CN101311189B - Antigen epitope for stimulating human body's protective immune response against Mycobacterium tuberculosis and use thereof - Google Patents

Abstract

The invention relates to the field of molecular immunology. In recent years, tuberculosis has resurged worldwide. Due to the emergence of drug-resistant bacterial strains, antibiotic treatment is often ineffective; the commonly used tuberculosis vaccine-BCG also has limitations. The purpose of the present invention is to screen out the molecular mimetic peptide of the antigenic epitope that can stimulate the protective immune response against Mycobacterium tuberculosis, to study the protective immune response mechanism of tuberculosis, and to further develop a new multi-unit multi-epitope tuberculosis vaccine. The present invention provides a molecular mimetic peptide of an antigenic epitope capable of stimulating human protective immune response against Mycobacterium tuberculosis, and the amino acid sequence of the peptide is shown in SEQ ID NO:14. The present invention also provides a screening method and application of the above-mentioned peptide. The invention will prevent and control the occurrence and development of tuberculosis more effectively.

Description

Antigen epitope for stimulating human body's protective immune response against Mycobacterium tuberculosis and use thereof

This application is a divisional application of the patent application filed on June 6, 2006 with the application number 200610027311.2 and the title of the invention is "antigen epitope for stimulating human body's protective immune response against Mycobacterium tuberculosis and its use".

Technical field:

The invention relates to the field of molecular immunology, in particular to a molecular mimetic peptide capable of stimulating a human body's protective immune response against Mycobacterium tuberculosis and its screening method and application.

Background technique:

In recent years, tuberculosis (Tuberculosis, TB) has resurged worldwide. According to the statistics of the World Health Organization, about 1/3 of the world's population and about 2 billion people are infected with Mycobacterium tuberculosis (Mtb), and there are about 9 million new cases every year, and the annual death toll is as high as 3 million. The World Health Organization has proposed a "global tuberculosis emergency" (global emergency). It can be seen that the impact and damage caused by tuberculosis on human health and economy is difficult to simply assess. The characteristics of tuberculosis epidemic in my country are: high prevalence rate, high drug resistance rate, high mortality rate, high infection rate and low decline rate, which have seriously affected the quality of life of individuals and families, social and national construction and economic development. The current strategy for controlling tuberculosis is mainly the direct use of antibiotics. However, multiple antibiotics need to be used in combination for at least 6 months, and due to the widespread emergence of Mtb drug-resistant strains, antibiotic treatment is often ineffective. The tuberculosis vaccine commonly used today, Bacillus Calmette-Guerin (BCG), has significant limitations: (1) Its effect on adult tuberculosis is not satisfactory; (2) It can cause severe disease and even death in immunocompromised individuals ; (3) It can cause a positive skin test, making it lose its diagnostic significance in Mycobacterium tuberculosis infection. Therefore, there is an urgent need to develop new and highly effective protective tuberculosis vaccines, and to develop new methods for detection, treatment and prevention of tuberculosis, so as to effectively control the occurrence and progression of tuberculosis. This research field has great significance and broad application prospects.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen whose control by the body relies on the recognition and destruction of infected cells. Studies in mouse models have shown that MHC class II-restricted CD4+ T cells, MHC class I-restricted CD8+ T cells, IFN-γ, TNF-α, γδ T cells, and CD1-restricted T cells have positive Protective effect. Different types of CD8+Mtb-specific T cells have been identified in humans, but their role in the pathogenesis of tuberculosis remains unclear. Macrophages: Macrophages play an important role in the body's immune response to Mtb. Mtb can survive in unactivated macrophages. When macrophages are activated, they can produce cytokines such as IFN-γ, induce the production of NO, reactive nitrate mediator (RNI) and NO synthase, thereby exerting immune effects. Another pathway by which macrophages exert antimicrobial activity is through phagosome fusion. However, Mtb infection of macrophages can inhibit the fusion and acidification of phagosomes, and Mtb can survive in macrophages. CD4+ T cells: Mtb antigens are presented to CD4+ T cells by MHC class II molecules. CD4+T cells can produce IFN-γ and other cytokines to play an effector role, induce apoptosis of infected cells, and assist the activation of B cells and CD8+T cells. However, Mtb infection of macrophages can lead to a decrease in the expression of MHC class II molecules in macrophages, thereby inhibiting the immune effect of CD4+ T cells. CD8+ T cells: Mtb is phagocytized by the phagosome of macrophages, and its antigen or its secreted protein enters the phagosome, and then enters the macrophage through the phagosome membrane, and then is digested and processed by MHC class I Molecule presentation to CD8+ T cells. CD8+ T cells can produce cytokines such as IFN-γ to exert effector effects, and can also directly exert cytotoxic effects through the Fas/Fasl pathway and granzyme B. Studies have shown that BCG cannot fully stimulate CD8+ T cells, which may be due to the loss of key membrane lysin in BCG, and the Mtb antigen cannot enter the cytosol of infected cells and be presented. Existing studies are mainly about the protective response of CD4+ T cells in tuberculosis, and the correlation of specific CD4+ T cell immune responses with different stages of the disease is under investigation. However, the mechanism of action of CD8+ T cells in tuberculosis has not yet been clarified. Activation of CD8+ T cells by a single antigenic peptide is dependent on many factors, among which it is well established that the presence of a high density of peptide/MHC complexes on the surface of APCs is key to 'naive' T cell activation in vivo or in vitro. Dendritic cells (DCs) express high levels of MHC and play an important role in the activation of "naive" T cells. Reports on the number of peptide/MHC complex molecules required to activate 'naive' T cells vary widely, from 300 to 10,000 per cell. But in general, "naive" T cell responses require 100-1000 times the number of peptide/MHC complex molecules required to activate memory cells. Moreover, studies have shown that 90% of the peptides recognized by CD8+ T cells bind with high affinity to their MHC class I molecules. Most of the peptides eluted from HLA class I molecules are 8-10 amino acids. Studies with synthetic peptides have shown that peptides with a length of 15-20 amino acids can also bind HLA class I molecules to activate T cells. The length of the peptides to which HLA class II molecules bind is quite flexible.

The current TB vaccines can be divided into 4 categories: subunit vaccines, DNA vaccines, whole bacterial vaccines and mixed vaccines. Subunit vaccines are based on the assumption that one or a few antigens are sufficient to induce a protective immune response. Subunit vaccines have been tested in mouse models, including culture-filtered proteins, single proteins, protein mixtures, and fusion proteins. Although such vaccines are safe, this mode of antigen presentation can only stimulate a limited number of specific T cells, mostly CD4+ T cells, and such responses are generally short-lived. DNA vaccines stimulate CD4+ and CD8+ T cell responses and are durable, but their safety concerns remain, including the risk of genomic integration. The most successful TB DNA vaccine tested to date is Ag85A. There are also many studies on whole bacterial vaccines. Removal of individual genes, such as virulence factors, from Mtb may reduce bacterial virulence but retain the ability to stimulate CD4+, CD8+, and non-traditional T cells. The safety of these mutants remains controversial. Hybrid vaccines are attempts to take advantage of the above strategies, eg BCG can be engineered to overexpress one or more immunogenic/protective proteins. For example, BCG has been engineered to secrete listerlysin, which allows the BCG protein to enter the cytoplasm of infected cells and enhance its ability to stimulate CD8+ T cells. Likewise, engineered BCG overexpressing Ag85 outperformed BCG alone in guinea pig experiments.

Mtb, as well as some other intracellular pathogens, also release proteins (medium leaching proteins) into the extracellular matrix when grown in culture medium. Some scholars analyzed Mtb culture filtrate by two-phase polyacrylamide agarose electrophoresis, and identified more than 200 different protein spots. Comparing intracellularly produced and culture-grown Mtb-producing proteins by two-phase polyacrylamide agarose electrophoresis revealed significant differences. Horwitz et al. purified and identified the six most abundant proteins in the Mtb culture filtrate, including antigen 85A, antigen 85B, superoxide dismutase, hsp71, Mpt51, and Mpt63. Immunization of guinea pigs with these purified proteins can make them immune to Mtb.

Although many antigens carry potential cytotoxic T lymphocyte (cytotoxic Tlymphocyte, CTL) epitopes, only a few can generate CTL responses in the initial reaction. The main epitopes are called immunodominant determinants, and the other epitopes are called Minor determinants or structural determinants. Minor determinants can be processed and presented, but in the presence of immunodominant determinants, minor determinants cannot elicit CTL responses. Structural epitopes cannot be presented and may be problematic during antigen processing, including the specificity of proteosome cleavage and due to inclusion of the epitope's flanking sequences. Factors affecting immune dominance include: (1) ineffective antigen processing by APCs; (2) lack of an appropriate TCR to recognize epitope/HLA complex molecules; (3) competition for antigen presentation by CD8+ T cells; Inhibition of responses to other determinants produced by immunodominant CD8+ T cells. These factors should be considered especially for TB vaccines, since nearly everyone is exposed to environmental bacteria, BCG and/or Mtb. While vaccine research has focused on immunodominant epitopes, studies of minor determinants may focus the immune response on epitopes that cannot escape immune recognition. It has been reported that immunizing mice with a minor determinant of ESAT-6 can induce a significant protective immune response against TB, while using some immunodominant epitopes cannot produce such a protective response. Therefore, it is necessary and urgent to study the antigenic epitopes that stimulate the protective immune response against Mycobacterium tuberculosis, including immunodominant determinants and minor determinants.

Invention content:

The purpose of the present invention is to screen out molecular mimic peptides that can stimulate the protective immune response against Mycobacterium tuberculosis in humans, to study the protective immune response mechanism of tuberculosis, and to further develop a novel multi-unit multi-epitope tuberculosis vaccine. More effectively prevent and control the occurrence and development of tuberculosis.

Comprehensive application of genome sequence, bioinformatics, functional genomics and immunology research, the present invention provides a molecular mimetic peptide of an antigenic epitope that can stimulate the human body's protective immune response against Mycobacterium tuberculosis, said peptide contains Amino acid sequence:

RLLALLCAAV (SEQ ID NO: 2),

KLILTQPFDV (SEQ ID NO: 5),

RLSQSADQYL (SEQ ID NO: 10),

FLTREMPAWL (SEQ ID NO: 12),

KLIANNTRV (SEQ ID NO: 14),

GLPVEYLQV (SEQ ID NO: 15).

The present invention also provides a method for screening molecular mimic peptides capable of stimulating the protective immune response against Mycobacterium tuberculosis in humans, characterized in that it comprises the following steps:

I Epitope Prediction and Molecular Mimetic Peptide Synthesis

Using the database SYFPEITHI to predict HLA-A*0201-restricted CTL epitopes for Mycobacterium tuberculosis Rv3840c, Rv0309, Rv0129c, and Rv0173, and synthesize the predicted molecular mimetic peptides by F-moc chemical method;

II The above-mentioned molecular mimic peptides are screened out through affinity analysis, stability analysis, cell proliferation experiment, cell activity detection and specific antibody detection. Molecular mimic peptides that can produce specific antibodies.

The present invention also provides the application of the above-mentioned molecular mimetic peptide capable of stimulating the protective immune response against Mycobacterium tuberculosis in preparing immunogens, immunizing animals, or generating immune protection.

The present invention also provides the use of the above-mentioned molecular mimetic peptide of the antigenic epitope capable of stimulating the protective immune response against Mycobacterium tuberculosis in the preparation of multiple multi-epitope tuberculosis vaccines.

The multi-unit multi-epitope tuberculosis vaccine contains one or more peptide segments directly or indirectly connected among the above-mentioned molecular mimic peptides SEQ ID NO: 2, 5, 10, 12, 14, and 15.

The invention greatly contributes to the prevention and treatment of tuberculosis, especially the control of multi-drug-resistant strains, and also facilitates the study of molecular and cellular mechanisms of tuberculosis, and promotes the resolution of the multi-drug-resistant problem that plagues clinical practice.

Description of drawings

Figure 1 The affinity and stability analysis results of predicted antigen peptides and HLA-A*0201

Figure 2 Proliferation response of PBMCs stimulated by Mycobacterium tuberculosis antigen peptide

Figure 3 Mycobacterium tuberculosis antigen peptide induces PBMC to produce specific CTL killing activity

Figure 4 Humoral immune response induced by tuberculosis antigen peptide

Detailed ways:

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example 1:

1 Materials and methods

1.1 Research object

200 cases of pulmonary tuberculosis were diagnosed, of which 100 were HLA-A*0201 positive and 100 were HLA-A*0201 negative, all of which were tested by radiology and clinical sputum culture. 100 HLA-A*0201 positive patients with other chronic lung diseases were the disease control group, and 100 HLA-A*0201 positive healthy volunteers were the normal control group.

1.2 Reagents and kits:

The lymphocyte separation liquid Lymphoprep ^TM was purchased from AXIS-SHIELD Company of Norway;

LPS, human recombinant IL-2, DMSO, BFA, and _β2 -mg were all purchased from Sigma;

Human recombinant GM-CSF and human recombinant IL-4 were purchased from R&D Company;

RPMI1640 medium and fetal bovine serum were purchased from Gibco; (Sigma);

FITC-labeled anti-CD3, anti-CD8, anti-HLA.A2 flow cytometric monoclonal antibodies and FITC-labeled antibodies for detecting intracellular factors (IFN-γ, TNF-α, IL-10) are all products of Caltag;

RNA extraction kit was purchased from Qiagen;

Time-resolved fluorescent DELFIA cell proliferation (AD0200) and cytotoxicity (AD0016) detection kits were purchased from Wallac Oy;

The peptide was synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.;

The T2 cell line was purchased from the ATCC cell bank in the United States.

1.3 Main instruments

Flow Cytometry (Coulter EPICS XL)

Time-resolved fluorescence detector (DELFIA 1235, Perkin Elmer)

1.4 Epitope prediction and peptide synthesis

Using the database SYFPEITHI to predict the HLA-A*0201 restricted CTL epitope of Mycobacterium tuberculosis Rv3840c, Rv0309, Rv0129c, Rv0173, and score according to whether the amino acid on the polypeptide is an anchor residue, an auxiliary residue or a dominant residue, the score Those with a score higher than 21 may be T cell epitopes. The peptides predicted above were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd. by F-moc chemical method. All the synthesized antigenic peptides were purified by high performance liquid chromatography (purity>95%) and identified by mass spectrometry. The freeze-dried powder was stored at 4°C, and the polypeptide (2 μg/ul) was resuscitated with sterile distilled water before use.

1.5 Antigen peptide affinity analysis

The principle of using T2 cell line to analyze the affinity of antigen peptide is to use the characteristics of T2 cell line. The expression of empty HLA-A2 molecules on the surface of T2 cells is extremely unstable and degrades soon after presentation. The combination of antigenic peptides stabilizes the expression of HLA-A2. The stronger the binding force between antigenic peptides and HLA-A2, the more T2 cells The higher the expression level of HLA-A2 molecules on the surface. Moreover, due to the lack of antigen polypeptide transporter (TAP) necessary in the endogenous antigen presentation pathway of T2 cells, the increase in the expression of HLA-A2 molecules on the surface of T2 cells directly reflects the combination of foreign antigen peptides and HLA-A2 force.

The specific method is as follows: T2 cells were pretreated with sodium citrate-phosphate buffer solution of pH 3.2 for 1 min to denature the MHC1 complexes on the cell surface, and immediately washed once with excess RPMI1640 (Gibco BRL) culture medium to adjust the cell Concentrate to 3×10 ⁵ , seed in 24-well cell culture plate at 1ml/well. Stimulate with antigen peptide (20ug/ml), supplement Brefeldin (BFA, 10ug/ml) and β2-MG (1ug/ml), incubate at 37°C with 5% CO ₂ saturated humidity for 4h, and use no antigen peptide as blank control well . Collect the cells, wash once with FACS (PBS containing 0.2% FCS and 0.1% sodium azide), stain with FITC-labeled mouse anti-human HLA-A*0201 monoclonal antibody, react for 30 min at room temperature in the dark, wash with FACS solution 2 times, suspended in the sheath fluid, and detected the average fluorescence intensity with a flow cytometer (Coulter EPICS XL). Each experiment was repeated 3 times, and the antigen peptide whose average fluorescence intensity was higher than the average fluorescence intensity of the blank control well + 3SD had a high affinity to HLA-A*0201.

1.6 Antigenic peptide stability analysis

T2 cells were pretreated as before, increasing the concentration of antigen peptide to 80 μg/ml, incubating at 37°C with 5% CO ₂ saturated humidity for 18 hours, supplementing with BFA to a final concentration of 10 ug/ml, and continuing to incubate for 3 hours. Cells were collected, and the average fluorescence intensity was measured as before. Antigen peptides whose average fluorescence intensity was higher than the average fluorescence intensity of blank control wells+3SD were used for cell proliferation and cytotoxicity experiments.

1.7 Antigen peptide-induced CTL preparation

Density gradient centrifugation was used to separate peripheral blood mononuclear cells (PBMCs) from venous blood anticoagulated with EDTA, and the isolated PBMCs were planted in 6-well cell culture plates in RPMI1640 medium containing 10% FCS , 37°C 5% CO ₂ and incubate for 1.5h. The suspended cells (mainly lymphocytes) were collected and stored in liquid nitrogen with 10% dimethyl sulfoxide and 90% calf serum for future use. Adherent cells are antigen-presenting cells-dendritic cells (DC), supplemented with RPMI1640 culture medium containing GM-CSF (1000U/ml, R&D), IL-4 (1000U/ml, R&D) and 10% FCS, Incubate at 37°C with 5% CO2 for 7 days, and supplement fresh culture medium when necessary. After 5 days, LPS (1 μg/ml, Sigma) was added and cultured for 2 days, and γ-irradiated to inactivate the proliferative activity. The irradiated DCs were co-incubated with the antigen peptide (10 μmol/L) in the culture medium at 37° C. 5% CO ₂ overnight. Seed 1×10 ⁵ DC loaded with antigen peptide and 1×10 ⁶ autologous PBMCs in a 24-well cell culture plate in RPMI1640 complete culture solution containing 10% FCS, mix culture at 37°C and 5% CO ₂ for 3 days, and then add rIL- 2 (20U/ml, Sigma) was cultured for 10 days, and the cell proliferation experiment and cell activity detection were carried out.

1.8 Cell Proliferation Experiment

Using the time-resolved fluorescent DELFIA cell proliferation detection kit (AD0200, Wallaroy), the irradiated 5×10 ³ antigen-loaded DC100ul and the 5× ¹⁰⁴ antigen peptide-induced autologous CTL100ul were seeded in a 96-well cell culture plate, and DCs not loaded with antigen peptide were used as negative control. Add 20ul of BrdU labeling solution and incubate for 10h, centrifuge once, add Eu-labeled anti-BrdU monoclonal antibody, incubate at room temperature for 2h, wash and fix. The fluorescence intensity was detected by a time-resolved fluorescence detector (DELFIA 1235). Three replicate wells were set up for each sample, and the average value was taken as the detection result. Proliferation index (SI) = average fluorescence intensity of the antigen-containing peptide/average fluorescence intensity of the negative control, and antigen peptides with SI>2 are regarded as capable of inducing lymphocyte proliferation.

1.9 Cytotoxicity analysis

Time-resolved fluorescent DELFIA EUTDA cytotoxicity assay (AD0116, Wallaroy) was used to detect the killing activity of CTL induced by antigen peptide as effector cells (E), and T2 cells loaded with antigen peptide as target cells (T). The operation was performed according to the instructions, the ratio of effector cells to target cells was 40:1, and cultured for 4 hours. A time-resolved fluorescence detector (DELFIA 1235) was used to detect the fluorescence intensity. Three replicate wells were set up for each sample, and the average value was taken as the test result. Cell killing activity=(fluorescence intensity of the well to be tested-fluorescence intensity of the natural release hole)/(fluorescence intensity of the maximum release hole-fluorescence intensity of the natural release hole)×100%.

1.10 Animal experiments

1.10.1 Animals and Grouping

Twenty C57BL/6 mice, 2 months old, weighing (18±2) g, male or female, were randomly divided into 4 groups, 5 mice in each group.

1.10.2 Immunization methods

The tuberculosis antigen peptide screened out in vitro was used as the immunogen and injected 3 times with an interval of 2 weeks each time. CFA was used as the adjuvant for the first time, and IFA was used as the adjuvant for booster immunization. The first and booster immunization doses of mice were 50 μg/mouse, and after being completely emulsified with 0.3ml adjuvant, the mice were injected at multiple points in the axilla, groin, subcutaneously in the armpit and in the abdominal cavity.

1.10.3 Collection and separation of specimens

21 days after the first immunization, the second immunization and the third immunization, blood was collected from the orbits of 5 mice/group respectively, the serum was separated, and frozen in a -20°C refrigerator for future use.

1.10.4 Detection of specific antibodies

The antibody titers in serum were measured by standard indirect ELISA method, and the specific antibody titers of each group of animals were measured respectively. Coat 100 wells of 96-well enzyme-linked plates with an antigen concentration of 20 μg/ml, overnight at 4°C; block with 5% BSA-PBS at 37°C for 120 minutes, wash 3 times, 3 minutes each time; 100 wells, incubated at 37°C for 90min. After washing 3 times, add HRP enzyme-labeled secondary antibody, 37°C for 30min; after washing 3 times, add DAB substrate, 37°C for 10min, to terminate the reaction. The absorbance A value was measured at 490nm by an enzyme-linked analyzer, and normal mouse serum was used as a negative control. The results are expressed as the mean A value of double-wells. The A value of the sample to be tested/the A value of the negative control > 2 is used as the positive standard, and the highest dilution with a positive reaction is used as the antibody titer in the sample.

1.10.5 Challenge protection experiment of immunized mice

The remaining 5 C57BL/6 mice in each group were immunized three times and then challenged at an interval of 8 weeks. The injection site of the challenge was the tail vein of the mice, the injection dose was 1×10 ⁶ CFU, and the challenge strain was H37Rv. The mice were killed 8 weeks after the challenge, and the lung and spleen homogenates were diluted and inoculated on solid Roche medium, and the viable bacteria were counted four weeks later.

2 results

2.1 Epitope prediction and peptide synthesis results

According to bioinformatics prediction, the following peptide sequences were selected for synthesis. The synthesized polypeptide is identified by mass spectrometry, and the purity is greater than 95% by high pressure liquid chromatography (HPLC).

Rv0309:

VLAVSLAV (SEQ ID NO: 1)

RLLALLCAAV (SEQ ID NO: 2)

SLAVVNPWFA (SEQ ID NO: 3)

VLAVSLAVV (SEQ ID NO: 4)

Rv0173:

KLILTQPFDV (SEQ ID NO: 5)

FLGKLDTFT (SEQ ID NO: 6)

VLLVLALLL (SEQ ID NO: 7)

RVMVADVWV (SEQ ID NO: 8)

YLVGALKLI (SEQ ID NO: 9)

RLSQSADQYL (SEQ ID NO: 10)

Rv0129c:

GLPVEYLQV (SEQ ID NO: 11)

FLTREMPAWL (SEQ ID NO: 12)

Rv3804c:

AIYGPQQFV (SEQ ID NO: 13)

KLIANNTRV (SEQ ID NO: 14)

GLPVEYLQV (SEQ ID NO: 15)

2.2 Antigen peptide affinity and stability analysis results

99% of cultured T2 cells express HLA-A*0201 molecules, but the average fluorescence intensity is weak. The binding activity of the above synthesized antigen peptides to HLA-A*0201 is shown in Table 1. The average fluorescence intensity of the cells treated with antigen peptides 1-15 increased, among which the increase of antigen peptides 2 and 14 was the most obvious. Growth factor of average fluorescence intensity of T2 cells treated with antigen peptide = average fluorescence intensity after antigen peptide treatment/average fluorescence intensity without antigen peptide, the highest was treated with antigen peptide 14, followed by antigen peptide 15. The results of stability analysis of the combination of antigen peptides and HLA-A*0201 molecules showed that after antigen peptides combined with HLA-A*0201 molecules on T2 cells, antigen peptides 2, 3, 5, 7, 8, 10, 12, 14 , 15 can stably up-regulate the expression of HLA-A*0201 molecules in T2 cells (see Figure 1). According to the binding force and stability results, antigenic peptides 2, 3, 5, 7, 8, 10, 12, 14, and 15 were selected to continue downstream experiments.

Table 1 Average fluorescence intensity of T2 cells HLA-A*0201 after antigen peptide stimulation

Antigenic peptide SEQ ID NO amino acid sequence Simulation source: amino acid position mean fluorescence intensity 1 VLAVSLAV Rv0309: aa.20～28 10.69 2 RLLALLCAAV Rv0309: aa.3～12 10.86 3 SLAVVNPWFA Rv0309: aa.25～34 10.73 4 VLAVSLAVV Rv0309: aa.20～29 9.18 5 KLILTQPFDV Rv0173: aa.302～311 11.54

6 FLGKLDTFT Rv0173: aa.191～199 8.08 7 VLLVLALLL Rv0173: aa.20～28 11.32 8 RVMVADVWV Rv0173: aa.69～77 12.46 9 YLVGALKLI Rv0173: aa.296～304 8.12 10 RLSQSADQYL Rv0173: aa.260～269 12.37 11 GLPVEYLQV Rv0129c: aa.51～59 10.65 12 FLTREMPAWL Rv0129c: aa.144～153 11.53 13 AIYGPQQFV Rv3804c: aa.178～186 8.29

6 FLGKLDTFT Rv0173: aa.191～199 8.08 14 KLIAN NTRV Rv3804c: aa.242～250 13.51 15 GLPVEYLQV Rv3804c: aa.48～56 12.84 negative control - - 4.21

2.3 Antigen peptide stimulates cell proliferation

The ability of antigen peptides 2, 3, 5, 7, 8, 10, 12, 14 and 15 to induce antigen-specific CTL in PBMCs of tuberculosis patients was evaluated by cell proliferation response, and further identified whether they were specific cell epitopes. Among them, 100 HLA-A*0201-positive tuberculosis patients, 100 HLA-A*0201-negative tuberculosis patients, 100 HLA-A*0201-positive patients with other chronic lung diseases were disease control groups, and 100 HLA-A*0201-positive healthy patients Volunteers were the normal control group. A positive proliferative response was defined as a stimulation index (SI) > 3. The results showed that the PBMCs of tuberculosis patients (HLA-A*0201 positive and negative patients) had strong positive proliferative responses to antigenic peptides 2, 5, 10, 12, 14 and 15, while those in the disease control group and healthy control group did not. A marked proliferative response occurred (see Figure 2).

2.4 Antigen peptides induce CTL cytotoxicity

T2 cells loaded with antigen peptides were used as target cells, CD8+ CTLs induced by antigen peptides were used as effector cells, and the effect-to-target ratio (E/T) was 20:1. CTL cytotoxic activity. The results showed that CD8+CTLs stimulated by antigenic peptides 2, 5, 10, 12, 14 and 15 were effector cells with strong cytotoxic activity (see Figure 3).

2.5 Dynamic determination of mouse antibody titer

Two weeks after the first immunization, blood was collected once a week, and the serum was separated, and the tuberculosis antigen peptide was used as the coating antigen, and the specific antibody titer was measured by ELISA indirect method. The specific antibody could be detected 3 weeks after the first immunization. During the measurement period, the antibody titer increased gradually. Eight weeks after the first immunization, the antibody titer reached a maximum of 1:64. Antibody titers increased with the number of immunizations and time, while no antibodies were detected in the negative control group (see Figure 4).

2.6 Analysis of the protective effect of the vaccine on immunized mice

The protective efficacy induced by each vaccine was studied by using mice 8 weeks after challenge as experimental materials. Experiments showed that the number of bacteria in the lungs and spleens of mice in each immunization group was significantly reduced. Its protection efficiency is shown in Table 2.

Table 2 Protective response ability induced by vaccines

*The calculation method of the protection rate is: the logarithm value of the bacteria number in the negative group minus the logarithm value of the bacteria number in the vaccine or BCG group. The larger the value, the higher the protection efficiency.

3 Conclusion

Comprehensive application of genome sequence, bioinformatics, functional genomics and immunology research, the results confirmed that the molecular mimic peptides of the following antigenic epitopes can stimulate the human body's protective immune response against Mycobacterium tuberculosis:

1. RLLALLCAAV (SEQ ID NO: 2), analog source Rv0309: aa.3～12;

2. KLILTQPFDV (SEQ ID NO: 5), analog source Rv0173: aa.302～311;

3. RLSQSADQYL (SEQ ID NO: 10), analog source Rv0173: aa.260～269;

4. FLTREMPAWL (SEQ ID NO: 12), analog source Rv0129c: aa.144～153;

5. KLIANNTRV (SEQ ID NO: 14), analog source Rv3804c: aa.242～250;

6. GLPVEYLQV (SEQ ID NO: 15), analog source Rv3804c: aa.48～56.

SEQUENCE LISTING

<110> The Second Military Medical University of the Chinese People's Liberation Army

<120> Antigen epitopes for stimulating human protective immune response against Mycobacterium tuberculosis and its use

<130> specification, claims

<160>15

<170>PatentIn version 3.1

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Claims (3)

1. The molecular mimetic peptide of the antigenic epitope that can stimulate the protective immune response of human body against Mycobacterium tuberculosis is characterized in that, the aminoacid sequence of described peptide is as shown in SEQ ID NO:14. 2. The application of the molecular mimetic peptide of the antigenic epitope capable of stimulating the protective immune response against Mycobacterium tuberculosis in the human body as claimed in claim 1 in the preparation of immunogens. 3. The use of the molecular mimetic peptide of the antigenic epitope capable of stimulating the protective immune response of human body against Mycobacterium tuberculosis as claimed in claim 1 in the preparation of multiple multi-epitope tuberculosis vaccines.

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