A multi-epitope peptide, a helicobacter pylori multi-epitope vaccine and application thereof

Abstract

The present application relates to a kind of polyepitope peptide, helicobacter pylori polyepitope vaccine and its application, belong to biological medicine technical field.The polyepitope peptide of the present application is composed of the dominant T / B cell epitope (UCVH) of Helicobacter pylori virulence factor UreB (urease B subunit), CagA (cytotoxin associated protein A), VacA (vacuolating cytotoxin A), HpaA (Helicobacter pylori adhesin A), further with cholera enterotoxin B subunit (CTB) jointly constructs helicobacter pylori polyepitope vaccine.Can effectively induce the strong humoral immunity and overall cellular immune response of organism, the synergistic effect of induced mucosal immunity and Th1 type cellular immunity has good immunogenicity, provides important foundation for the anti-infection protection of helicobacter pylori.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a multi-epitope peptide, a Helicobacter pylori multi-epitope vaccine and its application. Background Technology

[0002] Helicobacter pylori ( Helicobacter pylori, H. pylori *Helicobacter pylori* (Hp) is a Gram-negative microaerophilic bacterium that obligately colonizes the surface of human gastric mucosal epithelial cells. It is classified as a Group 1 carcinogen. H. pylori Long-term infection can gradually lead to chronic gastritis and peptic ulcers through mechanisms such as damaging the gastric mucosal barrier, inducing chronic inflammatory response, and regulating host cell signaling pathways. In severe cases, it can progress to gastric mucosa-associated lymphoid tissue lymphoma or even gastric cancer.

[0003] Currently, clinical treatment H. pylori The core treatment regimen for infection control is triple or quadruple therapy combining a proton pump inhibitor with two antibiotics, but this regimen has significant limitations. On the one hand, the irrational use of antibiotics has led to global... H. pylori The continuously rising drug resistance rate directly leads to an increased treatment failure rate. On the other hand, broad-spectrum antibiotics disrupt the balance of normal gut flora, causing adverse reactions such as diarrhea and indigestion, and have a high relapse rate after treatment. Furthermore, the applicability and safety of antibiotic therapy are further limited for asymptomatic carriers, children, and the elderly. Therefore, research and development... H. pylori Vaccines have become a means of prevention and control. H. pylori Helicobacter pylori is an ideal way to infect and reduce the burden of related diseases. However, due to the complex immune escape mechanisms and high variability of Helicobacter pylori, a single antigen is difficult to provide broad-spectrum protection; at the same time, the human body's resistance to Helicobacter pylori mainly relies on cellular immunity rather than antibodies, and the protective efficacy induced by traditional vaccines is not long-lasting. Therefore, providing a highly effective and safe Hp vaccine is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a multi-epitope peptide, a Helicobacter pylori multi-epitope vaccine and its application, which can effectively induce the body to produce a strong humoral immune response and a comprehensive cellular immune response, provide anti-infection protection against Helicobacter pylori, and block Helicobacter pylori infection.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a multi-epitope peptide, the amino acid sequence of which is obtained by sequentially linking amino acids 349-363 of the UreB subunit, 341-352 of the CagA subunit, 312-323 of the VacA subunit, 98-109 of the HpaA subunit, 407-421 of the UreB subunit, 178-192 of the CagA subunit, 411-425 of the VacA subunit, 124-138 of the HpaA subunit, 70-78 of the UreB subunit, 36-44 of the CagA subunit, 241-249 of the VacA subunit, and 124-138 of the HpaA subunit with short peptides.

[0006] Preferably, the short peptide includes AAY and / or KK.

[0007] Preferably, the amino acid sequence of the multi-epitope peptide is shown in SEQ ID NO.13.

[0008] This invention provides the application of the aforementioned multi-epitope peptide in the preparation of Helicobacter pylori multi-epitope vaccines.

[0009] This invention provides a Helicobacter pylori multi-epitope vaccine, comprising the aforementioned multi-epitope peptide.

[0010] Preferably, the active ingredient of the multi-epitope vaccine is a protein composed of a cholera enterotoxin B subunit and the multi-epitope peptide.

[0011] Preferably, the amino acid sequence of the cholera enterotoxin B subunit is shown in SEQ ID NO.1.

[0012] Preferably, the amino acid sequence of the protein is shown in SEQ ID NO.15.

[0013] This invention provides the use of the aforementioned multi-epitope peptide or the aforementioned multi-epitope vaccine in the preparation of a drug for preventing Helicobacter pylori infection.

[0014] Preferably, the drug also includes pharmaceutically acceptable excipients.

[0015] The beneficial effects of this invention are: This invention constructs multi-epitope peptides by selecting core antigens such as CagA, VacA, UreB, and HpaA, ensuring the functional relevance and immunogenicity of the epitopes. The fusion expression of multiple epitope antigens can simultaneously activate humoral and cellular immunity, avoiding a one-sided immune response and solving the problem of single-antigen vaccines being susceptible to the diversity of Hp antigens and immune escape. By rationally selecting epitopes and connection methods, spatial conformational interference of antigenic epitopes is avoided, ensuring the full immunogenicity of each epitope is achieved.

[0016] This invention further fuses CTB with multi-epitope antigens for expression, achieving synergistic delivery and avoiding the side effects of free adjuvants. The constructed Helicobacter pylori multi-epitope vaccine (CTB-mediated Hp multi-epitope vaccine, CTB-UCVH) has the following clinical translational advantages: First, the multi-epitope design enhances the vaccine's broad spectrum and functional targeting, inhibiting Hp at multiple stages such as adhesion, colonization, and virulence, addressing strain diversity and drug resistance issues; second, the synergistic effect of CTB adjuvant-induced mucosal immunity and Th1-type cellular immunity aligns with the mucosal defense requirements of Hp infection; third, the recombinant protein of the vaccine exhibits good stability, facilitating large-scale production and formulation development. Mouse models have clearly demonstrated that this vaccine can effectively induce specific mucosal and systemic immune responses, significantly increasing the levels of relevant specific antibodies and immune cell activation indicators, laying a solid immunological foundation for its potential protective effect against Hp infection. Attached Figure Description

[0017] Figure 1 This is a basic flowchart of the present invention for vaccine screening and validation using bioinformatics.

[0018] Figure 2 The diagram shows the results of the three-level structure validation of the CTB-UCVH vaccine; where A: Schematic diagram of the connection of the CTB-UCVH vaccine; B: UCVH vaccine model; C: CTB adjuvant model; D: Complete structure model of CTB-UCVH vaccine; E: Laplace plot of the CTB-UCVH vaccine model; F: Z-score of the CTB-UCVH vaccine model.

[0019] Figure 3 The image shows the identification results of each double-enzyme digested plasmid; where A: CTB; B: UCVH; C: CTB-UCVH; M represents protein standard, and 1 represents PET-28a. + ,2 indicates the target protein.

[0020] Figure 4 The graph shows the induction results of each protein expression; where A: CTB; B: UCVH; C: CTB-UCVH; 1 indicates no induction, 2 indicates after induction, M indicates protein standard, 3 indicates precipitated sample induced by 1.0mM IPTG at 37℃, and 4 indicates supernatant sample induced by 1.0mM IPTG at 37℃.

[0021] Figure 5 The image shows the results of recombinant protein analysis and identification; where A: SDS-PAGE analysis and identification results; B: Western Blot analysis and identification results; M represents protein standards.

[0022] Figure 6 CD4 +Flow cytometry analysis of T cell cytokine production; where A, C, and E are flow cytometry graphs of splenic lymphocyte cytokines IFN-γ, IL-4, and IL-17A, respectively; and B, D, and F are statistical graphs of flow cytometry analysis of splenic lymphocyte cytokines IFN-γ, IL-4, and IL-17A, respectively.

[0023] Figure 7 Figures showing the results of ELISPOT and ELISA analysis of cellular immune responses induced by various immunogens in mice; where A: ELISPOT spot map of IFN-γ secreting splenic lymphocytes; B: ELISPOT spot statistical map of IFN-γ secreting splenic lymphocytes; C: ELISA statistical map of IFN-γ secretion level in splenic lymphocyte supernatant; D: ELISA statistical map of IL-4 secretion level in splenic lymphocyte supernatant; E: ELISA statistical map of IL-17A secretion level in splenic lymphocyte supernatant.

[0024] Figure 8 The graph shows the results of ELISA detection of antigen-specific antibody titers; where A, B, and C are the results of serum antigen-specific IgG, IgG1, and IgG2a antibody titers, respectively; and D is the result of intestinal fecal antigen-specific sIgA antibody titers.

[0025] Figure 9 This diagram illustrates the construction of the Helicobacter pylori multi-epitope vaccine (CTB-UCVH) and the evaluation of its immunization effect in mice.

[0026] In the above figures, (P<0.05) (P<0.01) (P<0.001); In the efficacy verification experiment of the immunization regimen, PBS was used as a blank control; The statistical analysis was mainly carried out between the CTB group and each experimental group, and the results between the groups with significant differences were marked; There were no significant differences between the PBS group and the CTB group in each experimental result (NS), so they were not marked in the figure. Detailed Implementation

[0027] This invention provides a multi-epitope peptide, wherein the amino acid sequence of the multi-epitope peptide comprises amino acids 349-363 of the UreB subunit (SEQ ID NO. 2), amino acids 341-352 of the CagA subunit (SEQ ID NO. 3), amino acids 312-323 of the VacA subunit (SEQ ID NO. 4), amino acids 98-109 of the HpaA subunit (SEQ ID NO. 5), amino acids 407-421 of the UreB subunit (SEQ ID NO. 6), amino acids 178-192 of the CagA subunit (SEQ ID NO. 7), amino acids 411-425 of the VacA subunit (SEQ ID NO. 8), amino acids 124-138 of the HpaA subunit (SEQ ID NO. 9), amino acids 70-78 of the UreB subunit (SEQ ID NO. 10), and amino acids 36-44 of the CagA subunit (SEQ ID NO. 10). The amino acid sequences of the multi-epitope peptides are sequentially tandemly derived from amino acids 241-249 of the VacA subunit (SEQ ID NO. 12) and amino acids 124-138 of the HpaA subunit (SEQ ID NO. 9). The preferred short peptides include AAY and / or KK. The preferred amino acid sequence of the multi-epitope peptide is shown in SEQ ID NO. 13.

[0028] This invention selects core antigens such as CagA, VacA, UreB, and HpaA to construct multi-epitope peptides. These antigens are closely related to *Helicobacter pylori* colonization, virulence, and immunogenicity: UreB is a highly conserved urease catalytic subunit that can decompose urea to create a suitable environment for *Helicobacter pylori* survival, making it a key protein for colonization; CagA and VacA are the main virulence factors, with the former inducing activation of cell carcinogenesis-related pathways and the latter inducing apoptosis and inflammation; HpaA can mediate the binding of *Helicobacter pylori* to the gastric mucosa to facilitate long-term colonization and has strong immunogenicity, inducing antibodies to block adhesion. Selecting these antigens as epitope sources ensures the functional relevance and immunogenicity of the epitopes, providing better anti-infection protection against *Helicobacter pylori*.

[0029] This invention utilizes the fusion expression of multiple epitope peptides as antigens for Helicobacter pylori vaccines, addressing the issue of single-antigen vaccines being susceptible to the diversity of Hp antigens and immune escape. By rationally selecting epitopes and connection methods, spatial conformational interference of antigenic epitopes is avoided, ensuring the full immunogenicity of each epitope is achieved.

[0030] This invention provides the application of the aforementioned multi-epitope peptide in the preparation of Helicobacter pylori multi-epitope vaccines.

[0031] This invention provides a Helicobacter pylori multi-epitope vaccine, comprising the aforementioned multi-epitope peptide. The active ingredient of the multi-epitope vaccine is preferably a protein, which is preferably composed of a cholera enterotoxin B subunit and the multi-epitope peptide. The amino acid sequence of the cholera enterotoxin B subunit is preferably as shown in SEQ ID NO.1. The amino acid sequence of the protein is preferably as shown in SEQ ID NO.15. Preferably, the amino acid sequence of the protein further includes a 6×histidine tag added to the N-terminus of SEQ ID NO.15.

[0032] In the protein of this invention, CTB is linked to the N-terminus of a multi-epitope peptide using the EAAAK (SEQ ID NO.14) linker, ensuring the independent folding of the CTB pentamer structure and maximizing its adjuvant immunomodulatory effect. Verification has shown that CTB, as an adjuvant, can bind to the GM1 receptor on intestinal epithelial cells to mediate antigen-targeted delivery, activate antigen-presenting cells, and promote T / B cell activation. Furthermore, CTB is non-toxic and biocompatible, providing a safety guarantee for subsequent clinical applications. This invention also reveals that, compared to traditional aluminum adjuvants, CTB is more likely to induce a mucosal immune response, which is precisely the first line of defense against *H. pylori* infection. sIgA on the gastric mucosa surface can directly inhibit *H. pylori* colonization and pathogenicity by blocking the adhesion of *H. pylori* to gastric epithelial cells, neutralizing bacterial toxins, and mediating complement-dependent cytotoxicity.

[0033] This invention further fuses CTB with multi-epitope peptides for expression, achieving synergistic delivery and avoiding the side effects of free adjuvants. The constructed CTB-mediated *H. pylori* multi-epitope vaccine has the following clinical translational advantages: First, the multi-epitope design enhances the vaccine's broad spectrum and functional targeting, inhibiting *H. pylori* at multiple stages, including adhesion, colonization, and virulence, addressing strain diversity and drug resistance issues; second, the synergistic effect of CTB adjuvant-induced mucosal immunity and Th1-type cellular immunity aligns with the mucosal defense requirements of *H. pylori* infection; third, the recombinant protein of the vaccine exhibits good stability, facilitating large-scale production and formulation development. Mouse models have clearly demonstrated that this vaccine can effectively induce specific mucosal and systemic immune responses, significantly increasing the levels of relevant specific antibodies and immune cell activation indicators, laying a solid immunological foundation for its potential protective effect against *H. pylori* infection.

[0034] The preferred method for preparing the protein includes the following steps: cloning the nucleotide sequence (SEQ ID NO. 16) of the protein into a vector plasmid to obtain a recombinant expression plasmid; transforming and inducing expression using the recombinant expression plasmid; collecting bacterial cells and purifying the plasmid to obtain the protein. The protein is then further used as an active ingredient to prepare a vaccine. The preferred vector plasmid is pET-28a. +When the nucleotide sequence of the protein (SEQ ID NO. 16) is cloned into the vector plasmid, the preferred restriction enzyme sites are Nco I and Xho I. This invention does not specifically limit the steps for transformation, induction of expression, collection of bacterial cells, and purification of the recombinant expression plasmid; conventional steps in the art can be used. The preparation method of the multi-epitope peptide is the same as the protein preparation method, except that the nucleotide sequence of the protein is replaced with the nucleotide sequence of the multi-epitope peptide (SEQ ID NO. 18).

[0035] This invention provides the use of the aforementioned multi-epitope peptide or the aforementioned multi-epitope vaccine in the preparation of a drug for preventing Helicobacter pylori infection.

[0036] The multi-epitope peptides of the present invention and the vaccines prepared therefrom can effectively induce specific mucosal and systemic immune responses in the body, significantly increasing the levels of related specific antibodies and immune cell activation indicators, thereby playing a role in resisting Hp infection.

[0037] The drug preferably also includes pharmaceutically acceptable excipients. This invention does not specifically limit the type and source of the excipients; conventional types and sources in the art are acceptable.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the following embodiments are all conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Example 1 1. Design and bioinformatics analysis of multi-epitope vaccines 1.1 Collection of amino acid sequences The amino acid sequences of four dominant target antigens of Helicobacter pylori standard strain NCTC26695—UreB (GenBank: WP_000724295.1), CagA (GenBank: WP_000180747.1), VacA (GenBank: WP_000405496.1), and HpaA (GenBank: WP_000646667.1)—were obtained from the virulence factor database VFDB (http: / / www.mgc.ac.cn / VFs / main.htm). The amino acid sequence of CTB (GenBank: XQY95436.1) was obtained from the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.govd). The signal peptide amino acids at positions 1-21 of the N-terminus were removed, and the remaining core functional amino acid sequence was retained. The specific amino acid sequence of CTB is shown in SEQ ID NO.1.

[0042] TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN (SEQ ID NO. 1).

[0043] 1.2 Epitope Prediction SignalP 6.0 was used to detect signal peptides of four dominant antigen proteins (UreB, CagA, VacA, and HpaA) to clarify their presence. T-cell epitope prediction was performed using NetCTL 1.2 and NetMHCⅡpan-3.2. First, using the NetCTL 1.2 server, C-terminal cleavage and TAP transport efficiency weights were set to 0.15 and 0.05, respectively, to predict cytotoxic T lymphocyte (CTL) epitopes. The predicted CTL epitopes were validated through antigenicity assessment, toxicity testing, and sensitization assessment. Furthermore, the NetMHCⅡpan-3.2 server was used to screen for HLA-DRB, which has the highest infection rate among Chinese patients. 11501, HLA-DRB 11202, HLA-DRB1 Three alleles (0901, ...) were used, and conditions were set with peptide length of 15-18 mere molecule, a strong-binding peptide threshold of 2%, a weak-binding peptide threshold of 10%, and other parameters left default, to predict helper T lymphocyte epitopes. Epitopes with the highest scores were selected based on a comprehensive score. Simultaneously, the BCPred server was used to screen for B cell epitopes with scores higher than 0.9 under default parameters, and these were then validated through antigenicity, toxicity, and sensitization assessments. (See the basic flowchart of vaccine screening and validation using bioinformatics...) Figure 1 The relevant software and websites used are shown in Table 1. T cell and B cell epitope prediction results: CTL epitopes for four proteins were predicted using the NetCTL 1.2 server, and the top-ranked epitopes were selected based on their comprehensive scores. HTL epitopes for the four proteins were predicted using the NetMHCⅡpan-3.2 server, as shown in Table 2. B cell epitopes were predicted using the BCPred server, and epitopes with scores higher than 0.9 were selected, as shown in Table 2.

[0044] In the vaccine design and physicochemical property analysis stage, the screened T-cell epitopes and B-cell epitopes were first linked using AAY and KK short peptide linkers, respectively, to obtain a multi-epitope peptide (sequence shown in SEQ ID NO.13), denoted as UCVH. Then, the CTB pentamer was linked to the N-terminus of the multi-epitope peptide using the EAAAK (SEQ ID NO.14) linker to ensure that the CTB pentamer structure folds independently and exerts the best adjuvant immunization effect, resulting in the amino acid sequence of the vaccine (protein) (as shown in SEQ ID NO.15). The tandem structure of the vaccine is shown in the figure below. Figure 2 As shown in A, the vaccine is a Helicobacter pylori multi-epitope vaccine, denoted as CTB-UCVH. Subsequently, the physicochemical properties of the vaccine, including aliphatic coefficient, instability index, theoretical isoelectric point, molecular weight, half-life, amino acid composition, and grand average hydrophilicity (GRAVY), were evaluated using the online software ProtParam. An instability index less than 40 indicates a stable protein, the aliphatic coefficient reflects heat resistance, and a negative GRAVY value represents hydrophilicity while a positive value indicates hydrophobicity.

[0045] The results are shown in Table 3. ProtPara calculated that the vaccine protein contains 286 amino acids with a relative molecular weight of 31.95 KD (MW). Its theoretical isoelectric point is 9.68, its instability index (II) is 26.53 (less than the threshold of 40), classifying it as a stable protein, its aliphatic index is 75.77 (heat resistant), and its total average hydrophilicity (GRAVY) is -0.692, classifying it as a hydrophilic protein. Furthermore, its extinction coefficient in a 280 nm aqueous solution was calculated to be 20525. The half-life in different systems was: mammalian reticulocytes (in vitro) 7.2 hours, yeast (in vivo) >20 hours, and Escherichia coli (in vivo) >10 hours. These results indicate that the vaccine CTB-UCVH of this invention possesses good stability and hydrophilicity, which is beneficial for protein expression and purification, as well as the maintenance of antigen structure in vivo. Stable and hydrophilic antigen molecules can significantly enhance the immunogenicity of the vaccine and reduce the in vivo clearance rate.

[0046] SEQ ID NO.13 (underlined indicates the amino acid sequence of each antigenic epitope) TLHDMGIFSITSSDS AAY GYKDQQGNNVAT AAY NNANNNQQNSAQ AAY SVDSSDKDDLSF KK SKYTINP AIAHGISE KK GNQIRTDQKFMGVFD KK NLTGNITVDGPLRVN KK EIVLRPDPKRTIQKK KK ITNALIVDY KK KVDNV VASF KK RVNNQVGGY KK EIVLRPDPKRTIQKK SEQ ID NO.15 (underlined indicates the amino acid sequence of CTB and each antigenic epitope) TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMK DTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN EAAAK TLHDMGIFSITSSDS AAY GYKDQQGNNVAT AAY NNANN NQQNSAQ AAY SVDSSDKDDLSF KK SKYTINPAIAHGISE KK GNQIRTDQKFMGVFD KK NLTGNITVDGPLRVN KK EI VLRPDPKRTIQKK KK ITNALIVDY KK KVDNVVASF KK RVNNQVGGY KK EIVLRPDPKRTIQKK 1.3 Secondary and Tertiary Structure Analysis of the Vaccine (CTB-UCVH) The most common secondary structures of proteins are α-helices and β-sheets, followed by β-turns and random coils. The secondary structure of the vaccine was predicted using the SOPMA online server, with multiple alignments with sequences of proteins from the same family to improve prediction accuracy. Default values ​​were used for window width and similarity thresholds. A tertiary structure model of the vaccine was constructed using the SWISS-MODEL online server, and the quality of the protein's tertiary structure was validated. Laplace plots were generated using SWISS-MODEL to predict the chemical quality of the vaccine model. Furthermore, the Z-Score of the input structure was calculated using PROSA-Web; if this score exceeded the protein's characteristic range, the model may contain errors.

[0047] Secondary structure prediction results: In the secondary structure of the vaccine, α-helix involves 124 amino acids, accounting for 43.36%; β-sheet involves 53 amino acids, accounting for 18.53%; and random coil involves 109 amino acids, accounting for 38.11%.

[0048] Third-order structure validation results: In the SWISS-MODEL Workspace, the model with the highest coverage of multi-epitope peptide antigens and CTB was selected for homology modeling of the intact vaccine structure. The multi-epitope peptide model is as follows: Figure 2 As shown in B, Figure 2 C in the model represents the CTB adjuvant. Figure 2 The purple portion (D) represents a multi-epitope peptide, and the yellow portion represents CTB adjuvant. (The text then abruptly shifts to a seemingly unrelated topic: "Through...") Figure 2 The E-Laplace plot shows that 97.98% of the residues are located in allowed regions. Further structural identification was performed using Prosa-web, such as... Figure 2 As shown in F, the Z-score was -5.68, indicating that the vaccine protein CTB-UCVH has good stability and the vaccine structure is reasonable.

[0049] 2. Construction of recombinant plasmids, expression, purification and identification of proteins. The recombinant plasmid used is the high-copy vector pET-28a used in prokaryotic expression systems in genetic engineering. +Nco I and Xho I were selected as restriction enzyme sites. The Nco I base recognition site was added to the N-terminus of the target sequences (the nucleotide sequences corresponding to CTB, UCVH, and CTB-UCVH, as shown in SEQ ID NO. 16~18), and corresponding protective bases were added. To prevent frameshift mutations, GC was added after the Nco I base site to ensure that the correctness of the target sequence was not affected after restriction enzyme digestion. A 6×histidine tag (His-tag) was exogenously added to the C-terminus of the target sequence to facilitate protein purification. A stop codon and an Xho I restriction enzyme site were added to the C-terminus of each sequence. The plasmids pET-28a-CTB, pET-28a-UCVH, and pET-28a-CTB-UCVH were generated and further identified by PCR and double restriction enzyme digestion.

[0050] SEQ ID NO.16 (nucleotide sequence of CTB-UCVH) accccgcagaacattaccgatctgtgcgcggaatatcataacacccagattcataccctgaacgataaaatttttagctataccgaaagcctggcgggcaaacgcgaaatggcgattattacctttaaaaacggcgcgacctttcaggtggaagtgccgggcagccagcatattgatagccagaaaaaagcgattgaacgcatgaaagataccctgcgcattgcgtatctgaccgaagcgaaagtggaaaaactgtgcgtgtggaacaacaaaaccccgcatgcgattgcggcgattagcatggcgaacgaagcggcggcgaaaaccctgcatgatatgggcatttttagcattaccagcagcgatagcgcggcgtatggctataaagatcagcagggcaacaacgtggcgaccgcggcgtataacaacgcgaacaacaaccagcagaacagcgcgcaggcggcgtatagcgtggatagcagcgataaagatgatctgagctttaaaaaaagcaaatataccattaacccggcgattgcgcatggcattagcgaaaaaaaaggcaaccagattcgcaccgatcagaaatttatgggcgtgtttgataaaaaaaacctgaccggcaacattaccgtggatggcccgctgcgcgtgaacaaaaaagaaattgtgctgcgcccggatccgaaacgcaccattcagaaaaaaaaaaaaattaccaacgcgctgattgtggattataaaaaaaaagtggataacgtggtggcgagctttaaaaaacgcgtgaacaaccaggtgggcggctataaaaaagaaattgtgctgcgcccggatccgaaacgcaccattcagaaaaaa SEQ ID NO.17 (Nucleotide sequence of CTB) accccgcagaacattaccgatctgtgcgcggaatatcataacacccagattcataccctgaacgataaaatttttagctataccgaaagcctggcgggcaaacgcgaaatggcgattattacctttaaaaacggcgcgacctttcaggtggaagtgccgggcagccagcatattgatagccagaaaaaagcgattgaacgcatgaaagataccctgcgcattgcgtatctgaccgaagcgaaagtggaaaaactgtgcgtgtggaacaacaaaaccccgcatgcgattgcggcgattagcatggcgaac SEQ ID NO.18 (Nucleotide sequence of UCVH) accctgcatgatatgggcatttttagcattaccagcagcgatagcgcggcgtatggctataaagatcagcagggcaacaacgtggcgaccgcggcgtataacaacgcgaacaacaaccagcagaacagcgcgcaggcggcgtatagcgtggatagcagcgataaagatgatctgagctttaaaaaaagcaaatataccattaacccggcgattgcgcatggcattagcgaaaaaaaaggcaaccagattcgcaccgatcagaaatttatgggcgtgtttgataaaaaaaacctgaccggcaacattaccgtggatggcccgctgcgcgtgaacaaaaaagaaattgtgctgcgcccggatccgaaacgcaccattcagaaaaaaaaaaaaattaccaacgcgctgattgtggattataaaaaaaaagtggataacgtggtggcgagctttaaaaaacgcgtgaacaaccaggtgggcggctataaaaaagaaattgtgctgcgcccggatccgaaacgcaccattcagaaaaaa Construction results of recombinant expression vectors: The three plasmids obtained above were transformed into DH5α cells, and single colonies were picked for expansion culture. Plasmids were extracted from the bacterial culture, and double enzyme digestion identification revealed the presence of corresponding specific cloning fragments, namely CTB (amino acid sequence as shown in SEQ ID NO.1). Figure 3 A), UCVH (amino acid sequence as shown in SEQ ID NO. 13) Figure 3 B) and CTB-UCVH (amino acid sequence as shown in SEQ ID NO. 15) Figure 3 (C in the sequence). Sequence comparison analysis of the sequencing results revealed no gene mutations, and the inserted gene fragment was 100% identical to the target gene sequence, indicating successful construction of the recombinant plasmid.

[0051] The correctly sequenced recombinant expression plasmid (2 μL) was transformed into 100 μL of *E. coli* DE3 competent cells. After incubation on ice for 30 min, heat shock at 42°C for 90 s, and ice incubation for 2 min, 900 μL of LB medium was added and the cells were shaken at 37°C for 1 h. The bacterial culture was then spread onto LB agar plates containing 50 µg / mL Kan and incubated overnight at 37°C. Single colonies were picked and inoculated into 3 mL of LB medium, and cultured at 37°C for 10 h. 2 mL of the bacterial culture was then transferred to 200 mL of LB medium and shaken to OD. 600nm When the concentration reached 0.6-0.8, a final concentration of 1.0 mM IPTG was added, and expression was induced at 37℃ for 5 h. After collecting the bacterial cells and disrupting them with ultrasound, the supernatant and precipitate were separated by centrifugation (the precipitate was washed three times with inclusion body washing buffer and then dissolved in inclusion body dissolution buffer). Each sample was treated with 5×SDS-PAGE loading buffer in a boiling water bath, followed by electrophoresis with a 15% separating gel and a 5% stacking gel, and imaging analysis after Coomassie brilliant blue staining.

[0052] Purification was performed using a Ni-IDA affinity chromatography column. After pre-equilibration with binding buffer, the sample was loaded and washed with wash buffer containing 10 mM imidazole to remove contaminating proteins. The target protein was then collected with elution buffer containing 500 mM imidazole and dialyzed overnight at 4°C with PBS to remove imidazole. Purity of the purified product was verified by SDS-PAGE. For Western blot identification, the membrane was transferred, blocked with 5% skim milk powder, incubated overnight at 4°C with a 1:1000 dilution of His-tag monoclonal antibody, and incubated at room temperature for 2 h with a 1:1000 dilution of HRP-labeled secondary antibody. Specificity was confirmed by imaging after development.

[0053] Expression, purification, and identification results of vaccine antigen proteins: The constructed plasmid was transformed into BL21(DE3) cells, and bacteria were induced to the logarithmic growth phase at 37℃ using a final concentration of 1.0 mM IPTG. Bacterial cultures before and after induction, total protein after sonication, supernatant, and precipitate were collected. SDS-PAGE electrophoresis was used to detect the proteins before and after induction. The results showed (…). Figure 4 ), the three (CTB, Figure 4 A; UCVH, Figure 4 B in CTB-UCVH, Figure 4 In the C group, a specific band appeared at the precipitate after ultrasonic disruption, indicating that all proteins were expressed as inclusion bodies. To maintain the correct conformation and corresponding biological function, inclusion body proteins need to be diluted and refolded before purification via Ni-IDA column. The purified protein was then dialyzed to remove imidazole, and SDS-PAGE (…) was performed… Figure 5 A in the middle) and Western blot ( Figure 5 The results of identification B) showed that the recombinant protein band was single and no nonspecific bands were observed.

[0054] 3. Immunogenicity evaluation of the vaccine 3.1 Immunization regimen and sample collection Six- to eight-week-old SFP-grade female C57BL / 6 mice were randomly divided into four groups: PBS group (without immunogen), CTB group (using CTB as the immunogen), UCVH group (using UCVH as the immunogen), and CTB-UCVH group (using CTB-UCVH as the immunogen). CTB, UCVH, and CTB-UCVH were prepared in step 2 of this embodiment. Immunization was performed via nasal drop and subcutaneous administration. At weeks 0, 1, 2, 3, and 4, each group was immunized via nasal drop with 20 μg of the respective immunogen or an equal volume of PBS. At weeks 0, 2, and 4, each group was subcutaneously injected with 200 μL / mouse (20 µg) of the respective immunogen or an equal volume of PBS. Two weeks after the last immunization, blood samples from the eyeballs, spleen, and fecal samples from the intestines were collected for subsequent immunogenicity testing.

[0055] 3.2 Detection methods and results (1) Flow cytometry detection of mouse spleen antigen-specific CD4 ＋ T lymphocyte secretion Studies have shown that CD4 ＋ T cells dominate Helicobacter pylori infection, and Th1 / Th2 / Th17 cellular immunity constitutes the optimal defense. To investigate whether a designed Helicobacter pylori multi-epitope vaccine could enhance cellular immune responses, the types of Th cells were examined. The immune response efficiency of splenic lymphocytes in four mouse groups (PBS, CTB, UCVH, and CTB-UCVH) was evaluated two weeks after the final immunization.

[0056] The detection method is as follows: 3 × 10⁶ seeds are inoculated into each well of a 24-well plate. 6Splenic lymphocytes were stimulated with 5 μg UCVH in each group. After incubation for 12 h, 1 μL of 2 mM monensin was added to each well, and incubation continued for 5 h to block cytokine secretion. Cells were collected, centrifuged at 1500 rpm for 5 min at 4 °C, the supernatant was discarded, and the cells were washed twice with 2 mL of cold PBS. 1 μL of TruStainFcX™ PLUS antibody was added and the cells were blocked at 4 °C for 20 min. FITC anti-mouse CD3 (2 μL) and PE anti-mouse CD4 (1.5 μL) surface marker antibodies were added and stained at 4 °C for 30 min in the dark. After washing and centrifugation with PBS, 300 μL of FluoroFix™ Buffer was added and the cells were fixed at 4 °C for 30 min in the dark. After washing again, 10-fold diluted intracellular permeabilization buffer was added and the cells were incubated at 4 °C for 30 min in the dark. 5 μL of PECy7 anti-mouse IFN-γ and Brilliant Violet 421 anti-mouse antibodies were added in the dark. IL-17A (1 μL) and APC anti-mouse IL-4 (1 μL) were stained at 4°C in the dark for 30 min. Finally, the cells were washed with PBS, centrifuged, and mixed with 300 μL of 2% PFA. The cells were then analyzed by DxP Athena™ flow cytometer, and the data were analyzed using FlowJo software.

[0057] Flow cytometry results showed that, compared with the CTB and UCVH groups, the spleen CD4 count in the CTB-UCVH group was significantly higher. + IFN-γ in T cells + IL-17A + The level increased significantly ( Figure 6 The numbers A, B, E, and F in the table indicate that Th1 and Th17 immune responses were activated. The UCVH group alone showed higher IL-4 levels, significantly inducing a Th2 immune response, which was statistically different from the CTB-UCVH group. Figure 6 (C and D in the text). This indicates that the properties of CTB adjuvant alter the immune response bias, inhibiting the Th2 response and promoting a more balanced Th1 response. There was no difference in the secretion levels of the three cytokines between the CTB-only group and the PBS group, indicating that adjuvant alone does not have an immune response effect.

[0058] (2) ELISA detection of antigen-specific cytokine secretion To comprehensively evaluate the ability of the UCVH vaccine to trigger cellular immunity, the levels of various Helicobacter pylori immune-related cytokines in the supernatant of mouse spleen lymphocyte culture were measured.

[0059] The detection method is as follows: 3 × 10⁶ cells are seeded per well of a 24-well cell culture plate. 6Splenic lymphocytes were collected and stimulated with 15 μg UCVH in each group. After incubation at 37°C for 48 h, the cell culture supernatant was collected by centrifugation at 4000 rpm for 15 min in 1.5 mL EP tubes at 4°C. The procedure was performed according to the instructions of the commercial kit (DAKEWE, China): 100 μL of cytokine standard solution and the sample to be tested were added to each well. Biotin-labeled antibodies were used as primary antibodies, Streptavidin-HRP as secondary antibodies, and TMB substrate was used for color development. TMB blank wells were used as controls. OD was measured within 15 min after color development using a SpectraMax Mini microplate reader (Molecular Devices, Shanghai, China). 450 The absorbance values ​​of all standards and samples were subtracted from the absorbance values ​​of the blank wells. A standard curve was plotted with absorbance values ​​on the ordinate and concentration on the abscissa to derive the regression equation. The absorbance values ​​of the samples were then substituted into the equation to calculate the concentrations of each cytokine in the test samples.

[0060] Results: After stimulation with UCVH antigen, the levels of IFN-γ and IL-17A secreted by lymphocytes in the UCVH and CTB-UCVH groups were significantly higher than those in the PBS and CTB groups. Figure 7 C and E in the C group). The UCVH group had higher IL-4 levels and significantly induced a Th2 immune response, which was statistically different from the CTB-UCVH group, consistent with the flow cytometry results, indicating that the UCVH group secreted more IL-4. Figure 7 D). These results indicate that the CTB-UCVH vaccine not only induces adequate levels of IFN-γ and IL-17A, but more importantly, it can exhibit a Th1-dominant immune response.

[0061] (3) ELISPOT detection of antigen-specific IFN-γ secretion level ELISPOT is the gold standard for measuring antigen-specific T cell frequency after vaccination. Therefore, to confirm the ability of Helicobacter pylori multi-epitope vaccines to trigger cellular immunity, ELISPOT was used to detect IFN-γ secretion levels. Unlike ICS, which uses multiple immunomarkers to detect antigen-specific T cell frequency, ELISPOT primarily assesses the overall level of memory cells that secrete cytokines.

[0062] Detection Method: The antigen-specific IFN-γ secretion level was detected using a commercially available ELISPOT kit (DAKEWE, China). The specific steps were as follows: The kit was warmed to room temperature for 20 minutes before use; positive stimulants were dissolved in sterile PBS; the PVDF membrane at the bottom of the wells was activated with 200 μL of serum-free culture medium at room temperature for 10 minutes, then removed. Three parallel experimental groups of protein stimulation and positive / negative controls were set up, and samples were added according to the corresponding system (experimental group: 2 × 10⁻⁶).5 Cells + 5 μg UCVH; Positive control: 2 × 10⁻⁶ 5 Cells + 10 μL positive stimulus; negative control: only 2 × 10 5 Cells were added to RPMI 1640 complete medium to a final volume of 100 μL and incubated for 16 h. The wells were then cleaned, and the cells were lysed in 200 μL of ice-cold deionized water at 4°C for 10 min. The cells were then washed 6 times (1 min each time) with 250 μL of washing buffer. 100 μL of detection antibody and 100 μL of HRP were added sequentially, and the cells were incubated at 37°C for 1 h each time, with repeated washing after each incubation. The bottom and base of the membrane were washed and dried. The plate was capped and washed once before being plated. AEC chromogenic buffer was prepared, and 100 μL / well was added for chromogenic development at 37°C in the dark for 20 min. The reaction was terminated by washing 5-7 times with deionized water. After drying, the number of SFCs was counted using an IRISTM ELISPOT plate reader.

[0063] Results: After UCVH stimulation, the PBS group showed almost no response, while the CTB group showed a weak response. The CTB-UCVH group achieved the highest level of IFN-γ spotted cells (SFCs), which were significantly higher than those in other groups. Figure 7 (A) The UCVH group produced significantly higher levels of IFN-γ-SFC secretion than the PBS and CTB groups, second only to the CTB-UCVH group. Figure 7 (B in the original text). The above results indicate that the Helicobacter pylori multi-epitope vaccine designed in this invention can significantly enhance the ability of lymphocytes to secrete IFN-γ.

[0064] (4) ELISA detection of antigen-specific antibody secretion in mouse serum and intestinal feces To analyze antibody production in mice immunized with a Helicobacter pylori multi-epitope vaccine, mouse serum was collected two weeks after the last immunization to detect antigen-specific IgG and its subtypes, and mouse fecal samples were collected to detect antigen-specific sIgA.

[0065] The detection method was as follows: Mice were sacrificed two weeks after the last immunization, and blood was collected from the eyeballs. After standing at room temperature for 30 min, the serum was collected by centrifugation at 12000 rpm for 10 min. 50 mg of mouse feces was placed in a 1.5 mL EP tube, 300 μL of 1×PBS was added, and the tube was allowed to stand for 2 h. The feces were then crushed and mixed, and the mixture was centrifuged at 12000 rpm for 20 min. The supernatant was collected. 5 μg / well of UCVH was coated onto a 96-well plate and incubated overnight at 4 °C. After washing with PBST, 5% BSA was added and the plate was blocked at 37 °C for 2 h. After washing the plates, 100 μL of mouse serum was added per well, with concentration gradients of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and 1:51200 for mouse fecal intestinal samples. Concentration gradients of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, and 1:5120 were used for mouse fecal intestinal samples. The plates were incubated at 37°C for 1 hour. After washing, HRP (goat anti-mouse IgG), IgG1, IgG2a, and sIgA (1:5000) were added, and incubation continued for another hour. TMB substrate was added after washing the plates. After washing the plate, add TMB substrate and react for 10 min. Then add 100 μL of stop solution and measure the absorbance at 450 nm.

[0066] The results show that ( Figure 8 (A, B, and C in the original text) The levels of IgG, IgG1, and IgG2a antibodies in the CTB-UCVH group were significantly higher than those in the PBS, CTB, and UCVH groups, with statistically significant differences. IgA is crucial for clearing Helicobacter pylori from the mucosa, so the antibody titer of intestinal fecal antigen-specific sIgA was measured. Figure 8 (D) The UCVH group alone produced slightly more IgA than the PBS and CTB groups. The UCVH group alone produced slightly more sIgA than the PBS and CTB groups, while the CTB-UCVH group induced significantly the highest sIgA level, demonstrating that the vaccine has the effect of stimulating mucosal and humoral immunity.

[0067] 4. Statistical Analysis All data were analyzed using GraphPad Prism (San Diego, CA, USA) software. All results are expressed as mean ± standard deviation (SD). One-way ANOVA followed by Dunnett's test for multiple comparisons was used to determine whether significant differences existed between individual groups. Data were visualized using GraphPad Prism 9.0. A p-value < 0.05 was considered statistically significant. (P<0.05); (P<0.01); (P<0.001); (P<0.0001).

[0068] This invention constructs a CTB-mediated Hp multi-epitope vaccine and verifies its immunogenicity and immunizing effect using a mouse model. A schematic diagram illustrating the construction of the Helicobacter pylori multi-epitope vaccine and the evaluation of its immunizing effect in mice is shown below. Figure 9 .

[0069] The type and intensity of the immune response directly determine the protective effect of a vaccine. The immune protection mechanism of *Helicobacter pylori* (Hp) vaccines is centered on the synergistic effect of Th cell-mediated cellular immunity and antibody-mediated humoral immunity. In cellular immunity, Th1 cells, by secreting IFN-γ and IL-2, dominate the anti-Hp response, directly killing bacteria or activating effector cells; Th2 cells secrete IL-4 and IL-10 to help B cells produce antibodies, synergistically restoring immune balance with Th1 cells; Th17 cells, through IL-17, recruit neutrophils to participate in local defense, and their activity is regulated by miR-155, exhibiting a dual role of protection and inflammatory damage. Furthermore, Th1 / Th17 co-polarization is an important pathway for some adjuvants. At the humoral immune level, the vaccine induces the secretion of antibodies such as sIgA and IgG through mucosal immunity. sIgA can cross the epithelial barrier, bind to *Hp*, and inhibit its adhesion, while IgG participates in systemic immune defense.

[0070] In this invention, after subcutaneous and intranasal immunization of mice, the serum levels of *H. pylori*-specific IgG antibodies were significantly increased, with IgG2a and IgG1 levels significantly higher than in the control group, indicating that the vaccine induced a humoral immune response dominated by the Th1 type. *H. pylori*, as an intracellular parasite, relies on the participation of the Th1 immune response for its clearance process—IFN-γ secreted by Th1 cells can activate the phagocytic function of macrophages, enhancing intracellular bactericidal ability, while simultaneously promoting B cell differentiation into plasma cells, producing high-affinity IgG antibodies. Serum IgG can penetrate into the gastric mucosa through blood circulation, binding to *H. pylori* and mediating complement activation or antibody-dependent cytotoxicity (ADCC), accelerating bacterial clearance; the dominant expression of the IgG2a subtype further verifies the dominant role of the Th1 immune response, which is highly consistent with the immune defense requirements against *H. pylori* infection. Furthermore, the fecal sIgA level in the intestines of immunized mice was significantly increased, indicating that the CTB adjuvant successfully induced a mucosal immune response. SIgA, as the main local antibody in the gastric mucosa, can form an immune barrier on the surface of the gastric mucosa, preventing the adhesion of *Helicobacter pylori* (Hp) to gastric epithelial cells, while neutralizing the biological activity of toxins such as VacA and CagA, thus reducing inflammatory damage to the gastric mucosa. This dual response mode of "humoral immunity + mucosal immunity" is the core mechanism by which this vaccine exerts its protective effect.

[0071] Cellular immunity also plays a crucial role in the clearance of *H. pylori* infection. This invention further clarifies the characteristics of vaccine-induced cellular immunity using flow cytometry and ELISA: CD4+ + The levels of IFN-γ and IL-17A secreted by T cells were significantly higher in the CTB-UCVH group than in the PBS control group, the CTB adjuvant alone group, and the UCVH antigen alone group, while the IL-4 level showed a trend of being higher in the UCVH alone group than in the CTB-UCVH group.

[0072] The above results confirm that the vaccine of the present invention can effectively induce the body to produce specific mucosal and systemic immune responses, has good immunogenicity, and can play a role in resisting Hp infection.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multi-epitope peptide, characterized in that, The amino acid sequence of the multi-epitope peptide is obtained by sequentially linking amino acids 349-363 of the UreB subunit, 341-352 of the CagA subunit, 312-323 of the VacA subunit, 98-109 of the HpaA subunit, 407-421 of the UreB subunit, 178-192 of the CagA subunit, 411-425 of the VacA subunit, 124-138 of the HpaA subunit, 70-78 of the UreB subunit, 36-44 of the CagA subunit, 241-249 of the VacA subunit, and 124-138 of the HpaA subunit with short peptides.

2. The multi-epitope peptide according to claim 1, characterized in that, The short peptides include AAY and / or KK.

3. The multi-epitope peptide according to claim 1, characterized in that, The amino acid sequence of the multi-epitope peptide is shown in SEQ ID NO.

13.

4. The use of the multi-epitope peptide according to any one of claims 1 to 3 in the preparation of Helicobacter pylori multi-epitope vaccines.

5. A Helicobacter pylori multi-epitope vaccine, characterized in that, Includes the multi-epitope peptide according to any one of claims 1 to 3.

6. The multi-epitope vaccine according to claim 5, characterized in that, The active ingredient of the multi-epitope vaccine is a protein composed of a cholera enterotoxin B subunit and a multi-epitope peptide as described in any one of claims 1 to 3.

7. The multi-epitope vaccine according to claim 6, characterized in that, The amino acid sequence of the cholera enterotoxin B subunit is shown in SEQ ID NO.

1.

8. The multi-epitope vaccine according to claim 7, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

15.

9. The use of the multi-epitope peptide according to any one of claims 1 to 3 or the multi-epitope vaccine according to any one of claims 5 to 8 in the preparation of a drug for preventing Helicobacter pylori infection.

10. The application according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images