Protective antigen combination of mycobacterium tuberculosis and use thereof

Abstract

The present invention relates to a protective antigen combination of Mycobacterium tuberculosis and the use thereof. Specifically, the antigen is a combined antigen and contains at least the following antigens: Ag85B, Rv2465c, Rv2029c and Rv3406. The use is for the preparation of a vaccine against Mycobacterium tuberculosis.

Description

A protective antigen combination for Mycobacterium tuberculosis and its application Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a protective antigen combination for Mycobacterium tuberculosis and its application. Background Technology

[0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis and continues to pose a serious threat to global public health security. In 2022, there were 10.6 million new cases of tuberculosis and 1.3 million tuberculosis deaths worldwide, with an estimated one-quarter of the global population under latent tuberculosis infection. In 2022, my country estimated 748,000 new cases of tuberculosis, ranking third among the 30 countries with a high burden of tuberculosis (1).

[0003] Vaccination aims to generate lasting host immune memory and is a key means of global control and ultimate elimination of tuberculosis. The only licensed tuberculosis vaccine, Bacille Calmette-Guérin (BCG), has been the most widely used and time-tested. While it can prevent miliary tuberculosis and meningeal tuberculosis in children, it is not effective in preventing pulmonary tuberculosis in adolescents and adults, and it is ineffective in suppressing the reactivation of latent tuberculosis infection (2). Therefore, there is an urgent need for an effective vaccine targeting different stages of disease development in order to control tuberculosis in the long term. Over the past few decades, the world has been working to develop new vaccine strategies. To date, more than a dozen candidate vaccines have been developed in clinical trials, including live attenuated mycobacterial vaccines, inactivated mycobacterial vaccines, adjuvanted subunit vaccines, viral vector vaccines, and mRNA vaccines (3). For example, the viral vector vaccine MVA85A and the subunit vaccine M72 / AS01E have not shown enhanced protection or only partial efficacy in phase 2b clinical trials (4,5). Continued exploration of new vaccine construction remains necessary and urgent.

[0004] After infecting a host, Mycobacterium tuberculosis (MTBG) faces immune and environmental stresses, such as nutritional deficiencies and hypoxia. The bacteria's metabolism shifts from an active replication state to a quiescent, persistent state, becoming non-replicating, persistent, or dormant MTBG – a characteristic of latent tuberculosis infection (LTBI). This process is related to the differential expression of MTBG antigens (Ag). Some Ags, such as the Ag85 complex protein (A / B / C), are mainly produced during the acute phase of infection, while some ESX secretion system proteins (TB10.4, ESAT6, etc.) are produced throughout the infection process. Some antigens (RpfA / B / C / D / E) are expressed during the latent / dormant phase or during resuscitation (6,7,8). Latent-phase-dependent antigen expression is one of MTBG's immune evasion strategies, rendering vaccines that currently only express acute-phase antigens ineffective, particularly in preventing relapse of latent infection. Since first-line anti-tuberculosis drugs only target actively replicating bacteria, latent tuberculosis infection and its reactivation represent the greatest challenge in tuberculosis control and vaccine development.

[0005] Therefore, adopting an innovative perspective to advance the development of novel tuberculosis subunit vaccines is crucial for improving their efficacy in clinical practice. On the one hand, it is necessary to screen for novel antigens that can elicit more effective protective responses. These antigens may include latent Mycobacterium tuberculosis infection, adjuvant antigens, and immunodominant antigen-related antigens, or combinations thereof, in order to induce a more robust specific immune response. On the other hand, based on these screening results, suitable vaccine formulations should be developed to stimulate effective immune defense mechanisms in latent tuberculosis infection.

[0006] References:

[0007] 1.WHO.World Health Organization Global TB Report2023.

[0008] 2. Lange C, Aaby P, Behr MA, et al. 100 years of Mycobacterium bovis bacille Calmette-Guérin. Lancet Infect Dis. 2022; 22(1):e2-e12.

[0009] 3.Jeyanathan M,Yao Y,Afkhami S,Smaill F,Xing Z.New Tuberculosis Vaccine Strategies:Taking Aim at Un-Natural Immunity.Trends Immunol.2018;39(5):419-433.

[0010] 4.Van Der Meeren O,Hatherill M,Nduba V,et al.Phase 2b Controlled Trial of M72 / AS01E Vaccine to Prevent Tuberculosis.N Engl J Med.2018;379(17):1621-1634.

[0011] 5.Tameris MD,Hatherill M,Landry BS,et al.Safety and efficacy of MVA85A,a new tuberculosis vaccine,in infants previously vaccinated with BCG:a randomised,placebo-controlled phase 2b trial.Lancet.2013;381(9871):1021-1028.

[0012] 6.Andersen,P.&Scriba,T.J.Moving tuberculosis vaccines from theory to practice.Nat.Rev.Immunol.19,550–562(2019).

[0013] 7.Yousefi Avarvand,A.et al.The roles of latency-associated antigens in tuberculosis vaccines.Indian J.Tuberc.66,487–491(2019).

[0014] 8. Leyten, EMS et al. Human T-cell responses to 25 novel antigens encoded by genes of the dormancy regulon of Mycobacterium tuberculosis. Microbes Infect. 8, 2052–2060 (2006). Summary of the Invention

[0015] This invention relates to a protective antigen against Mycobacterium tuberculosis, wherein the antigen is a combination antigen and includes at least the following antigens: Ag85B, Rv2465c, Rv2029c, and Rv3406.

[0016] Furthermore, the protective antigen may also include 1-2 of the following antigens: RpfD, Rv2627c, LppZ, Rv2031c, EsxH.

[0017] Furthermore, the protective antigen is a fusion protein, in which each antigen is linked by a linker, preferably a P2A linker.

[0018] The amino acid sequence of Ag85B is shown in SEQ ID NO.1:

[0019] SEQ ID NO.1:

[0020] The amino acid sequence of Rv2465c is shown in SEQ ID NO.2:

[0021] SEQ ID NO.2:

[0022] The amino acid sequence of Rv2029c is shown in SEQ ID NO.3:

[0023] SEQ ID NO.3:

[0024] The amino acid sequence of Rv3406 is shown in SEQ ID NO.4:

[0025] SEQ ID NO.4:

[0026] The amino acid sequence of RpfD is shown in SEQ ID NO.5:

[0027] SEQ ID NO.5:

[0028] The amino acid sequence of Rv2627c is shown in SEQ ID NO.6:

[0029] SEQ ID NO.6:

[0030] The amino acid sequence of LppZ is shown in SEQ ID NO.7:

[0031] SEQ ID NO.7:

[0032] The amino acid sequence of Rv2031c is shown in SEQ ID NO.8:

[0033] SEQ ID NO.8:

[0034] The amino acid sequence of EsxH is shown in SEQ ID NO.9:

[0035] SEQ ID NO.9:

[0036] The amino acid sequence of the P2A linker is shown in SEQ ID NO.10:

[0037] SEQ ID NO.10:

[0038] The present invention also relates to a nucleic acid fragment encoding the protective antigen, wherein the nucleic acid fragment is a DNA fragment or an mRNA fragment.

[0039] The present invention also relates to the use of the protective antigen or the nucleic acid fragment encoding the protective antigen in the preparation of a vaccine against Mycobacterium tuberculosis.

[0040] The present invention also relates to a subunit vaccine against Mycobacterium tuberculosis, the subunit vaccine comprising: an immunologically effective amount of the protective antigen, and necessary immunoadjuvants and pharmaceutical excipients.

[0041] Preferably, the subunit vaccine is:

[0042] (1) Vaccines for the prevention of acute tuberculosis infection, and / or

[0043] (2) Vaccines to prevent post-exposure infection with latent tuberculosis, and / or

[0044] (3) Vaccines for the prevention or treatment of other mycobacterial infections.

[0045] Furthermore, the subunit vaccine is a subcutaneous injection vaccine or an inhaled vaccine.

[0046] This invention also relates to a subunit nucleic acid vaccine against Mycobacterium tuberculosis, said subunit nucleic acid vaccine comprising,

[0047] (1) A nucleic acid fragment expressing the protective antigen, preferably, the nucleic acid fragment is a DNA fragment or an mRNA fragment; and

[0048] (2) The necessary expression vector; preferably, the expression vector is an adenovirus vector, more preferably, the expression vector is the chimpanzee adenovirus vector pAdsimian.

[0049] Preferably, the subunit nucleic acid vaccine is:

[0050] (1) Vaccines for the prevention of acute tuberculosis infection, and / or

[0051] (2) Vaccines to prevent post-exposure infection with latent tuberculosis, and / or

[0052] (3) Vaccines for the prevention or treatment of other mycobacterial infections.

[0053] Furthermore, the subunit nucleic acid vaccine is a subcutaneous injection vaccine or an inhaled vaccine.

[0054] This invention also relates to the application of the aforementioned subunit vaccine or subunit nucleic acid vaccine in the preparation of combination vaccines, preferably, the combination vaccine further comprises: BCG vaccine.

[0055] The technical problem to be solved by this invention is:

[0056] A recombinant subunit vaccine expressing Mycobacterium tuberculosis protective antigens (including latent infection antigen, acute infection antigen, adjuvant antigen, and immunodominant antigen) was constructed, and the immunoprotective efficacy of the vaccine was verified in acute infection mouse models and latent infection mouse models, respectively.

[0057] The present invention also provides the use of the tuberculosis subunit vaccine in the preparation of vaccines for the prevention of acute tuberculosis and post-exposure tuberculosis vaccines for latent infection and / or in the preparation of novel vaccines for the prevention or treatment of other mycobacterial infection-related diseases.

[0058] The aforementioned uses can be achieved by using fusion antigen and subunit vaccines alone, or in combination with other tuberculosis vaccines.

[0059] The subunit vaccines are administered via intramuscular injection, subcutaneous immunization, and inhalation.

[0060] The beneficial effects of this invention are as follows:

[0061] (1) In this invention, latent infection antigens Rv2029c, RpfD, Hspx (Rv2031c), acute infection antigen Ag85B, adjuvant antigen Rv2465c, immunodominant antigens Rv3406, TB10.4 and novel protective antigens LppZ, Rv2627c were selected as potential candidate antigens.

[0062] (2) These antigens were fused to construct multiple combined antigens (B596, B5967, B596R, B59614 and B596Z). The combined antigens were fusion proteins. Based on this, a recombinant chimpanzee adenovirus vector vaccine expressing the fusion antigen was further constructed.

[0063] (3) The immunoprotective efficacy of these vaccines against tuberculosis infection induced by the mice infection model was evaluated.

[0064] The study found that B596Z-induced protection had the best efficacy, specifically:

[0065] (1) The ability of the recombinant viral vector vaccine ChAdB596Z, which expresses the protective antigen B596Z, to express these five antigens was verified by Western Blot.

[0066] (2) In mouse models, its immunogenicity in BCG-immunized and unimmunized mice was determined, and the immunization strategy was optimized. Finally, the immunoprotective efficacy of ChAdB596Z-induced anti-tuberculosis infection was verified using mouse models of acute and latent tuberculosis infection.

[0067] In summary, this invention has constructed a subunit tuberculosis vaccine that fusion-expresses five protective antigens, covering antigens highly expressed in different infection phases of Mycobacterium tuberculosis. The immunogenicity and protective efficacy against Mycobacterium tuberculosis infection were then verified using mouse immunization and infection models. The novel tuberculosis vaccine and its underlying concept provided by this invention are of significant importance for the development of novel tuberculosis vaccines. Attached Figure Description

[0068] Figure 1. Detailed information on the encoding genes of the candidate antigens related to the Mycobacterium tuberculosis combined antigens screened.

[0069] Figure 2. Methods for constructing Mycobacterium tuberculosis combinatorial antigen and protein expression of recombinant chimpanzee adenovirus subunit vaccine. Figure 2A. Chimeric sequence of the combinatorial antigen. The Kozak sequence is a nucleic acid sequence located after the 5' cap structure of eukaryotic mRNA, usually GCCACC, which binds to translation initiation factors to mediate translation initiation of mRNA containing the 5' cap structure. The P2A sequence itself contains a specific nucleic acid sequence, which is transcribed into a special peptide chain during translation. This peptide chain has an automatic cleavage function, which can cut the protein molecule into two parts. Figure 2B. Western blot results of the chimeric antigen protein in cell lysate using mouse antiserum to detect complete expression.

[0070] Figure 3. The therapeutic effects of five combined antigens in a mouse prophylactic immunization model; 3A. Experimental procedure and protocol; 3B. Bacterial load in mouse lungs after immunization with different preparations; 3C. Bacterial load in mouse spleen after immunization with different preparations.

[0071] Figure 4. Immunization interval, dosage, number of immunizations / route, and combined immunization effect of the combined antigen vaccine ChAdB596Z; 4A. Antigen-specific IFN-γ reaction detection procedure and results: the immune response was strongest after 3 weeks, and began to decline after 4 weeks; 4B. Detection procedure and protocol for dosage and immunization route; 4C. The immunization effect has a certain dose dependence; 4D. The immunization route has little effect on the immunization effect; 4E. Multiple immunization detection procedure and results: the antigen-specific immune response of the two-dose vaccine was the strongest; 4F. Compared with BCG vaccination alone, the combined antigen vaccine ChAdB596Z can significantly improve the immune level.

[0072] Figure 5. Detection of the immunoprotective efficacy induced by ChAdB596Z vaccine in a mouse acute infection model after BCG immunization; 5A. Experimental procedure; 5B. Bacterial load of Mycobacterium tuberculosis in the lungs of mice after vaccination; 5C. Bacterial load of Mycobacterium tuberculosis in the spleen of mice after vaccination.

[0073] Figure 6. Detection of the immunoprotective efficacy induced by ChAdB596Z vaccine using a mouse latent infection model; 6A. Experimental procedure; 6B. Bacterial load of Mycobacterium tuberculosis in the lungs of mice after vaccination; 6C. Bacterial load of Mycobacterium tuberculosis in the spleen of mice after vaccination; 6D. HE-stained sections (lung sections) to observe the inflammatory infiltration in each treatment group. Detailed Implementation

[0074] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0075] Example 1: Construction and validation of combined antigens (a combination of latent infection antigen, acute infection antigen, adjuvant antigen, and immunodominant antigen).

[0076] 1. Screening for candidate antigens for latent tuberculosis infection based on literature reports or predictions.

[0077] Based on literature reports on the screening of highly reactive antigens and the prediction of antigen structure in tuberculosis-infected populations, nine tuberculosis infection-related antigens were selected as candidate antigens; specific information on the candidate latent infection antigens is shown in Table 1.

[0078] Table 1. Latent Infection Antigen Information Table

[0079] 2. Construction of combined antigens

[0080] (1) Select latent infection antigens, acute infection antigens, adjuvant antigens and immunodominant antigens to construct combined antigens.

[0081] (2) Five antigen combinations were constructed by linking different antigens using a P2A linker: B596, B5967, B596R, B59614, and B596Z. The chimeric sequence of the combined antigens is shown in Figure 2A. The chimeric gene was constructed by linking the DNA sequences encoding the above genes using a P2A linker and then constructing the chimeric gene using a standard cloning procedure.

[0082] The coding nucleic acid sequence of the P2A linker is SEQ ID NO.11:

[0083] Example 2: Construction of a recombinant viral vector expressing combined antigens:

[0084] 1. Expression of combined antigens

[0085] A recombinant vaccine was constructed by inserting the DNA sequence of the chimeric antigen into the chimpanzee adenovirus vector Adsimian. The construction method was as follows:

[0086] (1) After the shuttle plasmid pSRK17 (constructed with the assistance of Zhejiang University of Science and Technology) was linearized by enzyme digestion, the chimeric gene was inserted downstream of the T7 promoter of the pSRK17 plasmid.

[0087] (2) The pSRK17 plasmid containing the chimeric gene was fused with the adenovirus plasmid pAdsimian (constructed with the assistance of Zhejiang Sci-Tech University) via seamless cloning to form a recombinant adenovirus plasmid, which was then transfected into HEK293A cells (transfection and culture conditions: cells were plated into 6-well plates at a density of 70-95% one day before transfection; 4 μg of plasmid DNA and 10 μL of liposome nucleic acid transfection reagent (catalog number 40802ES03, source: Yisheng Biotechnology (Shanghai) Co., Ltd.) were diluted with 250 μL of serum-free medium to prepare the liposome nucleic acid transfection reagent complex and incubated at room temperature for 20 min; the complex was directly added to the wells of the cell culture plate and gently mixed; the cells were cultured at 37℃ in a 5% CO2 incubator until transgenic expression analysis was performed.)

[0088] (3) Cell lysis and supernatant extraction after transfection: The specific steps are as follows: Remove the cell culture medium and wash once with PBS; add 250 μL LRIPA lysis buffer (catalog number 20118ES60, source: Yisheng Biotechnology (Shanghai) Co., Ltd.) to each well of a 6-well plate, and pipette several times to ensure that the lysis buffer and cells are in full contact. There should be no obvious cell precipitate after full lysis; after full lysis, centrifuge at 13000g for 5 min and collect the supernatant.

[0089] (4) The supernatant was used to detect the expression of the antigen, and Western blot experiment was performed using mouse antiserum. The results showed that after HEK293A was infected, the chimeric antigen protein was fully expressed in the cell lysate of recombinant adenovirus with different antigen combinations (Figure 2B).

[0090] Example 3: Screening for the optimal combination of antigens in a mouse prophylactic immunization model

[0091] Using a mouse prophylactic immunization model, BALB / c mice were immunized with two doses of recombinant adenovirus vaccine (dose: 1×10⁻⁶). 7 PFUs (administration method: intramuscular injection) were administered at 2-week intervals. Two weeks after the last immunization, mice were infected with Mycobacterium tuberculosis (H37Rv, 100 CFU / mouse). Four weeks after infection, colony smears were performed (i.e., under aseptic conditions, the lungs and lobes of mice were dissected, homogenized, and smeared on 7H11 solid medium, and colony counts were performed three weeks later) to determine the ability of each vaccine strain to fight Mycobacterium tuberculosis infection. PBS was used as a blank control, empty adenovirus was used as a negative control, and BCG (currently the only commercially available tuberculosis vaccine), MCG (a commercially available anti-tuberculosis immunizing agent), and AS01 / M72 (a protein tuberculosis vaccine developed by GSK, which has completed phase IIb clinical trials with an efficacy rate of nearly 50%) were used as positive controls (Figure 3A).

[0092] The results showed that all adenovirus combination antigens could reduce the bacterial load in the lungs (Fig. 3B) and spleen (Fig. 3C) of mice by more than 1 log, and the B596Z antigen combination had the lowest bacterial load in the lungs.

[0093] Example 4: Optimization of vaccination dosage, immunization schedule and immunization route

[0094] 1. Investigation of the change in immune response over time to ChAdB596Z (the recombinant adenovirus vaccine constructed in Example 2, where ChAd represents chimpanzee adenovirus).

[0095] BALB / c mice were immunized with muscle at a dose of 1×10⁻⁶. 7 PFUs (Plaque forming units) / animal were used to detect antigen-specific IFN-γ responses after 2, 3, and 4 weeks, with PBS and the recombinant adenovirus vector vaccine eGFP expressing irrelevant antigens as negative controls (Figure 4A).

[0096] The test results showed that the IFN-γ response was low at 2 weeks, the immune response was strongest at 3 weeks, and the response began to decline at 4 weeks (Figure 4A).

[0097] 2. Investigation of vaccination dosage and route of immunization

[0098] (1) At a low dose of 1×10 6 PFUs / animal, medium dose 1×10 7 PFUs / animal, high dose 1×10 8 PFUs / mouse, BALB / c mice were immunized intramuscularly; the same dose of adenovirus (1×10⁻⁶) was administered intramuscularly or intranasally. 7 PFUs / each (Figure 4B).

[0099] Figure 4C shows that the low-dose immunization effect was poor, and the high-dose immunization effect did not improve significantly, suggesting that the medium dose of 1×10 7 PFUs are even better.

[0100] (2) Comparing intramuscular injection and nasal inoculation, Figure 4D shows that there is no difference in IFN-γ response between the two. Considering the convenience of the inoculation method, the intramuscular injection route was selected in subsequent studies.

[0101] 3. Examination of the number of vaccinations.

[0102] BALB / c mice were immunized intramuscularly with one, two, and three injections, three weeks apart, at a dose of 1×10⁻⁶. 7 PFUs / animal (Figure 4E) showed that the antigen-specific immune response was strongest with two doses of the vaccine.

[0103] 4. To investigate the effect of this adenovirus vaccine on the immune response of mice after BCG vaccination.

[0104] BALB / c mice were vaccinated with BCG 4 weeks later and then revaccinated with ChAdB596Z (1×10⁻⁶ BCG). 5 CFUs / each, ChAdB596Z1×10 7 PFUs / animal were administered, and antigen-specific IFN-γ responses were detected after 3 weeks (Figure 4F). The results showed that ChAdB596Z significantly enhanced BCG-induced immunity compared with BCG-only vaccination.

[0105] Example 5: Detection of the immunoprotective efficacy of ChAdB596Z (acute mouse infection model)

[0106] BALB / c mice immunized with BCG were used to simulate adults who received BCG vaccination in infancy. Four weeks after BCG immunization, mice received two intramuscular injections of ChAdB596Z (low, medium, and high doses), three weeks apart. Three weeks after the last immunization, the mice were infected with Mycobacterium tuberculosis aerosol. Four weeks later, the mice were sacrificed, and lung and spleen homogenates were collected for plate smearing and counting. PBS, empty adenovirus, and single and double booster immunizations with BCG were used as controls (Figure 5A).

[0107] The results showed that, compared with PBS, BCG vaccination significantly reduced the bacterial load of Mycobacterium tuberculosis in the lungs (Fig. 5B) and spleen (Fig. 5C). Compared with BCG alone or BCG revaccination, the bacterial load in the lungs and spleen was further reduced in the BCG primary immunization-ChAdB596Z booster group, and the bacterial load decreased further with increasing ChAdB596Z dose. This result indicates that the recombinant adenovirus vaccine ChAdB596Z can enhance the immune protection of BCG as a booster vaccine.

[0108] Example 6: Detection of the immunoprotective efficacy of ChAdB596Z (mouse latent infection model)

[0109] BALB / c mice immunized with BCG were used to simulate adults who received BCG vaccination in infancy. Four weeks after BCG vaccination, the mice were infected with tuberculosis aerosols (100 CFU / mouse). After four weeks of infection establishment, the mice were treated with the anti-tuberculosis chemotherapy drugs isoniazid and pyrazinamide in their drinking water for four weeks to eliminate chemotherapy-sensitive bacteria and simulate the latent infection state in humans.

[0110] Immediately after chemotherapy, two doses of ChAdB596Z were administered (3 weeks apart) to induce an antigen-specific immune response and inhibit the reactivation of lingering tuberculosis bacteria.

[0111] Four weeks after the last immunization, mice were treated with dexamethasone three times (6 mg / kg, subcutaneous injection, three times in total, with each treatment two days apart) to suppress the host immune response, mimicking the resurgence of tuberculosis due to age-related decline in immunity in latently infected individuals. Eight weeks later, lungs and spleens of mice were homogenized, plated, and the bacterial load in the organs was assessed. HE staining of the lungs was also performed to evaluate lung pathology (Figure 6A).

[0112] The results showed that, compared with microcardiac and AS01 / M72, ChAdB596Z significantly reduced bacterial load in the lungs (Fig. 6B) and spleen (Fig. 6C) of a mouse model with latent infection. Furthermore, in HE-stained sections, significantly less inflammatory infiltration was observed in the ChAdB596Z group (Fig. 6D).

[0113] Therefore, the recombinant adenovirus vaccine ChAdB596Z significantly improved the ability of mice to resist latent tuberculosis infection.

[0114] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

Claims

1. A protective antigen against Mycobacterium tuberculosis, wherein the antigen is a combination antigen comprising at least the following antigens: Ag85B, Rv2465c, Rv2029c, and Rv3406.

2. The protective antigen according to claim 1, characterized in that, The protective antigens also include, optionally, one or two of the following antigens or combinations thereof: RpfD, Rv2627c, LppZ, Rv2031c, and EsxH.

3. The protective antigen according to claim 1 or 2, characterized in that, The protective antigen is a fusion protein, in which the antigens are linked by a linker; preferably, the linker is a P2A linker.

4. A subunit vaccine against Mycobacterium tuberculosis, characterized in that, The subunit vaccine contains: an immunogenic amount of the protective antigen as described in any one of claims 1-3, as well as necessary immunoadjuvants and pharmaceutical excipients. Preferably, the subunit vaccine is: (1) Vaccines for the prevention of acute tuberculosis infection, and / or (2) Vaccines to prevent post-exposure infection with latent tuberculosis, and / or (3) Vaccines for the prevention or treatment of other mycobacterial infections.

5. The subunit vaccine according to claim 4, characterized in that, The subunit vaccine is either a subcutaneous injection vaccine or an inhaled vaccine.

6. A subunit nucleic acid vaccine against Mycobacterium tuberculosis, said subunit nucleic acid vaccine comprising, (1) A nucleic acid fragment expressing any of the protective antigens described in claims 1-3, preferably, the nucleic acid fragment is a DNA fragment or an mRNA fragment; and (2) The necessary expression vector; preferably, the expression vector is an adenovirus vector, more preferably, the expression vector is the chimpanzee adenovirus vector pAdsimian. Preferably, the subunit nucleic acid vaccine is: (1) Vaccines for the prevention of acute tuberculosis infection, and / or (2) Vaccines to prevent post-exposure infection with latent tuberculosis, and / or (3) Vaccines for the prevention or treatment of other mycobacterial infections.

7. The subunit vaccine according to claim 6, characterized in that, The subunit nucleic acid vaccine is either a subcutaneous injection vaccine or an inhaled vaccine.

8. The application of the subunit vaccine of claim 4 or 5, or the subunit nucleic acid vaccine of claim 6 or 7, in the preparation of a combined vaccine, preferably, the combined vaccine further comprises: BCG vaccine.

9. A nucleic acid fragment encoding the protective antigen of any one of claims 1-3, characterized in that, The nucleic acid fragments mentioned are DNA fragments or mRNA fragments.

10. The use of the protective antigen of any one of claims 1-3 or the nucleic acid fragment of claim 9 in the preparation of a vaccine against Mycobacterium tuberculosis.

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