CN108752436A - Helicobacter pylori immunodominant epitope peptide L79-96And its application - Google Patents

Abstract

The present invention relates to an HLA-restricted CD4+ T cell epitope immunodominant peptide and core peptide of Helicobacter pylori Lpp20, the composition of which is shown in SEQ ID NO.10 and SEQ ID NO.30. The suitable CD4 ⁺ T cell epitopes (SEQ ID NO.10 and SEQ ID NO.30) involved in the present invention are reliable and accurate. More importantly, it also enables us to assess whether the identified epitopes are immunodominant or subdominant, which is more helpful for the design of epitope vaccines.

Description

Helicobacter pylori immunodominant epitope peptide L79-96 and its application

technical field

The invention belongs to bioengineering technology, and in particular relates to HLA-restricted CD4 ⁺ T cell immunodominant epitope peptide L _79-96 of Helicobacter pylori Lpp20 and application thereof.

Background technique

Helicobacter pylori (H. pylori) infects more than 50% of the world's population and can easily lead to chronic gastritis, peptic ulcer, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma. Accumulating evidence indicates that CD4 ⁺ T cell responses play a key role in resistance to H. pylori infection. Studies have confirmed that H. pylori infection increases the infiltration of T cells in the human stomach and exists in a typical Th1 phenotype. Acute H. pylori infection mainly causes Th1 responses in rhesus monkeys. Mouse vaccine-induced or host anti-H. pylori Natural immunity depends on Th1-type cellular responses. These studies suggest that Th1 responses are involved in host protective immunity against H. pylori infection.

Immunodominance refers to the fact that cellular immunity tends to be directed against a very limited number of antigenic epitopes in immunized or infected individuals. In general, immunodominant T cells are not only ubiquitous, but also provide better protection than dominant cells. Therefore, immunodominant T cells are considered to play a more effective and critical role in host adaptive immunity against pathogens, which has been demonstrated in many bacterial, viral and tumor systems. Many scientists in the field of vaccine research believe that immunodominant CD4 ⁺ T cell epitopes are critical in H. pylori vaccine development. Currently, although immunodominant CD4 ⁺ T cell epitopes of some H.pylori protective antigens have been identified, many H.pylori antigen immunodominance-specific CD4 ⁺ T cell responses and immunodominant CD4 ⁺ T cell epitopes have not been elucidated.

Lpp20 is H. pylori outer membrane lipoprotein, which is considered as a potential vaccine candidate antigen. Our previous studies identified a B-cell epitope of Lpp20 (L _10-119 ) that induces anti-Lpp20 serum in mice and two H-2 ^d -restricted CD4 ⁺ T-cell epitopes (L _{58- 72} and L _83-97 ), can induce BALB/C mice to secrete Th1 cytokines. In addition, epitope vaccines constructed with the above three antigenic epitopes can elicit specific high levels of Lpp20 antibodies and Th1-type cytokines, and significantly reduce H.pylori colonization in H.pylori-challenged mice, suggesting that Lpp20 epitopes Vaccine may be a promising vaccine against H. pylori infection. Due to differences in MHC molecules between mice and humans, H-2 ^d -restricted CD4 ⁺ T cell epitopes sometimes fail to generate strong immune responses in humans. Therefore, the identification of HLA-restricted CD4 ⁺ T cell epitopes of Lpp20 is very important for the design of human H. pylori vaccines.

Contents of the invention

Based on this, one of the objectives of the present invention is to provide a polypeptide of Helicobacter pylori HLA-restricted CD4 ⁺ T cell epitope.

The technical scheme for realizing the above-mentioned purpose is as follows:

An HLA-restricted CD4 ⁺ T cell epitope immunodominant peptide of Helicobacter pylori Lpp20, the composition of which is shown in SEQ ID NO.14.

An HLA-restricted CD4 ⁺ T cell epitope immune core peptide of Helicobacter pylori Lpp20, the composition of which is shown in SEQ ID NO.36.

Another object of the present invention is to provide the application of the above immunodominant peptide or immune core peptide in the preparation of Helicobacter pylori epitope vaccine.

Another object of the present invention is to provide a Helicobacter pylori epitope vaccine.

The technical scheme for realizing the above-mentioned purpose is as follows:

The active ingredient of the Helicobacter pylori epitope vaccine includes or is derived from the above immunodominant peptide or immune core peptide.

Currently, CD4 ⁺ T cell responses to many H. pylori antigens are unknown, although immunodominant epitopes for some H. pylori protective antigens have been identified. Our previous studies identified two H-2 ^d -restricted CD4 ⁺ T cell epitopes of Lpp20, and the epitope vaccines constructed with these epitopes were resistant to H. is closely related to Lpp20-specific CD4 ⁺ T cell responses. However, the MHC locus is the coding gene locus with the largest genetic variation in mammals, and the highly polymorphic nature of MHC molecules causes the immune systems of different species/objects to produce immune responses against different antigens/epitopes of the same pathogen/antigen. That's why many vaccine candidates succeed in mice but fail in clinical trials. Therefore, the present invention focused on finding HLA-restricted epitopes of Lpp20, rather than testing H-2 ^d -restricted epitopes in clinical trials. In this study, we identified CD4 ⁺ T cell epitopes of Lpp20 using PBMCs from H. pylori-infected individuals. To systematically and efficiently identify immunodominant epitopes, short-term in vitro T cell expansion was used to increase the proportion of Lpp20-specific T cells. This identification method also requires a large number of overlapping synthetic peptides, which is expensive and time-consuming, but in the end we found suitable CD4 ⁺ T cell epitopes (SEQ ID NO.14 and SEQ ID NO.36), which are reliable and accurate. More importantly it also allows us to assess whether the identified epitopes are immunodominant or subdominant.

The invention not only helps to further understand the protective immune mechanism against H. pylori infection, but also contributes to the design of epitope vaccines. However, H. pylori has a large genome encoding many protective antigens. Therefore, there is a need to better understand the properties of CD4 ⁺ T cell responses to other H. pylori-encoded antigens and to identify other HLA allele-restricted immunodominant CD4 ⁺ T cell epitopes.

Description of drawings

Figure 1 The response of Lpp20-specific CD4 ⁺ T cells in H.pylori-infected patients. The PBMCs of the subjects were isolated, stimulated with 0.2 μmol/L rLpp20 first, and then stimulated with Lpp20 overlapping synthetic peptide library (5 μmol/L) 13 days later. The proportion of CD4 ⁺ T cells secreting IFN-γ was detected by ICS method, and DMSO stimulation was used as a control. (A) H. pylori uninfected N1; (B) H. pylori infected H1. (C) PBMCs from 30 H.pylori uninfected (-) and 30 H.pylori-infected (+) individuals were isolated, and the proportion of CD4 ⁺ T cells secreting IFN-γ was detected by the method described above.

Fig. 2 Screening and identification of immunodominant CD4 ⁺ T cell epitopes of Lpp20. PBMCs from H.pylori-infected patients were first stimulated with 0.2 μmol/L rLpp20, and then stimulated with 27 18mer overlapping peptides (5 μmol/L) after 13 days. The proportion of CD4 ⁺ T cells secreting IFN-γ was detected by ICS method, AF Represent H. pylori infected persons H1, H6, H11, H16, H21 and H26, respectively.

Fig. 3 Screening and identification of core sequences of immunodominant epitopes L _55-72 and L _79-96 . PBMCs from H.pylor-susceptible H1 and H6 were isolated, first stimulated with 0.2 μmol/L rLpp20, and then stimulated with 5 μmol/L 13mer overlapping peptide 13 days later. The proportion of CD4 ⁺ T cells secreting IFN-γ was detected by ICS method. (A) Screening of the core sequence of L _55-72 using walking overlapping 13mer peptides. (B) Screening of the core sequence of L _79-96 using walking overlapping 13mer peptides. (C) Titrate L _55-72 , L _57-69 and L _59-71 (5×10 ^-9 mol/L～5×10 ^-5 mol/L) and compare the activities. (D) Titrate L _79-96 , L _83-95 and L _85-97 (5×10 ^-9 mol/L～5×10 ^-5 mol/L) and compare the activities.

Figure 4. Identification of immunodominant epitopes L _55-72 and L _79-96 HLA-restricted. Isolate the PBMCs of H6 and H1 from H. pylori-infected patients, expand in vitro to establish the specific T cell line of Lpp20, add anti-HLA-DR, HLA-DP or HLA-DQ antibody to treat for 30min, and use L _57-69 or L _{83- 95} stimulation, ICS method to detect the proportion of CD4 ⁺ T cells secreting IFN-γ. (A) HLA restriction analysis of L _55-72 . (B) HLA restriction analysis of L _83-95 . (C) A panel of BLCLs containing different HLA types used as APCs presenting 13mer peptides. (D) HLA-DR subtype analysis of L _55-72 . (E) HLA-DR subtype analysis of L _83-95 . Fig. 5 L _57-69 and L _83-95 are naturally processed and presented by autologous DC. (A) L _57-69 , rLpp20, HP-WCL or BSA stimulated DC for 24 hours, added monensin, then co-cultured with L _57-69 specific T cells of infected H6 for 5 hours, and measured the secretion of IFN-γ by ICS The proportion of CD4 ⁺ T cells. (B) The response of L _83-95 -specific T cells in infected H1 was measured by the same method. Figure 6 The specific CD4 ⁺ T cell responses of mice immunized with L _57-69 and L _83-95 . (A) Mice were immunized with L _57-69 and L _83-95 , isolated mononuclear cells, and stimulated with L _57-69 , L _83-95 , and rLpp20 in vitro to analyze lymphocyte proliferation. Control mice were immunized with PBS , each experiment was repeated 3 times, and all data are expressed as mean ± standard deviation. When SI ≥ 2, it was considered statistically significant, indicated by *. (B) Relative percentage of CD3+CD4+ T cells analyzed by flow cytometry. Mice were immunized with L _57-69 and L _83-95 , isolated mononuclear cells for staining, and the control group was immunized with PBS. Each experiment was repeated 3 times, and all data were expressed as mean ± standard deviation. When p<0.05, it indicated that the experimental group was statistically significant relative to the control group, indicated by *.

Figure 7 Immunodominant epitope peptide stimulates CD4 ⁺ T cells to secrete cytokines. (A) Isolate CD4 ⁺ T cells from mice immune to epitope peptides, incubate with epitope peptides L _57-69 and L _83-95 for 72 hours, collect the supernatant, detect IFN-γ and IL-4 by ELISA, control group does not add peptides . Each experiment was repeated 3 times, and the results were expressed as mean ± standard deviation. When there is a significant difference (p<0.05) between the experimental group and the control group, it is marked with *. (B) Immune epitope peptide mouse CD4 ⁺ T cells were isolated, the mRNA expressions of IFN-γ and IL-4 in CD4 ⁺ T cells were detected by fluorescent quantitative PCR, β-actin was used as an internal reference, and the control group was immunized with PBS. Each experiment was repeated 3 times, and the results were expressed as mean ± standard deviation. When there is a significant difference (p<0.05) between the experimental group and the control group, it is marked with *.

Detailed ways

The recombinant antigen and synthetic peptide of the present invention will be further described in detail in conjunction with specific examples below.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the technical field of the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In order to facilitate the understanding of the present invention, the following will describe the present invention more fully. However, the present invention can be embodied in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

The reagents or raw materials used in the following examples are all commercially available unless otherwise specified.

Example 1

1 Methods and materials

1.1 Patients and blood samples

Collection of samples was performed according to a protocol approved by the Human Ethics Review Board of Nanfang Hospital Affiliated to Southern Medical University, Guangzhou, and all participants gave written informed consent. The status of H.pylori infection in 30 gastric disease patients was confirmed by endoscopy and pathological diagnosis. Blood samples were collected, and peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Hypaque (TBDscience, Tianjin, China) density gradient method and then stored in liquid nitrogen. The HLA typing of PBMCs from H. pylori patients was identified by PCR-SBT, which was identified by Huada Genomics Research Institute (Shenzhen, China).

1.2 Recombinant antigens and synthetic peptides

The expression and purification of recombinant Lpp20 (rLpp20) of H. pylori NCTC11639 strain in E. coli refer to the article [Li Y, Ning YS, Wang YD, Hong YH, Luo J, Dong WQ and Li M. Production of mousemonoclonal antibodies against Helicobacter pylori Lpp20 and mapping the antigenic epitope by phage display library. J Immunol Methods. 2007; 325(1-2):1-8], stored at -70°C. The amino acid sequence of Lpp20 protein was obtained from NCBI protein database (No. AAZ13599, submitted by us). 18mer synthetic peptides covering Lpp20 protein, overlapping 12 amino acids (Table 1), and 13mer synthetic peptides, overlapping 11 amino acids (Table 2 and Table 3) were synthesized and purified (purity>95%) by Shanghai GL Biochem Company. Synthetic peptides were all dissolved in dimethyl sulfoxide (dimethyl sulfoxide, Sigma, Shanghai, China) and stored at -80°C.

Table 1 covers 18mer synthetic peptides of Lpp20 protein

Table 2 13mer synthetic peptides covering L _55-72

location sequence SEQ ID NO L53-65 FLVAKYEKYSGVF 28 L55-67 VAKYEKYSGVFLG 29 L57-69 KYEKYSGVFLGRA 30 L59-71 EKYSGVFLGRAED 31 L61-73 YSGVFLGRAEDLI 32

Table 3 covers L _79-96 13mer synthetic peptides

location sequence SEQ ID NO L77-89 DVDYSTNQATAKA 33 L79-91 DYSTNQATAKARA 34 L81-93 STNQATAKARANL 35 L83-95 NQATAKARANLAA 36 L85-97 ATAKARANLAANL 37

1.3 Expansion of Lpp20-specific CD4 ⁺ T cell lines from H. pylori-infected patients in vitro

Stimulate PBMC (1-2×10 ⁶ ) with 0.2 μmol/L Lpp20 (or 5 μmol/L immunodominant peptide), and culture in RPMI 1640 (Gibco) on a 48-well cell culture plate, containing 5% human AB serum, 2 mmol /L L-glutamine, 5×10 ^-5 mol/L 2-mercaptoethanol (2-mercaptoethanol, 2-ME) and antibiotics (penicillin 100U/mL, streptomycin 100μg/mL). On the fifth day, replace 50% of the culture medium with the above culture solution containing 10 U/mL recombinant human interleukin-2 (IL-2), and replace 50% of the culture medium with 25 U/mL IL-2 when the culture medium turns yellow. Medium. The cultured cells were split on the 10th day, and the cells were harvested on the 13th day.

1.4 Systematic screening of Lpp20CD4 ⁺ T cell epitopes

PBMCs were isolated from 200 mL of blood donated by H. pylori-infected individuals, stimulated with 0.2 μmol/L rLpp20, and after day 13, Lpp20-specific T cells were screened. 13mer synthetic peptides were subsequently screened to determine the core sequence of immunodominant CD4 ⁺ T cell epitopes.

1.5 Preparation and cultivation of Epstein-Barr virus transformed Blymphoblastoid cell lines (BLCLs)

Using the culture supernatant of Epstein-Barr virus-producing B95-8 cells, BLCLs were established in autologous PBMCs with 10% fetal bovine serum (GIBCO), 2mmol/L L-glutamine, 5× ^10-5 mol/L 2-mercaptoethanol and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL) RPMI-1640 (GIBCO) for cultivation.

1.6 Intracellular cytokine staining (ICS)

In the screening experiments of 18mer and 13mer peptides, a large number of cultured Lpp20-specific T cells were incubated with 5 μmol/L peptide in a medium containing monensin (Becton Dickinson Company, Shanghai, China) for 5 h. In the experiment of determining HLA restriction, BLCLs were first stimulated with 5 μmol/L peptide for 1 h, then monensin was added, and BLCLs and Lpp20-specific T cells were co-cultured at a ratio of 1:10 for 5 h. Cells were collected, first stained with anti-CD3-PE and anti-CD4-APC (Biolegend, Beijing) in 50 μl staining buffer (PBS containing 1% heat-inactivated FCS) for 30 min at 4°C, and fixed/permeabilized (BD Cat, No.554715) were washed and fixed for 20 min at 4°C, then washed, fixed and stained with anti-IFN-γ-FITC (Biolegend, Beijing). 1×10 ⁵ cells were obtained by FACSCanto II flow cytometer (Becton Dickinson), and the data were analyzed by FlowJo software (Tree Star, Ashland, OR). In antibody blocking experiments, antigen-presenting cells (APCs) were treated with 5 μg/mL HLA-DP (Abcam), HLA-DR (Biolegend) and HLA-DQ (Biolegend) before adding peptides and monensin. After incubation for 30 min, T cell activation was determined by ICS.

1.7 Preparation of H. pylori complete cell lysates (H pylori Whole Cell Lysates, HP-WCL)

Spread the H. pylori NCTC11637 strain on the brain-heart infusion (BHI) plate, containing 7% goat blood, polymyxin B (5 μg/mL), trimethoprim (5 μg/mL) and vancomycin (10 μg/mL), and then cultured in Brooke’s broth containing 5% fetal bovine serum, shaking gently at 37°C, and micro-aerobic (10% CO ₂ , 5% O ₂ , N ₂ 10%). After culturing for 1 day, collect viable bacteria, wash and lyse the cells, centrifuge the lysate at 10,000 g at 4°C for 20 min to remove complete cells and large debris, and the ultrasonic supernatant is the complete cell lysate of H.pylori (HP-WCL ). The protein content was measured with BCA protein assay kit (Beyotime, Shanghai, China), aliquoted and stored at -20°C.

1.8 Preparation of dendritic cells (DC) and co-culture with immunodominant epitope-specific T cells

Thaw PBMC, use CD14 ⁺ micro-magnetic beads (Miltenyi Biotec, Shanghai, China) to separate CD14 ⁺ cells from PBMC, in the medium containing 5% human AB serum, 10ng/mL interleukin-4 (interleukin-4, IL-4) and 20ng/mL Culture in mL granulocyte-macrophage colony-stimulating factor (GM-CSF) RPMI 1640, 5% CO ₂ , 37°C. After 6 days, DCs were collected and stimulated with 50 μg/mL immunodominant epitopes (L _57-69 or L _83-95 ), rLpp20, HP-WCL and BSA for 24 hours, respectively. Then the cells were washed, monensin was added, and DCs were co-cultured with epitope-specific T cells at a ratio of 1:10 for 5 h.

Immunization of BALB/c mice with 1.9rLpp20 and immunodominant epitope peptide

SPF grade female BALB/c mice aged 6-8 weeks were selected and immunized with rLpp20 plus complete Freund's adjuvant (complete Freund's adjuvant, CFA) subcutaneously for the first time at multiple points, 100 μg/0.5ml per mouse, and 3 weeks later The same dose of rLpp20 plus incomplete Freund's adjuvant (IFA) booster immunization once. Mice in the control group were immunized with PBS instead of rLpp20, and the immunization procedure was the same, with 5 mice in each group. BALB/c mice were immunized with L _57-69 and L _83-95 respectively. The first immunization was immunized with subcutaneous multi-point injection of IFA, each mouse was 100 μg/0.5ml, and the immunization was boosted once every 14 days. A total of three immunizations, each Group of 5 mice. In the control group, PBS was used to replace the epitope polypeptide, and the immunization procedure was the same, with 5 mice in each group.

1.10 Lymphocyte proliferation assay

The mice were killed by dislocation 14 days after the last immunization, the spleen was aseptically removed, and the single cell suspension was prepared by grinding on a steel mesh, and the mononuclear cells were separated with mouse lymphocyte separation medium, and the number of cells was adjusted to 5×10 with RPMI-1640 complete medium. ⁶ /mL in a 96-well culture plate, add 100 μL cells to each well, and add diluted epitope peptides L _57-69 , L _83-95 (1.25 μg/mL) and rLpp20 (15 μg/mL) at the same time, negative control wells do not Add any antigen, the final volume is 200μL/well, make 3 parallel wells for each group, culture in 37℃, 5% CO ₂ incubator for 5 days, add ³ H-TdR 12h before the end of culture, 37kBq/well, continue to culture for 12h Finally, the cells were collected on glass fiber filter paper, and after drying, the number of pulses per minute (cpm) was measured with a liquid scintillation counter, and the average value of 3 duplicate holes was calculated, and the results were represented by stimulation index (SI) (SI=mean value of cpm in the experimental group /mean cpm of the negative control group, the negative control refers to the well without antigen stimulation), SI≥2 is positive.

1.11 Flow Cytometry Analysis

The mice were killed by dislocation 14 days after the last immunization, the spleen was aseptically removed, and a single cell suspension was prepared. Mononuclear cells were separated with mouse lymphocyte separation medium, stimulated with L _57-69 and L _83-95 for 72 hours, and collected 1× 10 ⁶ cells were incubated with anti-mouse CD16/CD32 (clone 93, eBiosciensce) for 10 minutes to exclude non-specific Fc receptor-mediated binding. The cells were washed with cold PBS (2% bovine serum albumin), then double-stained with PE-CD4 (clone RM4-5, eBiosciensce) and FITC-CD3 (clone 145-2C11) on ice for 30 min, and the samples were collected for flow cytometry On-device analysis.

1.12 Detection of cytokines by ELISA and fluorescent quantitative PCR

The mice were killed by dislocation 14 days after the last immunization, the spleen was aseptically removed, and a single cell suspension was prepared. The mononuclear cells were separated with mouse lymphocyte separation medium, and CD4 ⁺ T cells were separated by magnetic beads, and suspended in a 24-well culture plate. 5×10 5 CD4 ⁺ T cells were added to each well, ⁵ ×10 ⁵ splenic mononuclear cells (as APC) were treated with mitomycin-C (500 μg/ml), and diluted immunodominant epitopes were added at the same time Peptide (final concentration 1.25 μg/mgl), no peptide was added to the negative control wells, the final volume was 0.5ml/well, and 3 parallel wells were made for each group. After 72 hours of stimulation, 400 μl of the supernatant was collected, and the cytokines IFN-γ and IL-4 were detected with an ELISA kit. In addition, total RNA of CD4 ⁺ T cells was extracted with Tripure reagent. Using SYBR Green method, fluorescent quantitative PCR (Q-PCR) was used to detect the expression levels of IFN-γ and IL-4 mRNA. The primers were as follows, and β-actin was used as an internal reference.

IFN-γ: upstream primer: 5'-AACTCAAGTGGCATAGATGTGG-3' SEQ ID NO.38

Downstream primer: 5'-GACCTCAAACTTGGCAATACTC-3' SEQ ID NO.39

IL-4: upstream primer: 5'-TGTCATCCTGCTCTTCTTTCTC-3' SEQ ID NO.40

Downstream primer: 5'-TGATGCTCTTTAGGCTTTCCAG-3' SEQ ID NO.41

β-actin: upstream primer: 5'-ATCCGTAAAGACCCTCTATGCCAACA-3'SEQ ID NO.42

Downstream primer: 5'-GTCGCCTTCACCGTTCCAGTTT-3' SEQ ID NO.43

1.13 Statistical Analysis

Differences between two groups were analyzed with paired Student's t-test, but when variances were unequal, unpaired Welch's t-test was used. Differences were considered statistically significant when p<0.05.

2 results

2.1 The frequency of Lpp20-specific CD4 ⁺ T cells in H.pylori-infected patients is higher than that in H.pylori-uninfected patients

In order to evaluate the response of Lpp20-specific CD4 ⁺ T cells in H. pylori-infected patients, PBMCs were isolated from the blood of H. pylori-infected patients, stimulated with 18mer peptides covering the full length of Lpp20, and the expression of Lpp20-specific CD4 ⁺ T cells was detected by ICS. frequency. However, the frequency of Lpp20-specific CD4 ⁺ T cells was too low to be detected in vitro (Fig. 1A). Cell expansion is often necessary for low frequency responses, especially for epitope identification. Therefore, rLpp20 was first used to stimulate PBMCs to expand the culture of Lpp20-specific CD4 ⁺ T cells, and the frequency of Lpp20-specific CD4 ⁺ T cells was significantly increased after 13 days of stimulation in vitro (Fig. 1B). In order to further confirm the presence of Lpp20-specific CD4 ⁺ T cell responses in H. pylori-infected patients but not in H. pylori-uninfected patients, Lpp20-specific CD4 ⁺ T cell responses were detected in 30 H. pylori-infected and 30 non-H. T cell response, the results showed that the frequency of Lpp20-specific CD4 ⁺ T cells was significantly higher in the H. pylori-infected group than in the H. pylori-uninfected group (Fig. 1C). These results suggest that rLpp20-stimulated cells can recognize antigen-primed CD4 ⁺ T cells in vitro.

2.2 Lpp20-specific CD4 ⁺ T cells mainly recognize two epitopes, L _55-72 and L _79-96

In order to systematically investigate the specificity and comprehensiveness of Lpp20-specific CD4 ⁺ T cell responses, isolate PBMCs from H. pylori-infected patients, stimulate them with rLpp20 first, and then stimulate them with 27 overlapping 18mer peptides for 13 days to screen for immunodominant epitopes. The results showed that Lpp20-specific CD4 ⁺ T cells mainly recognized two epitopes, L _55-72 and L _79-96 ( FIG. 2 ). Lpp20-specific CD4 ⁺ T cells from patients H6, H11 and H26 mainly recognized L _55-72 (Fig. 2B, 2C and 2F), whereas Lpp20-specific CD4 ⁺ T cells from patients H1, H16 and H21 mainly recognized L _79-96 (Fig. 2A, 2D and 2E). In summary, L _55-72 and L _79-96 are Lpp20 immunodominant CD4 ⁺ T cell epitopes.

2.3L _57-69 and L _83-95 are the core sequences of immunodominant epitopes L _55-72 and L _79-96 , respectively

In order to further determine the core sequences of the immunodominant CD4 ⁺ T cell epitopes L _55-72 and L _79-96 of Lpp20, the specific T cells of these two epitopes were expanded and cultured as described above, and a set of covering 13mer overlapping peptides of 18mer peptides and various N- and C-terminal extended or truncated 13mer peptides (Table 2 and Table 3) were further screened and titrated. For patient H6, L _55-72 -specific CD4 ⁺ T cells recognized two 13mer peptides (L _57-69 and L _59-71 ). The response of L _57-69 to stimulate T cells was stronger than that of L _59-71 , indicating that this _13mer peptide was the core sequence ( _{Fig .} ^-9 mol/L～5×10 ^{- 5} mol/L) titration also confirmed this conclusion (Fig. 3B). For patient H1, L _79-96 -specific CD4 ⁺ T cells recognized two 13mer peptides (L _83-95 and L _85-97 ). The response of L _83-95 to stimulate T cells was stronger than that of _{L 79-96} , indicating that this _13mer peptide was the core sequence (Fig. 3C) _. ^-9 mol/L～5×10 ^-5 mol/L) titration also confirmed this conclusion. In conclusion, the most potent and minimal immunodominant CD4 ⁺ T cell epitopes of Lpp20 are L _57-69 and L _83-95 .

2.4L _57-69 and L _83-95 reactive CD4 ⁺ T cells were restricted to HLA-DRB1 ^* 1501 and HLA-DRB1 ^* 1602, respectively

To determine the HLA restriction of L _57-69 and L _83-95 molecules, MHC-class II antibody blocking experiments were used to identify them. Isolate H6 PBMCs from H. pylori-infected patients, expand in vitro to establish Lpp20-specific T cell lines, add anti-HLA-DR, HLA-DP or HLA-DQ antibodies for treatment, stimulate with L _57-69 , and detect the secretion of IFN by ICS The proportion of -γ CD4 ⁺ T cells. As shown in Figure 4A, anti-HLA-DR antibodies inhibited L _57-69 -specific CD4 ⁺ T cells to secrete IFN-γ, while anti-HLA-DP and HLA-DQ antibodies had no such effect. Use the same method to determine the HLA restriction of L _83-95 , isolate the PBMC of H1 from H. pylori-infected patients, expand in vitro to establish the specific T cell line of Lpp20, and add anti-HLA-DR, HLA-DP or HLA-DQ Antibody treatment, stimulation with L _83-95 , and ICS experiments were performed. Anti-HLA-DR antibodies inhibited IFN-γ secretion by L _83-95 -specific CD4 ⁺ T cells, whereas anti-HLA-DP and HLA-DQ antibodies had no such effect (Fig. 4B). Therefore, epitopes L _57-69 and L _83-95 are both HLA-DR restricted.

In order to further clarify the HLA-DR subtype of L _57-69 , a group of BLCLs with different DR subtypes (Figure 4C) was used as APC, stimulated with 5 μmol/LL _57-69 , and L _57-69 -specific T cells were detected by ICS method Reaction. As shown in Figure 4D, the autologous _BLCLs (expressing HLA-DRB1 ^* 08031501 ⁾ of H. Can stimulate L _57-69 -specific T cells, while BLCLs of infected H22 and H27 both expressed HLA-DRB1 ^* 0803, but failed to present L _57-69 . Therefore, the restriction of L _57-69 is HLA-DRB1 ^* 1501. The HLA-DR restriction of L _83-95 was identified by the same method. A group of _BLCLs with different DR types (Fig. 4C) were used as APCs, stimulated with 5 μmol/LL _83-95 , and detected by ICS. Specific T cells secrete IFN-γ in response. The autologous BLCLs (expressing HLA-DRB1 ^* 0901 1602) of H1 infected with H. pylori effectively activated L _83-95 specific T cells, and the BLCLs of H16 and H21 infected persons expressed HLA-DRB1 ^* 1602, both of which could stimulate L _83-95 specific T cells, while the BLCLs of infected H17 and H22 both expressed HLA-DRB1*0901, but failed to present L _83-95 . Therefore, the restriction of L _83-95 is HLA-DRB1 ^* 1602 (Fig. 4E).

2.5L _57-69 and L _83-95 can be naturally processed and presented by antigen-presenting cells (APCs)

To assess whether L _57-69 and L _83-95 could be presented by APCs, the natural presentation properties of the epitopes were identified by APC loading rLpp20 or HP-WCL. Dendritic cells (DC) were first stimulated with rLpp20, HP-WCL, BSA or immunodominant epitope peptides for 24 hours, then monensin was added and epitope-specific T cells were cultured for 5 hours, and then detected by ICS and flow cytometry. The ability of epitope-specific CD4 ⁺ T cells to secrete IFN-γ was used to evaluate the ability of corresponding epitope-specific T cells to recognize APC. As shown in FIG. 5A , H. pylori patient H6 DCs stimulated by L _57-69 , rLpp20, and HP-WCL can present L _57-69 and stimulate L _57-69 -specific CD4 ⁺ T cell responses. However, DCs stimulated with BSA or DMSO stimulated _L57-69 -specific CD4 ⁺ T cells only to background levels. Using the same method, we also confirmed that L _79-96 can be naturally processed and presented by autologous DC (Fig. 5B).

2.6 Immune Dominant Epitope Peptide Immunized Mice to Stimulate CD4 ⁺ T Cell Proliferation

In order to detect whether the immunodominant epitope can induce the body to produce a specific CD4 ⁺ T cell immune response, and whether the induced immune response is directed against rLpp20. BALB/c mice were immunized with L _57-69 and L _83-95 respectively, mononuclear cells were isolated, and T lymphocyte proliferation was detected by ³ H-TdR method. As shown in Figure 6A, monocytes isolated from BALB/c mice immunized with L _57-69 and L _83-95 could elicit proliferative responses after stimulation with L _57-69 and L _83-95 and rLpp20, respectively, while Control mice did not respond to any stimulus, indicating that L _57-69 and L _83-95 -induced lymphocyte responses recognized the native antigen rLpp20. In addition, by flow cytometry analysis, it was found that the ratio of CD4 ⁺ CD3 ⁺ in mice immunized with L _57-69 and L _83-95 was higher than that in the control group (p<0.05, Fig. 6B ).

2.8 Immunodominant epitope peptide stimulates CD4 ⁺ T cells to differentiate into Th1 cells

In order to analyze the direction of differentiation of CD4 ⁺ T cells induced by the screened epitopes, CD4 ⁺ T cells from mice immunized with epitope peptides were isolated, co-cultured with epitope peptides, supernatants were collected, and IFN-γ and IL-4 were detected by ELISA. From the analysis of the results in Figure 7A, the L _57-69 and L _83-95 epitope peptides mainly caused the secretion of INF-γ. In addition, the mRNA expression levels of IFN-γ and IL-4 in CD4 ⁺ T cells were detected by fluorescent quantitative PCR. As shown in Figure 7B, the mRNA expression level of IFN-γ was higher than that of IL-4. The above results indicated that the immunodominant epitope peptide stimulated the differentiation of CD4 ⁺ T cells into Th1 cells.

In the present invention, the proportion of Lpp20-specific CD4 ⁺ T cells in the H. pylori-infected group was higher than that in the uninfected group ( FIG. 1C ). In fact, low levels of Lpp20-specific CD4 ⁺ T cell responses were found in some H. pylori-uninfected samples (Fig. 1C). We think this may be due to the false negative results of transient or mild H. pylori infection being diagnosed. Then, we systematically evaluated the response degree and strength of Lpp20-specific CD4 ⁺ T cells in H. pylori-infected patients by expanding cultured Lpp20-specific T cells and 18mer overlapping peptides in vitro, and the responses were mainly concentrated on the two 18mer peptides ( L _55-72 and L _79-96 ) (Figure 2). L _55-72 induced more T cell responses in patients H6, H11, H26, and L _79-96 induced more T cell responses in patients H1, H16H21 (Fig. 2). Subsequently, 13mer overlapping peptides were used to identify the core sequences of L ₅₅ -72 and L ₇₉ -96, which were L _57-69 and L _83-95 , respectively (Fig. 3).

Because MHC molecules are highly polymorphic in mammals, MHC alleles of the same species often present different peptides, let alone MHC alleles of different species. For example, none of the reported H. pylori UreB CD4 ⁺ T epitopes can be recognized by both murine and human CD4 ⁺ T cell responses. Therefore, identification of different HLA-restricted immunodominant T-cell epitopes is crucial for the rational design of H. pylori T-cell vaccines. However, only a few HLA-restricted UreB and HpaA antigen CD4 ⁺ T cell epitopes have been identified so far, and the immunodominant epitopes differ between individuals with different HLA alleles]. The allele frequency network database (http://www.allelefrequencies.net/default.asp) shows that the frequency of HLA-DRB1 ^* 1501 is relatively high (>10%) in the Chinese Han population, while HLA-DRB1 ^* 1404 and HLA The frequency of -DRB1*0803 is relatively low (<1%) in the Chinese Han population. However, HLA-DRB1 ^* 0803 is more common in some other populations, such as Australian Aboriginal people (>20%), Papua New Guinea and Taiwanese natives (>10%). Chen et al. demonstrated that HpaA _88-100 -specific CD4 ⁺ T cell responses were immunodominant in individuals expressing HLA-DRB1*1501 and were strongly associated with a reduction in severe gastric disease. Interestingly, HLA-DRB1*0901-specific CD4 ⁺ T cell responses were most immunodominant in the HLA-DRB1*1501-negative group. In the present invention, L _57-69 and L _83-95 can mainly induce CD4 ⁺ T cell responses in patients with HLA-DR genotype, and the HLA-DR type is further determined by antibody blocking experiment. Using BLCLs of different HLA alleles as APC-presenting epitope peptides to react with CD4 ⁺ T cells, we found that L _57-69 and L _83-95 were restricted to HLA-DRB1 ^* 1501 and HLA-DRB1 ^* 1602, respectively , can not only recognize autologous DCs loaded with epitope peptides, but also DCs loaded with rLpp20 and HP-WCL, indicating that L _57-69 and L _83-95 can be naturally processed and presented by APCs. Therefore, HLA-DRB1 ^* 1501-restricted L _57-69 and HLA-DRB1 ^* 1602-restricted L _83-95 may have potential application value for the development of H.pylori vaccine. We immunized mice with immunodominant epitope peptides, and found that CD4 ⁺ T cells could proliferate and secrete IFN-γ, indicating that epitope peptides have good immunogenicity and promote the differentiation of CD4 ⁺ T cells into Th1 cells.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

sequence listing

<110> Southern Medical University

<120> Helicobacter pylori immunodominant epitope peptide L_79-96 and its application

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<170> SIPOSequenceListing 1.0

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Met Lys Asn Gln Val Lys Lys Ile Leu Gly Met Ser Val Ile Ala Ala

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Met Val

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Lys Ile Leu Gly Met Ser Val Ile Ala Ala Met Val Ile Val Gly Cys

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Ser His

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Val Ile Ala Ala Met Val Ile Val Gly Cys Ser His Ala Pro Lys Ser

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Gly Ile

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Ile Val Gly Cys Ser His Ala Pro Lys Ser Gly Ile Ser Lys Ser Asn

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Lys Ala

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Ala Pro Lys Ser Gly Ile Ser Lys Ser Asn Lys Ala Tyr Lys Glu Ala

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Thr Lys

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Ser Lys Ser Asn Lys Ala Tyr Lys Glu Ala Thr Lys Gly Ala Pro Asp

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Trp Val

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Tyr Lys Glu Ala Thr Lys Gly Ala Pro Asp Trp Val Val Gly Asp Leu

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Glu Lys

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Gly Ala Pro Asp Trp Val Val Gly Asp Leu Glu Lys Val Ala Lys Tyr

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Glu Lys

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Val Gly Asp Leu Glu Lys Val Ala Lys Tyr Glu Lys Tyr Ser Gly Val

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Phe Leu

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Val Ala Lys Tyr Glu Lys Tyr Ser Gly Val Phe Leu Gly Arg Ala Glu

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Asp Leu

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Tyr Ser Gly Val Phe Leu Gly Arg Ala Glu Asp Leu Ile Thr Asn Asn

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Asp Val

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Gly Arg Ala Glu Asp Leu Ile Thr Asn Asn Asp Val Asp Tyr Ser Thr

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Asn Gln

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Ile Thr Asn Asn Asp Val Asp Tyr Ser Thr Asn Gln Ala Thr Ala Lys

1 5 10 15

Ala Arg

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Asp Tyr Ser Thr Asn Gln Ala Thr Ala Lys Ala Arg Ala Asn Leu Ala

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Ala Asn

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Ala Thr Ala Lys Ala Arg Ala Asn Leu Ala Ala Asn Leu Lys Ser Thr

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Leu Gln

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Ala Asn Leu Ala Ala Asn Leu Lys Ser Thr Leu Gln Lys Asp Leu Glu

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Asn Glu

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Leu Lys Ser Thr Leu Gln Lys Asp Leu Glu Asn Glu Lys Thr Arg Thr

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Val Asp

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Lys Asp Leu Glu Asn Glu Lys Thr Arg Thr Val Asp Ala Ser Gly Lys

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Arg Ser

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Lys Thr Arg Thr Val Asp Ala Ser Gly Lys Arg Ser Ile Ser Gly Thr

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Asp Thr

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Ala Ser Gly Lys Arg Ser Ile Ser Gly Thr Asp Thr Glu Lys Ile Ser

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Gln Leu

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Ile Ser Gly Thr Asp Thr Glu Lys Ile Ser Gln Leu Val Asp Lys Glu

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Leu Ile

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Glu Lys Ile Ser Gln Leu Val Asp Lys Glu Leu Ile Ala Ser Lys Met

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Leu Ala

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Val Asp Lys Glu Leu Ile Ala Ser Lys Met Leu Ala Arg Tyr Val Gly

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Lys Asp

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Ala Ser Lys Met Leu Ala Arg Tyr Val Gly Lys Asp Arg Val Phe Val

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Leu Val

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Arg Tyr Val Gly Lys Asp Arg Val Phe Val Leu Val Gly Leu Asp Lys

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Gln Ile

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Arg Val Phe Val Leu Val Gly Leu Asp Lys Gln Ile Val Asp Lys Val

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Arg Glu

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Gly Leu Asp Lys Gln Ile Val Asp Lys Val Arg Glu Glu Leu Gly Met

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Val Lys Lys

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Phe Leu Val Ala Lys Tyr Glu Lys Tyr Ser Gly Val Phe

1 5 10

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Val Ala Lys Tyr Glu Lys Tyr Ser Gly Val Phe Leu Gly

1 5 10

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Lys Tyr Glu Lys Tyr Ser Gly Val Phe Leu Gly Arg Ala

1 5 10

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Glu Lys Tyr Ser Gly Val Phe Leu Gly Arg Ala Glu Asp

1 5 10

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Tyr Ser Gly Val Phe Leu Gly Arg Ala Glu Asp Leu Ile

1 5 10

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Asp Val Asp Tyr Ser Thr Asn Gln Ala Thr Ala Lys Ala

1 5 10

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Asp Tyr Ser Thr Asn Gln Ala Thr Ala Lys Ala Arg Ala

1 5 10

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Ser Thr Asn Gln Ala Thr Ala Lys Ala Arg Ala Asn Leu

1 5 10

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Asn Gln Ala Thr Ala Lys Ala Arg Ala Asn Leu Ala Ala

1 5 10

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Ala Thr Ala Lys Ala Arg Ala Asn Leu Ala Ala Asn Leu

1 5 10

<210> 38

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<212>DNA

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aactcaagtg gcatagatgt gg 22

<210> 39

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gacctcaaac ttggcaatac tc 22

<210> 40

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<212>DNA

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tgtcatcctg ctcttctttc tc 22

<210> 41

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<212>DNA

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<400> 41

tgatgctctt taggctttcc ag 22

<210> 42

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<212>DNA

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<400> 42

atccgtaaag acctctatgc caaca 25

<210> 43

<211> 22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

gtcgccttca ccgttccagt tt 22

Claims (6)

1. An HLA-restricted CD4 ⁺ T cell epitope immunodominant peptide of Helicobacter pylori Lpp20, characterized in that its composition is shown in SEQ ID NO.14. 2. An HLA-restricted CD4 ⁺ T cell epitope immune core peptide of Helicobacter pylori Lpp20, characterized in that its composition is shown in SEQ ID NO.36. 3. The application of the immunodominant peptide according to claim 1 in the preparation of Helicobacter pylori epitope vaccine. 4. The application of the immune core peptide described in claim 2 in the preparation of Helicobacter pylori epitope vaccine. 5. A Helicobacter pylori epitope vaccine, characterized in that its active ingredient comprises or is derived from the immunodominant peptide of claim 1. 6. A Helicobacter pylori epitope vaccine, characterized in that its active ingredient comprises or is derived from the immune core peptide of claim 2.