foundation for developing more effective CCHFV treatment and containment strategies. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a preferred epitope peptide at the C-terminus of the envelope glycoprotein of Crimean-Congo hemorrhagic fever virus, a tandem multi-epitope vaccine, and its application. The preferred epitope peptide and the tandem multi-epitope vaccine have demonstrated good cellular immunogenicity in various mouse models.

[0008] The specific technical solution provided by this invention is as follows: In a first aspect, the present invention provides a preferred epitope peptide of CCHFV Gc, the amino acid sequence of which is shown in any one of SEQ ID NO.1 to SEQ ID NO.21.

[0009] SEQ ID NO.1: TSLSIEAPW; SEQ ID NO.2: LEERTGISW; SEQ ID NO.3: MYSPVFEYL; SEQ ID NO.4: LHKEWPHSR; SEQ ID NO.5: WPHSRNWRC; SEQ ID NO.6: RNWRCNPTW; SEQ ID NO.7: DVKDLFTDY; SEQ ID NO.8: FVKWKVEYI; SEQ ID NO. NO.9: VEYIKTEAI; SEQ ID NO.10: TEAIVCVEL; SEQ ID NO.11: RFNLGPVTI; SEQ ID NO.12; IEEGFFDLM; SEQ ID NO.13: EPHFNTSWM; SEQ ID NO.14: MGDWPSCTY; SEQ ID NO.15: EPDELTVHV; EQ ID NO.16: SLCFYIVER; SEQ ID NO.17: QPQSILIEH; SEQ ID NO.18: ILIEHKGTI; SEQ ID NO.19: MLSGIFGNV; SEQ ID NO.20; APFILLILF; SEQ ID NO.21: RRTRGLFKY.

[0010] The 21 preferred epitope peptides provided by this invention not only possess the comprehensive advantages of high affinity, strong immunogenicity, broad-spectrum conservation, and low safety risk, but also successfully overcome a series of key challenges in existing epitope screening technologies, such as narrow HLA coverage, easy escape, low prediction reliability, unclear safety, and lack of structural basis, through a systematic strategy of multi-algorithm cross-validation, multi-dimensional evaluation, and experimental verification. This provides core candidate molecules and a reliable design foundation for developing safe, efficient, and broad-spectrum CCHFV epitope vaccines. The preferred epitope peptides provided by this invention exhibit superior immunogenicity compared to conventional epitope peptides in the field that only possess high affinity.

[0011] Secondly, this invention provides a tandem multi-epitope vaccine comprising preferred epitope peptides of the CCHFV Gc. The tandem multi-epitope vaccine is obtained by linking sixteen peptide segments on the CCHFV Gc containing the amino acid sequences described in SEQ ID NO.1 to SEQ ID NO.21, as shown in Table 7, via linkers. This tandem multi-epitope vaccine, by integrating multiple technical advantages such as multi-target targeting, broad coverage, anti-escape, and strong immunity, successfully overcomes the key bottlenecks in existing CCHFV vaccine development, including single epitope screening, strong HLA restriction, viral mutation, and insufficient immune response. Furthermore, it eliminates the sensitization and toxicity risks associated with single epitopes, further improving safety. This provides an innovative molecular entity and technical pathway for developing safe, efficient, and broad-spectrum CCHFV epitope vaccines.

[0012] Preferably, the amino acid sequence of the tandem multi-epitope vaccine is shown in SEQ ID NO.22.

[0013] Preferably, the nucleotide sequence of the tandem multi-epitope vaccine is shown in SEQ ID NO.24.

[0014] Thirdly, the present invention provides an expression vector pVAX1-CCHFV-Gc21p, comprising the nucleotide sequence shown in SEQ ID NO.24.

[0015] Preferably, the expression vector pVAX1-CCHFV-Gc21p is prepared by the following method: synthesizing the nucleotide sequence shown in SEQ ID NO. 24, then cloning it into the pVAX1 vector, transforming it into bacteria, and obtaining the recombinant plasmid through screening and verification. Preferably, the bacteria are Escherichia coli.

[0016] Fourthly, the present invention provides the use of the preferred epitope peptide of CCHFV Gc, the tandem multi-epitope vaccine, or the expression vector in the preparation of products for the prevention or treatment of CCHFV infection.

[0017] Preferably, the product is obtained by combining a preferred epitope peptide of the CCHFV Gc, a tandem multi-epitope vaccine, or an expression vector as the active substance with a vaccine adjuvant. The vaccine adjuvant is an aluminum adjuvant, an oil-emulsion adjuvant, or a cytokine adjuvant.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention, for the first time, successfully predicted and validated the preferred immunogenic epitope peptides of the Crimean-Congo hemorrhagic fever virus (CCHFV) glycoprotein using the ELISpot assay. These peptides, composed of the amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 21, exhibited significant protective properties in various viral isolates worldwide. This invention further explored the binding affinity and immunogenicity of these preferred epitope peptides to MHC class I molecules, providing important evidence for a deeper understanding of the immunobiology of CCHFV. Based on these findings, this invention offers important reference for the development of CCHF vaccines and also provides valuable reference and guidance for research in other virological fields.

[0019] This invention presents a tandem multi-epitope vaccine designed based on a precisely screened epitope library. By integrating core advantages such as multi-target, broad coverage, anti-escape, and strong immunity, it systematically solves the key technical challenges currently faced in CCHFV vaccine development, including strong HLA restriction, easy viral escape, and single and insufficient immune response. Furthermore, it eliminates the risks of sensitization and toxicity associated with single epitopes, further improving safety. This lays a crucial molecular foundation and design paradigm for developing next-generation, highly effective, broad-spectrum, and safe CCHFV epitope vaccines. Attached Figure Description

[0020] Figure 1 This represents the interaction between CCHFV Gc9 peptide and pan-MHC-I subtypes. Red indicates strong affinity, and blue indicates weak affinity.

[0021] Figure 2 This is a simulation of the interaction between the CCHFV Gc9 peptide epitope and the MHC molecule. The background shows a thermal map of the maximum binding free energy of molecular docking.

[0022] Figure 3 This is a scatter plot analysis of potential toxicity and sensitization risks.

[0023] Figure 4 This shows the distribution of toxicity scores.

[0024] Figure 5 The results show the distribution of sensitization scores.

[0025] Figure 6Variation analysis of CCHFV Gc epitopes. A, WebLogo diagram of variation analysis of preferred epitope peptides. B, Mutation frequency and impact range of high-frequency mutation sites. C, Cluster analysis of the difference in MHC-I affinity before and after the aa1257-aa1265 mutation, where blue indicates mutations leading to decreased affinity and red indicates mutations leading to increased affinity. D, Scatter plot of the difference in MHC-I affinity before and after the aa1257-aa1265 mutation, where each data point in the scatter plot corresponds to an analyzed epitope, and points within the gray area indicate that the epitope has high affinity.

[0026] Figure 7 The results represent the ELISpot assay for 21 high-affinity, highly immunogenic epitopes that are conserved both interspeciesly and intraspeciesly. Results are presented as per 10 6 The average number of speckle units (SFU) formed by each spleen cell is represented. The vertical axis represents the difference between the experimental group and the control group (SUFs / 10). 6 (Spleen cells), the horizontal axis represents the different peptides used for stimulation.

[0027] Figure 8 These are the results of pathological observation.

[0028] Figure 9 In the diagram, A represents the structure of the expression vector pVAX1-CCHFV-Gc21p, and B represents the results of protein-level validation using Western blotting. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0030] Example 1: Screening of preferred C-terminal epitope peptides of Crimean-Congo hemorrhagic fever virus envelope glycoprotein Step 1: Amino acid sequence retrieval The glycoprotein of Crimean-Congo hemorrhagic fever virus (GP, accession number: AF467768.2) was obtained from the NCBI GenBank database and used as input for various bioinformatics tools for epitope prediction, conservation analysis, molecular docking, and experiments.

[0031] Step 2: Epitope Prediction For H2-Db, HA-Dd, H2-Kb, H2-Kd, H2-Kk, and H2-Ld epitope prediction, network-based tools are recommended, such as the IEDB method (http: / / tools.iedb.org / mhci / ), SMMPMBEC from the immune epitope database (http: / / tools.immuneepitope.org / mhci / ), NetMHCpan 4.1 (http: / / www.cbs.dtu.dk / services / NetMHCpan / ), SYFPEITHI (http: / / www.syfpeithi.de / bin / MHCServer.dll / EpitopePrediction.htm), and Rankpep (http: / / imed.med.ucm.es / Tools / rankpep.html). Select the top 2% of predicted epitopes from the total prediction results. Finally, epitopes predicted by at least three prediction tools are selected for further in-depth analysis; that is, in this invention, if a specific epitope appears more than or equal to three times in the analysis results of the above five tools, then the epitope is included in the next step of analysis.

[0032] Human major leukocyte antigen (HLA)-I supertype allele epitopes, such as HLA-A1 (HLA-A*2601, -A*3001, -A*3002), HLA-A2 (HLA-A*0201, -A*0203, -A*0206, -A*6802), HLA-A3 (HLA-A*0301, -A*1101, -A*3001, -A*3101, -A*3301, -A*6801), and HLA-A24 (HLA-A*2301, -A*2402). The HLA-B7 (HLA-B*0702, -B*3501, -B*5101, -B*5301), HLA-B8 (HLA-B*0801), HLA-B15 (HLA-B*1501, -A*0101), HLA-B44 (HLA-B*4001, -B*4402, -B*4403), and HLA-B58 (HLA-B*5701, -B*5801) prediction tools were used to predict the supertypes of HLA-B7, HLA-B8, HLA-B15 (HLA-B*1501, -A*0101), HLA-B44 (HLA-B*4001, -B*4402, -B*4403), and HLA-B58 (HLA-B*5701, -B*5801), covering more than 97% of the population. Finally, the top 2% epitopes of each predicted supertype were selected and sequentially studied.

[0033] For the mouse H-2 subtype, epitopes appearing three or more times in five prediction software programs were considered dominant epitopes. For the human HLA-I subtype, all nine peptides appearing in the top 2% of prediction results were considered dominant epitopes. As described above, bioinformatics analyses were performed using various computational tools to predict potential MHC-I epitopes with different binding affinities across CCHFV Gc. Alleles from mice (H2-Db, HA-Dd, H2-Kb, H2-Kd, H2-Kk, and H2-Ld) and major HLA-I subtypes were analyzed. Statistical analysis showed that 94 dominant epitopes were identified in the human HLA-I haplotype and 37 dominant epitopes were identified in the mouse H2 haplotype. Tables 1 and 2 show the number of dominant epitopes identified by the prediction tools. After recalculation to exclude duplicate nine peptides, we obtained 41 dominant epitopes in the H-2 subtype and 133 dominant epitopes in the HLA-I subtype. In the H-2 subtype, H2-Kd and H2-Kk have the most dominant epitopes, with 8 and 10 respectively. In the HLA-I subtype, HLA-A3 and HLA-B44 have the most dominant epitopes, with 21 and 25 respectively, while other haplotypes have fewer dominant epitopes.

[0034] Table 1. Number of dominant epitopes in the H-2 subtype of CCHFV Gc mice Note: " / " indicates that there is no data for this item.

[0035] Table 2 Number of dominant epitopes in CCHFV Gc human HLA-I subtype Note: " / " in the table indicates that the item does not exist.

[0036] Step 3: Immunogenetic Analysis 3.1 Cluster analysis of 9-peptide epitope affinity In epitope vaccine design, MHC binding to peptides is the most selective factor in determining T-cell epitopes; therefore, predicting peptide-MHC binding is fundamental to T-cell epitope prediction. Among mainstream T-cell epitope prediction tools, we selected the aforementioned five bioinformatics platforms. These platforms are based on a combination of sequence patterns, structures, pattern matrices, quantitative affinity matrices, and artificial neural network models, as well as binding prediction, supertype, TAP binding, and proteasome cleavage functions. Using the models and algorithms of different prediction tools, we predicted comprehensive data on the binding affinity of different MHC class I epitopes to CCHFV Gc. Based on the prediction results, we analyzed and statistically analyzed the alleles of mouse H-2 subtypes (H2-Db, H2-Dd, H2-Kb, H2-Kd, H2-Kk, and H2-Ld) and major human HLA class I subtypes. Using the algorithm, we evaluated that CCHFV Gc can generate 565 different MHC class I antigenic epitopes and obtained epitope affinity scores based on ranking values.

[0037] Figure 1 Hierarchical clustering analysis of the binding affinity of all 9 peptides of CCHFV Gc to MHC-I molecules. The direction of the arrows indicates the affinity from strong to weak. Figure 1 The horizontal coordinates at the bottom, from left to right, are: HLA-A68:01, HLA-A31:01, HLA-A33:01, HLA-A30:01, HLA-A3:01, HLA-A11:01, HLA-A02:03, HLA-A02:01, HLA-A02:06, HLA-A68:02, HLA-B51:01, H-2-Kb, H-2-Db, H-2-Dd, HLA-B08:01, HLA-B07:0 2. H-2-Ld, HLA-A01:01, HLA-A26:01, HLA-B35:01, HLA-B53:01, HLA-A30:02, HLA-B15:01, HLA-A32:01, HL A-B57:01, HLA-B58:01, H-2-Kd, HLA-A23:01, HLA-A24:02, HLA-B44:02, HLA-B44:03, HLA-B40:01, H-2-Kk.

[0038] The 33 MHC class I subtypes were divided into three clusters, including the HLA-I subtype group and two cross-reactivity groups (HLA-major and H2-major), indicating the similarities and differences in antigen presentation capabilities among different MHC molecules. Within the HLA-I subtype group, the scores of HLA-A3 (-A6801, -A3101, -A3301, -A3001) were similar to those of HLA-A2 (-A0201, -A0202, -A0206, and -A6801) and higher than those of other HLA-A1 superfamily members, suggesting the presence of HLA-A3-like characteristics during CCHFV Gc presentation. The antigen presentation results for HLA-B7 (-B3501, -B5301) and HLA-A1 (-A2601, -A0101) were similar; the antigen presentation results for HLA-A1 (-A3002), HLA-B15 (-B1501), HLA-A3201, and HLA-B58 (-B5701, -B5801) were also similar. In the cross-reactivity group (H-2 major), the H2-Ld score was similar to that of HLA-B7 (-B0702) and HLA-B8 (-B0801); H2-Db, H2-Dd, and H2-Kb were presented together in an H-2-specific manner. However, in the cross-reactivity group (HLA major), the H2-Kk score was similar to that of HLA-B44 (-B4001, -B4402, -B4403). The similarity in the presentation of epitopes in the above-mentioned MHC molecules suggests that these genotypes exhibit cross-genotype, cross-population, cross-regional, and even cross-species cross-immunoreactivity.

[0039] 3.2 Immunogenicity Analysis: High-affinity peptides may not adequately induce an immune response. In addition to immunoreactivity, antigens should also be immunogenic. Therefore, we performed immunogenicity analysis on all GP 9-peptide epitopes. The immunogenicity of the 9-peptide epitopes was calculated using IEBDB (Class Immunogenicity (iedb.org)). A score >0.5 was considered positive, and otherwise, the peptide was not considered immunogenic. The results showed that 54 of the 94 human high-affinity epitopes were considered highly immunogenic, and 23 of the 37 mouse epitopes were considered highly immunogenic.

[0040] 3.3 Conservatism Analysis To ensure high conservation and stability of epitopes, we used the BLASP (BLAST: Basic LocalAlignment Search Tool (nih.gov)) tool to analyze the evolutionary conservation of major epitopes screened in the initial stage to protect the predicted high-affinity 9-peptide. The interspecific conservation criteria, except for CCHFV (taxid:1980519), were all from the genus *Narrovirus* (taxid:1980517); the intraspecific conservation criteria were CCHFV (taxid:1980519), except for CCHFV IbAr10200 strain (taxid:652961). In the analysis results, the conservation E-value was less than 10. -5 Conserved peptide sequences between CCHFV and humans (taxid:9606) or mice (taxid:10088) were excluded. Therefore, based on conservation, major epitopes can be classified into four categories: interspecific-intraspecific, interspecific-intraspecific+, interspecific+intraspecific-, and interspecific+intraspecific+, where "+" indicates a conserved epitope and "-" indicates a non-conserved epitope. Multiple sequence alignments determined the conservation status of the dominant CCHFV Gc epitope. Based on the conservation analysis of pan-MHC-I restriction epitopes, Figure 2 The statistical results of the conservation analysis of all dominant epitopes are listed. Figure 6 Variation analysis of CCHFV Gc epitopes.

[0041] The statistical results of the analysis of dominant epitopes of pan-MHC class I are as follows: Figure 7 The results show that pan-HLA I-restricted dominant epitopes are more conserved than H-2-restricted dominant epitopes. Statistically, human HLA-restricted high-affinity 9-peptides are much more conserved than mouse epitopes, especially HLA-B7, which has the most (7 epitopes). Because the algorithms required to identify H2-restricted epitopes are more complex, only some interspecific and intraspecific conserved epitopes were detected in H2-Kd, H2-Kk, and H2-Ld. Table 3 shows the interspecific and intraspecific conservation results of CCHFV dominant epitopes.

[0042] Table 3. Conservation of dominant epitopes of CCHFV between and within species (unit: epitopes) Example 2: Validation of the immunogenicity and application prospects of the preferred C-terminal epitope peptide of the envelope glycoprotein of Crimean-Congo hemorrhagic fever virus Step 1: Peptide-MHC molecular docking: In T-cell-mediated immune responses, epitopes are mostly short peptides lacking well-defined three-dimensional structures. The structural basis of MHC antigen presentation provides valuable insights into antigen peptide design, enabling more effective induction of T-cell-mediated responses. Therefore, structural biology methods can determine the structures of antigen proteins and antigen-antibody complexes. Following the aforementioned benchmark screening, we identified dominant epitopes with high immunoreactivity and immunogenicity and analyzed their evolutionary conservation. In structural biology, we used docking modeling of dominant epitopes with the molecular conformation of each MHC-I subtype to obtain modeling and docking thresholds for pan-MHC-I and dominant epitopes.

[0043] We used MHC-I alleles (HLA-A1 (HLA-A0206 (3OXR), HLA-A0101 (4NQV)), HLA-B7 (HLA-B0702 (5EO1), HLA-B3501 (1A9E), HLA-B5101 (1E28), HLA-B5301 (1A1N)), HLA-B8 (HLA-B0801 (4QRP)), HLA-B15 (HLA-B1501 (1XR9)), HLA-B44 (HLA-B4402 (3KPL)), H2-Ld (6L9M), H2-Kb (6JQ3), and H2-Db (1JUF)) for docking simulation. The simulations were performed using PEPDOCK 2.0. Inputting a 9-peptide dominant epitope sequence into (hust.edu.cn) allows for blind protein-peptide docking using the 9-peptide dominant epitope sequence, allele interactions, and a hierarchical algorithm. This yields docking simulation models and thresholds for analysis and screening. The resulting modeling and threshold docking tests produced 100 simulation models, with the top 10 selected as high-confidence docking results.

[0044] Molecular docking simulations and functional scoring were performed using HPEPDOCK 2.0 to estimate the binding affinity between the major epitope and the 3D structure of the MHC-I molecule. After molecular docking, a binding model was generated using the 3D structure and the binding conformation of the major epitope, depicting the cluster of binding sites on the MHC-I molecule. Following comprehensive evaluation, seven epitopes with high affinity, strong immunogenicity, and conservation in both inter- and intra-species variation were selected; these epitopes represent the most representative candidates among the 21 identified epitopes. The simulation results for molecular docking and binding sites are shown below. Figure 2As shown. Each docking model provides the first 10 epitopes of the MHC-I molecule and their corresponding binding models. The main docking epitopes include LEERTGISW, MYSPVFEYL, VEYIKTEAI, RFNLGPVTI, APFILLILF, FVKWKVEYI, and RNWRCNPTW. Figure 2 In the process, 10 optimal binding conformations were simulated through docking calculations, and the maximum binding free energy of the binding reflected the energy of the optimal ligand-receptor interaction.

[0045] Visualized 3D models of molecular docking cannot fully represent the binding affinity strength between the same MHC and different epitopes. Therefore, we performed cluster analysis on the states with the lowest molecular binding energy (i.e., optimal docking) to present the docking results in a more intuitive way. Figure 2 (Background section). Comparison of epitopes showed that, in their respective binding conformations, the algorithm-defined epitopes RNWRCNPTW and FVKWKVEYI exhibited superior ligand-receptor affinity with MHC-I molecules. Regarding comparisons between MHC-I molecules, for most epitopes, the optimal binding conformations of the three alleles HLA-B*0702, HLA-B*4402, and H2-Db showed weaker binding affinity than other groups. Furthermore, it was found that, according to the algorithm, other groups exhibited superior binding affinity compared to these three allele groups.

[0046] Step 2: Peptide Synthesis Based on the predicted peptide sequences of SEQ ID NO.1 to SEQ ID NO.21, the corresponding peptides were synthesized. All peptides used in the experiment were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., with a purity of 95%, totaling 10 mg. After centrifugation, the synthesized peptide powder was first dissolved in 20 μL of dimethyl sulfoxide (DMSO), then mixed with 480 μL of sterile ddH2O and stored at -20℃ for later use.

[0047] Step 3: Immune Validation Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of specific antibodies against Crimean-Congo hemorrhagic fever virus (CCHFV) Gc protein in immune serum. The specific steps were as follows: Synthetic Gc protein peptides were used as solid-phase coating antigens, with a concentration of 10 µg / mL and a density of 100 µL per well. The wells were then blocked with PBST solution containing 1% bovine serum albumin. Mouse serum samples collected on day 56 after the third immunization (i.e., 2 weeks post-immunization) were serially diluted using a universal antibody diluent, preferably at a dilution ratio of 1:400. 100 μL of each diluted sample was added to the blocked antigen-coated wells and incubated at 37°C for 1 hour. Positive and negative control wells were included: the positive control was coated with 10 μg / mL pVAX-Gc, ​​and the negative control was coated with 10 μg / mL BSA (with a background absorbance of 0.05-0.08). Each sample was tested in triplicate (replication 1, replication 2, and replication 3). HRP-labeled goat anti-mouse antibody and goat anti-rabbit antibody were used as secondary antibodies in the assay. Both primary and secondary antibodies were diluted using a universal antibody diluent. The result was considered statistically significant if the absorbance value measured at 450 nm was greater than 0.1 and the positive / negative ratio was greater than 2.1.

[0048] Results: BALB / c mice: APFILLILF (epitope 20) induced the strongest antibody response, consistent with ELISA results; FVKWKVEYI (epitope 8) was the second strongest, as shown in Table 4. C57BL / 6 mice: FVKWKVEYI (epitope 8) induced the strongest antibody response; LEERTGISW (epitope 2) showed a significantly enhanced response, as shown in Table 5. Conserved dominant epitopes: LEERTGISW, MYSPVFEYL, and RFNLGPVTI all performed well in both strains; Positive / negative controls: Positive OD > 1.2, negative OD < 0.08, signal-to-noise ratio > 10, indicating experimental validity; Data variation: CV% controlled within 5-7%, meeting the repeatability requirements of ELISA experiments.

[0049] Table 4. ELISA results of BALB / c mice (H-2d haplotype) Table 5. ELISA results of C57BL / 6 mice (H-2b haplotype) Note: In Tables 4 and 5, "-" indicates a negative result.

[0050] Step 4: Toxicity and Allergenicity Analysis The potential toxicity and sensitization of peptides may affect their efficacy in disease treatment and prevention. We used computational analysis tools to assess the potential toxicity and sensitization of selected epitopes. Toxicity screening was performed using the ET + MERCI model in ToxinPred 3.0, and peptides with analysis scores higher than the non-toxicity threshold of 0.7 were classified as potentially toxic peptides. Simultaneously, sensitization analysis was performed using the MERCI + BLAST model in AlgPred 2.0, and peptides with scores exceeding the non-sensitization threshold of 0.4 were identified as potential allergens.

[0051] In the first stage, we screened 21 preferred epitopes with high affinity and interspecies and intraspecies conservation. To ensure the safety of these pre-screened Gc MHCI 9 peptides as vaccine candidates and to eliminate potential toxicity and sensitization risks in subsequent vaccine design, we performed toxicity and sensitization analyses on the 21 Gc 9 peptides screened by bioinformatics network algorithms. The scores given in the analysis results include sequence header information, ML score, MERCI score, BLAST score, mixed score, and comprehensive analysis of the query sequence (Table 6). The results show that, under the default thresholds (negative toxicity threshold of 0.7; negative sensitization threshold of 0.4), among the previously screened dominant epitopes, WPHSRNWRC, RNWRCNPTW, TEAIVCVEL, MGDWPSCTY, and SLCFYIVER were identified by the algorithm as having toxicity risks; while in the AlgPred 2.0 algorithm analysis of Gc, the dominant epitopes VEYIKTEAI and QPQSILIEH indicated sensitization risks. However, risk analysis showed that TSLSIEAPW, MYSPVFEYL, and MLSGIFGNV had lower risk factors for toxicity and sensitization than other dominant epitopes.

[0052] To further eliminate the potential toxicity and sensitization risks of single peptide fragments, this invention employs a peptide tandem strategy to optimize and reconstruct preferred epitopes. Taking SEQ ID NO.22 (test38) as an example, 16 peptide fragments containing 21 epitope peptides (SEQ ID NO.1~SEQ ID NO.21) on CCHFV Gc, as shown in Table 7, were randomly arranged in different orders and then ligated using AAY to obtain 225 sequences, forming random tandem sequence groups (test38-263). Among these, SEQ ID NO.22 (test38) retains the amino acid sequence of the original CCHFV Gc protein to the greatest extent possible. Calculation and analysis verified that this strategy demonstrates a significant improvement in safety: Figure 3As shown in the scatter plot analysis, the toxicity and sensitization scores of the random tandem sequence group (test38-263) shifted to the lower left compared to the 21 peptide groups (21 preferred epitopes of SEQ ID NO.1~SEQ ID NO.21) and the 16 peptide groups (see Table 7, used to connect and form the amino acid sequence shown in SEQ ID NO.22). The data points were concentrated in the safe region of low toxicity (<0.7) and low sensitization (<0.4), far from the high-risk quadrants. The violin plot distribution further confirmed that the peak value of the toxicity score distribution of the tandem peptides was significantly lower than the threshold of 0.7. Figure 4 The sensitization score distribution also showed a significant improvement, with its peak value far below the threshold of 0.4, indicating a substantial decrease in overall risk level. Figure 5 These results strongly demonstrate that epitope tandem design can not only effectively avoid the residual risks of individual dominant epitopes (such as WPHSRNWRC and VEYIKTEAI), but also achieve synergistic risk reduction through sequence reconstruction. This allows epitopes such as TSLSIEAPW, MYSPVFEYL, and MLSGIFGNV to maintain high affinity while achieving superior safety profiles, providing safer and more reliable candidate molecules for subsequent vaccine development. These more accurate and safer dominant epitopes have significant application potential in various designs, such as epitope vaccines. Figure 3 and Figure 4 The results also demonstrate that the amino acid sequence shown in SEQ ID NO.22 of this invention has higher sensitization and safety than other randomly tandem peptides.

[0053] Table 6. Comprehensive Assessment Table of Dominant Epitope Toxicity and Allergenicity Example 3: Construction and application of tandem multi-epitope vaccines Step 1: Construction of a tandem multi-epitope vaccine To address the key technical challenges in current CCHFV vaccine development, such as strong HLA restriction, easy viral escape, and insufficient and singular immune response, and to eliminate the risks of sensitization and toxicity associated with single epitopes, this embodiment designs a tandem multi-epitope vaccine based on 16 peptides on the CCHFV Gc containing the aforementioned 21 preferred epitopes, as shown in Table 7. To maintain normal protein folding, AAY is used to connect the various peptides. The amino acid sequence of the tandem multi-epitope vaccine is shown in SEQ ID NO. 22.

[0054] TSLSIEAPWAAYLEERTGISWAAYMYSPVFEYLAAYLHKEWPHSRNWRCNPTWAAYDVKDLFTDYAAYFVKWKVEYIKTEAIVCVELAAYRFNLGPVTIAAYIEEG FFDLMAAYEPHFNTSWMAAYMGDWPSCTYAAYEPDELTVHVAAYSLCFYIVERAAYQPQSILIEHKGTIAAYMLSGIFGNVAAYAPFILLILFAAYRRTRGLFKY.

[0055] Table 7. 16 peptides involved in the 21 preferred epitopes on CCHFV Gc Further human-biased codon optimization was performed on the amino acid sequence shown in SEQ ID NO.22 using the JCat tool. After optimization, the codon adaption index (CAI) increased to 0.95. The optimized sequence length is 633 base pairs. The nucleotide sequence of the tandem multi-epitope DNA vaccine is shown in SEQ ID NO.23.

[0056] .

[0057] Step 2: Preparation and identification of expression vector pVAX1-CCHFV-Gc21p EcoRI and KpnI restriction sites were introduced upstream of the sequence SEQ ID NO.23, and XhoI and XbaI restriction sites were introduced downstream. A Kozak sequence and a start codon ATG were added to the N-terminus, and a 3×FLAG tag was appended to the C-terminus to facilitate subsequent identification of vaccine antigen expression, resulting in the nucleotide sequence shown in SEQ ID NO.24. The nucleotide sequence of SEQ ID NO.24 was synthesized according to a standard gene based on the designed sequence and inserted between the EcoRI and XhoI sites of the pVAX1 vector containing kanamycin resistance. This was then transformed into E. coli TOP10 strains and delivered as the expression vector pVAX1-CCHFV-Gc21p. Figure 9 As shown in A, this constitutes a tandem multi-epitope DNA vaccine. SEQ ID NO.24:

[0058] GAATTCGGTACCGCCGCCACCATGACCAGCCTGAGCATCGAGGCCCCCTGGGCCGCCTACCTGGAGGAGCGCACCGGCATCAGCTGGGCCGCCTACATGTACAGCCCCGTGTTCGAGTACCTGGCCGCCTACCTGCACAAGGAGTGGCCCCACAGCCGCAACTGGCGCTGCAACCCCACCTGGGCCGCCTACGACGTGAAGGACCTGTTCACCGACTACGCCGCCTACTTCGTGAAGTGGAAGGTGGAGTACATCAAGACCGAGGCCATCGTGTGCGTGGAGCTGGCCGCCTACCGCTTCAACCTGGGCCCCGTGACCATCGCCGCCTACATCGAGGAGGGCTTCTTCGACCTGATGGCCGCCTACGAGCCCCACTTCAACACCAGCTGGATGGCCGCCTACATGGGCGACTGGCCCAGCTGCACCTACGCCGCCTACGAGCCCGACGAGCTGACCGTGCACGTGGCCGCCTACAGCCTGTGCTTCTACATCGTGGAGCGCGCCGCCTACCAGCCCCAGAGCATCCTGATCGAGCACAAGGGCACCATCGCCGCCTACATGCTGAGCGGCATCTTCGGCAACGTGGCCGCCTACGCCCCCTTCATCCTGCTGATCCTGTTCGCCGCCTACCGCCGCACCCGCGGCCTGTTCAAGTACGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGTAACTCGAGTCTAGA。

[0059] The expression vector pVAX1-CCHFV-Gc21p was verified by electrophoresis, showing a single vector fragment with a size consistent with the theoretical size.

[0060] The integrity of the expression vector pVAX1-CCHFV-Gc21p sequence was verified by sequencing. Purification of the expression vector pVAX1-CCHFV-Gc21p was performed using the TIANGEN plasmid large-scale extraction kit (Beijing), and the purified plasmid was stored at -20℃. The accuracy of the plasmid was confirmed by agarose gel electrophoresis and Sanger sequencing. Further protein extraction after transfection into HEK293T and HeLa cells was performed for protein level verification using Western blotting. Figure 9 As shown in Figure B, the accuracy of the plasmid is confirmed.

[0061] Step 3: Immunization Program Eight-week-old female BALB / c and SJL mice free of specific pathogens were purchased from the Experimental Animal Center of Air Force Medical University and randomly divided into two groups (n=6 per group): an experimental group and a control group. Mice in the experimental group were inoculated with 30 μg of the expression vector pVAX1-CCHFV-Gc21p into the gastrocnemius muscle, while mice in the control group were inoculated with an equal amount of sterile ddH2O. The mice were immunized three times via injection into the gastrocnemius muscle on days 1, 21, and 42. Serum samples were collected from the tail vein two weeks after each immunization. Blood and organ collection was performed on day 56. All animals were well cared for, and all procedures followed standard animal experimental protocols.

[0062] Step 4: ELISpot test verification: Synthetic CCHFV Gc 9 peptide (SEQ ID NO.1~SEQ ID NO.21) was diluted with sterile PBS to a final concentration of 20 μg / mL for ELISpot assays. IL-2 specific capture antibody was first diluted with sterile PBS to a final concentration of 5 μg / mL, then added to each well of a 96-well plate and incubated overnight at 4°C. The 96-well plates were then blocked for 2 hours at room temperature with RPMI 1640 medium containing 10% fetal bovine serum. After lysing red blood cells from collected mouse spleen cell suspensions, the spleen cells were diluted with RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin (hereinafter the same), and added to each well at a concentration of 1×10⁶ cells / well. 6Add one number of spleen cells to each well of a 96-well plate. Add an equal volume of diluted 9- or 15-peptide solution and incubate at 37°C with 5% CO2 for 24 hours. The negative control was RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-antibody solution; the positive control was 10 μg / mL concanavalin A (Con A). After incubation, wash the plates with double-distilled water and PBST. After washing, add 100 μL of biotinylated anti-IL-2 antibody to each well (final concentration 2 μg / mL) and incubate at room temperature for 2 hours. After washing with PBST, add streptavidin-horseradish peroxidase and incubate at room temperature for 1 hour. After washing with PBST, add AEC substrate for color development and wash with water to terminate the reaction. After the 96-well plate has air-dried, count the spots using a plate counter. All results are shown as per 10-10. 6 The average number of spotted forming units (SFU) per spleen cell. Bar charts were generated using GraphPad Prism 8.0.1. Results are as follows... Figure 7 The text is displayed as every 10 6 The average number of spotted forming units (SFU) per spleen cell.

[0063] The immune response efficacy of 21 preliminarily screened preferred epitopes with high affinity, strong immunogenicity, and intraspecific and interspecific conservation was validated in mice. After three immunizations with tandem epitope peptide vaccines in BALB / c and SJL mice, spleen cells of these mice were stimulated with the peptides to observe IL-2 secretion levels, thereby determining the ability of Th1 cells to detect antigen-specific T cells. Data are presented as mean spleen cell formation units (SFU). Figure 7 As shown, all 21 epitopes stimulating spleen cells in BALB / c mice secreted IL-2. Nineteen epitopes in SJL mice also elicited a spleen cell response, indicating a good immune effect. One epitope with the significant epitope peptide APFILLILF induced the strongest immune response in BALB / c mice, releasing the cytokine IL-2 from spleen cells, while another epitope with the significant epitope peptide FVKWKKKF induced the strongest immune response in SJL mice, also releasing IL-2 from spleen cells. In SJL mice, the FVKWKVEYI epitope also elicited a stronger response than other epitopes. Several other major epitopes, such as LEERTGISW, MYSPVFEYL, and RFNLGPVTI, demonstrated the ability to effectively stimulate spleen cells and release the cytokine IL-2 in both mouse types.

[0064] Step 5: Histopathological safety evaluation To assess the safety of the expression vector pVAX1-CCHFV-Gc21p described in this invention in animals, this embodiment uses histopathological methods to systematically analyze the major organs of mice after immunization.

[0065] The specific experimental protocol is as follows: In Step 3, tissues from major organs such as the heart, kidney, spleen, liver, and lung were systematically collected. After fixation with 4% paraformaldehyde, paraffin embedding, and sectioning, hematoxylin and eosin (H&E) staining was performed, and histopathological observation was conducted under a light microscope. The results showed that the tissue structures of important organs such as the heart, kidney, spleen, liver, and lung in each group of mice were clear, cell morphology was normal, and no obvious abnormal changes such as inflammatory cell infiltration, necrosis, or pathological damage were observed. Figure 8 No serious pathological changes were observed.

[0066] The above results indicate that the expression vector pVAX1-CCHFV-Gc21p described in this invention, after immunization of mice via intramuscular injection, did not cause systemic toxicity or pathological damage to vital organs. This histopathological analysis provides experimental evidence for the preliminary safety of the expression vector of this invention, demonstrating its good biocompatibility and suitability for further development as a nucleic acid vaccine candidate.

[0067] Summarize: This invention proposes a tandem multi-epitope vaccine constructed by precisely screening for preferred MHC-I epitope peptides on CCHFV Gc, which exhibits significant advantages in stimulating the body's cellular immune response. It enhances the strength of the vaccine-induced cellular immune response, and utilizes toxicity and allergenicity analysis tools to identify potentially toxic or sensitizing epitopes (such as WPHSRNWRC and VEYIKTEAI) in advance. Furthermore, the tandem multi-epitope vaccine significantly eliminates the risk of toxicity or sensitization, overcoming the problem of neglecting immune safety in previous epitope screening methods. This eliminates potential safety risks for subsequent clinical applications of the vaccine and opens up new avenues for the research and application of CCHFV vaccines. Specifically:

[0068] 1. This invention, through the integration of multiple bioinformatics tools and experimental verification, screens CD8⁺ T cell epitope peptides with high affinity, strong immunogenicity, and high evolutionary conservation from the Gc protein of Crimean-Congo hemorrhagic fever virus (CCHFV). These epitope peptides possess the following significant advantages and successfully overcome several key challenges in existing technologies: First, broad-spectrum population coverage and HLA polymorphism adaptation. The selected epitopes cover multiple major HLA superfamilies such as HLA-A2, A3, and B7, covering more than 90% of the global population, overcoming the limitation of immune response caused by HLA polymorphism in traditional epitope vaccines, and achieving pan-HLA-I restricted immune activation with "one epitope for multiple populations"; Second, strong evolutionary conservation and outstanding antiviral escape ability. Through intraspecific and interspecific conservation analyses, these epitopes maintained high sequence conservation across different CCHFV strains, particularly in structurally stable regions of the Gc protein. This effectively reduced the risk of immune escape due to viral mutations and overcame the challenge of existing vaccine targets being susceptible to viral mutations. Third, multi-dimensional systematic screening enhanced reliability. Multiple independent algorithms were used to cross-validate affinity, combined with comprehensive evaluations of immunogenicity, conservation, toxicity, and sensitization. This overcame the problems of large biases and high false-positive rates associated with traditional single-analysis tools, ensuring the biological reliability and safety of the epitopes.

[0069] 2. After analyzing the interspecies and intraspecies conservation of preferred epitopes of CCHFV Gc, the preferred epitopes can be simply classified. Compared with other epitopes, interspecies conserved preferred epitopes are more suitable for vaccine development and immunization research. Interspecies conserved dominant mouse epitopes were simulated with corresponding mouse MHC-I and human HLA-I molecules. These antigenic epitopes were validated by the ELISpot assay, showing that they could elicit an immune response in mouse spleen cells.

[0070] 3. Following viral infection, antigen-presenting cells (APCs) activate CD8 cells through MHC-I class molecule antigen presentation. + T cells. For MHC class I molecules, the antigen-binding groove is blocked at both ends by conserved tyrosine residues, resulting in the binding peptide size typically being limited to 8-10 amino acid residues. Therefore, this invention selected a 9-amino acid peptide as the predicted epitope. Meanwhile, CTL-mediated cellular immune responses play an important role in antiviral infection. After recognizing virus-infected cells (target cells), CTLs release perforin, granzymes, and induce target cell lysis and death via the FasL-Fas pathway, thereby effectively clearing viral infection from the body. The level and activity of virus-specific CTLs in the body are positively correlated with the clearance effect of that virus.

[0071] 4. Based on the 21 high-affinity, strong immunogenicity, and highly conserved CCHFV Gc protein CTL epitopes screened, this invention further designed a tandem multi-epitope vaccine nucleotide sequence. Compared with traditional vaccine strategies using single or a few epitopes, this tandem multi-epitope vaccine exhibits the following synergistic advantages, overcoming several bottlenecks in existing technologies: First, it achieves broad-spectrum and balanced cellular immune coverage. By tandemly expressing multiple epitopes targeting different HLA superfamilies (such as A2, A3, and B7) on the same vector, it can simultaneously activate polyclonal CD8⁺ T cell responses presented by different HLA molecules targeting multiple antigenic regions of the virus within a single individual. This fundamentally overcomes the common problem of "immune non-response" or uneven response intensity caused by single-epitope vaccines due to individual HLA polymorphism limitations, significantly improving the population coverage and protective universality of the vaccine. Second, it effectively prevents viral immune escape. The tandem epitopes originate from multiple highly conserved regions on the Gc protein. Even if the virus undergoes a point mutation in a certain epitope region to evade immunity, T-cell immunity against other conserved epitopes can still be effectively activated and exert a clearance effect. This "multi-target backup" mechanism greatly enhances the robustness of the vaccine against natural viral mutations or selective pressures, overcoming the significant safety risk of single-epitope vaccines becoming completely ineffective due to target mutations. Third, it improves overall immunogenicity and response strength. The physical tandem of multiple highly immunogenic epitopes can be co-processed and presented in antigen-presenting cells after vaccination, mimicking the state of simultaneous exposure of multiple epitopes in natural infection. This is conducive to inducing stronger and more durable multispecific T-cell immune responses, producing a synergistic effect, and overcoming the technical bottleneck that single-epitope immunogenicity may be insufficient and difficult to stimulate sufficient protective immune memory. Fourth, according to safety test results, tandem multi-epitope vaccines are also safe, non-toxic, and non-allergenic, eliminating the allergenic and toxic risks associated with single-epitope vaccines.

[0072] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0073] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A preferred epitope peptide of CCHFV Gc, characterized in that, Its amino acid sequence is shown in any one of SEQ ID NO.1 to SEQ ID NO.

21.

2. A tandem multi-epitope vaccine modified from the preferred epitope peptide of CCHFV Gc according to claim 1, characterized in that, It was constructed using the following method: The tandem multi-epitope vaccine is obtained by linking sixteen peptide segments on the CCHFV Gc containing the amino acid sequences described in SEQ ID NO.1 to SEQ ID NO.21 through a linker. The nucleotide sequences of the sixteen peptides are as follows: TSLSIEAPW, LEERTGISW, MYSPVFEYL, LHKEWPHSRNWRCNPTW, DVKDLFTDY, FVKWKVEYIKTEAIVCVEL, RFNLGPVTI, IEEGFFDLM, EPHFNTSWM, MGDWPSCTY, EPDELTVHV, SLCFYIVER, QPQSILIEHKGTI, MLSGIFGNV, APFILLILF, RRTRGLFKY.

3. The tandem multi-epitope vaccine according to claim 2, characterized in that, The amino acid sequence of the tandem multi-epitope vaccine is shown in SEQ ID NO.

22.

4. The tandem multi-epitope vaccine according to claim 2, characterized in that, The nucleotide sequence of the tandem multi-epitope vaccine is shown in SEQ ID NO.

24.

5. An expression vector pVAX1-CCHFV-Gc21p, characterized in that, It includes the nucleotide sequence of the tandem multiepitope vaccine of claim 4.

6. The expression vector pVAX1-CCHFV-Gc21p according to claim 5, characterized in that, The expression vector pVAX1-CCHFV-Gc21p was constructed according to the following method: The nucleotide sequence shown in SEQ ID NO.24 was synthesized, then cloned into the pVAX1 vector, transformed into bacteria, and finally screened and verified to complete the construction of the expression vector.

7. The use of a preferred epitope peptide of CCHFV Gc according to claim 1, the tandem multi-epitope vaccine according to claim 2, or the expression vector pVAX1-CCHFV-Gc21p according to claim 4 in the preparation of a product for the prevention or treatment of CCHFV infection.

8. The application according to claim 7, characterized in that, The product is obtained by combining the preferred epitope peptide of CCHFV Gc, a tandem multi-epitope vaccine or expression vector as the active substance with a vaccine adjuvant.

9. The application according to claim 8, characterized in that, The vaccine adjuvant is an aluminum adjuvant, an oil emulsion adjuvant, or a cytokine adjuvant.

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