Lipopeptides and applications for the prevention of Helicobacter pylori infection

Abstract

This invention provides a lipopeptide for preventing Helicobacter pylori infection and its application, comprising a Helicobacter pylori immunodominant epitope peptide and Pam2Cys covalently bound to the N-terminus of the Helicobacter pylori immunodominant epitope peptide. This invention also provides a pharmaceutical composition with this lipopeptide as the active ingredient and its application. The lipopeptide provided by this invention can efficiently enhance specific cellular and mucosal immune responses and preventively reduce the amount of Helicobacter pylori colonizing the stomach, and its in vitro safety is good.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to Helicobacter pylori self-adjuvant lipopeptides that can enhance cellular and mucosal immune responses and their applications. Background Technology

[0002] Helicobacter pylori (H. pylori) colonizes the digestive epithelium of approximately 50% of the world's population and is a leading cause of peptic ulcers, chronic gastritis, gastric cancer, and gastric MALT lymphoma. Triple or quadruple therapy with antibiotics combined with proton pump inhibitors is the first-line treatment for H. pylori infection. However, due to rising drug resistance rates, the effectiveness of antibiotic-based treatments is declining. There is an urgent need to find alternative or complementary therapies to antibiotic therapy. Developing an effective vaccine to prevent or treat H. pylori infection is the most economical and effective option. H. pylori virulence factors, or adhesins, have been shown to be potential vaccine candidate antigens. Among them, urease (Ure) is essential for H. pylori to overcome the highly acidic environment of the stomach, and cytotoxin-associated gene A (CagA) is one of the most typical toxins of H. pylori, associated with an increased risk of gastritis, peptic ulcers, and gastric cancer. In addition, other candidate antigens include heat shock protein 60 (HSP60), lipoprotein 20 (Lpp20), and Helicobacter pylori adhesin A (HpaA). Combined immunization using these antigens and mucosal adjuvants has been shown to provide protection against Helicobacter pylori infection.

[0003] Given that subunit vaccines containing intact antigens contain non-protective epitopes that can lead to immunosuppression or even immune damage, epitope vaccines composed of immunodominant epitopes of protective antigens offer advantages over intact antigen vaccines, including specific immune responses, fewer side effects, and better targeting. However, a major challenge in epitope vaccine development is their low immunogenicity. The most common strategy is to combine them with potent adjuvants to help the antigen be recognized by the innate immune system and further initiate an adaptive immune response. Because protection against Helicobacter pylori infection relies primarily on vaccine-specific mucosal immunity and T-cell responses, aluminum adjuvants, which enhance humoral responses and Th2 bias, are not suitable as adjuvants for H. pylori vaccines. In fact, few adjuvants have been found to induce both cellular and mucosal immune responses safely and effectively; therefore, exploration in this area may be key to improving the immunogenicity of Helicobacter pylori epitope vaccines. Summary of the Invention

[0004] This invention synthesizes a lipopeptide vaccine with self-adjuvant activity by chemically linking Pam2Cys to an immunodominant epitope from a protective antigen of Helicobacter pylori. The resulting lipopeptide vaccine has been validated to activate dendritic cells (DCs) and provide protection against Helicobacter pylori infection.

[0005] The present invention first provides a lipopeptide for preventing Helicobacter pylori infection, comprising a Helicobacter pylori immunodominant epitope peptide and Pam2Cys covalently bound to the N-terminus of the Helicobacter pylori immunodominant epitope peptide.

[0006] In one embodiment of the present invention, the Helicobacter pylori immunodominant epitope peptide is selected from polypeptides with the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10; preferably, the Helicobacter pylori immunodominant epitope peptide is selected from polypeptides with the amino acid sequences SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:10.

[0007] In one embodiment of the present invention, the Pam2Cys structure is as follows:

[0008] .

[0009] The present invention also provides a pharmaceutical composition in which the active ingredient comprises at least one lipopeptide as described above, and a pharmaceutically acceptable excipient.

[0010] In one embodiment of the present invention, the active ingredient of the pharmaceutical composition comprises a lipopeptide formed by covalently binding Pam2Cys to a polypeptide with the amino acid sequence of SEQ ID NO:4 and / or SEQ ID NO:10 of the Helicobacter pylori immunodominant epitope peptide. Its advantage is that it is hydrophilic at one end and hydrophobic at the other end, and can easily self-assemble into nanoparticles.

[0011] The present invention also provides the use of the above-described lipopeptide or pharmaceutical composition in the preparation of a medicament for the prevention or treatment of Helicobacter pylori infection.

[0012] The beneficial effects of the above-described technical solution of the present invention are as follows:

[0013] The Helicobacter pylori self-adjuvant lipopeptide provided by this invention is composed of Pam2Cys and different Helicobacter pylori immunodominant epitope peptides, which can efficiently enhance cellular immune response and mucosal immune response. It has good in vitro safety, a particle size of about 50 nm, which is similar in size to the pathogen and is easily recognized and phagocytosed by antigen-presenting cells. It can stimulate the maturation of dendritic cells by activating multiple pattern recognition receptors.

[0014] The Helicobacter pylori self-adjuvant lipopeptide provided by this invention, when used alone or in combination, can induce high levels of cellular and mucosal immune responses after nasal immunization in experiments, and reduce the colonization of Helicobacter pylori in the stomach of mice. This indicates that both lipopeptide vaccines and their combination can prevent Helicobacter pylori infection to a certain extent. It has significant market application value and broad application prospects. Attached Figure Description

[0015] Figure 1A : Synthesis route diagram of lipopeptide vaccines;

[0016] Figure 1B Schematic diagrams of the structures of Hp2, Hp4, Hp7, and Hp10;

[0017] Figure 1C In vitro TLR2 activation activity of Hp2, Hp4, Hp7, and Hp10;

[0018] Figure 1D Transmission electron microscopy images of Hp4 and Hp10;

[0019] Figure 1E This is a graph showing the results of a cytotoxicity assay.

[0020] Figure 1F Image showing the results of the hemolysis test.

[0021] Figure 1A Data in -F are expressed as mean ± standard deviation, n = 3, **** indicates P < 0.001 relative to the control group, and **** indicates P < 0.0001 relative to the control group.

[0022] Figure 2A The results are from transcriptome sequencing and bioinformatics analysis after co-incubation of BMDCs with Hp4 or Hp10. Principal component analysis showed significant differences between the Hp4, Hp10 lipopeptide vaccine groups and the PBS control group.

[0023] Figure 2B Volcano plot of differentially expressed genes (DEGs) in the Hp4-treated group. Upregulated genes are marked in red (LFC>1, P<0.05), and downregulated genes are marked in blue (LFC<-1, P<0.05). Representative gene names with significant changes are highlighted.

[0024] Figure 2C Volcano plot of differentially expressed genes (DEGs) in the Hp10 treatment group. Upregulated genes are marked in red (LFC>1, P<0.05), and downregulated genes are marked in blue (LFC<-1, P<0.05). Representative gene names with significant changes are highlighted.

[0025] Figure 2D This is a heatmap of unsupervised clustering of differentially expressed genes in the sample.

[0026] Figure 2E Venn diagram to show the overlap and differences of upregulated genes in Hp4 and Hp10 treated samples.

[0027] Figure 2F To use Metscape to cluster and label Hp4-upregulated genes, Hp10-upregulated genes, Hp4-only upregulated genes, Hp10-only upregulated genes, and co-upregulated genes, and further fold redundant labels into immune-related and irrelevant ones.

[0028] Figure 2G To cluster and annotate genes that are jointly upregulated by Hp4 and Hp10 using Metascape, redundant labels were folded and visualized using Cytoscape.

[0029] Figure 3A This study analyzed the pathways of genes co-regulated by Hp4 and Hp10 treatment in BMDCs, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG).

[0030] Figure 3B A heatmap showing the core genes of enriched signaling pathways. Gene expression levels are measured in transcript fractions per million (TPM).

[0031] Figure 3C The graph shows the results of gene set enrichment analysis (GSEA) for the Hp4-treated group, indicating significant enrichment of gene features related to Toll-like receptors (TLRs), NOD-like receptors (NLRs), and human retinoic acid-induced gene-I-like receptors (RLRs) (P<0.05). The y-axis represents the enrichment score (top) and the ranking list (bottom). The x-axis represents individual genes in the gene set shown.

[0032] Figure 3D The graph shows the results of gene set enrichment analysis (GSEA) for the Hp10 treatment group, indicating significant enrichment of TLR, NLR, and RLR-related gene features (P<0.05). The y-axis represents the enrichment score (top) and the ranking list (bottom). The x-axis represents individual genes in the gene set shown.

[0033] Figure 3E Confocal microscopy images of BMDCs incubated with P4, P10, Hp4, and Hp10 for 24 hours, respectively. The cytoskeleton was stained with TRITCphalloidin, and the nuclei were visualized using DAPI. P4, P10, Hp4, and Hp10 were labeled with Abfluor™ 680.

[0034] Figure 4AThe GSEA enrichment results show the enrichment of the mature gene set of Lindstedt DCs in Hp4-treated (left) and Hp10-treated (right) BMDCs.

[0035] Figure 4B Gene heatmaps of co-stimulatory molecules, cytokines, and chemokines. Gene expression is measured in parts per million (TPM).

[0036] Figure 4C A representative figure showing the expression of CD40, CD80, and CD86 in a CD11c+ cell population after BMDCs were co-incubated with PBS, Hp4, or Hp10 for 48 hours.

[0037] Figure 4D This is a statistical graph showing the expression of CD40, CD80, and CD86 in the CD11c+ cell population after BMDCs were co-incubated with PBS, Hp4, or Hp10 for 48 hours.

[0038] Figure 4E The levels of representative cytokines that induce CD4+ T cell differentiation toward Th1 in the culture supernatant of BMDCs were detected by ELISA.

[0039] Figure 4F The levels of representative cytokines that induce CD4+ T cell differentiation toward Th2 in the culture supernatant of BMDCs were detected by ELISA.

[0040] Figure 4G The levels of representative cytokines that induce CD4+ T cell differentiation toward Th17 in the culture supernatant of BMDCs were detected by ELISA.

[0041] Figure 4A Data in -G are expressed as mean ± standard deviation, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared with the PBS control group;

[0042] Figure 5A For the immunization and sampling schedule.

[0043] Figure 5B The figure shows the results of significant specific cellular immune responses induced by both Hp4 and Hp10. Fourteen days after the last immunization, the number of IFN-γ, IL-4, and IL-17 spot-forming cells in spleen lymphocytes stimulated by P4 or P10 was detected using the ELISpot method.

[0044] Figure 5C A statistical graph showing the levels of IFN-γ, IL-4, and IL-17 in the supernatant of spleen lymphocytes after stimulation, as determined by ELISA.

[0045] Figure 5D This is a representative graph and statistical chart showing the proportion of central memory T cells (Tcm) in CD4+ T cells.

[0046] Figure 5E This is a representative graph and statistical chart showing the proportion of Tcm in CD8+ T cells.

[0047] Figure 5A Data in -E are expressed as mean ± variance, n = 5. * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0048] Figure 6A Four weeks after mice were immunized with Hp10 lipopeptide vaccine, the colonization level of Hp10 in their stomachs was measured by real-time quantitative PCR. The results showed that the bacterial colonization in the stomach of the Hp10 lipopeptide vaccine-immunized group was significantly reduced.

[0049] Figure 6B This is a map showing the serum IgG levels in mice detected using the ELISA method during the immunization process.

[0050] Figure 6C To determine the IgG subtypes in Hp10-immunized mice.

[0051] Figure 6D This is a map showing the serum IgA levels in mice detected by ELISA during the immunization process.

[0052] Figure 6E Two weeks after the last immunization, gastric homogenate was collected and its specific secretory IgA (sIgA) level profile was determined by ELISA.

[0053] Figure 6F Two weeks after the last immunization, intestinal lavage fluid was collected and its specific sIgA level profile was determined by ELISA.

[0054] Figure 6G This is a saliva sample, showing the changes in sIgA at the mucosal site during the immunization process.

[0055] Figure 6H This is a vaginal lavage fluid sample, showing the changes in sIgA in the mucosal region during the immunization process;

[0056] Figure 6A Data in -F are expressed as mean ± standard deviation, n = 5. * * P<0.01, ****P<0.0001.

[0057] Figure 7AFour weeks after mice were immunized with Helicobacter pylori-infected lipopeptide vaccine, the colonization level of Helicobacter pylori in their stomachs was measured by real-time quantitative PCR. The results showed that the bacterial colonization in the stomach of the Hp4+Hp10 lipopeptide vaccine combined immunization group decreased more significantly.

[0058] Figure 7B The levels of IFN-γ, IL-4, and IL-17 in the supernatant of spleen lymphocytes after stimulation were detected by ELISA.

[0059] Figure 7C A representative diagram showing the proportion of Tcm in CD4+ T cells and CD8+ T cells.

[0060] Figure 7D A statistical graph showing the proportion of Tcm in CD4+ T cells and CD8+ T cells.

[0061] Figure 7E The graph shows the serum IgG antibody levels in mice measured using the ELISA method during the immunization process.

[0062] Figure 7F A graph showing the IgG subclass levels in mice immunized with Hp4+Hp10.

[0063] Figure 7G Gastric homogenate was collected 2 weeks after the last immunization, and its specific sIgA level was detected by ELISA.

[0064] Figure 7H Intestinal lavage fluid was collected 2 weeks after the last immunization, and its specific sIgA level was detected by ELISA.

[0065] Figure 7I A graph showing changes in sIgA in the mucosal region during the immunization process in vaginal irrigation samples.

[0066] Figure 7J The serum of mice immunized with Hp4+Hp10, as well as the gastric homogenates of mice immunized with Hp10 and Hp4+Hp10, both significantly inhibited bacterial adhesion to AGS cells in vitro, with the gastric homogenate showing a stronger inhibitory effect.

[0067] Figure 7A Data in -J are expressed as mean ± standard deviation, n = 5. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0068] Figure 8 The mass spectrum of Hp2;

[0069] Figure 9 Mass spectrum of Hp4;

[0070] Figure 10 Mass spectrum of Hp7;

[0071] Figure 11 This is the mass spectrum of Hp10. Detailed Implementation

[0072] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0073] Reagents and materials:

[0074] Unless otherwise specified, all reagents and materials involved in this invention were obtained through legal commercial channels.

[0075] The mice were BALB / c strain and purchased from Beijing Huafukang.

[0076] Helicobacter pylori Sydney strain HpSS1 was purchased from the NTCC Type Culture Collection (accession number: NTCC502694).

[0077] The IFN-γ, IL-4, and IL-17A cytokine kits were purchased from Dakota (catalog numbers: 1210002; 1210402; 1211702).

[0078] FITC anti-mouse CD11c antibody was purchased from Biolegend (catalog number: 117306).

[0079] The PE anti-mouse CD40 antibody was purchased from Biolegend (catalog number: 124610).

[0080] Percp anti-mouse CD86 antibody was purchased from Biolegend (catalog number: 105114).

[0081] APC anti-mouse CD80 antibody was purchased from Biolegend (catalog number: 104713).

[0082] The PE anti-mouse CD3 antibody was purchased from Biolegend (catalog number: 100205).

[0083] FITC anti-mouse CD4 antibody was purchased from Biolegend (catalog number: 130308).

[0084] PerCP / Cy5.5 anti-mouse CD8a antibody was purchased from Biolegend (catalog number: 100734).

[0085] The PE / Cy7 anti-mouse CD44 antibody was purchased from Biolegend (catalog number: 103029).

[0086] APC anti-mouse CD62L antibody was purchased from Biolegend (catalog number: 104411).

[0087] Goat pAb Anti-Mouse IgG / IgG1 / IgG2a / IgG2b / IgG2c / IgG3 / IgA (HRP) were purchased from Abogen (product numbers: ab6789; ab97240; ab97245; ab97250; ab97255; ab97260; ab97235).

[0088] Fetal bovine serum was purchased from GIBCO (product number: 10099141).

[0089] 1% penicillin / streptomycin was purchased from Sigma (product number: V900929).

[0090] GM-CSF cytokine was purchased from Pipetek (catalog number: 315-03).

[0091] IL-4 cytokine was purchased from Pipertec (catalog number: 214-14).

[0092] The trypsin inhibitor was purchased from Shanghai Sangon Biotech (product number: A003570-0100).

[0093] EDTA-2Na was purchased from Bio-Sens (item number: D10185).

[0094] PMSF was purchased from Boaosen (item number: D10411).

[0095] CCK-8 was purchased from White Shark Easy (item number: BS350B).

[0096] Pilocarpine was purchased from Yuanye Biotechnology (product number: B20843).

[0097] Sodium pentobarbital was purchased from Beijing Baihao Biotechnology Co., Ltd. (Catalog No.: P6031).

[0098] Phosphotungstic acid was purchased from Solarbio (item number: P9761).

[0099] PBS was purchased from White Shark Easy (item number: BL601A).

[0100] Example 1: Preparation and screening of Helicobacter pylori (H. pylori) self-adjuvanted lipopeptide vaccine

[0101] 1. Screening of epitope peptides

[0102] Dominant epitopes with immunoprotective effects were obtained through screening, as shown in Table 1.

[0103] Table 1. Sequences and sources of dominant epitope peptides

[0104]

[0105] 2. Synthesis of lipopeptide vaccines:

[0106] Pam2Cys was covalently bound to the N-terminus of a selected epitope using a solid-phase synthesis method to obtain a lipopeptide vaccine (hereinafter referred to as Hp1-Hp10). The synthetic route is as follows: Figure 1A As shown. The structural diagrams of the four synthesized lipopeptides Hp2, Hp4, Hp7, and Hp10 are shown in the figure. Figure 1B The purities of Hp2, Hp4, Hp7, and Hp10 determined by HPLC were 98.66%, 98.34%, 98.97%, and 98.48%, respectively. Mass spectrometry further confirmed the structure of the lipopeptide vaccine; the mass spectra are shown below. Figures 8-11 As shown.

[0107] 3. In vitro TLR2 activity screening of lipopeptide vaccines:

[0108] The TLR2 activity of the synthetic lipopeptide vaccine was detected using HEK-Blue™-mTLR2 cells. The synthetic lipopeptide vaccine, endotoxin-free water (negative control), and Pam2CSK4 (hereinafter referred to as P2CSK4) (positive control) were respectively mixed with HEK-Blue™-assay-concentrated HEK-Blue. TM mTLR2 cells were incubated at 37°C for 14 h, and then SEAP activity was measured at 620 nm using a spectrophotometer. The results are shown in Figure 1C, indicating that lipopeptides Hp4 and Hp10 can activate TLR2 cells in vitro.

[0109] 4. In vitro characterization of self-adjuvanted lipopeptide vaccines:

[0110] Self-adjuvanted lipopeptide vaccines Hp4 and Hp10 were dissolved in deionized water to prepare 0.1 mg / mL solutions, which were then ultrasonically dispersed. The solutions were dropped onto a copper mesh covered with a carbon film. The sample solutions were negatively stained with 2% phosphotungstic acid, and the morphology of the lipopeptides was observed using a transmission electron microscope (JEOL JEM-1230, Tokyo, Japan). The results are shown in Figure 1D. Hp4 and Hp10 both self-assembled into regular spherical particles with a diameter of approximately 50 nm in aqueous solution.

[0111] 5. In vitro safety studies of self-adjuvanted lipopeptide vaccines:

[0112] The safety of the lipopeptide vaccine was preliminarily evaluated using mouse erythrocyte hemolysis and CCK-8 cytotoxicity assays. A 2% mouse erythrocyte suspension was mixed with different concentrations of lipopeptide vaccine, equal volumes of deionized water (positive control), and physiological saline (negative control). After incubation at 37°C for 3 hours, the OD value in the supernatant was measured. 570 nm Hemolysis rate (%) = (OD) 570 nm Sample - OD 570 nm (Negative control) / (OD) 570 nmPositive control - OD 570 nm (Negative control)*100%. Cytotoxicity assay: After BMDCs adhered to the culture medium, lipopeptide vaccines Hp4 and Hp10 were added, and the cells were cultured at 37°C and 5% CO2 for 24 hours. The culture medium was used as a blank control, and the cell suspension was used as a negative control. 10 μl of CCK-8 solution was added to each well, and OD was measured after 1-4 hours. 450 nm Cell viability (%) = (OD) 450 nm Sample - OD 450 nm Blank control) / (OD) 450 nm Negative control - OD 450 nm (Blank control)*100%. The results showed that Hp4 and Hp10 exhibited extremely high safety in vitro, with cell viability exceeding 98% (Figure 1 E). The hemolysis rate of mouse erythrocytes was less than 2% (Figure 1 F).

[0113] Example 2: The ability of Hp4 and Hp10 to promote the maturation of BMDCs

[0114] 1. Acquisition and cultivation of BMDCs

[0115] Bone marrow cells from the tibia and femur of SPF mice were aseptically isolated. Cells were induced into dendritic cells using directional differentiation medium (RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, 20 ng / mL GM-CSF, and 10 ng / mL IL-4). Half of the medium was replaced with fresh medium on days 3 and 5, and cells were collected on day 7.

[0116] 2. Transcriptome sequencing (RNA-seq)

[0117] BMDCs were incubated with PBS, Hp4, or Hp10 for 48 hours, and then resuspended in 1 ml of Trizol before collection. Raw RNA-seq library data generated and sequenced by Shanghai Sangon Biotech were evaluated using FastQC. Trimmomatic was used for data processing to obtain relatively accurate and efficient results.

[0118] 3. Bioinformatics Analysis

[0119] Gene expression was assessed using StringTie and known gene models. Differential gene expression analysis was performed using DESeq2, and the results were visualized using heatmaps. KEGG pathway and gene ontology (GO) enrichment analyses were performed using ClusterProfiler and Metascape. GSEA enrichment analysis was used to compare our gene set with annotated or experimentally validated gene sets. Bioinformatics showed that the immune process of BMDCs was fully initiated. Figure 2A-G), which may be because lipopeptide vaccines activate multiple pattern recognition receptors, including toll-like receptors (TLRs), nod-like receptors (NLRs), and retinoic acid-inducible gene (RIG-i)-like receptors (RLRs). Figure 3A -D). Further enrichment of the mature phenotype of BMDCs after lipopeptide vaccine stimulation was achieved using GSEA. Figure 4A And the heatmap showed a significant upregulation of co-stimulatory molecules, chemokines, and cytokines. Figure 4B ).

[0120] 4. Confocal Laser Scanning Microscope (CLSM)

[0121] BMDCs cultured for 7 days were collected and added to confocal microscopy dishes, and co-incubated for 24 hours with Abfluor™ 680-labeled lipopeptide or polypeptide solutions (10 μg / ml, both). Before observation using a laser confocal microscope, the cells were fixed and permeabilized, and the nuclei and cytoskeleton were stained with DAPI and TRITC phalloidin, respectively. The results showed that P4 and P10 were only adsorbed onto the surface of BMDCs, while Hp4 and Hp10 were internalized by BMDCs (Figure 3E), indicating that the activation and endocytosis of PRRs are specific processes triggered by Pam2CS fragments rather than epitopes.

[0122] 5. In vitro maturation assay of BMDCs

[0123] After co-culturing BMDCs with stimulants (Hp4 or Hp10, 10 μg / mL) for 48 hours, cells and supernatant were separated by centrifugation at 1800 rpm / min. Cells were washed three times with PBS and then incubated with FITC anti-mouse CD11c antibody, PE anti-mouse CD40 antibody, Percp anti-mouse CD86 antibody, and APC anti-mouse CD80 antibody at 4°C in the dark for 30 min. Fluorescently labeled cells were analyzed using FACS Verse flow cytometry (BD Biosciences, San Jose, CA). Simultaneously, the levels of IL-1, IL-2, IL-4, IL-6, IL-12, and IFN-γ cytokines in the supernatant were measured using a pre-coated kit. The results showed that the proportions of CD40+ / CD11c+, CD80+ / CD11c+, and CD86+ / CD11c+ cells were significantly higher in the Hp4 or Hp10 treatment groups than in the control group (Figure 4 CD), demonstrating that both Hp4 and Hp10 have a strong ability to stimulate DC cell maturation. Furthermore, the secretion levels of IL-1, IL-2, IL-12, and IFN-γ were significantly increased in the Hp4 and Hp10 treatment groups (Fig. 4 E), and these cytokines induce Th1-oriented cell differentiation. In addition, Hp4 or Hp10 can also increase the secretion levels of IL-2 (Fig. 4 F) and IL-6 (Fig. 4 G), promoting the differentiation of Th2 and Th17 cells.

[0124] Example 3: Both Hp4 and Hp10, self-adjuvanted lipopeptide vaccines, enhance cellular immune responses.

[0125] 1. Animal grouping and immunization

[0126] Mice were randomly divided into 7 groups based on body weight. After anesthesia with sodium pentobarbital, they were administered drugs intranasally (peptide, peptide + P2CSK4, lipopeptide) or an equal volume of PBS (control group). Immunization was performed every two weeks for a total of three times. Two weeks after the third immunization, mice were sacrificed and serum, spleen lymphocytes, and gastrointestinal homogenate were collected (Figure 5A).

[0127] 2. ELIspot assay to detect the levels of IFN-γ, IL-4, and IL-17A secreted by mouse spleen cells.

[0128] The spleen of a mouse was removed, and spleen cells were ground in 3 mL of erythrocyte lysis buffer to obtain a suspension. After centrifugation and discarding the supernatant, the cells were resuspended in complete culture medium and the spleen lymphocyte concentration was adjusted to 1 × 10⁻⁶. 6Mouse spleen lymphocyte suspension (Hp4 or P10, 10 μg / mL) pre-coated with IFN-γ, IL-4, and IL-17A cytokines was added to 96-well plates (Mabtech) and co-cultured with peptides (P4 or P10, 10 μg / mL) for 48 h. Peptide-free cells served as a negative control. Spot counts were read using an ELISpot Reader (AID). The results showed that the levels of IFN-γ, IL-4, and IL-17A secreted by spleen lymphocytes stimulated with P4 or P10 epitope peptides were significantly higher in the Hp4 and Hp10 groups than in the other control groups, indicating that the lipopeptide vaccines Hp4 and Hp10 can induce a strong cellular immune response (Figure 5B).

[0129] 3. ELISA was used to detect the levels of IFN-γ, IL-4, and IL-17A secreted by mouse spleen cells.

[0130] The spleen of a mouse was removed, and spleen cells were ground in 3 mL of erythrocyte lysis buffer to obtain a suspension. After centrifugation and discarding the supernatant, the cells were resuspended in complete culture medium and the spleen lymphocyte concentration was adjusted to 1 × 10⁻⁶. 7 Cells / mL were added to 24-well plates at 1 mL / well; a complete culture medium solution containing 1 mg / mL peptide (P4 or P10) was prepared, filtered through a 0.22 μm filter for sterilization, and 10 μL of the cell suspension was added to each well. The plates were incubated at 37°C for 48 hours; the supernatant was collected by centrifugation at 1500 rpm for 10 minutes; the concentrations of each cytokine in the cell culture supernatant were quantitatively determined according to the method described in the IFN-γ, IL-4, and IL-17 assay kit instructions. The results showed that the levels of IFN-γ, IL-4, and IL-17A cytokines in the spleen lymphocyte culture supernatant were significantly higher in the Hp4 and Hp10 groups than in the other control groups, indicating that the lipopeptide vaccines Hp4 and Hp10 can induce a strong cellular immune response. Figure 5C ).

[0131] 4. Flow cytometry was used to determine the level of central memory T cells (Tcm) in mouse splenic lymphocytes.

[0132] Mouse spleen lymphocytes were collected after co-culturing with peptides (P4 or P10, 10 μg / ml) for 48 h. After separation from the supernatant, cells were added to PBST containing 2% rat serum to block nonspecific sites. Cells were stained with PE anti-mouse CD3 antibody, FITC anti-mouse CD4 antibody, PerCP / Cy5.5 anti-mouse CD8a antibody, PE / Cy7 anti-mouse CD44 antibody, and APC anti-mouse CD62L antibody at 4°C for 30 min in the dark. Fluorescently labeled cells were analyzed using FACS Verse flow cytometry (BDBiosciences, San Jose, CA). Results showed that immunization with Hp4 or Hp10 vaccines significantly increased the level of mouse central memory T cells (Tcm). In CD4+ T cells, the proportion of Tcm in the lipopeptide vaccine group was as high as 20%, which was 4 times that of the control group (Figure 5D). A similar trend was observed in CD8+ T cells (Figure 5E). In addition, the proportion of Tcm in CD8+ T cells was lower than that in CD4+ T cells, indicating that the lipopeptide vaccine induced a stronger response in CD4+ memory T cells.

[0133] Example 4: Self-adjuvanted lipopeptide vaccine Hp10 enhances mucosal immunity and resists H. pylori infection

[0134] 1. Establishment of H. pylori infection model and detection of colonization rate

[0135] Mice were randomly divided into 7 groups based on body weight. After anesthesia with sodium pentobarbital, they were administered intranasally with either peptides, peptides + P2CSK4, or lipopeptides, or an equal volume of PBS (control group). Immunization was performed every two weeks for a total of three times. Serum from the tail vein, vaginal irrigation fluid, and saliva were collected during immunization. Two weeks after the last immunization, mice were administered 1*10g of PBS via gavage. 7 HpSS-1 (pre-treated with gastric acid-neutralizing solution). Four weeks after bacterial infection, mice were sacrificed, and gastric tissue was removed. Bacterial DNA was extracted from the stomach using a bacterial genome extraction kit and quantified using real-time quantitative PCR probe method.

[0136] Primers:

[0137] Forward, 5'TTTGTTAGAGAAGATAATGACGGTATCTAAC-3'; (SEQ ID NO: 11)

[0138] Reverse, 5'-CATAGGATTTCACACCTGACTGACTATC-3'; (SEQ ID NO:12)

[0139] Probe:

[0140] 5'-FAM-CGTGCCAGCAGCCGCGGT-TAMRA-3' (SEQ ID NO: 13).

[0141] The results showed that intranasal immunization of mice with Hp10 instead of Hp4 significantly reduced the colonization of Helicobacter pylori in the stomach (Figure 6A).

[0142] 2. Detection of total amount, subtype and IgA level of antigen-specific IgG antibodies in serum

[0143] The levels of specific IgG, IgG1, IgG2a, IgG2b, IgG2c, IgG3 and specific secreted IgA were detected by indirect ELISA. Coating: Antigens (P4, P10) were diluted with 1× coating buffer and coated at 10 μg / mL 100 μL / well on 96-well removable microplates. The plates were incubated overnight at 4°C and then washed with an automated plate washer. Blocking: Non-specific antigenic epitopes were blocked with 1% bovine serum albumin. Blocking was performed at 37°C for 2 hours, followed by washing with an automated plate washer. Sample addition: Mouse blood samples were incubated overnight at 4°C. Serum was separated at 3000 rpm / min and transferred to new EP tubes. Serial dilution with antibody diluent (PBST) was performed, and 100 μL / well was added to each well of the 96-well plate. Specific antibodies in the samples bound to the antigens. Binding was performed at 37°C for 1 hour, followed by washing with an automated plate washer. Secondary antibody addition: 100 μL of Goat pAb Anti-Mouse IgG / IgG1 / IgG2a / IgG2b / IgG2c / IgG3 / IgA diluted 1:10000 with antibody diluent was added to each well. (HRP), incubate at 37℃ for 40 min, wash plates with an automated plate washer; add substrate for color development, and stop the reaction with stop solution after 5-10 min. The absorbance of the solution at 450 nm is read using a microplate reader. Each wash in the process requires thorough washing to remove non-specific bindings. The OD of the control group mice is used as an example. 450nm Antibody titers were calculated using a cut-off value of 2.1 times the mean. Results showed that only intranasally immunized mice with Hp10 produced an antibody response, with antigen-specific IgG levels beginning to rise after the second immunization (Figure 6B). In the Hp10 group, there was no significant difference in IgG1 and IgG2a levels, indicating that Hp10 enhanced both Th1 and Th2 immune responses (Figure 6C). Furthermore, an increase in specific IgA levels was observed in the serum of Hp10-immunized mice after three immunizations. Figure 6D ).

[0144] 3. Detection of antigen-specific sIgA levels in gastric homogenate and small intestinal lavage fluid

[0145] Sample processing:

[0146] A PBS solution containing 0.1 mg / mL trypsin inhibitor, 50 mM EDTA-2Na, and 1 mM PMSF was prepared as the small intestinal lavage fluid. The small intestine was dissected along its length and immersed in the lavage fluid. The solution was vortexed at 4°C for 30 minutes to fully dissolve the intestinal contents. The mixture was centrifuged at 4000 rpm for 20 minutes, and the supernatant was centrifuged again at the highest speed at 4°C for 10 minutes. The supernatant was collected as the small intestinal lavage fluid sample. Mouse stomach tissue was isolated and placed in a tissue homogenate tube. 1 mL of PBS was added to the tube containing the stomach tissue, and the stomach tissue specimen was homogenized using a mini-bead tissue homogenizer. The mixture was centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected as the gastric homogenate. The homogenate was coated and blocked in 96-well plates, and the following samples were added: gastric homogenate and small intestinal lavage fluid. Secondary antibodies were added: 100 μL of Goat pAb Anti-Mouse IgA (HRP) diluted 1:10000 with antibody dilution buffer was added to each well. The colorimetric termination procedure was the same as described above. OD in control mice 450nm Antibody titers were calculated using a cut-off value of 2.1 times the mean. The results showed that sIgA levels were significantly elevated in both the gastric mucosa (Fig. 6 E) and intestinal mucosa (Fig. 6 F) of the Hp10 group, suggesting that Hp10 can induce a strong mucosal immune response.

[0147] 4. Detection of antigen-specific sIgA antibody levels in vaginal douche fluid

[0148] Sample processing:

[0149] The samples were collected on days 13, 27, and 41 after the start of immunization. 75 μL of antibody dilution was used to rinse the mouse vagina, repeated four times, and the solution was placed in centrifuge tubes. The tubes were centrifuged at 8000 rpm for 5 min at 4°C to separate the supernatant, which was then used as the vaginal irrigation fluid. 96-well plates were coated and blocked, and the following samples were added: vaginal irrigation fluid; secondary antibody was added: 100 μL of GoatpAb Anti-Mouse IgA (HRP) diluted 1:10000 with antibody dilution was added to each well; the colorimetric termination procedure was the same as described above. The control group mice's OD... 450nm Antibody titers were calculated using a cut-off value of 2.1 times the mean. The results showed that the sIgA level in the vaginal mucosa was significantly increased in the Hp10 group (Figure 6 G), suggesting that Hp10 can induce a strong mucosal immune response.

[0150] 5. Detection of antigen-specific sIgA antibody levels in saliva

[0151] Sample processing:

[0152] Dilute 1% pilocarpine, prepared and stored at 4℃, 10-fold to obtain a 0.1% solution. Inject 100 μL of 0.1% pilocarpine intraperitoneally per mouse. When mice begin to salivate, use a 200 μL pipette to collect the saliva into centrifuge tubes. Coat and block 96-well plates, add sample: saliva; add secondary antibody: add 100 μL of GoatpAb Anti-Mouse IgA (HRP) diluted 1:10000 with antibody dilution buffer to each well; the colorimetric termination procedure is the same as described above. Use the OD of control mice as an example. 450nm Antibody titers were calculated using a cut-off value of 2.1 times the average value. Results showed that the Hp10 group had significantly higher sIgA levels in saliva ( Figure 6H This suggests that Hp10 can induce a strong mucosal immune response.

[0153] Example 5: Combined immunization with self-adjuvanted lipopeptide vaccines Hp4 and Hp10 induces a higher protective immune response.

[0154] 1. Animal grouping and immunization

[0155] Mice were randomly divided into four groups based on body weight. After anesthesia with sodium pentobarbital, they were administered intranasally (P4+P10, P4+P10+P2CSK4, Hp4+Hp10) or an equal volume of PBS (control group). Immunization was performed every two weeks for a total of three times. Serum, saliva, and vaginal lavage fluid were collected during the immunization process as before. Two weeks after the third immunization, mice were sacrificed, and serum, spleen lymphocytes, and gastrointestinal homogenate were collected. Figure 5A ).

[0156] 2. Establishment of H. pylori infection model and colonization detection

[0157] Mice were randomly divided into four groups based on body weight. After anesthesia with sodium pentobarbital, they were administered intranasally (P4+P10, P4+P10+P2CSK4, Hp4+Hp10) or an equal volume of PBS (control group). Immunization was performed every two weeks for a total of three times. Two weeks after the last immunization, mice were administered 1*10g of PBS via gavage. 7 HpSS-1 (pre-treated with gastric acid-neutralizing solution). Four weeks after bacterial infection, mice were sacrificed, and gastric tissue was removed. Bacterial DNA was extracted from the stomach using a bacterial genome extraction kit and quantified using real-time quantitative PCR probe method.

[0158] Primers:

[0159] Forward, 5′-TTTGTTAGAGAAGATAATGACGGTATCTAAC-3′; (SEQ ID NO:14)

[0160] Reverse, 5′-CATAGGATTTCACACCTGACTGACTATC-3′; (SEQ ID NO:15)

[0161] Probe:

[0162] 5'-FAM-CGTGCCAGCAGCCGCGGT-TAMRA-3' (SEQ ID NO: 16).

[0163] The results showed that the colonization of Helicobacter pylori in the Hp4+Hp10 group was reduced by about 10 times compared with the PBS group and by about 3 times compared with the Hp10 alone group, indicating that combined immunization better protected mice against Helicobacter pylori infection (Figure 7A).

[0164] 3. ELISA was used to detect the levels of IFN-γ, IL-4, and IL-17A secreted by mouse spleen cells.

[0165] The detection method was the same as described above. The results showed that the levels of IFN-γ, IL-4, and IL-17A cytokines in the splenic lymphocyte culture supernatant were significantly higher in the Hp4+Hp10 group than in the other control groups, indicating that the combined immunization with the lipopeptide vaccine Hp4+Hp10 can induce a strong cellular immune response. Figure 7B ).

[0166] 4. Flow cytometry was used to determine the level of central memory T cells (Tcm) in mouse splenic lymphocytes.

[0167] The detection method was the same as described above. The results showed that in the Hp4+Hp10 group, Tcm was present in CD4+ T cells (Figure 7C) and CD8+ T cells (Figure 7C). Figure 7D The proportions of all of them increased significantly.

[0168] 5. Detection of total antigen-specific IgG antibody levels, subtypes, and sIgA levels at various mucosal sites in serum.

[0169] The detection method is the same as described above. The results show that the total amount of antigen-specific IgG antibodies in the serum ( Figure 7E ), IgG subclass ( Figure 7F ) and stomach (Figure 7 G), small intestine ( Figure 7H ),vaginal( Figure 7I The significantly elevated levels of antigen-specific sIgA at various mucosal sites, including cellular and mucosal immune responses, confirm that effective responses to Helicobacter pylori infection may mediate protection against the infection.

[0170] 6. AGS cell in vitro adhesion experiment

[0171] Dilute Helicobacter pylori to 1*10 in DMEM / F12 medium. 8CFU / mL, and then treated with serum or gastric homogenate at 4°C for 1 hour without treatment. AGS cells were incubated with the pretreated bacterial suspension at 37°C for 1 hour (multiple of infection = 100), followed by washing three times with PBS to remove unbound bacteria. Finally, real-time quantitative PCR was used to detect the bacterial load on AGS cells. The results showed that serum from Hp4+Hp10 immunized mice, as well as gastric homogenates from Hp10 and Hp4+Hp10 immunized mice, significantly inhibited bacterial adhesion in vitro. Figure 7J Furthermore, the inhibitory effect of gastric homogenate is even stronger.

[0172] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles 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 lipopeptide for preventing Helicobacter pylori infection, characterized in that, It consists of a Helicobacter pylori immunodominant epitope peptide and a Pam2Cys peptide covalently bound to the N-terminus of the Helicobacter pylori immunodominant epitope peptide, wherein the Helicobacter pylori immunodominant epitope peptide is selected from a polypeptide with the amino acid sequence SEQ ID NO:

10. The lipopeptide structure is as follows: .

2. A pharmaceutical composition, characterized in that, The active ingredient contains the lipopeptide as described in claim 1, as well as pharmaceutically acceptable excipients.

3. The use of the lipopeptide of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for the prevention of Helicobacter pylori infection.

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