Non-canonical ifn-gamma-dependent mycobacterial peptide epitope p15, coding sequences and uses, anti-tuberculosis vaccine

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a non-classical IFN-gamma-dependent mycobacterium tuberculosis peptide epitope P15, a coding sequence and application thereof, and an anti-tuberculosis vaccine. The amino acid sequence of the mycobacterium tuberculosis peptide epitope P15 is shown as SEQ ID NO. 1. The application is found through research that the mycobacterium tuberculosis peptide epitope P15 has good immunogenicity, in-vivo immunity has a significant anti-tuberculosis protection effect, and the protection effect is not dependent on the host cell IFN-gamma response, and can be used as an ideal target antigen of a tuberculosis vaccine, and is made into an epitope vaccine, and has a good application prospect.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to a non-classical IFN-γ dependent Mycobacterium tuberculosis peptide epitope P15, its coding sequence and application, and an anti-tuberculosis vaccine. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), and it is a single-pathogen infection. Given its high mortality rate and increasing drug resistance, developing more effective vaccines has become crucial for global TB control.

[0003] However, while the existing BCG vaccine, the only approved tuberculosis vaccine, is effective in preventing tuberculous meningitis and disseminated tuberculosis in infants and young children, its protective efficacy against pulmonary tuberculosis in adolescents and adults is limited. Furthermore, recent results indicate that BCG revaccination is ineffective in preventing primary Mycobacterium tuberculosis infection and persistent tuberculosis infection in adolescents. This inefficiency highlights the urgent need for the development of novel tuberculosis vaccines. Summary of the Invention

[0004] The purpose of this application is to provide a non-classical IFN-γ-dependent tuberculosis peptide epitope P15 and its coding sequence and application, as well as an anti-tuberculosis vaccine, to solve the technical problem of how to prevent tuberculosis.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides a non-classical IFN-γ-dependent tuberculosis peptide epitope P15, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] Secondly, this application provides a coding sequence for a tuberculosis peptide epitope P15, the coding sequence encoding the tuberculosis peptide epitope P15 provided in the first aspect of this application.

[0007] In some embodiments, the nucleotide sequence of the coding sequence is as shown in SEQ ID NO.2.

[0008] Thirdly, this application provides an application, namely the application of the tuberculosis peptide epitope P15 provided in the first aspect of this application in the preparation of an anti-tuberculosis vaccine.

[0009] In some embodiments, the protective effect of the tuberculosis peptide epitope P15 is independent of the host cell IFN-γ response.

[0010] In some embodiments, the anti-tuberculosis vaccine includes an immune adjuvant.

[0011] Fourthly, this application provides an anti-tuberculosis vaccine, said anti-tuberculosis vaccine comprising an epitope vaccine having the tuberculin peptide epitope P15 provided in the first aspect of this application.

[0012] In some embodiments, the protective effect of the tuberculosis peptide epitope P15 is independent of the host cell IFN-γ response.

[0013] In some embodiments, the anti-tuberculosis vaccine includes any one of DNA vaccines, mRNA vaccines, short peptide vaccines, and recombinant viral vector vaccines; And / or, the anti-tuberculosis vaccine further includes at least one of BCG vaccine and clinically experimental IFN-γ-guided vaccine.

[0014] In some embodiments, the anti-tuberculosis vaccine includes an immune adjuvant.

[0015] The first aspect of this application provides a tuberculosis peptide epitope P15, the amino acid sequence of which is shown in SEQ ID NO.1. Through research, this application has found that the tuberculosis peptide epitope P15 possesses good immunogenicity, exhibits significant anti-tuberculosis protective effects in vivo, and its protective effect is independent of the host cell IFN-γ response. Therefore, it can serve as an ideal target antigen for tuberculosis vaccines, and the formulation of epitope vaccines shows great promise for application.

[0016] A second aspect of this application provides a coding sequence that can encode the tuberculosis peptide epitope P15 provided in the first aspect of this application. This coding sequence can be applied to the preparation of the tuberculosis peptide epitope P15 and related epitope vaccines.

[0017] The third aspect of this application provides an application based on the fact that the tuberculosis peptide epitope P15, as shown in the amino acid sequence SEQ ID NO.1, has good immunogenicity, exhibits significant anti-tuberculosis protective effects in vivo, and its protective effect does not depend on the host cell IFN-γ response. Therefore, an anti-tuberculosis vaccine can be prepared, and the preparation method is simple and suitable for industrial production.

[0018] The fourth aspect of this application provides an anti-tuberculosis vaccine comprising an epitope vaccine having the tuberculosis peptide epitope P15 provided in the first aspect of this application. Based on the fact that the tuberculosis peptide epitope P15 can induce a Th1-type immune response in the host and promote the secretion of IFN-γ by lymphocytes, it exhibits good immunogenicity. Furthermore, the epitope vaccine based on the short peptide tuberculosis peptide epitope P15 shows comparable protective efficacy to BCG in preventing primary infection with Mycobacterium tuberculosis. Moreover, experiments have demonstrated that the epitope vaccine still provides significant protection in IFN-γ receptor-deficient mice, offering a good complement to current tuberculosis subunit vaccines based on IFN-γ secretion. Compared to traditional vaccines, epitope vaccines do not integrate into the host genome, do not carry the risk of pre-existing immunity, and are convenient to synthesize with a short development cycle, making them particularly suitable for addressing the rapid mutation and drug resistance issues of Mycobacterium tuberculosis, and are easily mass-produced.

[0019] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is the result of detecting the intracellular tuberculosis load in macrophages stimulated by the tuberculosis peptide epitope P15 in Example 1 of this application; Figure 2 This is the result of detecting the IFN-γ secretion level of splenic lymphocytes in mice immunized with the tuberculosis peptide epitope P15 in Example 2 of this application; Figure 3 This is the result of measuring the tuberculosis bacterial load in the lung tissue of wild-type mice immunized with the tuberculosis peptide epitope P15 epitope vaccine in Example 3 of this application 4 weeks after tuberculosis infection; Figure 4 This is the lung tissue HE staining assessment result of wild-type mice immunized with the tuberculosis peptide epitope P15 epitope vaccine in Example 3 of this application, 4 weeks after tuberculosis infection. Figure 5 The results of measuring the tuberculosis load in lung tissue of mice immunized with the tuberculosis peptide epitope P15 epitope vaccine in Example 4 of this application are as follows: Detailed Implementation

[0022] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0023] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0024] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0025] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0026] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0027] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0028] The terms "first" and "second" are used only to describe purpose, to distinguish purposes such as substances from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.

[0029] A peptide epitope, also known as an epitope peptide, is a short peptide consisting of a continuous amino acid sequence that corresponds to an antigenic determinant (epitaxe) on a pathogen protein. It can be specifically recognized by antibodies, B cell receptors, and T cell receptors and is the smallest polypeptide unit that mediates specific immunity.

[0030] Epitope vaccines, also known as peptide epitope vaccines, are vaccine formulations made with peptide epitopes as the core immunogen, which can be combined with adjuvants, buffer solutions, and other excipients. They select only the short peptide fragments of pathogen proteins responsible for immune recognition, discarding the complete pathogen and full-length protein. They rely on B / T cell epitopes to induce specific protective immunity and belong to the subunit vaccine category.

[0031] "Anti-tuberculosis vaccine," also known as tuberculosis vaccine, is a vaccine against Mycobacterium tuberculosis (TB) used for the prevention or adjuvant treatment of tuberculosis. It is divided into two main categories: commercially available vaccines and new vaccines under clinical investigation. Functionally, it is categorized into three types: primary prevention, booster prevention, and adjuvant therapy. Currently, the only basic preventative vaccine globally is BCG (Bacillus Calmette-Guérin).

[0032] The selection of the core antigen in a vaccine has always been crucial to its protective efficacy. Existing tuberculosis vaccines, such as BCG, offer limited protection, particularly against adult pulmonary tuberculosis. Currently, many novel subunit tuberculosis vaccines in clinical trials use the ability to induce IFN-γ secretion from T cells as a screening criterion. However, clinical studies have found that some tuberculosis patients, even with high IFN-γ levels, are unable to effectively clear the pathogen. This indicates that IFN-γ production is not directly equivalent to Mtb clearance, suggesting that Th1 immunity is not the sole determinant of anti-tuberculosis immunity. Multiple clinical trials have provided direct evidence of the limitations of Th1 immunity. For example, the MVA85A vaccine enhanced IFN-γ-mediated T-cell responses in preclinical studies but failed to provide significant protection in phase III clinical trials. Similarly, while Th1-guided vaccines H56:IC31 and H4:IC31 induced strong IFN-γ production in phase I / II trials, their protective efficacy in population studies remains limited. These cases demonstrate that vaccine development strategies relying solely on IFN-γ as a single immune evaluation indicator have shortcomings. There is an urgent need to discover novel tuberculosis vaccine target antigens that are not IFN-γ-oriented, in order to improve vaccine efficacy. Current research on tuberculosis vaccines, especially peptide epitope vaccines, is incomplete; the immune mechanisms of candidate vaccine epitopes are not sufficiently understood; the application of peptide epitope vaccines in the field of tuberculosis has not been fully developed; and there is a lack of validated, highly effective antigen targets.

[0033] Based on this, this application, through experiments, first discovered that a non-classical IFN-γ-dependent Mycobacterium tuberculosis peptide epitope P15 possesses excellent immunogenicity and protective efficacy. Furthermore, using an IFNγ receptor-deficient mouse model of Mycobacterium tuberculosis infection, it was confirmed that the anti-tuberculosis protective effect of Mycobacterium tuberculosis peptide epitope P15 is independent of IFN-γ, making it an ideal target antigen for tuberculosis vaccines. The technical solution is described in detail below.

[0034] In a first aspect, embodiments of this application provide a non-classical IFN-γ-dependent tuberculosis peptide epitope P15. Specifically, the amino acid sequence of the tuberculosis peptide epitope P15 in embodiments of this application is shown in SEQ ID NO.1.

[0035] SEQ ID NO. 1: HMHNYEGPETHTSLR.

[0036] The aforementioned Mycobacterium tuberculosis peptide epitope P15 exhibits good immunogenicity and significant anti-tuberculosis protective effects in vivo. Furthermore, its protective effect is independent of the host cell IFN-γ response, making it an ideal target antigen for tuberculosis vaccines. The formulation of epitope vaccines shows great promise for application.

[0037] Secondly, embodiments of this application provide a coding sequence for the tuberculosis peptide epitope P15. Specifically, the coding sequence of this application encodes the amino acid sequence shown in SEQ ID NO.1 for the tuberculosis peptide epitope P15.

[0038] The coding sequence of this application embodiment can be applied to the preparation of tuberculosis peptide epitope P15 and related epitope vaccines, for example, using genetic engineering methods to prepare tuberculosis peptide epitope P15.

[0039] In some embodiments, the nucleotide sequence encoding the sequence is shown in SEQ ID NO.2.

[0040] SEQ ID NO. 2: CATATGCATAACTATGAAGGTCCGGAAACCCATACCAGCCTGCGT.

[0041] Thirdly, this application provides an application of the tuberculosis peptide epitope P15, whose amino acid sequence is shown in SEQ ID NO.1, in the preparation of an anti-tuberculosis vaccine.

[0042] Based on the fact that the tuberculosis peptide epitope P15, whose amino acid sequence is shown in SEQ ID NO.1, has good immunogenicity and has a significant anti-tuberculosis protective effect in vivo, and its protective effect does not depend on the host cell IFN-γ response, it is possible to prepare an anti-tuberculosis vaccine. Moreover, the preparation method is simple and suitable for industrial production.

[0043] In some embodiments, the protective effect of the tuberculosis peptide epitope P15 is independent of the host cell IFN-γ response.

[0044] In some embodiments, the anti-tuberculosis vaccine includes an immune adjuvant.

[0045] Fourthly, embodiments of this application provide an anti-tuberculosis vaccine. Specifically, the anti-tuberculosis vaccine includes an epitope vaccine having the tuberculin peptide epitope P15 of embodiments of this application.

[0046] Based on the ability of the Mycobacterium tuberculosis peptide epitope P15 to induce a Th1-type immune response in the host and promote the secretion of IFN-γ by lymphocytes, it exhibits good immunogenicity. Furthermore, epitope vaccines based on the short peptide Mycobacterium tuberculosis peptide epitope P15 show comparable protective efficacy to BCG in preventing primary infection with Mycobacterium tuberculosis. Moreover, experiments have demonstrated that epitope vaccines still provide significant protection in IFN-γ receptor-deficient mice, offering a good complement to current IFN-γ secretion-guided tuberculosis subunit vaccines. Compared to traditional vaccines, epitope vaccines do not integrate into the host genome, eliminating the risk of pre-existing immunity. Additionally, peptide epitope synthesis is convenient, and the development cycle is short, making them particularly suitable for addressing the rapid mutation and drug resistance issues of Mycobacterium tuberculosis, and they are easily mass-produced.

[0047] In some embodiments, the protective effect of the tuberculosis peptide epitope P15 is independent of the host cell IFN-γ response. The anti-tuberculosis vaccine of this application can induce a Th1 / Th17 immune response, and the induced immune protection effect is independent of the IFN-γ response.

[0048] In some embodiments, the anti-tuberculosis vaccine further includes at least one of BCG and a clinically experimental IFN-γ-guided vaccine. For example, an epitope vaccine containing the tuberculin peptide epitope P15 can be used in combination with the existing tuberculosis vaccine BCG to form a primary-booster immunization strategy, or an epitope vaccine containing the tuberculin peptide epitope P15 can be used in combination with a clinically experimental IFN-γ-guided vaccine to form a combined supplementary immunization strategy to further improve protective efficacy.

[0049] In some embodiments, the anti-tuberculosis vaccine includes an immune adjuvant. The anti-tuberculosis vaccine comprises an epitope vaccine formed from the tuberculosis epitope P15 described in the embodiments of this application and an immune adjuvant. This immune adjuvant may be Complete Freund's Adjuvant (CFA), whose composition includes an oil (such as paraffin oil) water-in-oil emulsion containing heat-inactivated, inactive Mycobacterium tuberculosis. As an inducer of Th1-type immune responses and a TLR ligand, it can stimulate a strong and durable immune response. In the anti-tuberculosis vaccine, the mass-to-volume ratio of the tuberculosis epitope peptide P15 to Freund's adjuvant may be 1 microgram (μg): 9-11 microliters (μL). For example, 10 micrograms of tuberculosis epitope peptide P15 are mixed with 100 microliters of Freund's adjuvant.

[0050] The anti-tuberculosis vaccine of this application embodiment can be of various forms, such as any one of DNA vaccine, short peptide vaccine, recombinant viral vector vaccine, and mRNA vaccine.

[0051] In summary, the epitope vaccine based on the tuberculin peptide epitope P15 of this application can be well used for the prevention and treatment of tuberculosis. It has the following significant advantages: (1) Good immunogenicity: The tuberculin peptide epitope P15 can induce the host's Th1 immune response and promote the secretion of IFN-γ by lymphocytes, which is crucial for controlling Mycobacterium tuberculosis infection. (2) Protective effect comparable to BCG: Experiments have shown that the epitope vaccine based on the tuberculin peptide epitope P15 has a protective effect comparable to that of BCG, which has been used for a century, in preventing primary infection of Mycobacterium tuberculosis. (3) Protective effect independent of IFN-γ: Experiments have shown that the epitope vaccine based on the tuberculin peptide epitope P15 still has a significant protective effect in immunizing IFN-γ receptor-deficient mice, which has a good complementary effect to the current tuberculosis subunit vaccines based on IFN-γ secretion. (4) High safety and short R&D cycle: Compared with traditional vaccines, peptide epitope vaccines do not need to be integrated into the host genome, do not have the risk of pre-existing immunity, and peptide epitope synthesis is convenient and the R&D cycle is short, making them particularly suitable for dealing with the rapid mutation and drug resistance of Mycobacterium tuberculosis. (5) Easy to scale up production: The production process of epitope vaccines is relatively standardized and is not limited by biological preparation processes such as cell culture, which facilitates large-scale production and quality control. (6) Strong compatibility: Epitope vaccines based on Mycobacterium tuberculosis peptide epitope P15 can be used in combination with existing tuberculosis vaccines (such as BCG) and IFN-γ-guided vaccines in clinical trials to form a primary-booster immunization or combined supplementary immunization strategy, further improving the protective effect.

[0052] This application has undergone multiple experiments, and some of the experimental results are presented here for reference to further describe the application in detail. The following is a detailed description in conjunction with specific embodiments.

[0053] Example 1 The intracellular clearance effect of Mycobacterium tuberculosis peptide epitope P15 was investigated using the following experimental steps: 1) Mouse bone marrow-derived cells were induced in vitro for 6 days with macrophage colony-stimulating factor (M-CSF) at a concentration of 10 ng / ml to differentiate into small bone marrow-derived macrophages (BMDM). Cell counting and plating were then performed on day 7 in 24-well plates, yielding approximately 2.5 × 10⁶ cells. 5 One cell per well.

[0054] 2) On the second day after plating, Mycobacterium tuberculosis Mtb H37Rv (ATCC27294, preserved in BSL-3 laboratory of Shenzhen University) was used for infection with a multiplicity of infection (MOI) of 5. After 4-6 hours of infection, the extracellular bacteria were washed away, the medium was changed, and the cells were stimulated with 10 μg / well / ml of Mycobacterium tuberculosis peptide epitope P15 (amino acid sequence: SEQ ID NO.1) for incubation. After 72 hours of infection, the cells were lysed for intracellular Mycobacterium tuberculosis load determination.

[0055] Experimental results are as follows Figure 1 As shown in the figure: the horizontal axis represents the group stimulated by the tuberculosis peptide epitope P15, and the Mock group represents the group without the addition of the tuberculosis peptide epitope P15; the vertical axis represents the colony-forming unit (CFU). The results indicate that the tuberculosis peptide epitope P15 does not promote intracellular bacterial clearance in macrophages, and there was no significant difference compared to the group without the addition of the tuberculosis peptide epitope P15.

[0056] Example 2 The immunogenicity evaluation experiment for the Mycobacterium tuberculosis peptide epitope P15 epitope vaccine was conducted as follows: 1) Animal immunization: 6-8 week old female C57BL / 6 mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were randomly divided into the following 3 groups (n=5): Experimental group: Epitope vaccine of Mycobacterium tuberculosis peptide epitope P15 (10μg / dose).

[0057] Negative control group: Freund's adjuvant (MCE, HY-153808B).

[0058] The steps for preparing the epitope vaccine of Mycobacterium tuberculosis peptide epitope P15 in the experimental group included: before immunization, 60 μg of Mycobacterium tuberculosis peptide epitope P15 was diluted to 300 μL with sterile ultrapure water, and 300 μL of Freund's adjuvant was mixed and emulsified (ice bath ultrasonic emulsification) to obtain the epitope vaccine emulsifier. The immunization dose for each mouse was 10 μg / 100 μL of emulsifier.

[0059] Immunization regimen: Subcutaneous injections on day 0 and day 14, for a total of 2 times.

[0060] 2) Cellular immune response detection: Mice were sacrificed 14 days after the last immunization, and spleens were harvested to prepare single-cell suspensions. The single-cell suspensions were evenly added to IFN-γ-coated Elispot assay plates (96-well plates) (MabTech, 3321-4APW), with 100,000 cells / 100 μL per well. Spleen cells were stimulated with tuberculosis peptide epitope P15 (2 μg / mL) for 20 hours, and the IFN-γ secretion capacity of spleen cells was detected according to the Elispot instructions.

[0061] Experimental results are as follows Figure 2 As shown in the figure: the horizontal axis represents the experimental group of the Mycobacterium tuberculosis peptide epitope P15 epitope vaccine, and the Mock group represents the negative control group with Freund's adjuvant; the vertical axis represents the IFN-γ secretion capacity of spleen cells. The results indicate that the Mycobacterium tuberculosis peptide epitope P15 epitope vaccine induced a certain proportion of peptide epitope-specific IFN-γ secretory T cells.

[0062] Example 3 The evaluation steps for the anti-tuberculosis protective efficacy of the tuberculosis peptide epitope P15 epitope vaccine are as follows: Mycobacterium tuberculosis challenge experiment: C57BL / 6 wild-type mice purchased from Yaokang Biotechnology were immunized according to Example 2, with BCG immunization added as a positive control at the first peptide epitope assay (subcutaneous immunization, 10×). 6 Two weeks after the last immunization with the peptide epitope vaccine (CFU / mouse, immunized only once), all mice were exposed to Mycobacterium tuberculosis strain H37Rv (approximately 200 CFU) via aerosol infection. Four weeks post-infection, mice were sacrificed: lung tissue was harvested, homogenized, and spread on Michaelis-Menten 7H10 agar plates; colony-forming units (CFU) were counted, representing the tuberculosis load in the lungs. A portion of lung tissue was taken for pathological examination to assess the degree of inflammation and granuloma formation.

[0063] Experimental results are as follows Figure 3 As shown in the figure: the horizontal axis represents the experimental group of the epitope vaccine containing the tuberculosis peptide epitope P15, and CFA represents the Freund's adjuvant negative control group; the vertical axis represents the tuberculosis bacterial load in the mouse lungs (colony-forming units, CFU, in lung homogenate). The results indicate that the bacterial load in the lung tissue of mice immunized with the epitope vaccine was reduced by 0.6 log [missing value] compared to the adjuvant negative control group. 10 CFU (p<0.001), comparable to the positive control group immunized with BCG. Additionally, the results of HE staining of lung tissue were as follows: Figure 4 As shown, pathological examination revealed a significant reduction in pulmonary inflammatory infiltration and lesion extent in the epitope vaccine experimental group.

[0064] Example 4 The efficacy of the tuberculosis peptide epitope P15 epitope vaccine against IFN-γ receptor-deficient mice was evaluated using the following steps: Mycobacterium tuberculosis challenge experiment: Two weeks after the last immunization, IFN-γ receptor-deficient mice (C57BL / 6 background, purchased from Saiye Biotechnology) were exposed to Mycobacterium tuberculosis H37Rv strain (approximately 200 CFU) via aerosol infection. Four weeks post-infection, mice were sacrificed: lungs and spleens were harvested, homogenates were prepared, spread on Michaelis 7H10 agar plates, and colony-forming units (CFU) were counted. A portion of lung tissue was taken for pathological examination to assess the degree of inflammation and granuloma formation.

[0065] Experimental results are as follows Figure 5 As shown in the figure: the horizontal axis represents the experimental group of the epitope vaccine containing the tuberculosis peptide epitope P15, and CFA represents the Freund's adjuvant negative control group; the vertical axis represents the tuberculosis bacterial load in the mouse lungs (colony-forming units, CFU, in lung homogenate). The results indicate that the bacterial load in the lung tissue of mice in the epitope vaccine experimental group was reduced by 0.6 log [missing value] compared to the adjuvant negative control group. 10 CFU (p<0.0001).

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and specification of this application. In particular, as long as there is no structural conflict, the various technical features mentioned in the embodiments can be combined in any way. This application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A non-classical IFN-γ-dependent tuberculosis peptide epitope P15, characterized in that, The amino acid sequence of the tuberculosis peptide epitope P15 is shown in SEQ ID NO.

1.

2. A coding sequence for the tuberculosis peptide epitope P15, characterized in that, The coding sequence encodes the tuberculosis peptide epitope P15 as described in claim 1.

3. The encoded sequence as described in claim 2, characterized in that, The nucleotide sequence of the coding sequence is shown in SEQ ID NO.

2.

4. The use of the tuberculosis peptide epitope P15 as described in claim 1 in the preparation of an anti-tuberculosis vaccine.

5. The application as described in claim 4, characterized in that, The protective effect of the tuberculosis peptide epitope P15 is independent of the host cell IFN-γ response.

6. The application as described in claim 4 or 5, characterized in that, The anti-tuberculosis vaccine includes an immune adjuvant.

7. An anti-tuberculosis vaccine, characterized in that, The anti-tuberculosis vaccine includes an epitope vaccine having the tuberculin peptide epitope P15 as described in claim 1.

8. The anti-tuberculosis vaccine as described in claim 7, characterized in that, The protective effect of the tuberculosis peptide epitope P15 is independent of the host cell IFN-γ response.

9. The anti-tuberculosis vaccine as described in claim 7, characterized in that, The anti-tuberculosis vaccine includes any one of DNA vaccines, mRNA vaccines, short peptide vaccines, and recombinant viral vector vaccines; And / or, the anti-tuberculosis vaccine further includes at least one of BCG vaccine and clinically experimental IFN-γ-guided vaccine.

10. The anti-tuberculosis vaccine according to any one of claims 7-9, characterized in that, The anti-tuberculosis vaccine includes an immune adjuvant.

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