Design method for improving population coverage rate of multi-epitope vaccine

Abstract

The invention provides a design method for improving the population coverage rate of a multi-epitope vaccine, which comprises the following steps: carrying out epitope prediction on B cells and T cells according to candidate proteins to obtain B cell epitopes and T cell epitopes; setting a sliding window for the candidate antigen protein; judging whether the needed epitope exists in the sliding window or not; if the needed epitope exists, shortening the length of the window by one amino acid, and judging whether the specifically bound MHC-I / II allele is reduced or not; if the MHC-1 / II allele is reduced, the window shortening operation is revoked, and overlapping epitopes which are specifically combined with the maximum MHC-1 / II allele are reserved, so that candidate overlapping epitopes are obtained. According to the invention, a three-type epitope maximization strategy based on a sliding window is adopted, and population coverage rate maximization and dual immune recognition are realized in a vaccine construct with a compact structure by recognizing the number of specific binding MHC-I / II alleles and the number of partially overlapped B cell epitope amino acids.

Description

Technical Field

[0001] This invention relates to the field of vaccine development technology, and more specifically to a design method for improving the population coverage of multi-epitope vaccines. Background Technology

[0002] The major histocompatibility complex (MHC) alleles (commonly known as the human leukocyte antigen (HLA) system in humans) possess unique peptide-binding motifs. As of 2023, the IMGT / HLA database contains over 27,000 HLA class I (A, B, C) and over 7,000 class II (DRB1, DQA1, DQB1, DPB1, etc.) alleles, exhibiting the highest level of polymorphism in the human genome. The high polymorphism of the MHC leads to significant differences in the specific binding and presentation capabilities of different populations for the same antigen. Therefore, when designing multi-epitope vaccines, it is crucial to fully consider the overall coverage of candidate epitopes in the target population, i.e., the proportion of individuals in the target population whose epitopes contained in the vaccine can be effectively presented by one or more HLA molecules.

[0003] Existing prediction tools NetMHCpan-4.1 and NetMHCIIpan-4.1 have integrated MHC frequencies from over 150 populations worldwide using deep learning. Their output population coverage metric quantifies the theoretical protection rate of any epitope combination in the target population. This data enables multi-epitope vaccine design to take allele frequencies into account. Therefore, fully considering MHC polymorphisms and optimizing epitope composition based on allele frequency data is crucial for achieving multi-epitope vaccine coverage of specific or global populations.

[0004] Traditional multi-epitope vaccine design typically treats B-cell epitopes and T-cell epitopes as independent and mutually exclusive predictors, screening them separately before linear assembly. The sequential epitope screening strategy, an improvement on this approach, involves some adjustments: first, manually searching for B-cell epitopes in long sequences; then, screening for MHC-II epitopes embedded within B-cell epitopes; and finally, screening for MHC-I epitopes. However, this strategy fails to consider the partial overlap between MHC-I / II epitope motifs and B-cell epitopes, potentially leading to truncation of functional MHC-binding regions. Furthermore, the sequential epitope screening strategy assumes B-cell epitopes must have relatively long sequences (≥9 amino acids), while studies have shown that linear B-cell epitopes of only 5 amino acids still possess immunogenicity and can serve as building blocks for conformational B-cell epitopes. Therefore, this sequential epitope screening strategy ignores the naturally occurring partial overlap between epitopes, resulting in lengthy vaccine constructs, low immunogenicity density, and insufficient specifically binding MHC alleles, hindering population coverage.

[0005] To improve the above-mentioned technical problems, it is necessary to comprehensively consider partially overlapping B-cell epitopes and T-cell epitopes within the limited sequence length of the vaccine to increase the compactness of the vaccine construct, and to comprehensively consider the polymorphism of human leukocyte antigen MHC (i.e., HLA). Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a design method to improve the population coverage of multi-epitope vaccines, thereby overcoming the deficiencies in existing technologies.

[0007] The technical solution adopted in this invention is as follows: Firstly, a method for searching overlapping epitopes to improve population coverage of multi-epitope vaccines is provided, comprising the following steps: Multiple immunogenic proteins were pre-screened to obtain candidate protein sequences; The candidate protein sequences were searched for overlapping regions of B-cell epitopes and T-cell epitopes using the sliding window method to obtain candidate overlapping epitopes.

[0008] Furthermore, the candidate protein sequences were searched for overlapping regions of B-cell and T-cell epitopes using a sliding window method to obtain candidate overlapping epitopes, including: Step 1: Predict B-cell epitopes and T-cell epitopes based on candidate protein sequences to obtain B-cell epitopes and T-cell epitopes; Step 2: Set a sliding window for the candidate protein sequences; Step 3: Determine whether there are any overlapping epitopes that meet the overlapping epitope screening criteria; Step 4: If there are overlapping epitopes that meet the screening criteria, shorten the window length by one amino acid and determine whether the number of specifically bound MHC-I or MHC-II alleles has decreased; if there are no overlapping epitopes that meet the screening criteria, slide the window by one amino acid. If the judgment results of steps 5 and 4 are a reduction, then cancel the window shortening operation, retain the overlapping epitopes that specifically bind to the most MHC-1 / II alleles, and obtain candidate overlapping epitopes; then slide the window one amino acid; repeat steps S2 to S5 until all candidate overlapping epitopes are obtained; If the result of step 4 is no reduction, then the window length is shortened by one amino acid, and then the specific binding MHC-I or MHC-II alleles are judged again until the specific binding MHC-I or MHC-II alleles are reduced.

[0009] Furthermore, the candidate overlapping epitopes must simultaneously meet the following overlapping epitope screening criteria: It contains at least one MHC-I binding motif; It contains at least one MHC-II binding motif; It overlaps with B cell epitopes by at least 5 amino acids.

[0010] Furthermore, when predicting B-cell epitopes based on candidate protein sequences, B-cell epitopes are predicted in either conformational or linear smooth mode, and a threshold is set to ensure specificity. The top-scoring B-cell epitope amino acids are selected, and B-cell epitopes with a predicted length shorter than the preset number of amino acids are removed.

[0011] Furthermore, when predicting T-cell epitopes based on candidate protein sequences, a percentile ranking threshold is set as the basis for identifying "epitaxy-MHC-I / II specific binding"; and high-frequency MHC-I / II alleles in the IEDB reference set are selected.

[0012] Furthermore, the sliding window method employs a sliding window with an initial length of at least 9 amino acids.

[0013] In a second aspect, a bacterial vesicle multiepitope vaccine is provided, which is designed using the overlapping epitope search method described in the first aspect to improve the population coverage of multiepitope vaccines. The bacterial vesicle multiepitope vaccine can be carried and displayed using Staphylococcus aureus vesicles and can also be used to prevent Vibrio vulnificus pathogen infection.

[0014] Furthermore, in the preparation of vaccines for Vibrio vulnificus pathogens, Freund's adjuvant is mixed with purified bacterial vesicles.

[0015] Furthermore, when using Staphylococcus aureus membrane vesicles for mounting and display, multiple Vibrio vulnificus antigenic epitope fragments are linked together by linker peptides and inserted into the C-terminal display site of the Staphylococcus aureus PdhB protein.

[0016] Furthermore, firstly, the overlapping epitope coding sequence was spliced ​​with the upstream and downstream homologous arms of the pdhB gene of Staphylococcus aureus using overlapping PCR technology to form the recombinant sequence uppdhB-overlapping epitope combination-downpdbB. After double digestion with BamHI / SacI, the recombinant sequence fragment was cloned into the multiple cloning site of the temperature-sensitive shuttle vector pBT2 using Gibson assembly to form the recombinant plasmid pBT2-uppdhB-5Epi-downpdhB. After electroporation into Staphylococcus aureus RN4220-Δagr, the fusion expression cassette was seamlessly inserted into the pdhB gene site on the chromosome through temperature-controlled homologous recombination, enabling stable expression of the PdhB-overlapping epitope protein.

[0017] As can be seen from the above technical solution, the beneficial technical effects of the present invention are as follows: 1. The vaccine design method adopts a sliding window-based three-epitope maximization strategy, which breaks through the limitations of sequential epitope screening strategies. By identifying partially overlapping B-cell epitope amino acids embedded in MHC-I / II epitopes, it achieves dual immune recognition (humoral immunity and cellular immunity) and maximizes population coverage in a compact vaccine construct.

[0018] 2. Vaccine design methods can increase the number of MHC-I and MHC-II alleles that specifically bind to partially overlapping sequences and improve binding affinity, thereby further increasing the population coverage of MHC-I, MHC-II and their combined analysis.

[0019] 3. The total number of overlapping epitopes in the final construct is limited to 5, and the cumulative length of overlapping epitopes is 100 amino acids, which effectively ensures the compactness of the vaccine construct. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0021] Figure 1 This is a flowchart of the method for searching overlapping epitopes in an embodiment of the present invention; Figure 2 This is an integrated analysis diagram of the distribution of MHC-I / II class alleles and B-cell epitope prediction in an embodiment of the present invention; Figure 3 This is a schematic diagram illustrating the construction of a Vibrio vulnificus multiepitope protein vaccine based on the vaccine design method of this application in an embodiment of the present invention; Figure 4 This is a schematic diagram of the carrier construction scheme based on the Staphylococcus aureus engineered bacteria membrane vesicle display platform in an embodiment of the present invention; Figure 5 This is a prediction graph showing the population coverage and related indicators of the vaccine construct that specifically binds to MHC-I / II alleles in the embodiments of the present invention. Figure 6 This is a distribution diagram of the predicted linear B-cell epitope fractions on the vaccine construct in an embodiment of the present invention. Detailed Implementation

[0022] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0023] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Example The inventors of this patent discovered through research that antigen display efficiency and carrier space utilization are key factors determining immunization efficacy in vesicle vaccine development. *Staphylococcus aureus* RN4220-Δagr can express PdhB-overlapping epitope fusion proteins, thus using its naturally generated membrane vesicles (MVs) as vaccines. Furthermore, overlapping epitope combinations from *Vibrio vulnificus* can replace the display of single antigen proteins on the vesicle surface, improving the theoretical antigen display efficiency and carrier space utilization of *Vibrio vulnificus* multi-epitope vesicle vaccines. Based on the above research findings, this patent utilizes a proposed "sliding window - maximizing three types of epitopes" method to screen overlapping epitope combinations, achieving highly efficient vesicle display. The specific process is described below in the section on five overlapping epitope tandems.

[0025] The design method for improving population coverage of multi-epitope vaccines described in the patent, such as Figure 1 As shown, the specific steps are as follows: Step S1: Pre-screen multiple immunogenic proteins to obtain candidate protein sequences. In this step, B cell and T cell epitopes are predicted using the Immune Epitope Database and Analysis Resource (IEDB) web server. Specifically, third-party epitope prediction tools can be used, including linear B cell epitope prediction tools (e.g., BepiPred 3.0), MHC-I allele epitope prediction tools (e.g., NetMHCpan 4.1 EL), and MHC-II allele epitope prediction tools (e.g., NetMHCIIpan 4.1 EL).

[0026] The types of immunogenic proteins are not limited, including but not limited to xenobiotic serum proteins, enzyme proteins, bacterial exotoxins, toxoids, flagellin proteins, and capsular proteins.

[0027] Step S2: Using the sliding window method, search for overlapping regions of B-cell epitopes and T-cell epitopes in the candidate protein sequences to obtain candidate overlapping epitopes. This step includes the following sub-steps: Step S21: Predict B-cell epitopes based on candidate protein sequences to obtain B-cell epitopes. In a specific implementation, the BepiPred 3.0 tool is used to predict B-cell epitopes in either conformational or linear smoothing mode, with a default threshold of 0.151 to ensure specificity. Amino acids ranking in the top 20% (meeting the BepiPred 3.0 high-confidence threshold) and exceeding the 0.1512 threshold are preferentially selected, while epitopes with a predicted length of less than 5 amino acids are discarded.

[0028] Step S22: Predict T cell epitopes based on candidate protein sequences to obtain T cell epitopes. In a specific implementation, T-cell epitopes are predicted using MHC-I and MHC-II epitope prediction tools in IEDB. Specifically, NetMHCpan 4.1 EL (for MHC-I epitope prediction) and NetMHCIIpan 4.1 EL (for MHC-II epitope prediction), recommended by IEDB (September 2023 version), are used for binding affinity prediction. Binding affinity analysis is based on the MHC-I / II allele reference set recommended by IEDB, with the prediction threshold for strong binding epitopes set to the default critical values: percentile rank of MHC-I binding epitopes < 0.5%, and percentile rank of MHC-II binding epitopes < 2%. During MHC-I epitope prediction, only frequently occurring MHC-I / II alleles in the IEDB reference set are selected; the default length of the predicted MHC-II epitope composition is set to 15 amino acids.

[0029] In a specific implementation, the execution order of steps S21 and S22 is not important.

[0030] Step S23: Use the sliding window method to search for overlapping regions of B-cell epitopes and T-cell epitopes to obtain candidate overlapping epitopes. The sliding window method in this step uses a sliding window with an initial length of at least 9 amino acids, decreasing by 1 amino acid in each iteration, thus minimizing the vaccine construct size while preserving epitope density. The specific implementation is as follows: Step S23-1: Set sliding windows for B cell epitopes and T cell epitopes; Step S23-2: Determine whether there are overlapping epitopes that meet the overlapping epitope screening criteria. The screening strategy must meet all of the following criteria: (1) it contains at least one MHC-I binding motif (9 amino acids); (2) it contains a 9-amino acid core peptide of an MHC-II binding epitope; and (3) it overlaps with the B cell epitope composition by at least 5 amino acids. The above screening strategy can achieve dual targeting of humoral immunity and cellular immunity, which is crucial for pathogens with immune escape mechanisms, such as Vibrio vulnificus.

[0031] Step S23-3: If there are overlapping epitopes that meet the screening criteria, shorten the window length by one amino acid and determine whether the number of specifically bound MHC-I or MHC-II alleles has decreased; if there are no overlapping epitopes that meet the screening criteria, slide the window by one amino acid. Step S23-4: Shorten the window length by one amino acid. If the number of MHC-I or MHC-II alleles that specifically bind decreases, cancel the window shortening operation and retain the overlapping epitopes that specifically bind to the most MHC-1 / II alleles to obtain candidate overlapping epitopes. Then slide the window by one amino acid and repeat steps S23-1 to S23-4 until all candidate overlapping epitopes are obtained.

[0032] If the result is no reduction, the window length is shortened by one amino acid, and then the specific binding MHC-I or MHC-II alleles are checked again until the specific binding MHC-I or MHC-II alleles are reduced.

[0033] In a specific implementation, the above screening process can be implemented using a Python script. This script employs a series of lookup functions to systematically process and integrate the three types of epitopes from BepiPred 3.0, NetMHCpan 4.1 EL, and NetMHCIIpan 4.1 EL. By integrating allele distribution with BepiPred scores through a biaxial graph, the overlapping regions of B-cell epitopes, MHC-I epitopes, and MHC-II epitopes are visualized, such as... Figure 2 As shown.

[0034] Figure 2 The horizontal axis represents protein motif coordinates, used to locate the distribution of T-cell and B-cell epitopes within protein motifs; the vertical axis (left) represents the number of predicted epitopes specifically binding to MHC alleles, and the horizontal axis (right) represents the score of predicted linear B-cell epitopes. These epitopes were predicted using NetMHCpan 4.1, NetMHCIIpan 4.1, and BepiPred-3.0, respectively.

[0035] Combining the sliding window search strategy, in Figure 2 Overlapping epitopes were extracted using markers. Compared to other overlapping regions, the overlapping epitopes shown specifically bind to more MHC-I / II alleles and contain potential B-cell epitope fragments, thereby effectively increasing the population coverage of the vaccine and achieving dual immune recognition (humoral and cellular immunity). The extracted overlapping epitopes are compiled into Table 1, where * indicates a unique identifier for the protein in the RefSeq database. Table 1. Summary of overlapping epitopes in multi-protein sequence searches The meta-analysis of Table 1 revealed several epitope fragments with high immunogenic potential, including fragments derived from flagellin (FlgF, FlgG) and virulence-associated outer membrane proteins (TolC, GspD). These fragments exhibited a relatively broader and more specific binding capacity to MHC-I / II alleles.

[0036] The results of the analysis of epitope fragment features are shown in Table 2. In Table 2, * indicates that if an epitope is predicted as a potential antigen by VaxiJenv2.0 (antigenicity score > 0.4), then the epitope is marked in bold. In Table 2, ** indicates that all candidate overlapping epitopes are predicted as non-toxic by ToxinPred3.0 because their scores are lower than the preset threshold of 0.38.

[0037] Table 2 Summary of Predictive Attributes of Overlapping Epitopes Table 2 shows that: (1) there are significant differences in antigenicity scores (calculated by VaxiJen v2.0) among different epitope fragments, with WP_011078330.1 (204-223) and WP_017421053.1 (188-205) having extremely high scores (1.7514 and 1.5212, respectively), indicating that they have strong immunogenic potential; (2) AllerTOP v2.1 prediction shows that most epitope fragments (15 out of 22) may be allergens; (3) toxicity prediction (by ToxinPred 3.0) shows that the epitope fragments have low toxicity risk, with 11 fragments scoring 0 and the remaining fragments scoring below 0.25; (4) there are significant differences in solubility, with 10 soluble fragments and 12 insoluble fragments identified, which may affect their applicability in vaccine formulations. These computational evaluation results provide preliminary data support for decision-making based on immunogenicity, safety, and efficacy during vaccine development.

[0038] The calculation and evaluation results in Table 2 show that the seven shortlisted epitope fragments are antigenic, non-sensitizing, and non-toxic. Among them, three fragments were predicted to be soluble: WP_017421280.1 (61-80), WP_038963342.1 (156-175), and WP_224654715.1 (154-173); the other four fragments were insoluble: WP_011078336.1 (38-57), WP_011078339.1 (187-206), WP_103155281.1 (376-395), and WP_158122315.1 (136-155). Furthermore, the scheme containing the epitope combination “WP_017421280.1 (61-80), WP_038963342.1 (156-175), WP_224654715.1 (154-173), WP_011078336.1 (38-57), WP_011078339.1 (187-206)” exhibited the highest population coverage, with an average of 8.06 matches and a 90% population match rate (PC90) of 3.92. Simultaneously, by tandemly connecting the three soluble fragments with the two selected insoluble fragments, potential hydrophobic interactions were mitigated; therefore, this epitope combination was used to construct the final vaccine construct.

[0039] Based on the above-mentioned method for searching overlapping epitopes to improve the population coverage of multi-epitope vaccines, in some embodiments, bacterial vesicle multi-epitope vaccines can be designed. These vaccines can be carried and displayed using Staphylococcus aureus vesicles and can also be used to prevent Vibrio vulnificus pathogen infection.

[0040] In some embodiments, when preparing a bacterial vesicle multiepitope vaccine for Vibrio vulnificus pathogen, Freund's adjuvant is mixed with purified bacterial vesicles.

[0041] In some embodiments, when a bacterial vesicle multiepitope vaccine is mounted and displayed using Staphylococcus aureus vesicles, the vaccine construction scheme of the Staphylococcus aureus engineered vesicle display platform is as follows: Figure 3 , Figure 4As shown, five Vibrio vulnificus antigenic epitope fragments (Epitope Segments 1 to 5) were tandemly linked by a rigid linker peptide (GPGPG)2 and inserted into the C-terminal display site of the Staphylococcus aureus PdhB protein. This 140-amino acid overlapping epitope combination can be precisely integrated using the temperature-sensitive shuttle vector pBT2. The assembly process is as follows: First, the overlapping epitope coding sequence was spliced ​​with the upstream and downstream homologous arms (uppdhB / downpdhB) of the pdhB gene of the engineered Staphylococcus aureus strain RN4220-Δagr using overlap PCR technology, forming a recombinant sequence "uppdhB-epitope combination-downpdbB". After double digestion with BamHI / SacI, this fragment was assembled and cloned into the multiple cloning site (MCS) of the pBT2 vector via Gibson, forming the recombinant plasmid pBT2-uppdhB-5Epi-downpdhB. After electroporation into the engineered bacterium RN4220-Δagr, the fusion expression cassette was seamlessly inserted into the pdhB gene locus on the chromosome via temperature-controlled homologous recombination, enabling stable expression of the PdhB-overlapping epitope fusion protein. Figure 4 In this context, Epitope Segment represents Vibrio vulnificus antigenic epitope, and Linker represents linker peptide.

[0042] By comparing and analyzing the epitope segmentation strategies in this embodiment for different pathogens, it is shown that the binding affinity and coverage of each strategy differ for different pathogens, as shown in Table 3 below. Figure 5 , Figure 6 : Table 3 Comparison of the total length (amino acids) of multi-epitope vaccines constructed using different epitope selection strategies From Table 3 and Figure 5 , Figure 6 It can be seen that the performance of the non-overlapping strategy is significantly lower, with a population coverage rate of only 24.31%; in contrast, the overall population coverage rate of the overlapping strategy is higher, ranging from 53.89% to 99.80%. The strategy of manually searching for T-cell epitopes embedded in linear B-cell epitopes (Examples 4-7) can achieve a medium-to-high level of population coverage (53.89% to 99.39%). In two independently implemented studies, the population coverage rates of the partially overlapping parallel processes reached 99.80% (based on the algorithm in this embodiment) and 94.71% (based on the manual search in Example 2), respectively.

[0043] The Vibrio vulnificus multiepitope protein vaccine sequence constructed according to the vaccine design method provided in this embodiment is as follows: Figure 3As shown, among different strategies, the method exhibits the highest population coverage rate of 99.80% and a PC90 index of 3.84, surpassing the partially overlapping epitope search strategy and significantly outperforming other overlapping epitope search strategies. Therefore, the vaccine design method provided in this embodiment can increase the number of specifically binding MHC-I / II alleles by searching for partially overlapping sequences, thereby further improving the population coverage associated with MHC-I / II alleles.

[0044] The vaccine design method provided in this embodiment employs a sliding window-based three-epitope maximization strategy, overcoming the limitations of sequential epitope screening strategies. By identifying the number of partially overlapping B-cell epitopes (≥5 amino acids) within the sliding window, it achieves dual immune recognition (humoral and cellular immunity) and maximizes population coverage within a compact vaccine construct. This method, combined with dynamic window length adjustment, effectively controls the length of individual overlapping epitopes, while limiting the total number of overlapping epitopes in the final construct to 5, with a cumulative length of only 100 amino acids, effectively ensuring the compactness of the vaccine construct.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for searching overlapping epitopes to improve population coverage of multi-epitope vaccines, characterized in that, Includes the following steps: Multiple immunogenic proteins were pre-screened to obtain candidate protein sequences; The candidate protein sequences were searched for overlapping regions of B-cell epitopes and T-cell epitopes using the sliding window method to obtain candidate overlapping epitopes.

2. The method for searching overlapping epitopes to improve population coverage of multi-epitope vaccines according to claim 1, characterized in that, The candidate protein sequences were searched for overlapping regions of B-cell and T-cell epitopes using a sliding window method to obtain candidate overlapping epitopes, including: Step 1: Predict B cell and T cell epitopes based on candidate protein sequences to obtain B cell epitopes and T cell epitopes; Step 2: Set a sliding window for the candidate protein sequences; Step 3: Determine whether there are any overlapping epitopes that meet the overlapping epitope screening criteria; Step 4: If there are overlapping epitopes that meet the screening criteria, shorten the window length by one amino acid and determine whether the number of specifically bound MHC-I or MHC-II alleles has decreased; if there are no overlapping epitopes that meet the screening criteria, slide the window by one amino acid. If the judgment results of steps 5 and 4 are a reduction, then cancel the window shortening operation, retain the overlapping epitopes that specifically bind to the most MHC-1 / II alleles, and obtain candidate overlapping epitopes; then slide the window one amino acid; repeat steps S2 to S5 until all candidate overlapping epitopes are obtained; If the result of step 4 is no reduction, then the window length is shortened by one amino acid, and then the specific binding MHC-I or MHC-II alleles are judged again until the specific binding MHC-I or MHC-II alleles are reduced.

3. The method for searching overlapping epitopes to improve population coverage of multi-epitope vaccines according to claim 2, characterized in that, The candidate overlapping epitopes must simultaneously meet the following overlapping epitope screening criteria: It contains at least one MHC-I binding motif; It contains at least one MHC-II binding motif; It overlaps with B cell epitopes by at least 5 amino acids.

4. The method for searching overlapping epitopes to improve population coverage of multi-epitope vaccines according to claim 2, characterized in that, When predicting B-cell epitopes based on candidate protein sequences, B-cell epitopes are predicted in either conformational or linear smooth mode, and a threshold is set to ensure specificity. The top-scoring B-cell epitope amino acids are selected, and B-cell epitopes with predicted lengths shorter than the preset value are removed.

5. The method for searching overlapping epitopes to improve population coverage of multi-epitope vaccines according to claim 2, characterized in that, When predicting T-cell epitopes based on candidate protein sequences, a percentile ranking threshold is set as the basis for determining epitope-MHC-I / II specific binding; high-frequency MHC-I / II alleles in the IEDB reference set are selected.

6. The method for searching overlapping epitopes to improve population coverage of multi-epitope vaccines according to claim 2, characterized in that, The sliding window method uses a sliding window with an initial length of at least 9 amino acids.

7. A bacterial membrane vesicle multi-epitope vaccine, characterized in that, The bacterial vesicle multiepitope vaccine, designed using the method for searching overlapping epitopes to improve population coverage of multiepitope vaccines according to any one of claims 1-6, can be carried and displayed using Staphylococcus aureus vesicles and can also be used to prevent Vibrio vulnificus pathogen infection.

8. The bacterial vesicle multiepitope vaccine according to claim 7, characterized in that, When preparing vaccines for Vibrio vulnificus pathogens, Freund's adjuvant is mixed with purified bacterial vesicles.

9. The bacterial vesicle multiepitope vaccine according to claim 7, characterized in that, When using Staphylococcus aureus membrane vesicles for mounting and display, multiple Vibrio vulnificus antigenic epitope fragments are linked together by linker peptides and inserted into the C-terminal display site of the Staphylococcus aureus PdhB protein.

10. The bacterial membrane vesicle multiepitope vaccine according to claim 9, characterized in that, First, the overlapping epitope coding sequence was spliced ​​with the upstream and downstream homologous arms of the pdhB gene of Staphylococcus aureus using overlapping PCR technology to form a recombinant sequence uppdhB-overlapping epitope combination-downpdbB. After double digestion with BamHI / SacI, the recombinant sequence fragment was cloned into the multiple cloning site of the temperature-sensitive shuttle pBT2 via Gibson assembly, forming the recombinant plasmid pBT2-uppdhB-5Epi-downpdhB. After electroporation into Staphylococcus aureus RN4220-Δagr, the fusion expression cassette was seamlessly inserted into the chromosomal pdhB gene site via temperature-controlled homologous recombination, enabling stable expression of the PdhB-overlapping epitope protein.

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