Vaccine for preventing and treating mycobacterium tuberculosis infection and preparation method therefor

Abstract

The present invention relates to a Mycobacterium tuberculosis vaccine. Specifically, the vaccine comprises an immune composition, and the immune composition comprises an antigen component and a particle protein component. The particle protein component comprises a nanoparticle protein, and the antigen component and the particle protein component are covalently combined by means of a binding peptide 1 and a binding peptide 2 to form an immunogenic complex. The vaccine can induce or stimulate humoral and cellular immune responses. Also disclosed is a preparation method for the Mycobacterium tuberculosis vaccine. The method is simple, cost-effective, and suitable for large-scale production.

Description

A vaccine for preventing and treating mycobacterium tuberculosis infection and a preparation method thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an immune composition product for preventing and treating mycobacterium tuberculosis infection and a production method thereof. BACKGROUND

[0002] Tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis (Mtb) and other mycobacterium infections. The main transmission route of the disease is droplet transmission, and it can occur at any age, and the immunocompromised are more susceptible to infection. Infected individuals may have no typical symptoms for a long time, but after the onset, they show acute lung inflammation, symptoms such as dry cough and fever, and if not treated in time, it can directly lead to serious complications and death.

[0003] At present, tuberculosis is a major disease in developing countries, widely distributed in the global range, and is becoming more and more serious. Tuberculosis mainly affects people in low-income countries, and the risk of tuberculosis in poor people with poor living and working conditions and malnutrition is often the highest. Globally, as many as one-fourth of the population are latent tuberculosis infections, and this group of patients are infected with tuberculosis without any symptoms, but can develop into active tuberculosis.

[0004] At present, antibiotic treatment using isoniazid, rifampicin, pyrazinamide and other drugs can control tuberculosis, but this treatment cannot effectively prevent the spread of the disease, and individuals after infection do not develop for a period of time, but have certain infectivity. In addition, long-term use of antibiotics for treatment poses a huge challenge to the medication compliance of patients, and even some patients cannot complete effective treatment, and long-term antibiotic treatment may develop drug resistance.

[0005] Effective vaccination and accurate early diagnosis are important means to control tuberculosis. At present, the BCG (Bacillus Calmette-Guerin) vaccine (BCG) for vaccination of infants and young children is prepared from avirulent Mycobacterium bovis. The wide use of BCG vaccination has provided great help for the global tuberculosis prevention and control. BCG can protect infants and young children from suffering from severe systemic tuberculosis, but the protection effect on pulmonary tuberculosis of adolescents and adults is very limited, so there is an urgent need to develop a new tuberculosis vaccine to replace or enhance BCG. The recombinant subunit tuberculosis vaccine has been proved to be effective in humans, and gradually becomes a hotspot for the development of new tuberculosis vaccines. However, the Mtb genome expresses about 4000 genes, and the functions and roles of most gene products in the life cycle of Mtb have not been characterized, so screening of antigens with strong immunogenicity is the key and difficulty of TB vaccine research. At present, the recombinant subunit TB vaccine is still in the clinical trial stage, and it still faces great challenges to develop a universally effective vaccine that can replace or enhance the immune effect of BCG.

[0006] At present, nanoparticle vaccines are a new generation of vaccine design methods, which can not only induce strong neutralizing antibodies, but also improve the level of cellular immune response, including human HPV vaccine, hepatitis B vaccine, and PCV2 vaccine for animals. There are many natural proteins (Ferritin, Lumazine Synthase, AP205, etc.) in nature that can self-assemble into nanoparticles, and can induce strong immune response after loading antigens on the surface. At present, they have been widely studied and applied. The body mainly relies on cellular immune mechanisms to resist Mycobacterium tuberculosis infection, and after vaccination, effective Th1 CD4 + T and CD8 + T cells can be produced, and long-term cellular immune memory is the key point of the development of new tuberculosis vaccines, so self-assembled nanoparticles are an ideal carrier and have feasibility in the research of tuberculosis vaccines. SUMMARY

[0007] In order to solve the current technical and supply shortage of tuberculosis subunit vaccines, especially the current situation of basically blank supply of tuberculosis nanoparticle vaccines, the present application provides a tuberculosis nanoparticle vaccine and a preparation method thereof.

[0008] In a first aspect, the present application provides one or more isolated Mycobacterium tuberculosis (M. tuberculosis, abbreviated as Mtb in the present application) antigens, wherein the Mtb antigens include wild-type antigens and mutant antigens.

[0009] In some embodiments, the Mtb antigen sequence of the present application is derived from H37RV strain.

[0010] In some embodiments, the Mtb wild type antigens of the present application are derived from the antigen molecules shown in Table 1 below:

[0011] Table 1 Mtb antigen related information of the present application

[0012] In some embodiments, the amino acid and gene sequences of the Mtb antigens of the present application are derived from GenBank: AL123456.3.

[0013] In some embodiments, the Mtb mutant antigens of the present application are obtained by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of the Mtb wild type antigens. The Mtb mutant antigens can improve the stability of the protein; and / or inhibit, reduce or decrease the protein aggregation, such as the formation of intermolecular dimers, during recombinant expression; and / or improve the expression level of the protein; and / or facilitate the soluble expression of the protein; and / or improve the immunogenicity of the antigen molecules.

[0014] In some embodiments, the Mtb antigens of the present application can be obtained by recombinant expression or chemical synthesis, such as chemical synthesis using an automated polypeptide synthesizer.

[0015] The positions of the mutation sites of the Mtb mutant antigens of the present application are determined according to the Mtb wild type antigen sequence in GenBank: AL123456.3, i.e., the starting methionine is regarded as the first amino acid residue. Those skilled in the art can select to delete or retain the methionine start codon according to the expression needs, such as fusion expression, to avoid incorrect translation start sites.

[0016] In some embodiments, the Mtb mutant antigens of the present application comprise one or more amino acid mutations relative to the wild type antigens, and the amino acid mutations can disrupt the formation of disulfide bonds. Preferably, one or more Cys in the wild type Mtb antigens is mutated to Ala, thereby obtaining the Mtb antigen mutants of the present application.

[0017] In some embodiments, the Mtb mutant antigens of the present application comprise one or more amino acid mutations relative to the wild type antigens, and the amino acid mutation sites are located in the enzyme active center.

[0018] In some embodiments, the Mtb mutant antigens of the present application have deletions at the N-terminus or C-terminus relative to the wild type antigens, and the obtained Mtb mutant antigens can reduce the hydrophobic polymerization of the protein, and / or facilitate the soluble expression of the protein, and / or improve the immunogenicity of the antigen molecules.

[0019] In some embodiments, the present application provides the following Mtb mutant antigens:

[0020] (1) Mtb32a mutant comprising a mutation of S177A relative to wild-type Mtb32a, designated "Mtb32a(S177A)";

[0021] (2) Mtb39a mutant lacking at least 166 amino acid residues at the N-terminus relative to wild-type Mtb39a; preferably, the Mtb39a mutant is wild-type from amino acid 167-391, designated "Mtb39a(L167-G391)";

[0022] (3) Ag85a mutant lacking at least 42 amino acid residues at the N-terminus relative to wild-type Ag85a; preferably, wild-type Ag85a lacks 42 residues at the N-terminus, designated "Ag85a ΔN"; optionally, the obtained truncation further comprises one or more amino acid mutations at positions 127-129, such as L127G, S128G / A / E / L / W, M129G; preferably, the mutation sites are S128G and M129G, designated "Ag85a ΔN(127LGG129)";

[0023] (4) RV2660c mutant comprising a mutation of C66A relative to wild-type RV2660c, designated "RV2660c(C66A)";

[0024] (5) EspC mutant comprising a mutation of C48A relative to wild-type EspC, designated "EspC(C48A)";

[0025] (6) Hrp1 mutant lacking at least 8 amino acid residues at the C-terminus relative to wild-type Hrp1; preferably, wild-type Hrp1 lacks 8 residues at the C-terminus, designated "Hrp1M1"; optionally, further comprising one or more amino acid mutations; preferably, comprising mutations of C14A and / or C39A;

[0026] (7) RpfD mutant comprising one or more amino acid mutations relative to wild-type RpfD; preferably, comprising mutations of C18A, C32A, C60A, C121A, C124A and / or C148A;

[0027] (8) Mpt64 mutant comprising mutations of C16A, C17A, C29A and / or C41A relative to wild-type Mpt64; preferably, comprising mutations of C16A, C17A, C29A and C41A, designated "Mpt64M1";

[0028] (9) Mpt70 mutants comprising a mutation of C38A and / or C172A relative to wild-type Mpt70; preferably, a mutation of C38A and C172A;

[0029] (10) Mpt83 mutants comprising a mutation of C25A, C64A and / or C198A relative to wild-type Mpt83; preferably, a mutation of C25A, C64A and C198A.

[0030] In some embodiments, the Mtb mutant antigens of the present application have an amino acid sequence selected from any one of:

[0031] (1) Mtb32a mutants having an amino acid sequence with 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to SEQ ID NO: 1;

[0032] (2) Mtb39a mutants having an amino acid sequence with 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to SEQ ID NO: 3;

[0033] (3) Ag85a mutants having an amino acid sequence with 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to SEQ ID NO: 5 or 7;

[0034] (4) RV2660c mutants having an amino acid sequence with 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to SEQ ID NO: 9;

[0035] (5) EspC mutants having an amino acid sequence with 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to SEQ ID NO: 11;

[0036] (6) Hrp1 mutants having an amino acid sequence with 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to SEQ ID NO: 13;

[0037] (7) Mpt64 mutants having an amino acid sequence with 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to SEQ ID NO: 15.

[0038] In some embodiments, the Mtb antigen mutants of the present application have an amino acid sequence selected from any one of:

[0039] (1) the amino acid sequence of the Mtb32a mutant is shown as SEQ ID NO: 1; (2) the amino acid sequence of the Mtb39a mutant is shown as SEQ ID NO: 3; (3) the amino acid sequence of the Ag85a mutant is shown as SEQ ID NO: 5 or 7; (4) the amino acid sequence of the RV2660c mutant is shown as SEQ ID NO: 9; (5) the amino acid sequence of the EspC mutant is shown as SEQ ID NO: 11; (6) the amino acid sequence of the Hrp1 mutant is shown as SEQ ID NO: 13; (7) the amino acid sequence of the Mpt64 mutant is shown as SEQ ID NO: 15.

[0040] In some embodiments, the ESAT6 of the present application has an amino acid sequence shown as SEQ ID NO: 17, or the CFP10 has an amino acid sequence shown as SEQ ID NO: 19, or the TB10.4 has an amino acid sequence shown as SEQ ID NO: 21, or the PE13 has an amino acid sequence shown as SEQ ID NO: 23, or the EsxC has an amino acid sequence shown as SEQ ID NO: 25, or the EsxD has an amino acid sequence shown as SEQ ID NO: 27, or the EsxV has an amino acid sequence shown as SEQ ID NO: 29, or the EsxW has an amino acid sequence shown as SEQ ID NO: 31.

[0041] In some embodiments, the Mtb antigens of the present application further include immunogenic fragments of wild-type antigens and mutant antigens, which refer to an isolated polypeptide capable of stimulating the immune system to produce an immune response.

[0042] In a second aspect, the present application provides a Mtb antigen composition comprising one or more Mtb antigens of the present application or immunogenic fragments thereof, which include wild-type and mutant antigens.

[0043] In some embodiments, the Mtb antigen composition of the present application comprises 2-18 Mtb antigens of the present application; preferably, the Mtb antigen composition of the present application comprises 4-15 Mtb antigens of the present application; more preferably, 4, 5 or 6 Mtb antigens of the present application.

[0044] In some embodiments, the Mtb antigen composition of the present application comprises active phase, latent phase and resuscitation phase antigens selected from Table 1.

[0045] In some embodiments, the Mtb antigen composition of the present application comprises active phase, latent phase and resuscitation phase antigens selected from Table 1.

[0046] In some embodiments, the Mtb antigen composition of the application comprises active phase, latent phase and multi-stage antigens selected from Table 1.

[0047] In some embodiments, the Mtb antigen composition of the application comprises active phase, resuscitation phase and multi-stage antigens selected from Table 1.

[0048] In some embodiments, the Mtb antigen composition of the application comprises multi-stage antigens selected from Table 1, and an antigen selected from active phase or latent phase or resuscitation phase.

[0049] Preferably, the Mtb antigen composition of the application comprises 2, 3 or 4 multi-stage antigens selected from Table 1; or comprises 1 active phase antigen and 1 multi-stage antigen selected from Table 1; or comprises 1 active phase antigen and 1 latent phase antigen selected from Table 1; or comprises 1 latent phase antigen and 1 multi-stage antigen selected from Table 1; or comprises 2, 3 or 4 active phase antigens selected from Table 1.

[0050] In some embodiments, the Mtb antigen composition of the application comprises ESAT6 and / or CFP10, and optionally other antigens of Table 1.

[0051] In some embodiments, the Mtb antigen composition of the application comprises Ag85a, and optionally other antigens of Table 1.

[0052] In some embodiments, the Mtb antigen composition of the application comprises Mpt83, and optionally other antigens of Table 1.

[0053] In some embodiments, the Mtb antigen composition of the application comprises EsxC, and optionally other antigens of Table 1.

[0054] In some embodiments, the Mtb antigen composition of the application comprises EsxD, and optionally other antigens of Table 1.

[0055] In some embodiments, the Mtb antigen composition of the application comprises EsxV, and optionally other antigens of Table 1.

[0056] In some embodiments, the Mtb antigen composition of the application comprises EsxW, and optionally other antigens of Table 1.

[0057] Preferably, the Mtb antigen composition of the application comprises ESAT6, CFP10 and Mtb39a; or comprises ESAT6, CFP10 and Mtb32a; or comprises ESAT6, CFP10 and Ag85a; or comprises ESAT6, CFP10, Mtb39a and Ag85a.

[0058] In some embodiments, the Mtb antigen composition of the application comprises ESAT6, CFP10 and Mtb39a, and can further comprise optional additional antigens of Table 1.

[0059] In some embodiments, the Mtb antigen composition of the application comprises ESAT6, CFP10 and Mtb32a, and can further comprise optional additional antigens of Table 1.

[0060] In some embodiments, the Mtb antigen composition of the application comprises ESAT6, CFP10 and Ag85a, and can further comprise optional additional antigens of Table 1.

[0061] Preferably, the Mtb antigen composition of the application comprises ESAT6, CFP10, Ag85a, RV2660c and TB10.4.

[0062] Preferably, the Mtb antigen composition of the application comprises ESAT6, CFP10, Mtb32a, Ag85a, RV2660c and TB10.4.

[0063] In some embodiments, the Mtb antigen composition of the application comprises ESAT6, CFP10, Mtb39a and Ag85a, and can further comprise optional additional antigens of Table 1.

[0064] In some embodiments, the Mtb antigen composition of the application comprises Mtb32a and / or Mtb39a, and can further comprise optional additional antigens of Table 1.

[0065] Preferably, the Mtb antigen composition of the application comprises Mtb32a and Ag85a; or comprises Mtb39a and Ag85a; or comprises Mtb32a, EsxC, EsxD, EsxV and EsxW; or comprises Mtb39a, EsxC, EsxD, EsxV and EsxW.

[0066] In some embodiments, the Mtb antigen composition of the application comprises Mtb32a and Ag85a, and can further comprise optional additional antigens of Table 1.

[0067] In some embodiments, the Mtb antigen composition of the application comprises Mtb39a and Ag85a, and can further comprise optional additional antigens of Table 1.

[0068] In some embodiments, the Mtb antigen composition of the application comprises Mtb32a, EsxC, EsxD, EsxV and EsxW, and can further comprise optional additional antigens of Table 1.

[0069] In some embodiments, the Mtb antigen compositions of the application comprise Mtb39a, EsxC, EsxD, EsxV, and EsxW, and can also comprise optional additional antigens of Table 1.

[0070] In some embodiments, the Mtb antigen compositions of the application comprise EspC and / or PE13, and can also comprise optional additional antigens of Table 1.

[0071] Preferably, the Mtb antigen compositions of the application comprise EspC, PE13, and Mtb32a; or comprise EspC, PE13, and Mtb39a; or comprise EspC, PE13, and Ag85a; or comprise EspC, PE13, ESAT6, and CFP10.

[0072] In some embodiments, the Mtb antigen compositions of the application comprise EspC, PE13, and Mtb32a, and can also comprise optional additional antigens of Table 1.

[0073] In some embodiments, the Mtb antigen compositions of the application comprise EspC, PE13, and Mtb39a, and can also comprise optional additional antigens of Table 1.

[0074] In some embodiments, the Mtb antigen compositions of the application comprise EspC, PE13, and Ag85a, and can also comprise optional additional antigens of Table 1.

[0075] In some embodiments, the Mtb antigen compositions of the application comprise EspC, PE13, ESAT6, and CFP10, and can also comprise optional additional antigens of Table 1.

[0076] In some embodiments, the Mtb antigen compositions of the application comprise RV2660c and / or TB10.4, and can also comprise optional additional antigens of Table 1.

[0077] Preferably, the Mtb antigen compositions of the application comprise RV2660c, TB10.4, and Mtb32a; or comprise RV2660c, TB10.4, and Mtb39a.

[0078] In some embodiments, the Mtb antigen compositions of the application comprise RV2660c, TB10.4, and Mtb32a, and can also comprise optional additional antigens of Table 1.

[0079] In some embodiments, the Mtb antigen compositions of the application comprise RV2660c, TB10.4, and Mtb39a, and can also comprise optional additional antigens of Table 1.

[0080] In some embodiments, the Mtb antigen compositions of the application comprise HRP1 and / or Mpt64, and can also comprise optional additional antigens of Table 1.

[0081] Preferably, the Mtb antigen compositions of the application comprise HRP1, Mpt64 and Mtb32a; or HRP1, Mpt64 and Mtb39a.

[0082] In some embodiments, the Mtb antigen compositions of the application comprise HRP1, Mpt64 and Mtb32a, and can also comprise optional additional antigens of Table 1.

[0083] In some embodiments, the Mtb antigen compositions of the application comprise HRP1, Mpt64 and Mtb39a, and can also comprise optional additional antigens of Table 1.

[0084] In some embodiments, the Mtb antigen compositions of the application comprise RpfD and / or Mpt70, and can also comprise optional additional antigens of Table 1.

[0085] Preferably, the Mtb antigen compositions of the application comprise RpfD, Mpt70 and Mtb32a; or RpfD, Mpt70 and Mtb39a.

[0086] In some embodiments, the Mtb antigen compositions of the application comprise RpfD, Mpt70 and Mtb32a, and can also comprise optional additional antigens of Table 1.

[0087] In some embodiments, the Mtb antigen compositions of the application comprise RpfD, Mpt70 and Mtb39a, and can also comprise optional additional antigens of Table 1.

[0088] In some embodiments, the Mtb antigen composition of the application comprises an antigen selected from the group consisting of ESAT6, CFP10, Mtb39a, Ag85a, Mtb32a, EsxC, EsxD, EsxV, EsxW, EspC, PE13, RV2660c, TB10.4, Hrp1, Mpt64. Preferably, the Mtb antigen composition of the application comprises 2, 3, 4, 5, 6 or 7 of the antigens ESAT6, CFP10, Mtb39a, Ag85a, Mtb32a, EsxC, EsxD, EsxV, EsxW, EspC, PE13, RV2660c, TB10.4, Hrp1, Mpt64; more preferably, the Mtb antigen composition of the application comprises 4, 5 or 6 of the antigens ESAT6, CFP10, Mtb39a, Ag85a, Mtb32a, EsxC, EsxD, EsxV, EsxW, EspC, PE13, RV2660c, TB10.4, Hrp1, Mpt64.

[0089] In some embodiments, the Mtb antigen composition of the application comprises ESAT6, CFP10, Mtb39a, Ag85a, Mtb32a, EspC, PE13, EsxC, EsxD, EsxV, EsxW, Hrp1, Mpt64. Preferably, the Mtb antigen composition of the application consists of the 13 antigens ESAT6, CFP10, Mtb39a, Ag85a, Mtb32a, EspC, PE13, EsxC, EsxD, EsxV, EsxW, Hrp1 and Mpt64. More preferably, the Mtb39a is a Mtb39a mutant, such as Mtb39a(L167-G391); the Ag85a is a Ag85a mutant, such as the aforementioned Ag85a ΔN or Ag85a ΔN(127LGG129); the Mtb32a is a Mtb32a mutant, such as Mtb32a(S177A); the EspC is a EspC mutant, such as the aforementioned EspC(C48A); the Mpt64 is the aforementioned mutant Mpt64M1; the Hrp1 is the aforementioned mutant Hrp1M1.

[0090] In some embodiments, the Mtb antigen composition of the application comprises ESAT6, CFP10, Mtb39a, Ag85a, Mtb32a, EspC, PE13, Hrp1 and Mpt64. Preferably, the Mtb antigen composition of the application consists of the following 9 antigens: ESAT6, CFP10, Mtb39a, Ag85a, Mtb32a, EspC, PE13, Hrp1 and Mpt64. More preferably, the Mtb39a is a Mtb39a mutant, such as Mtb39a(L167-G391); the Ag85a is a Ag85a mutant, such as the aforementioned Ag85a ΔN or Ag85a ΔN(127LGG129); the Mtb32a is a Mtb32a mutant, such as Mtb32a(S177A); the EspC is a EspC mutant, such as the aforementioned EspC(C48A); the Mpt64 is the aforementioned mutant Mpt64M1; the Hrp1 is the aforementioned mutant Hrp1M1.

[0091] In some embodiments, the application provides a Mtb antigen composition comprising the following optional groups: (1) Mtb32a; (2) Mtb39a; (3) Ag85a; (4) RV2660c and TB10.4; (5) EspC and PE13; (6) Mpt64 and Hrp1; (7) EsxC, EsxD, EsxV and EsxW; (8) ESAT6 and CFP10. Preferably, the Mtb39a is a Mtb39a mutant, such as Mtb39a(L167-G391); the Ag85a is a Ag85a mutant, such as the aforementioned Ag85a ΔN or Ag85a ΔN(127LGG129); the Mtb32a is a Mtb32a mutant, such as Mtb32a(S177A); the RV2660c is a RV2660c mutant, such as RV2660c(C66A); the EspC is a EspC mutant, such as the aforementioned EspC(C48A); the Mpt64 is the aforementioned mutant Mpt64M1; the Hrp1 is the aforementioned mutant Hrp1M1. Preferably, the Mtb antigens in the above groups are present in the form of Mtb fusion proteins as described in the application, such as groups (1) and (3) are present in the form of Mtb fusion proteins, or groups (2) and (8) are present in the form of fusion proteins, and the Mtb antigens in groups (4), (5), (6), (7) are present in the form of Mtb fusion proteins.

[0092] In some embodiments, the present application provides a Mtb antigen composition comprising Group (1), and optionally other groups. In some embodiments, the present application provides a Mtb antigen composition comprising Group (2), and optionally other groups. In some embodiments, the present application provides a Mtb antigen composition comprising Group (3), and optionally other groups. In some embodiments, the present application provides a Mtb antigen composition comprising Group (4), and optionally other groups. In some embodiments, the present application provides a Mtb antigen composition comprising Group (5), and optionally other groups. In some embodiments, the present application provides a Mtb antigen composition comprising Group (6), and optionally other groups. In some embodiments, the present application provides a Mtb antigen composition comprising Group (7), and optionally other groups. In some embodiments, the present application provides a Mtb antigen composition comprising Group (8), and optionally other groups.

[0093] In some embodiments, the Mtb antigen composition of the present application comprises any two, three or four of Groups (1), (2), (3) and (8), and optionally other groups. Preferably, the Mtb antigen composition of the present application comprises Groups (1) and (3); or Groups (2) and (8); or Groups (1), (2), (3) and (8).

[0094] In some embodiments, the Mtb antigen composition of the present application comprises any two, three or four of Groups (1), (3), (4) and (8), and optionally other groups. Preferably, the Mtb antigen composition of the present application comprises Groups (1), (3), (4) and (8).

[0095] In some embodiments, the Mtb antigen composition of the present application comprises any two, three, four or five of Groups (1), (2), (3), (4) and (8), and optionally other groups. Preferably, the Mtb antigen composition of the present application comprises Groups (1), (2), (3), (4) and (8).

[0096] In some embodiments, the Mtb antigen composition of the present application comprises Groups (5) and (6), and optionally other groups. Preferably, the Mtb antigen composition of the present application comprises Groups (5) and (6).

[0097] In some embodiments, the Mtb antigen composition of the present application comprises Groups (5) and (7), and optionally other groups. Preferably, the Mtb antigen composition of the present application comprises Groups (5) and (7).

[0098] In some embodiments, the Mtb antigen composition of the present invention comprises any two, three, four, five, or six groups from groups (2), (3), (5), (6), (7), and (8), and optionally, further comprises other optional groups. Preferably, the Mtb antigen composition of the present invention comprises groups (2), (3), (5), and (8); or comprises groups (2), (3), (6), and (8); or comprises groups (2), (3), (7), and (8).

[0099] Thirdly, the present invention provides an Mtb fusion protein, wherein the fusion protein refers to at least two heterologous Mtb antigens linked by peptide bonds. The Mtb fusion protein comprises the Mtb antigen described in this invention or an immunogenic fragment thereof, wherein the Mtb antigen includes the Mtb antigen mutant described in this invention and the wild-type Mtb antigen. The linking order of the Mtb antigens in the Mtb fusion protein of the present invention is arbitrary.

[0100] In some embodiments, the Mtb fusion protein of the present invention is formed by fusing any two or more Mtb antigens from the Mtb antigen composition described in the present invention. Optionally, the Mtb fusion protein of the present invention further includes Mtb antigens other than those described in the present invention.

[0101] In some embodiments, the fusion protein of the present invention comprises at least two Mtb antigens, or at least three Mtb antigens, or at least four Mtb antigens, or at least five Mtb antigens, or at least six Mtb antigens, or at least seven Mtb antigens, or at least eight Mtb antigens. Preferably, the fusion protein of the present invention comprises two, three, or four Mtb antigens.

[0102] In some embodiments, the fusion protein of the present invention comprises two Mtb antigens, such as ESAT6 and CFP10, such as RV2660c and TB10.4, such as EspC and PE13, or such as Ag85a and Mtb32a.

[0103] In some embodiments, the fusion protein of the present invention comprises three Mtb antigens, such as ESAT6, CFP10 and Mtb39a, or Mtb32a, RpfD and HRP1.

[0104] In some embodiments, the fusion protein of the present invention comprises four Mtb antigens, such as EsxC, EsxD, EsxV and EsxW, as well as Ag85a, ESAT6, CFP10 and Mtb39a.

[0105] In some embodiments, the fusion protein of the present invention comprises five Mtb antigens, such as EsxC, EsxD, EsxV, EsxW, and EspC.

[0106] In some embodiments, the fusion protein of the present application comprises six Mtb antigens, such as Ag85a, ESAT6, CFP10, Mtb39a, RV2660C and TB10.4, such as Ag85a, ESAT6, CFP10, Mtb39a, EspC and PE13, such as EsxC, EsxD, EsxV, EsxW, RV2660C and TB10.4, such as EsxC, EsxD, EsxV, EsxW, EspC and PE13.

[0107] In some embodiments, the fusion protein of the present application comprises seven Mtb antigens, such as EsxC, EsxD, EsxV, EsxW, RV2660C, TB10.4 and EspC.

[0108] In some embodiments, the fusion protein of the present application comprises eight Mtb antigens, such as EsxC, EsxD, EsxV, EsxW, RV2660C, TB10.4, EspC and PE13, such as EsxC, EsxD, EsxV, EsxW, EspC, PE13, RV2660C and TB10.4.

[0109] The present application provides fusion proteins comprising the following structures, from N-terminus to C-terminus as shown in the following table:

[0110] Table 2 Structures of Mtb fusion proteins of the present application

[0111] Preferably, in the Mtb fusion proteins of the present application, the Mtb39a is a Mtb39a mutant, such as Mtb39a(L167-G391); the Ag85a is a Ag85a mutant, such as the aforementioned Ag85a AN or Ag85a AN(127LGG129); the Mtb32a is a Mtb32a mutant, such as Mtb32a(S177A); the RV2660c is a RV2660c mutant, such as RV2660c(C66A); the EspC is a EspC mutant, such as the aforementioned EspC(C48A); the Mpt64 is the aforementioned mutant Mpt64M1; the Hrp1 is the aforementioned mutant Hrp1M1.

[0112] Preferably, the Mtb fusion protein of the application comprises the following structure from N-terminus to C-terminus: Ag85a AN(127LGG129)-Mtb32a(S177A); ESAT6-CFP10-Mtb39a(L167-G391); EsxC-EsxD-EsxV-EsxW; EspC(C48A)-PE13; Mpt64M1-Hrp1M1; RV2660c(C66A)-TB10.4.

[0113] In some embodiments, the Mtb fusion protein of the application comprises the following structure from N-terminus to C-terminus: Ag85a AN(127LGG129)-Mtb32a(S177A); ESAT6-CFP10-Mtb39a(L167-G391); EsxC-EsxD-EsxV-EsxW; EspC(C48A)-PE13; Mpt64M1-Hrp1M1; RV2660c(C66A)-TB10.4.

[0114] In the fusion proteins provided by the application, the individual antigens can be covalently linked directly or indirectly via an amino acid linker. The linker can be from 1 amino acid to 100 amino acids in length. The individual antigens forming the fusion protein are typically linked C-terminus to N-terminus, but they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. Regardless of how presented or recited, the antigens can be linked in any order.

[0115] Preferably, the Mtb antigens of the application are linked by a linking peptide; optionally, the linking peptide is selected from the group consisting of (GSG) n , (GGGGS) n , n can be an integer greater than 0 and less than or equal to 5.

[0116] In some embodiments, the fusion protein of the application has an amino acid sequence that is more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% identical to SEQ ID NO: 33, 35, 37, 39, 41, or 43.

[0117] In some embodiments, the fusion protein of the application has an amino acid sequence selected from the group consisting of SEQ ID NO: 33, 35, 37, 39, 41, or 43.

[0118] To enable the production of the Mtb antigens or fusion proteins of the application, they can comprise a suitable purification tag to allow purification from, for example, a recombinant expression system. Thus, in one embodiment of the application, the fusion protein comprises a purification tag.

[0119] Another embodiment of the present application relates to the fusion protein as described herein, wherein the purification tag is selected from the group consisting of His-tag, chitin binding protein (CBP), maltose binding protein (MBP) and glutathione-S-transferase (GST).

[0120] In some embodiments, the purification tag used in the present application is selected from the group consisting of 6xHis tag or PSR tag. Preferably, the amino acid sequence of the PSR tag is set forth in SEQ ID NO: 45.

[0121] The Mtb antigens and fusion proteins of the present application also contain a signal peptide. Preferably, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 46.

[0122] The present application provides a Mtb antigen composition comprising the fusion protein of the present application. Alternatively, the Mtb fusion protein of the present application can be replaced by a combination of corresponding individual Mtb antigens.

[0123] In some embodiments, the Mtb antigen composition of the present application comprises two or three Mtb fusion proteins of the present application.

[0124] In some embodiments, the Mtb antigen composition of the present application comprises both the Mtb antigen and the Mtb fusion protein of the present application.

[0125] In a fourth aspect, the present application provides a Mtb immunogenic complex comprising

[0126] a: any one of the Mtb antigens of the present application; or b: any one of the Mtb fusion proteins of the present application.

[0127] In some embodiments, the Mtb immunogenic complex of the present application comprises a particulate protein component, said particulate protein component comprising a particulate protein for displaying antigens such that the same antigens are presented in a geometric repeating array, said array being capable of triggering an immune response compared to soluble antigens.

[0128] In some embodiments, the Mtb antigen or fusion protein of the present application can be displayed by any particulate protein.

[0129] In some embodiments, the particulate protein component of the present application can be natural or non-natural, capable of chemical synthesis or produced by biological methods. Particulate proteins of natural origin include components derived from viruses, bacteria and the like, such as virus-like particles (VLPs) or recombinant forms thereof. The component can be a structural protein of a virus, such as a viral envelope or capsid protein or surface antigen. Examples of structural proteins include matrix Ml protein and viral envelope M2 protein from influenza virus, HBsAg from hepatitis B virus, E. coli bacteriophage AP205 virus coat protein (CP3), hemagglutinin-neuraminidase from various viruses including mumps, and the like. Suitable viral structural proteins will be known to those skilled in the art.

[0130] In some embodiments, the VLP of the present application can comprise or consist of: a recombinant rotavirus polypeptide; a recombinant norovirus polypeptide; a recombinant alphavirus polypeptide; a recombinant protein that can form a bacterial pilus or pilus-like structure; a recombinant foot-and-mouth disease virus polypeptide; a recombinant measles virus polypeptide; a recombinant Sindbis virus polypeptide; a recombinant retrovirus polypeptide; a recombinant hepatitis B virus polypeptide (e.g. HBcAg); a recombinant tobacco mosaic virus polypeptide; a recombinant avian encephalomyelitis virus polypeptide; a recombinant human papillomavirus polypeptide; a recombinant polyomavirus polypeptide, in particular a recombinant human polyomavirus polypeptide, in particular a recombinant BK virus polypeptide; a recombinant bacteriophage polypeptide, a recombinant RNA bacteriophage polypeptide; a recombinant Ty polypeptide; a recombinant fr-bacteriophage polypeptide, a recombinant GA-bacteriophage polypeptide, a recombinant AP205-bacteriophage polypeptide, in particular a recombinant Qβ-bacteriophage polypeptide. The virus-like particle can also comprise or consist of one or more fragments of these polypeptides and variants of these polypeptides. Variants of polypeptides are at least 80%, 85%, 90%, 95%, 97% or 99% identical at the amino acid level to their wild-type counterparts.

[0131] In some embodiments, the VLP of the present application comprises, consists essentially of or consists of a recombinant protein or fragment thereof of an RNA-bacteriophage. Preferably, the RNA-bacteriophage is selected from the group consisting of: bacteriophage Qβ, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriophage MS2, bacteriophage Ml l, bacteriophage MXl, bacteriophage NL95, bacteriophage f2, bacteriophage PP7.

[0132] In some embodiments, the particulate protein component of the present application can be a protein or peptide, including a multimerization domain forming a nanoparticle such as IMX313, or a computer designed derived particle such as I3-01 and mi3.

[0133] Preferably, the VLPs used in the present application include, but are not limited to, HBsAg VLP, HBcAg VLP, HPV 18 L1 VLP, HPV 16 L1 VLP, HEV P239 VLP, CuMV VLP, CCMV VLP, RHDS VLP, PPV VLP, CPV VLP, Norwalk VLP, influenza HA VLP, and the like.

[0134] In some embodiments, the nanoparticle protein used in any of the immunogenic complexes provided by the present application can also be selected from the group consisting of: AP205, NPM particle, Ferritin, I53-50 particle, Lumazine Synthase (LS) particle, RC_I_1, RC_I_2, and the like.

[0135] In some embodiments, the nanoparticle protein I53-50 particle used in any of the immunogenic complexes provided by the present application is composed of two subunits of I53-50A and I53-50B.

[0136] In some embodiments, the immunogenic complex provided by the present application comprises the following (1) antigen component and (2) particle protein component: (1) an antigen component comprising any of the Mtb antigens or fusion proteins described in the present application; (2) a particle protein component comprising a nanoparticle protein; the antigen component and the particle protein component form an immunogenic complex.

[0137] In some embodiments, the immunogenic complex provided by the present application comprises the following (1) antigen component and (2) particle protein component: (1) an antigen component comprising a1: any of the Mtb antigens described in the present application and a binding peptide 1, the Mtb antigen and the binding peptide 1 form a fusion protein; or b1: any of the fusion proteins described in the present application and a binding peptide 1, the fusion protein and the binding peptide 1 form a fusion protein; (2) a particle protein component comprising a nanoparticle protein and a binding peptide 2; the nanoparticle protein and the binding peptide 2 form a fusion protein; the antigen component and the particle protein component are covalently bound through a peptide binding pair, such as Spytag-Spycatcher, thereby forming an immunogenic complex. In the present application, the binding peptide 1 is selected from Spytag, and the binding peptide 2 is selected from Spycatcher.

[0138] In other embodiments, the peptide binding pair used in the immunogenic complex of the present application also includes SpyTag002 / SpyTag002RG T3H-SpyCatcher; SpyTag / SpyTag002 / SpyTag002 RG T3H-SpyCatcher002; SpyTag / SpyTag002 / SpyTag002 RG T3H-SpyCatcher002(D5A, A92P, Q100D); SnoopTag / SnoopTagJr-SnoopCatcher; RrgATag / RrgATag2 / DogTag-RrgACatcher; Isopeptag-Pilin-C; Isopeptag-N-Pilin-N; PsCsTag-PsCsCatcher; SnoopTagJr-DogTag, etc.

[0139] In other embodiments, the binding peptide 1 of the present application can also be selected from any one of SnoopTag, RrgATag, RrgATag2, DogTag, IsopepTag, IsopepTagN, PsCsTag and SnoopTagJr. Correspondingly, the binding peptide 2 of the present application can also be selected from any one of SnoopCatcher, RrgACatcher, Pilin-C, Pilin-N, PsCsCatcher and DogTag.

[0140] In some embodiments, the antigen component in any one of the immunogenic complex provided by the present application is formed by fusing the Mtb antigen or immunogenic fragment thereof described by the present application at the C-terminus with the binding peptide 1 via the linker peptide 1.

[0141] In some embodiments, the granulin component in any one of the immunogenic complex provided by the present application is formed by fusing the nanogranulin at the N-terminus with the binding peptide 2 via the linker peptide 2.

[0142] In some embodiments, the antigen component in any one of the immunogenic complex provided by the present application is formed by fusing the Mtb antigen or immunogenic fragment thereof described by the present application at the C-terminus with the binding peptide 1 via the linker peptide 1.

[0143] In some embodiments, the antigen component and / or the granulin component in any one of the immunogenic complex provided by the present application comprises a purification tag, such as a histidine tag.

[0144] The present application provides an immunogenic complex comprising: (1) an antigen component comprising a Mtb antigen or an immunogenic fragment thereof according to the present application, a linker peptide 1; and (2) a granulin component comprising a subunit of a nanoparticle protein.

[0145] In some embodiments, the Mtb antigen or an immunogenic fragment thereof according to the present application is linked to one subunit of a nanoparticle protein to form a fusion protein, which is further linked to another subunit of the nanoparticle protein.

[0146] In some embodiments, any of the immunogenic complexes provided by the present application, the linker peptide 1 comprises an amino acid sequence as set forth in SEQ ID NO: 47. In some embodiments, any of the immunogenic complexes provided by the present application, the linker peptide 2 comprises an amino acid sequence as set forth in SEQ ID NO: 48.

[0147] In some embodiments, any of the immunogenic complexes provided by the present application, the linker peptide 1 comprises an amino acid sequence of (GSG) n , (GGGGS) n or (EAAAK) n , wherein n is an integer greater than 0 and less than or equal to 5. In some embodiments, any of the immunogenic complexes provided by the present application, the linker peptide 1 is preferably GSG GSG (SEQ ID NO: 49).

[0148] In some embodiments, any of the immunogenic complexes provided by the present application, the linker peptide 2 comprises an amino acid sequence of (GGS) n , (SGGSGG) n or (GSGGSGGSG) n , wherein n is an integer greater than 0 and less than or equal to 10. In some embodiments, any of the immunogenic complexes provided by the present application, the linker peptide 2 is preferably GGSGGSGGSGGS (SEQ ID NO: 50).

[0149] Specifically, the Mtb antigen in the Mtb antigen component of the present application is selected from the Mtb antigens or fusion proteins described in the present application, the Mtb antigen or fusion protein is connected with the binding peptide 1 (i.e. Spytag, also referred to as "4T" in the present application) at the C-terminal through a specific linker 1, and a purification tag (such as 6xHis) can be added at the C-terminal of the fusion protein; the coding gene encoding the Mtb antigen or fusion protein is inserted into a prokaryotic cell expression vector (such as pET21a), and the expression is carried out in E. coli BL21 (DE3) cells to obtain a fusion protein formed by the Mtb antigen / fusion protein-binding peptide 1, and the antigen component, i.e. the Mtb antigen / fusion protein-4T, is obtained by nickel column affinity chromatography and molecular sieve chromatography and the like to obtain a high-purity protein.

[0150] In some embodiments, the antigen component in any one of the immunogenic complexes provided by the present application comprises, from N-terminal to C-terminal, the Mtb antigen / fusion protein, the linker 1 and the binding peptide 1 described in the foregoing of the present application.

[0151] In some embodiments, the antigen component in any one of the immunogenic complexes provided by the present application comprises, from N-terminal to C-terminal, the Mtb fusion protein, the linker 1 and the binding peptide 1 shown in Table 2.

[0152] Preferably, the antigen component in the immunogenic complex provided by the present application comprises the following structure (from N-terminal to C-terminal): Ag85a-Mtb32a-4T; ESAT6-CFP10-Mtb39a-4T; Mpt64M1-Hrp1M1-4T; EsxC-EsxD-EsxV-EsxW-4T; EspC-PE13-4T; RV2660c-TB10.4-4T.

[0153] In some embodiments, the antigen component provided by the present application has an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to the amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 54.

[0154] Preferably, the antigen component provided by the present application has an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 54.

[0155] In some embodiments, the present application provides any one of the above-mentioned immunogenic complexes, wherein the granule protein component is a fusion protein of the N- terminal of the nanoparticle protein and the binding peptide 2 via the linker peptide 2; preferably, the nanoparticle protein is mi3 (named NPM in the present application), AP205 capsid protein 3 (AP205) or Ferritin protein. Specifically, in some alternatives, the binding peptide 2 (i.e. SpyCatcher, also named "4C" in the present application) is connected to the coding gene of the nanoparticle protein via the linker peptide 2, inserted into a prokaryotic expression vector (e.g. pET-28a(+), pET-30a(+)), expressed in E. coli cells, and the fusion protein of the binding peptide 2 and the nanoparticle protein is obtained, which can be purified by chromatography, such as anion exchange chromatography, hydrophobic chromatography, to obtain the product. The nanoparticle protein is preferably NPM, AP205 or Ferritin; and the formed granule protein component is named NPM-4C, AP205-4C, Ferritin-4C.

[0156] Specifically, in some alternatives, any one of the above-mentioned antigen components is conjugated with the granule protein component under suitable reaction conditions to form a covalent bond through the binding of the binding peptide 1 of the antigen component and the binding peptide 2 of the granule protein component, thereby forming the immunogenic complex. Different immunogenic complexes can be formed by using different nanoparticle proteins, which are named Mtb antigen-NPM, Mtb antigen-AP205 or Mtb antigen-Ferritin, respectively.

[0157] In some embodiments, the present application provides an immunogenic complex, which comprises: (1) an antigen component comprising the Mtb antigen, the linker peptide 1 and the binding peptide 1; and (2) a granule protein component comprising the nanoparticle protein, the linker peptide 2 and the binding peptide 2.

[0158] The linker peptide 1 is any commonly used linker peptide in the art, including but not limited to the amino acid sequence of (GSG)n, (GGGGS)n, or (EAAAK)n, wherein n is an integer greater than 0 and less than or equal to 5, and preferably GSGGSG; the linker peptide 2 is any commonly used linker peptide in the art, including but not limited to the amino acid sequence of (GGS)n, (SGG)n, or (GSGGSGGSG)n, wherein n is an integer greater than 0 and less than or equal to 10, and preferably GGSGGSGGSGGS. The nanoparticle protein is NPM, AP205 or Ferritin. n n n n n n The nanoparticle protein is NPM, AP205 or Ferritin.​​​​​

[0159] Preferably, in any one of the immunogenic complexes provided by the present application, the granulin component comprises NPM-4C, as set forth in SEQ ID NO: 52, which is a fusion protein obtained by linking the binding peptide 2 as set forth in SEQ ID NO: 48 to the nanoparticle protein NPM via the linking peptide 2 as set forth in SEQ ID NO: 50.

[0160] In some embodiments, the present application provides an immunogenic complex comprising: (1) an antigen component comprising a Mtb antigen and a linking peptide 1 according to the present application; and (2) a granulin component comprising a nanoparticle protein subunit; preferably, the nanoparticle protein subunit is I53-50A and / or I53-50B subunit.

[0161] In some embodiments, the present application provides an immunogenic complex comprising: (1) an antigen component comprising a Mtb antigen and a linking peptide 1 according to the present application; and (2) a granulin component comprising a nanoparticle protein I53-50; preferably, the nanoparticle protein I53-50 comprises I53-50A and / or I53-50B subunit.

[0162] In some embodiments, the present application provides an immunogenic complex comprising: (1) an antigen component comprising a Mtb antigen and a linking peptide 1 according to the present application; and (2) a granulin component comprising a nanoparticle protein I53-50; preferably, the nanoparticle protein I53-50 comprises I53-50A and / or I53-50B subunit.

[0163] In some embodiments, the present application provides an immunogenic complex comprising any one or more of (1)-(7) as follows:

[0164] (1) the amino acid sequence of the Mtb antigen is as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43;

[0165] (2) the amino acid sequence of the linking peptide 1 is as set forth in SEQ ID NO: 49;

[0166] (3) the amino acid sequence of the binding peptide 1 is as set forth in SEQ ID NO: 47;

[0167] (4) the nanoparticle protein is selected from NPM, AP205 or Ferritin;

[0168] (5) the nanoparticle protein subunit is selected from I53-50A and / or I53-50B;

[0169] (6) the linker peptide 2 comprises an amino acid sequence of (GGS)n n , (SGGSGG)n n or (GSGGSGGSG)n n , n can be an integer greater than 0 and less than or equal to 10; preferably, the amino acid sequence of the linker peptide 2 is shown in SEQ ID NO: 50;

[0170] (7) the amino acid sequence of the binding peptide 2 is shown in SEQ ID NO: 48.

[0171] Further, the present application also provides a preparation method of any one of the above immunogenic complexes, comprising the following steps:

[0172] (1) connecting the encoding genes of the antigen component and the particle protein component into expression vectors respectively, constructing into expression recombinant plasmids and expression host strains, expressing the target proteins, and purifying; and / or (2) co-incubating the antigen component and the particle protein component obtained in step (1) to obtain the immunogenic complex.

[0173] The present application provides a preparation method of an immunogenic complex for preventing or treating Mycobacterium tuberculosis virus related diseases:

[0174] (1) connecting the encoding genes of the Mtb antigen component and the particle protein component into expression vectors respectively, constructing into recombinant expression plasmids;

[0175] (2) constructing recombinant strains capable of expressing the Mycobacterium tuberculosis virus antigen component and the particle protein component in host cells;

[0176] (3) expressing the fusion protein using the recombinant strain, and purifying the fusion protein;

[0177] (4) co-incubating the antigen component and the particle protein component to occur a conjugation reaction, and obtaining the immunogenic complex.

[0178] Preferably, the immunogenic complex obtained in the above step (4) is purified to obtain a vaccine stock solution.

[0179] Preferably, in the method for preparing the immunogenic complex for preventing or treating the mycobacterium tuberculosis virus related disease, the plasmid for expressing the Mtb antigen component in step (1) can be pET21a, and the plasmid for expressing the granular protein component can be pET-28a(+) or pET-30a(+).

[0180] In the method for preparing the immunogenic complex for preventing or treating the mycobacterium tuberculosis virus related disease, the host cell for expressing the Mtb antigen in step (2) is E. coli, and the host cell for expressing the vector of the granular protein component is E. coli.

[0181] The immunogenic complex for preventing or treating the mycobacterium tuberculosis virus related disease comprises a fusion protein formed by the Mtb antigen / fusion protein-binding peptide 1.

[0182] In the immunogenic complex for preventing or treating the mycobacterium tuberculosis virus related disease, the high-purity Mtb antigen / fusion protein obtained by molecular sieve purification is mixed according to a BCA protein concentration ratio of 6:1, 50% sucrose mother liquor is added to about 25% sucrose final concentration, and 1M Tris-HCl Mtb mother liquor accounting for 10% of the total reaction volume is added to stabilize the pH. The binding reaction is carried out at 22°C, and the reaction time is 48 hours. The endotoxin detection is less than 100 EU / ml, which meets the requirements of large-scale production.

[0183] In a fifth aspect, the present application provides derivative products of the Mtb antigen mutant, the fusion protein, and the immunogenic complex, including encoding nucleic acids, vectors, and host cells.

[0184] The present application provides encoding nucleic acids of the Mtb antigen component in the Mtb antigen mutant, the fusion protein, and the immunogenic complex, and the specific nucleotide sequence can be easily obtained by codon table and other conventional means by those skilled in the art.

[0185] The present application also provides encoding nucleic acids of the granular protein component and the nanoparticle protein, and the specific nucleotide sequence can be easily obtained by codon table and other conventional means by those skilled in the art.

[0186] Preferably, the present application provides an encoding nucleic acid sequence of the Mtb antigen component in the immunogenic complex, which is shown in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, or 64.

[0187] The present application also provides vectors containing the nucleic acid sequence of the present application, including cloning or expression vectors, and host cells transformed with the vectors.

[0188] In some embodiments, the vector used in the present application is selected from the group consisting of pET21a, pET-28a(+), pET-30a(+).

[0189] In some embodiments, the host cell used in the present application is selected from the group consisting of mammalian cell, insect cell, yeast cell or bacterial cell; alternatively, the mammalian cell comprises HEK293 cell or CHO cell, and the bacterial cell comprises E. coli cell. Preferably, the host cell expressing the antigen component vector in the present application is E. coli, and the host cell expressing the granulin vector in the present application is E. coli.

[0190] In a sixth aspect, the present application provides a Mtb immunological composition comprising a Mtb immunological component, wherein the Mtb immunological component is selected from the group consisting of Mtb antigen, fusion protein and immunogenic complex as described in the present application, and the Mtb antigen comprises Mtb antigen mutant and wild type antigen as described in the present application.

[0191] In some embodiments, the Mtb immunological composition of the present application comprises Mtb antigen or corresponding immunogenic complex of the present application.

[0192] In some embodiments, the Mtb immunological composition of the present application comprises Mtb fusion protein or corresponding immunogenic complex selected from Table 2.

[0193] In some embodiments, the Mtb immunological composition of the present application can comprise both single Mtb antigen and Mtb fusion protein or corresponding immunogenic complex.

[0194] In some embodiments, the Mtb immunological composition provided in the present application comprises two or three Mtb immunological components.

[0195] In some embodiments, the Mtb immunological composition of the present application comprises two Mtb immunological components: component 1 and component 2.

[0196] In some embodiments, component 1 is selected from the group consisting of Mtb antigen as described in the present application or Mtb fusion protein as shown in Table 2:

[0197] (1-1) Ag85a mutant or immunogenic complex related to Ag85a mutant;

[0198] (1-2) fusion protein D or immunogenic complex related to fusion protein D;

[0199] (1-3) fusion protein F1 or F2, or immunogenic complex related to fusion protein F1 or F2;

[0200] (1-4) fusion protein G1 or G2, or immunogenic complex related to fusion protein G1 or G2;

[0201] (1-5) fusion protein H1, H2 or H3, or an immunogenic complex involving fusion protein H1, H2 or H3;

[0202] (1-6) fusion protein I1 or I2, or an immunogenic complex involving fusion protein I1 or I2;

[0203] (1-7) fusion protein J1, J2 or J3, or an immunogenic complex involving fusion protein J1, J2 or J3;

[0204] (1-8) fusion protein K1 or K2, or an immunogenic complex involving fusion protein K1 or K2;

[0205] (1-9) fusion protein L1, L2 or L3, or an immunogenic complex involving fusion protein L1, L2 or L3;

[0206] (1-10) fusion protein Q1 or Q2, or an immunogenic complex involving fusion protein Q1 or Q2;

[0207] (1-11) fusion protein E1 or E2, or an immunogenic complex involving fusion protein Q1 or Q2.

[0208] Component 2 is selected from the Mtb antigens described herein or the Mtb fusion proteins shown in Table 2:

[0209] (2-1) fusion protein D or an immunogenic complex involving fusion protein D;

[0210] (2-2) Mtb32a mutant or an immunogenic complex involving Mtb32a mutant;

[0211] (2-3) fusion protein J1, J2 or J3, or an immunogenic complex involving fusion protein J1, J2 or J3;

[0212] (2-4) fusion protein K1 or K2, or an immunogenic complex involving fusion protein K1 or K2;

[0213] (2-5) fusion protein L1, L2 or L3, or an immunogenic complex involving fusion protein L1, L2 or L3;

[0214] (2-6) fusion protein E1 or an immunogenic complex involving fusion protein E1;

[0215] (2-7) fusion protein E2 or an immunogenic complex involving fusion protein E2;

[0216] (2-8) fusion protein F1 or F2, or an immunogenic complex involving fusion protein F1 or F2;

[0217] (2-9) Fusion protein A or immunogenic complexes involving fusion protein A;

[0218] (2-10) Fusion protein B or immunogenic complexes involving fusion protein B;

[0219] (2-11) Fusion protein C or immunogenic complexes involving fusion protein C;

[0220] (2-12) Fusion protein G1 or G2, or immunogenic complexes involving fusion protein G1 or G2;

[0221] (2-13) Fusion proteins H1, H2, or H3, or immunogenic complexes involving fusion proteins H1, H2, or H3;

[0222] (2-14) Fusion protein I1 or I2, or immunogenic complexes involving fusion protein I1 or I2;

[0223] (2-15) Fusion protein N1 or N2, or immunogenic complexes involving fusion protein N1 or N2;

[0224] (2-16) Fusion proteins O1, O2, or O3, or immunogenic complexes involving fusion proteins O1, O2, or O3;

[0225] (2-17) Fusion proteins M1, M2 or M3, or immunogenic complexes involving fusion proteins M1, M2 or M3;

[0226] (2-18) Fusion protein N1 or N2, or immunogenic complexes involving fusion protein N1 or N2;

[0227] (2-19) Fusion protein R1 or R2, or immunogenic complexes involving fusion protein R1 or R2;

[0228] (2-20) Fusion protein S, and immunogenic complexes involving fusion protein S;

[0229] (2-21) Fusion protein P1 or P2, or an immunogenic complex involving fusion protein P1 or P2.

[0230] In some embodiments, when component 1 is the Ag85a mutant or its immunogenic complex as described in this invention, component 2 is selected from the fusion protein or its immunogenic complex described in (2-1), (2-2), (2-3), (2-4), (2-5), (2-6) or (2-7).

[0231] In some embodiments, when component 1 is fusion protein D or an immunogenic complex thereof according to the application, component 2 is selected from the fusion proteins according to (2-2), (2-3), (2-4), (2-5), (2-6), (2-7), (2-8), (2-9), (2-10), (2-11) or (2-20) or an immunogenic complex thereof.

[0232] In some embodiments, when component 1 is fusion protein F1 or F2 or an immunogenic complex thereof according to the application, component 2 is selected from the fusion proteins according to (2-9), (2-10) or (2-11) or an immunogenic complex thereof.

[0233] In some embodiments, when component 1 is fusion protein G1 or G2 or an immunogenic complex thereof according to the application, component 2 is selected from the fusion proteins according to (2-9), (2-10), (2-11) or (2-13) or an immunogenic complex thereof.

[0234] In some embodiments, when component 1 is fusion protein H1, H2 or H3 or an immunogenic complex thereof according to the application, component 2 is selected from the fusion proteins according to (2-9), (2-10) or (2-11) or an immunogenic complex thereof.

[0235] In some embodiments, when component 1 is fusion protein I1 or I2 or an immunogenic complex thereof according to the application, component 2 is selected from the fusion proteins according to (2-3), (2-4), (2-5), (2-15), (2-16) or (2-17) or an immunogenic complex thereof.

[0236] In some embodiments, when component 1 is fusion protein J1, J2 or J3 or an immunogenic complex thereof according to the application, component 2 is selected from the fusion proteins according to (2-15), (2-16) or (2-21) or an immunogenic complex thereof.

[0237] In some embodiments, when component 1 is fusion protein K1 or K2 or an immunogenic complex thereof according to the application, component 2 is selected from the fusion proteins according to (2-16) or (2-17) or an immunogenic complex thereof.

[0238] In some embodiments, when component 1 is fusion protein L1, L2 or L3 or an immunogenic complex thereof according to the application, component 2 is selected from the fusion proteins according to (2-17) or (2-18) or an immunogenic complex thereof.

[0239] In some embodiments, when component 1 is a fusion protein Q1 or Q2 as described herein, or an immunogenic complex thereof, component 2 is selected from a fusion protein or an immunogenic complex thereof as described in (2-6), (2-7), (2-9), (2-10) or (2-11).

[0240] In some embodiments, when component 1 is a fusion protein E1 or E2 as described herein, or an immunogenic complex thereof, component 2 is selected from a fusion protein or an immunogenic complex thereof as described in (2-9), (2-10) or (2-11).

[0241] In some embodiments, the Mtb immunological composition of the present application comprises three Mtb immunological components: component 1, component 2 and component 3, wherein the candidate antigens of component 3 and component 2 are the same. The three components are shown as follows:

[0242] (1) Component 1 is an Ag85a mutant or an immunogenic complex involving an Ag85a mutant as described herein, and component 2 is a fusion protein D or an immunogenic complex involving a fusion protein D as described herein, and component 3 is selected from a fusion protein or an immunogenic complex thereof as described in (2-2), (2-3), (2-4), (2-5), (2-6) or (2-7).

[0243] (2) Component 1 is a fusion protein F1 or F2 as described herein, or an immunogenic complex involving a fusion protein F1 or F2, and component 2 is a fusion protein D or an immunogenic complex involving a fusion protein D as described herein, and component 3 is selected from a fusion protein or an immunogenic complex thereof as described in (2-9), (2-10) or (2-11).

[0244] (3) Component 1 is a fusion protein G1 or G2 as described herein, or an immunogenic complex involving a fusion protein F1 or F2, and component 2 is a fusion protein H1, H2 or H3 as described herein, or an immunogenic complex involving a fusion protein H1, H2 or H3, and component 3 is selected from a fusion protein or an immunogenic complex thereof as described in (2-9), (2-10) or (2-11).

[0245] (4) Component 1 is a fusion protein I1 or I2 as described herein, or an immunogenic complex involving a fusion protein I1 or I2, and component 2 is a fusion protein J1, J2 or J3 as described herein, or an immunogenic complex involving a fusion protein J1, J2 or J3, and component 3 is selected from a fusion protein or an immunogenic complex thereof as described in (2-15) or (2-16).

[0246] (5) Component 1 is fusion protein II or I2 as described in the present application, or an immunogenic complex involving fusion protein II or I2, and component 2 is fusion protein K1 or K2 as described in the present application, or an immunogenic complex involving fusion protein K1 or K2, and component 3 is selected from the group consisting of fusion proteins described in (2-16) or (2-17) or immunogenic complexes thereof.

[0247] (6) Component 1 is fusion protein II or I2 as described in the present application, or an immunogenic complex involving fusion protein II or I2, and component 2 is fusion protein L1, L2 or L3 as described in the present application, or an immunogenic complex involving fusion protein L1, L2 or L3, and component 3 is selected from the group consisting of fusion proteins described in (2-15) or (2-17) or immunogenic complexes thereof.

[0248] (7) Component 1 is fusion protein Q1 or Q2 as described in the present application, or an immunogenic complex involving fusion protein Q1 or Q2, and component 2 is fusion protein E1 or E2 as described in the present application, or an immunogenic complex involving fusion protein E1 or E2; and component 3 is selected from the group consisting of fusion proteins described in (2-9), (2-10) or (2-11) or immunogenic complexes thereof.

[0249] In some embodiments, the Mtb immunological composition of the present application comprises fusion protein PI or P2 as described in the present application, or an immunogenic complex involving fusion protein PI or P2. In some embodiments, the Mtb immunological composition of the present application comprises fusion protein R1 or R2 as described in the present application, or an immunogenic complex involving fusion protein R1 or R2. In some embodiments, the Mtb immunological composition of the present application comprises fusion protein S as described in the present application, or an immunogenic complex involving fusion protein S. Preferably, the Mtb immunological composition of the present application comprises an immunogenic complex of fusion protein PI or P2, an immunogenic complex of fusion protein R1 or R2 and an immunogenic complex of fusion protein S; optionally, further comprising an immunogenic complex of fusion protein A or C.

[0250] In a preferred embodiment, the Mtb immunological composition of the present application comprises the following components:

[0251] (1) Mtb32a, Ag85a, ESAT6-CFP10, RV2660c-TB10.4;

[0252] (2) Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, RV2660c-TB10.4-NPM;

[0253] (3) ESAT6-CFP10-Mtb39a, Ag85a-Mtb32a;

[0254] (4) ESAT6-CFP10-Mtb39a-NPM, Ag85a-Mtb32a-NPM;

[0255] (5) EsxC-EsxD-EsxV-EsxW, EspC-PE13;

[0256] (6) EsxC-EsxD-EsxV-EsxW-NPM, EspC-PE13-NPM;

[0257] (7) Mpt64-Hrp1, EspC-PE13;

[0258] (8) Mpt64-Hrp1-NPM, EspC-PE13-NPM;

[0259] (9) Mtb32a, Ag85a, ESAT6-CFP10-Mtb39a, RV2660c-TB10.4; or

[0260] (10) Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-Mtb39a-NPM, RV2660c-TB10.4-NPM.

[0261] Preferably, in the immunological composition described above, the amino acid sequence of Mtb32a is as shown in SEQ ID NO: 1, the amino acid sequence of Ag85a is as shown in SEQ ID NO: 5 or 7, the amino acid sequence of RV2660c-TB10.4 is as shown in SEQ ID NO: 33, the amino acid sequence of ESAT6-CFP10-Mtb39a is as shown in SEQ ID NO: 39; the amino acid sequence of Ag85a-Mtb32a is as shown in SEQ ID NO: 42, the amino acid sequence of EsxC-EsxD-EsxV-EsxW is as shown in SEQ ID NO: 35, the amino acid sequence of EspC-PE13 is as shown in SEQ ID NO: 37, the amino acid sequence of Mpt64-Hrp1 is as shown in SEQ ID NO: 43; the amino acid sequence of ESAT6-CFP10 is as shown in SEQ ID NO: 53.

[0262] Optionally, the Mtb immunological composition of the present application further comprises a pharmaceutically acceptable carrier, which can be a freeze-dried preparation, an injection preparation, an oral preparation or a spray preparation.

[0263] The present application also provides an immunological composition comprising any of the immunogenic complexes described above and a pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier comprises a stabilizer, an excipient, a surfactant, a buffer, a pH regulator, the stabilizer is sucrose, arginine, the excipient is mannitol, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, the pH regulator is hydrochloric acid. Preferably, the pharmaceutically acceptable carrier is 20 mM Tris-HCl, pH 7.4.

[0264] In some embodiments, the immunological composition provided by the present application comprises the immunogenic complex in an amount of 0.1-50 μg / dose, preferably 0.1-0.5 μg / dose or 10-50 μg / dose, more preferably 10 μg / dose, 15 μg / dose, 20 μg / dose, 25 μg / dose, 30 μg / dose, 35 μg / dose, 40 μg / dose, 45 μg / dose, 50 μg / dose, and more preferably 40 μg / dose.

[0265] In some embodiments, the immunological composition provided by the present application is an injection solution or a lyophilized preparation, preferably a lyophilized preparation.

[0266] In some embodiments, the immunological composition provided by the present application is a lyophilized preparation comprising the Mtb antigen-NPM immunogenic complex, a stabilizer, an excipient, a surfactant, a buffer, a pH regulator; preferably, the stabilizer is sucrose, arginine, the excipient is mannitol, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, the pH regulator is hydrochloric acid.

[0267] In some embodiments, the immunological composition provided by the present application is a lyophilized preparation comprising the Mtb antigen-NPM immunogenic complex, sucrose, arginine, mannitol, Tween 80, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, hydrochloric acid.

[0268] In some embodiments, the immunological composition provided by the present application is an injection solution comprising the Mtb antigen-NPM immunogenic complex, a stabilizer, a surfactant, a buffer, a pH regulator; preferably, the stabilizer is sucrose, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, the pH regulator is hydrochloric acid.

[0269] In some embodiments, the immunological composition provided by the present application is an injection solution comprising the Mtb antigen-NPM immunogenic complex, sucrose, Tween 80, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, hydrochloric acid.

[0270] In a seventh aspect, the present application provides a vaccine for preventing and treating Mtb infection or Mtb-caused diseases, which comprises the Mtb immunizing composition of the present application.

[0271] The vaccine provided by the present application includes a subunit vaccine, a nucleic acid vaccine and a viral vector vaccine.

[0272] The present application provides a nucleic acid vaccine for preventing and treating Mtb infection or Mtb-caused diseases, which comprises a nucleic acid sequence encoding the Mtb vaccine composition of the present application.

[0273] In some embodiments, the nucleic acid vaccine of the present application is a DNA vaccine or an RNA vaccine.

[0274] The present application provides a viral vector vaccine for preventing and treating Mtb infection or Mtb-caused diseases, which comprises a nucleic acid sequence encoding the Mtb vaccine composition of the present application.

[0275] In some embodiments, the viral vector used in the present application is selected from one or more of the following: an adenovirus vector, a poxvirus vector, an influenza virus vector, an adeno-associated virus vector.

[0276] The present application further provides a Mtb vaccine, which comprises any of the Mtb immunizing compositions of the present application and an adjuvant selected from at least one of the following: an aluminum salt adjuvant, a Freund's complete adjuvant, a propolis adjuvant, a water-in-oil adjuvant, a cytokine, a CpG DNA, a genetically engineered attenuated toxin, an immunostimulatory complex, a liposome.

[0277] In some embodiments, the Mtb vaccine provided by the present application comprises a water-in-oil adjuvant. Preferably, the water-in-oil adjuvant is a squalene adjuvant comprising squalene.

[0278] In some embodiments, the Mtb vaccine provided by the present application comprises a liposome adjuvant.

[0279] In some embodiments, the liposome adjuvant used in the present application comprises the following components (per 0.5 ml): 50 μg Quillaja saponaria saponin QS-21, 50 μg 3-O-deacylated-4'-monophosphoryl lipid A (3D-MPL), 1 mg dioleoyl phosphatidylcholine (DOPC), 0.25 mg cholesterol.

[0280] In some embodiments, the liposome adjuvant used in the present application comprises the following components (per 0.5 ml): 25 μg Quillaja saponaria saponin QS-21, 25 μg 3-O-deacylated-4'-monophosphoryl lipid A (3D-MPL), 0.5 mg dioleoyl phosphatidylcholine (DOPC), 0.125 mg cholesterol.

[0281] In some embodiments, the adjuvant used in the present application is a CAF adjuvant, including CAF01, CAF09b and CAF10b. Preferably, the CAF adjuvant is CAF01 adjuvant.

[0282] The Mycobacterium tuberculosis vaccine of the present application contains the immunogenic complex in an amount of 10-50 μg / dose, preferably 40 μg / dose per unit dose of vaccine for human use.

[0283] The squalene adjuvant of the present application contains (w / w) squalene 0.5%-5%, Span 85 0.05%-1%, Tween 80 0.05%-1%, 10 mM citrate buffer.

[0284] The squalene adjuvant of the present application preferably contains (w / w) squalene 2%-4.5%, Span 85 0.2%-0.5%, Tween 80 0.2%-0.5%, 10 mM citrate buffer. More preferably, the squalene is used in an amount of 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4% (w / w), the Span 85 is more preferably used in an amount of 0.3%-0.4% (w / w), and the Tween 80 is more preferably used in an amount of 0.3%-0.4% (w / w).

[0285] As described above, the amount of Mtb immunogenic complex and adjuvant used for human and mice is different, and the corresponding relationship is that the amount of Mtb immunogenic complex and adjuvant used for human is 5-10 times that used for mice, for example, 0.4 μg / dose of Mtb immunogenic complex is used for mice, and 40 μg / dose is used for human, and so on.

[0286] The present application provides a kit of parts, which contains the Mycobacterium tuberculosis vaccine of the present application, and the instruments and containers required for vaccination of the vaccine.

[0287] The present application provides a Mycobacterium tuberculosis vaccine, which contains Mtb immunogenic complex (i.e. an immune combination of Mtb immunogenic complex, which can be prepared into a freeze-dried preparation or an injection liquid preparation) and adjuvant (which is a liquid). The immune combination formed by various Mtb immunogenic complexes and the adjuvant are packaged in separate bottles.

[0288] In an eighth aspect, the present application provides a method for preventing and treating Mycobacterium tuberculosis infection. The prevention includes preventing the infection with Mycobacterium or delaying the risk of infection with Mycobacterium.

[0289] The present application provides the use of a Mtb immunogenic complex, immunological composition or vaccine for the manufacture of a medicament for the prevention or treatment of tuberculosis.

[0290] The present application provides a therapeutically effective amount of an immunogenic complex, immunological composition or vaccine for the prevention or treatment of diseases associated with Mycobacterium tuberculosis infection. The therapeutically effective amount is a dosage sufficient to achieve the intended use. When referring to a prophylactic use, the term means a dosage sufficient to prevent or delay the occurrence and / or establishment of an infection with Mycobacterium (e.g. Mtb infection). For therapeutic use, the composition is administered to a subject already infected with a Mycobacterium species, with the goal of treating active disease or preventing reactivation of a latent infected individual, eventually in combination with one or more conventional treatment modalities described herein. In particular, the therapeutically effective amount used in the present application can be the amount necessary to induce or stimulate the immune system (e.g. leading to the production of innate and / or specific responses) in the subject to which it is administered.

[0291] The appropriate dosage can be adjusted in function of various parameters, in particular the active agent comprised in the composition, the mode of administration, the age, health and weight of the subject, the nature and extent of the symptoms, the type of concurrent treatment, the frequency of the treatment and / or the need for prevention or treatment. Further precise calculations necessary to determine the appropriate dosage for treatment are usually performed by the physician in charge according to the circumstances.

[0292] The immunogenic complex, immunological composition or vaccine of the present application is suitable for various modes of administration. Any conventional route of administration is suitable in the context of the present application, including systemic, local or mucosal routes.

[0293] Systemic administration includes, for example, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, intravascular, intra-arterial injection and skin breach. The injection can be performed using conventional syringes and needles, or any other suitable device available in the art (e.g. electroporation). Mucosal administration includes, but is not limited to, oral / dietary, intranasal, intratracheal, intrapulmonary, intraleaf sheath or intrarectal routes. Respiratory administration can be performed by nebulization or aerosolization of liquid droplets, spraying or dry powder compositions using suitable dispersers. Local administration can also be performed using transdermal means (e.g. patches, etc.). Intramuscular, intradermal and subcutaneous routes, as well as intranasal, intratracheal and intrapulmonary administration are particularly preferred in the context of the present application.

[0294] The Mtb immunogenic complex, immunological composition or vaccine of the present application can be used in combination with one or more conventional therapies, such as one or more chemotherapeutic agents effective against Mycobacterium infection.

[0295] In some embodiments, the Mtb immunogenic complex, immunological composition or vaccine of the present application can be used in combination with isoniazid, rifamycin (i.e. rifampin, rifapentine and rifabutin), ethambutol, streptomycin, pyrazinamide and fluoroquinolones; or ofloxacin, ciprofloxacin, ethionamide, aminosalicylic acid, cycloserine, ambutonium, kanamycin and capreomycin.

[0296] In some embodiments, the Mtb immunogenic complex, immunological composition or vaccine of the present application can be administered prior to, concurrently with or after administration of one or more of the above-mentioned chemotherapeutic drugs.

[0297] The present application also provides a reagent for diagnosing Mycobacterium tuberculosis infection or diseases caused by Mycobacterium tuberculosis infection, which comprises the Mtb antigen mutant, fusion protein, immunogenic complex and composition of the present application.

[0298] Compared with the prior art, the present application has the following advantages:

[0299] 1. The present application uses E. coli expression system to express Mtb antigen-4T in a fusion expression form, and at the same time, NPM-4C nanoparticles are prepared by means of E. coli expression system. The Mtb antigen-NPM nanoparticles of the present application have uniform particle size, uniform distribution and no aggregation, stable product performance, qualified endotoxin, and are suitable for non-clinical development and antibody immunogenicity test, thereby suitable for use as a tuberculosis vaccine.

[0300] 2. The Mtb antigen-4T is covalently coupled with NPM-4C nanoparticles respectively for the first time to prepare nanoparticle antigen Mtb antigen-NPM, which has stable production process, low cost and great industrialization potential.

[0301] 3. The Mtb vaccine provided by the present application can induce IgG, IFN-γ, TNFα and IL-2 in mice when used alone or in various combinations, which indicates that the Mtb antigen, fusion protein and immunogenic complex of the present application all have good immunogenicity and can stimulate cellular immunity and humoral immunity. Compared with mice immunized with normal saline, the Mtb load of the spleen and lung of mice immunized with the Mtb vaccine or combination thereof of the present application is reduced.

[0302] 4. The various Mtb antigen-NPM compositions of the present application can inhibit Mycobacterium tuberculosis infection, including reducing the Mycobacterium tuberculosis load in the lung of infected mice, which has important development value.

[0303] 5. In this invention, the particulate protein component is prepared using E. coli fermentation and chromatographic purification, while the Mtb antigen-4T antigen component can be prepared using cell reactor culture and chromatographic purification. Both methods are suitable for large-scale industrial production and offer advantages such as high expression levels, stable processes and yields, and simple operation. The amount of one batch of recombinant particulate protein component can be used to bind multiple batches of Mtb antigen-4T antigen component, improving production efficiency and saving costs associated with large-scale production.

[0304] 6. The method for preparing recombinant particulate protein components provided by this invention is suitable for industrial production, can reduce the cost of large-scale industrial production, is simple to operate, and reduces the amount of organic solvent used in subsequent chromatography purification; the protein products prepared using the method for preparing recombinant particulate protein components provided by this invention effectively reduce the side effects caused by the residues of impurities, host proteins, organic solvents, exogenous DNA, antibiotics, bacterial endotoxins, etc. in the particles, and improve safety. Attached Figure Description

[0305] Figure 1: Western Blot identification results of 014 molecules. Lane 1 represents the supernatant of 014 molecules, and lane 2 represents the precipitate of 014 molecules.

[0306] Figure 2A: Western Blot identification results of molecules 072, 076 and 077. Lane 1 represents the supernatant of molecule 072, lane 2 represents the precipitate of molecule 072, lane 3 represents the supernatant of molecule 076, lane 4 represents the precipitate of molecule 076, lane 5 represents the supernatant of molecule 077, and lane 6 represents the precipitate of molecule 077.

[0307] Figure 2B: Western Blot identification results of 117-1 molecules. Lane 1 represents the supernatant of 117-1 molecules, and lane 2 represents the precipitate of 117-1 molecules.

[0308] Figure 3: Western Blot identification results of 072M7 molecules. Lane 1 represents the supernatant of 072M7 molecules, and lane 2 represents the precipitate of 072M7 molecules.

[0309] Figure 4: Western Blot identification results of 076M3 and 105 molecules. Lane 1 represents the supernatant of 076M3 molecules, lane 2 represents the precipitate of 076M3 molecules, lane 3 represents the supernatant of 105 molecules, and lane 4 represents the precipitate of 105 molecules.

[0310] Figure 5: Western Blot identification results of 099 molecules. Lane 1 represents the supernatant of 099 molecules, and lane 2 represents the precipitate of 099 molecules.

[0311] Figure 6: SDS-PAGE identification results of 014 molecule, lane 1 is 014 molecule before purification, lanes 2-5 are 014 molecule after purification;

[0312] Figure 7A: SDS-PAGE identification results of 072 molecule; Figure 7B: SDS-PAGE identification results of 117-1 molecule;

[0313] Figure 8: SDS-PAGE identification results of 076 molecule;

[0314] Figure 9: SDS-PAGE identification results of 077 molecule;

[0315] Figure 10: SDS-PAGE identification results of 072 molecule and its mutants M1-M11, the expression amount is M7>M6>M1>M9>M4>M5>M3>M2>M8>M11>M10>072;

[0316] Figure 11: SDS-PAGE identification results of 072M7 molecule;

[0317] Figure 12A: SDS-PAGE identification results of 076M1 after SEC purification; Figure 12B: SDS-PAGE identification results of 076M2 after SEC purification;

[0318] Figure 12C: SDS-PAGE identification results of 076M3 after SEC purification;

[0319] Figure 13: SDS-PAGE identification results of 099 molecule;

[0320] Figures 14A-14C: SDS-PAGE identification results of 105 molecule before and after mutation under DTT- / DTT+ conditions; Figure A is the SDS-PAGE identification results of 105 molecule before mutation (DTT+), lanes 1-4: EspC-PE13-4T before mutation, no dimer; Figure B is the SDS-PAGE identification results of 105 molecule (DTT+), lanes 1-5: EspC(C48A)-PE13-4T, no dimer; Figure C is the SDS-PAGE identification results of 105 molecule and 105 molecule before mutation under DTT- / DTT+ conditions, lane 1: 105 molecule EspC(C48A)-PE13-4T (DTT-), lane 2: 105 molecule EspC(C48A)-PE13-4T (DTT+), lane 3: 105 molecule EspC-PE13-4T before mutation (DTT-), lane 4: 105 molecule EspC-PE13-4T before mutation (DTT+);

[0321] Figure 15: SDS-PAGE identification results of 077 before and after mutation, lane 1 : RV2660C-TB10.4-4T before mutation (DTT+), lane 2: RV2660C-TB10.4-4T before mutation (DTT-), lane 3: RV2660C-TB10.4-4T after mutation (DTT+), lane 4: RV2660C-TB10.4-4T after mutation (DTT-).

[0322] Figure 16: Purification results of M72-4T protein molecular sieve separation, M represents protein molecular marker, lanes 1-8 represent M72-4T protein;

[0323] Figure 17: Purification results of NPM-4C protein Octyl Bestarose 4FF separation;

[0324] Figure 18: SDS-PAGE identification results of 014-NPM;

[0325] Figure 19A: SDS-PAGE identification results of 072-NPM; Figure 19B: SDS-PAGE identification results of 117-1-NPM;

[0326] Figure 20: SDS-PAGE identification results of 076-NPM;

[0327] Figure 21: SDS-PAGE identification results of 077-NPM;

[0328] Figure 22: SDS-PAGE identification results of 072M7-NPM;

[0329] Figure 23: SDS-PAGE identification results of 076M3-NPM;

[0330] Figure 24: SDS-PAGE identification results of 099-NPM;

[0331] Figure 25: SDS-PAGE identification results of 105-NPM.

[0332] Figure 26: Negative staining electron microscopy detection results of 014-NPM nanoparticles;

[0333] Figure 27A: Negative staining electron microscopy detection results of 072-NPM nanoparticles; Figure 27B: Negative staining electron microscopy detection results of 117-1-NPM nanoparticles;

[0334] Figure 28: Negative staining electron microscopy detection results of 076-NPM nanoparticles;

[0335] Figure 29: Negative staining electron microscopy detection results of 077-NPM nanoparticles;

[0336] Figure 30: Negative staining electron microscopy detection results of 072M7-NPM nanoparticles;

[0337] Figure 31: Negative staining electron microscopy detection results of 076M3-NPM nanoparticles;

[0338] Figure 32: Negative staining electron microscopy detection results of 099-NPM nanoparticles;

[0339] Figure 33: Negative staining electron microscopy detection results of 105-NPM nanoparticles;

[0340] Figures 34A-34H: Particle size analysis results of 014-NPM, 072-NPM, 076-NPM, 077-NPM, 076M3-NPM, 072M7-NPM, 099-NPM, and 117-1-NPM, respectively;

[0341] Figures 35A-35B: Particle size analysis results of NPM nanoparticles prepared before and after mutation of RV2660c-TB10.4. Figure A is NPM nanoparticles prepared before mutation of RV2660c-TB10.4; Figure B is NPM nanoparticles prepared after mutation of RV2660c-TB10.4;

[0342] Figures 36A-36B: Particle size analysis results of NPM nanoparticles prepared before and after mutation of 105. Figure A is NPM nanoparticles prepared before mutation of 105; Figure B is NPM nanoparticles prepared after mutation of 105;

[0343] Figures 37A-37E: Detection of total binding antibody IgG levels of D20 corresponding group serum using 072, 076, 077, 014, and M72 proteins as coating antigens, respectively. Figures A-D are, in turn, corresponding IgG antibody levels detected by 072, 076, 077, and 014 antigens as coating antigens, respectively, of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and physiological saline); Figure E corresponds to IgG antibody levels detected by M72 antigen;

[0344] Figures 38A-38H: Figures A-D are, in turn, IFN-γ, IL-2, TNFα, and IL-4 levels detected by 072 antigen as specific stimulating antigen of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and physiological saline); Figures E-H are, in turn, IFN-γ, IL-2, TNFα, and IL-4 levels detected by 076 antigen as specific stimulating antigen of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and physiological saline);

[0345] FIG. 39A-FIG. 39H: FIG. A-D are in turn the levels of IFN-γ, IL-2, TNFα and IL-4 detected in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, saline) using 077 antigen as specific stimulating antigen; FIG. E-H are in turn the levels of IFN-γ, IL-2, TNFα and IL-4 detected in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, saline) using 014 antigen as specific stimulating antigen;

[0346] FIG. 40: The levels of IFN-γ, IL-2, TNFα and IL-4 specificities in D20 splenocytes detected using M72 protein as stimulating antigen;

[0347] FIG. 41: The results of challenge protection experiment of vac-1, vac-2, vac-3, vac-4, vac-5 and vac-6;

[0348] FIG. 42A-FIG. 42C: The levels of total binding antibody IgG of serum of D20 corresponding groups detected using 076M3, 117-1, M72 protein as coating antigen respectively;

[0349] FIG. 43A-FIG. 43E: FIG. A-B are in turn the levels of IFN-γ and IL-2 detected in different groups using 076M3 antigen as specific stimulating antigen, FIG. C-D are in turn the levels of IFN-γ and IL-2 detected in different groups using 117-1 antigen as specific stimulating antigen, FIG. E is the levels of IFN-γ and IL-2 specificities in D20 splenocytes detected using M72 protein as stimulating antigen;

[0350] FIG. 44: The ICS results of vac-10 and M72 groups;

[0351] FIG. 45: The results of challenge protection experiment of vac-7, vac-8, vac9 and vac-10;

[0352] FIG. 46A-FIG. 46C: The levels of total binding antibody IgG of serum of D20 corresponding groups detected using 099, 105, M72 protein as coating antigen respectively;

[0353] FIG. 47A-FIG. 47E: FIG. A-B are in turn the levels of IFN-γ and IL-2 detected in different groups using 099 antigen as specific stimulating antigen, FIG. C-D are in turn the levels of IFN-γ and IL-2 detected in different groups using 105 antigen as specific stimulating antigen, FIG. E is the levels of IFN-γ and IL-2 specificities in D20 splenocytes detected using M72 protein as stimulating antigen;

[0354] FIG. 48: The results of challenge protection experiment of vac-11, vac-12, vac13 and vac-14;

[0355] Figure 49A-49B: Detection of total binding antibody IgG levels in D20 corresponding group sera using 072M7 and M72 protein as coating antigen respectively;

[0356] Figure 50A-50C: Figure A-B are in turn detection of IFN-γ and IL-2 levels in different groups corresponding to 072M7 antigen as specific stimulating antigen, Figure C is detection of specific IFN-γ and IL-2 levels in D20 splenocytes using M72 protein as stimulating antigen;

[0357] Figure 51: Results of challenge protection experiment of vac-15, vac-16, vac17 and vac-18. DETAILED DESCRIPTION

[0358] The principles and features of the present application are described below in conjunction with examples, which are only used to explain the present application and are not intended to limit the scope of the present application. Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the present application. The test methods in the following examples are not specified, and are generally carried out under conventional conditions or under the conditions recommended by the manufacturers. When the examples give numerical ranges, it should be understood that, unless otherwise specified by the present application, both endpoints of each numerical range and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, devices, materials used in the examples, any method, device and material of the prior art similar or equivalent to those described in the embodiments of the present application can also be used to implement the present application according to the prior art mastered by those skilled in the art and the description of the present application. The experimental materials used in the following examples, unless otherwise specified, are purchased from conventional reagent companies.

[0359] Example 1: Construction and expression of Mtb antigen molecules

[0360] 1. Construction of Mtb antigen molecules

[0361] The Mtb antigen molecules of the present application are constructed in two stages (Phase 1 and Phase 2), and the structures of the antigen molecules are shown in the following table:

[0362] Table 3 Structure of Mtb antigen molecules

[0363] The antigen molecules shown in Table 3 were constructed by antigen engineering and fusion with reference to the wild-type sequence of the structural protein of the Mycobacterium tuberculosis H37RV strain (GenBank: AL123456.3). The specific methods for constructing the Mtb antigen molecules are as follows:

[0364] 1) 014 molecule:

[0365] The wild-type Mtb32a sequence was mutated at S177A to obtain Mtb32a(S177A) (SEQ ID NO: 1). A linker, 4T (AHIVMVDAYKPTK), and a 6His sequence were sequentially added to the C terminus of the mutant, and a stop codon was added at the end. The obtained antigen molecule was named 014.

[0366] 2) 072 molecule:

[0367] The N terminus of the wild-type Ag85a sequence was truncated, and the specific sequence of the N terminus that was truncated was QLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAG, to obtain the mutant Ag85aΔN (SEQ ID NO: 5). A linker, 4T, and 6His were sequentially added to the C terminus of the mutant Ag85aΔN, and a stop codon was added at the end. The obtained antigen molecule was named 072.

[0368] 3) 076 molecule:

[0369] ESAT6 was placed at the N terminus of the fusion protein, and CFP10 protein was placed at the C terminus of the fusion protein, and a linker (GSGGSG) sequence was used for connection therebetween to obtain ESAT6-CFP10 (SEQ ID NO: 53), and a linker, 4T, and 6His sequence were sequentially added to the C terminus, and a stop codon was added at the end. The obtained antigen molecule was named 076.

[0370] 4) 077 molecule:

[0371] The wild-type sequence of RV2660c was mutated at C66A to obtain RV2660c(C66A) (SEQ ID NO: 9). The mutated RV2660c(C66A) was placed at the N terminus of the fusion protein, and TB10.4 protein was placed at the C terminus of the fusion protein, and a linker sequence was used for connection therebetween to obtain RV2660c(C66A)-TB10.4 (SEQ ID NO: 33), and a linker, 4T, and 6His sequence were sequentially added to the C terminus, and a stop codon was added at the end. The obtained antigen molecule was named 077.

[0372] 5) 072M7 molecule:

[0373] In Phase 2, single point mutations or combined mutations were made to the residues at positions 127-129 of Ag85aΔN, obtaining a series of mutant Ag85aM1-M11. The C-terminus of the mutant was sequentially added with a linker, 4T and 6His, obtaining 072M1-M11, as shown in the following table:

[0374] Table 4: Mutants of 072 molecules

[0375] 6) 076M3 molecule:

[0376] In Phase 2, the PPE family protein Mtb39a was selected, and the L167-G391 fragment was obtained, obtaining Mtb39a(L167-G391) (SEQ ID NO: 3). Mtb39a(L167-G391) was placed at the N-terminus, middle or C-terminus of ESAT6 and CFP10, to construct three fusion proteins: Mtb39a(L167-G391)-ESAT6-CFP10, ESAT6-Mtb39a(L167-G391)-CFP10, ESAT6-CFP10-Mtb39a(L167-G391) (SEQ ID NO: 39), and the C-terminus was sequentially added with a linker, 4T and 6His sequences, and ended with a stop codon. The following antigen molecules were obtained: Mtb39a(L167-G391)-ESAT6-CFP10-4T (076M1), ESAT6-Mtb39a(L167-G391)-CFP10-4T (076M2), ESAT6-CFP10-Mtb39a(L167-G391)-4T (076M3).

[0377] 7) 099 molecule:

[0378] In Phase 2, the ESAT-6-like family antigens EsxC, EsxD, EsxV and EsxW were selected, and fused in the order of EsxC, EsxD, EsxV and EsxW from the N-terminus, with a linker used to connect EsxC, EsxD, EsxV, and no linker between EsxV and EsxW, directly connected, obtaining the fusion protein EsxC-EsxD-EsxV-EsxW (SEQ ID NO: 35). The C-terminus of the fusion protein was sequentially added with a linker, 4T and 6His sequences, and ended with a stop codon, and the obtained antigen molecule was named 099.

[0379] 8) 105 molecule:

[0380] In Phase2, antigen EspC and PE13 were added. Mutation of C48A was performed on EspC, obtaining EspC(C48A) (SEQ ID NO: 11). EspC(C48A) was expressed in fusion with PE13, in which EspC(C48A) was located at the N terminus and PE13 was located at the C terminus, and a linker was used in between, obtaining EspC(C48A)-PE13 (SEQ ID NO: 37). The C terminus of the fusion protein was sequentially added with a linker, 4T and 6His sequences, and ended with a stop codon at the end. The obtained antigen molecule was named 105.

[0381] 9) 107-2 molecule:

[0382] In Phase2, antigens Hrp1 and Mpt64 were added. Among them, the sequence CSPMALAS at the C terminus of wild-type Hrp1 was truncated, and the obtained mutant was named Hrp1M1 (SEQ ID NO: 13). Mutations of C16A, C17A, C29A and C41A were performed on wild-type Mpt64, obtaining the mutant Mpt64(C16, 17, 29, 41A), named Mpt64M1 (SEQ ID NO: 15). Further, Mpt64M1 was placed at the N terminus of Hrp1M1, and after fusion, Mpt64M1-Hrp1M1 (SEQ ID NO: 43) was obtained. The C terminus was sequentially added with a linker, 4T and 6His, and ended with a stop codon at the end. The obtained antigen molecule was named 107-2.

[0383] 10) 117-1 molecule:

[0384] In Phase2, the core antigen Ag85aΔN(127LGG129) of 072M7 was expressed in fusion with Mtb32a(S177A), in which Ag85aΔN(127LGG129) was located at the N terminus and Mtb32a(S177A) was located at the C terminus, and a linker (GSGGSG) was used in between, obtaining Ag85aΔN(127LGG129)-Mtb32a(S177A) (SEQ ID NO: 41). The C terminus of the fusion protein was sequentially added with a linker, 4T and 6His sequences, and ended with a stop codon at the end. The obtained antigen molecule was named 117-1.

[0385] 11) Control molecule M72-4T

[0386] Referring to the sequence of the M72 fusion protein in the patent (Patent No. CN 103249431B) published by GSK, 6His, 4T and linker are sequentially added at the N-terminus of the original sequence, and a stop codon is added at the end to obtain an amino acid sequence as shown in SEQ ID NO: 55.

[0387] 2. Recombinant expression of Mtb antigen molecules

[0388] The expression vectors of the antigen molecules shown in Tables 3-4 were prepared according to conventional techniques of molecular cloning, and the corresponding BL21(DE3) expression bacteria were obtained. After overnight culture at 37°C on LB agar plates (containing 50 μg / ml ampicillin), single colonies were picked into 10 ml of TB medium containing 50 μg / ml ampicillin, and incubated at 37°C, 220 rpm / min overnight.

[0389] The bacteria were inoculated into TB medium containing 50 μg / ml ampicillin at a ratio of 1 / 100, and incubated at 37°C, 220 rpm / min for 2-3 hours.

[0390] The OD600 of the bacteria was determined using a Nanodrop. When the OD value reached 0.6-0.8, the bacteria were transferred to a 18°C shaker to cool down, and IPTG was added for overnight induction for about 16 hours, with a final concentration of 500 μM.

[0391] The bacteria were collected and centrifuged at 6000g, 4°C for 15 minutes, and the supernatant was discarded.

[0392] The bacteria were again centrifuged at 6000g, 4°C for 3 minutes, and the culture medium in the supernatant was removed. The bacteria were stored in a -80°C freezer.

[0393] Those skilled in the art can adjust the relevant condition parameters according to conventional techniques of molecular cloning.

[0394] Example 2: Western blot identification of recombinant antigens

[0395] 1) The expression bacteria of the Mtb antigen molecules of Example 1 were resuspended with 20 ml of 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 buffer, and after ultrasonic disruption, centrifuged at 13000g / min, 4°C for 30 min. The supernatant and precipitate (20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 buffer suspension) were taken separately.

[0396] 2) Western blot identification was performed using LDS sample loading buffer (4x) with reducing agent DTT, heating at 70℃ for 5 min, cooling to room temperature, centrifuging at 10000 rpm for 20 s, vortex mixing, and finally loading 0.5 μg.

[0397] 3) The sample to be analyzed and the pre-stained protein molecular weight marker were loaded into a 4-12% Bis-Tris gel, and MES electrophoresis buffer was used, and the voltage was set to 150 V, and the electrophoresis lasted for about 60 min.

[0398] 4) The membrane transfer was performed using a Turbo instrument and corresponding reagents, the incubation was performed using an iBind instrument, an anti-his mouse monoclonal antibody and a goat anti-mouse secondary antibody coupled with AP enzyme, and then color development was performed using a color developing solution, and a GelDoc Go was used to take a picture.

[0399] The Western blot identification results are as follows (M in the figure represents a protein molecular marker): Fig. 1: lane 1 is the supernatant of 014 molecules, and lane 2 is the precipitate of 014 molecules; Fig. 2A: lane 1 is the supernatant of 072 molecules, lane 2 is the precipitate of 072 molecules, lane 3 is the supernatant of 076 molecules, lane 4 is the precipitate of 076 molecules, lane 5 is the supernatant of 077 molecules, and lane 6 is the precipitate of 077 molecules. Fig. 2B: lane 1 is the supernatant of 117-1 molecules, and lane 2 is the precipitate of 117-1 molecules. Fig. 3: lane 1 is the supernatant of 072M7 molecules, and lane 2 is the precipitate of 072M7 molecules; Fig. 4: lane 1 is the supernatant of 076M3 molecules, lane 2 is the precipitate of 076M3 molecules, lane 3 is the supernatant of 105 molecules, and lane 4 is the precipitate of 105 molecules; Fig. 5: lane 1 is the supernatant of 099 molecules, and lane 2 is the precipitate of 099 molecules.

[0400] In the above Western blot identification results, the supernatant band shows that the Mtb antigen molecules can be expressed in a soluble manner, and the precipitate band shows that the Mtb antigen molecules form inclusion bodies.

[0401] It can be seen that the Mtb antigen molecules screened by the present application can realize soluble expression and inclusion body expression, and the soluble expression amount is higher than the inclusion body expression amount, which lays a good foundation for large-scale production, simplification of production process and improvement of safety of Mtb vaccine.

[0402] Example 3: Purification of recombinant antigens, SDS-PAGE analysis of purified antigens

[0403] 1. The Mtb antigen molecules of Example 1 obtained by expression of Escherichia coli were purified by nickel column affinity chromatography and molecular sieve chromatography to obtain high-purity proteins. The specific steps are as follows: ​

[0404] 1) Pre-treatment before purification

[0405] The Mtb antigen molecule expression slurry (corresponding to 200 mL of expression broth) of Example 1 was resuspended with 20 mL of 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 buffer and subjected to ultrasonic disruption; after cell disruption, centrifugation was performed at 4°C, 13000 g / min for 30 min, the precipitate was discarded, and the supernatant was retained.

[0406] 2) Nickel ion affinity chromatography

[0407] Affinity purification was completed using a nickel ion affinity filler chromatography column. The column volume was 10 mL, the chromatography flow rate was 5 mL / min, and the sample volume was 70 mL.

[0408] Chromatography procedure: Ni-Bestarose Fast Flow, sterilization, capture buffer 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 equilibration column, sample, 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 solution washing, 20 mM Trsi-HCl, 150 Mm NaCl, 2% Triton–x100, pH 7.4 solution endotoxin washing, 20 mM imidazole + 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 buffer washing off impurities, 500 mM imidazole + 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 buffer elution to obtain affinity chromatography product.

[0409] 3) Molecular sieve SEC purification

[0410] Fine purification was performed using HiLoad 16 / 600 Superdex 200 pg, the molecular sieve column volume was 120 mL, and the sample volume of the affinity chromatography product of step 2) was controlled at about 4%.

[0411] Chromatography procedure: Superdex 200 pg; sterilization; equilibration buffer (20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4) equilibration column, sample, TBS solution washing, and collection of molecular sieve purification product.

[0412] 2, SDS-PAGE analysis of purified antigen

[0413] SDS-PAGE analysis of the molecular sieve SEC purified product. The sample to be analyzed is prepared with LDS sample loading buffer (4x) plus reducing agent DTT, heated at 70°C for 5 min, cooled to room temperature, centrifuged at 10000 rpm for 20 s, vortexed, and finally loaded with 5 μg. The sample to be analyzed and the non-pre-stained protein molecular weight marker are loaded into a 4-12% Bis-Tris gel, matched with MES running buffer, set at a voltage of 150 V, and electrophoresed for about 60 min. After electrophoresis, the gel is removed and placed in a clean container, and an appropriate amount of Coomassie brilliant blue staining solution is added to cover the gel. The gel is stained on a shaker for 2 h. After staining, the staining solution is poured off, and the gel is soaked to decolorize. The gel is continuously decolorized on a shaker until the gel background is completely decolorized. The gel is photographed using a GelDoc Go gel imager.

[0414] The SDS-PAGE identification results are as follows (M in the figure represents protein molecular marker): Figure 6: SDS-PAGE identification results of 014 molecules, lane 1 is 014 molecules before purification, and lanes 2-5 are 014 molecules after purification; Figure 7A: SDS-PAGE identification results of 072 molecules; Figure 7B: SDS-PAGE identification results of 117-1 molecules; Figure 8: SDS-PAGE identification results of 076 molecules; Figure 9: SDS-PAGE identification results of 077 molecules;

[0415] Figure 10: SDS-PAGE identification results of 072 and its mutants M1-M11, the expression amount M7>M6>M1>M9>M4>M5>M3>M2>M8>M11>M10>072;

[0416] Figure 11: SDS-PAGE identification results of 072M7 molecules;

[0417] Figure 12A-C: SDS-PAGE identification results of 076M1-M3 molecules, 076M1 is severely degraded or broken during expression, and the broken bands cannot be effectively separated by SEC after purification, and cannot be used for NPM binding experiments; 076M2 also appears to be broken during expression, and after binding with NPM, it precipitates during SEC separation and cannot be effectively separated; 076M3 peaks are correct, and after binding with NPM, it can be effectively separated using SEC. Therefore, 076M3 is selected as a candidate antigen molecule.

[0418] Figure 13: SDS-PAGE identification results of 099 molecules;

[0419] Figure 14: A is SDS-PAGE identification result of wild type 105 molecule (DTT+), lane 1-4: EspC-PE13-4T before mutation, no dimer; B is SDS-PAGE identification result of 105 molecule (DTT+), lane 1-5: EspC(C48A)-PE13-4T, no dimer; C is SDS-PAGE identification result of 105 and WT 105 under DTT- / DTT+ conditions after mutation, lane 1: 105 molecule EspC(C48A)-PE13-4T (DTT-), lane 2: 105 molecule EspC(C48A)-PE13-4T (DTT+), lane 3: wild type WT 105 molecule EspC-PE13-4T (DTT-), lane 4: wild type WT 105 molecule EspC-PE13-4T (DTT+). It can be seen that the mutation of C48A to wild type EspC can avoid the formation of dimer in EspC(C48A)-PE13-4T, so that EspC(C48A)-PE13-4T remains a soluble monomer form (single band under SDS-PAGE analysis in reduced and non-reduced state after mutation).

[0420] Figure 15: SDS-PAGE identification result of 077 molecule before and after mutation, lane 1: RV2660C-TB10.4-4T before mutation (DTT+), lane 2: RV2660C-TB10.4-4T before mutation (DTT-), lane 3: RV2660C-TB10.4-4T after mutation (DTT+), lane 4: RV2660C-TB10.4-4T after mutation (DTT-). It is accidentally found through experiment that the mutation of C66A to wild type sequence of RV2660c can avoid the formation of dimer in RV2660c-TB10.4-4T, so that RV2660c(C66A)-TB10.4-4T remains a soluble monomer form (single band under SDS-PAGE analysis in reduced and non-reduced state after mutation).

[0421] The above SDS-PAGE results show that the Mtb antigen molecules of Example 1 can be purified to obtain, and the purity can reach more than 90%.

[0422] Example 4: Expression and purification preparation of M72 fusion protein

[0423] 1) Pre-treatment before purification

[0424] Following the method in Example 1, M72-4T protein was expressed and bacterial sludge was collected. The M72-4T expression bacterial sludge (corresponding to 200 mL of expression bacterial solution) was resuspended in 20 mL of 20 mM Trsi-HCl, 150 Mm NaCl, and pH 7.4 buffer and sonicated. After cell disruption, the cells were centrifuged at 13000 g / min for 30 min at 4 °C, the supernatant was discarded, and the precipitate was retained. The precipitate was resuspended and dissolved in 20 mM Trsi-HCl, 150 Mm NaCl, 8 M Urea, and pH 7.4, and centrifuged at 13000 g / min for 30 min at 4 °C, and the supernatant was retained.

[0425] 2) Nickel ion affinity chromatography

[0426] Affinity purification was performed using a nickel ion affinity chromatography column. The column volume was 10 mL, the flow rate was 5 mL / min, and 70 mL of sample was loaded. Chromatographic procedure: Ni-Bestarose Fast Flow, sterilization, column equilibration with capture buffer 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH 7.4, sample loading, washing with 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH 7.4 solution, washing with 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, 2% Triton-X100, pH 7.4 solution to remove endotoxin, washing with 20mM imidazole + 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH 7.4 buffer to remove contaminating proteins, eluting M72 antigen components with 500mM imidazole + 220mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH 7.4 buffer.

[0427] 3) Membrane-encapsulated ultrafiltration refolding

[0428] Using a 10 kDa ultrafiltration membrane, the M72-4T antigen purified by nickel ion affinity chromatography was concentrated by tangential flow ultrafiltration. Then, 20 mM Trsi-HCl, 150 Mm NaCl, and pH 7.4 buffer were continuously added to the concentrated protein solution to gradually remove Urea from the M72-4T antigen, and the M72 antigen was completely renatured in 20 mM Trsi-HCl, 150 Mm NaCl, and pH 7.4 buffer.

[0429] 4) Molecular sieve purification

[0430] The refolded M72-4T antigen was purified using a HiLoad 16 / 600 Superdex 200pg filter with a molecular sieve column volume of 120mL. The loading volume of the M72-4T affinity purified sample was controlled at approximately 4%.

[0431] Chromatography procedure: Superdex 200pg; sterilization; equilibration buffer (20mM Tris-HCl, 150Mm NaCl, pH7.4) equilibration column, sample loading, 20mM Tris-HCl, 150Mm NaCl, pH7.4 solution washing, M72-4T antigen component collection. Figure 16 shows the results of M72-4T protein molecular sieve separation and purification (M represents protein molecular marker, lanes 1-8 represent M72-4T protein).

[0432] Example 5: Construction, expression and purification of the gene encoding the peptide 2-NPM fusion protein

[0433] The E. coli expression host codon optimization was performed on the NPM-4C protein sequence, and gene synthesis and subcloning were performed, and the coding gene of the fusion protein was constructed into pET30a, expressed in E. coli BL21 (DE3), and after harvesting the bacterial cells, the target protein needed to be released by high-pressure homogenization crushing and liquid clarification, the main purpose was to remove bacterial debris and impurity proteins. The amino acid of NPM-4C is shown as SEQ ID NO: 52, and the nucleotide of NPM-4C is shown as SEQ ID NO: 65.

[0434] The protein purification was performed according to the pre-treatment before chromatography and chromatography purification method in the patents CN114395015B or CN117462666B.

[0435] It was found through purity detection that after refining by chromatography medium combination, the purity of the product could reach more than 99.0%, and the SDS-PAGE analysis results of NPM-4C protein Octyl Bestarose 4FF separation and purification could be seen in Figure 17 (M: protein molecular marker; lanes 1-2: NPM-4C protein).

[0436] Example 6: Binding of Mtb antigen molecules and NPM-4C, purification of the binding product, and particle characterization

[0437] 1. Binding of Mtb antigen and NPM-4C

[0438] The high purity Mtb antigens obtained from molecular sieve purification in Example 3 were mixed with NPM-4C according to a BCA protein concentration ratio of 6:1, and 50% sucrose mother liquor was added to a final concentration of about 25% sucrose, and 10% of the total reaction volume of 1M Tris-HCl Mtb mother liquor was added to stabilize the pH. The binding reaction was carried out at 22°C, and the reaction time was 48 hours. As an example, the Mtb32a-NPM binding system can be specifically: Mtb32a-4T (1 mg / mL) 6 mL, NPM-4C (1 mg / mL) 1 mL, 50% sucrose 8.75 mL, 1M Tris-HCl 7.4 1.75 mL, total volume 17.5 mL.

[0439] 2. Purification of the binding product

[0440] Cytiva HiLoad 16 / 600 Superdex 200pg (column volume 120 mL) or Cytiva Superdex 200Increase 10 / 300 GL (column volume 23 mL) was used for purification of Mtb antigen-NPM binding products, and unbound Mtb antigens to NPM-4C were separated and removed. If the molecular sieve HiLoad 16 / 600 Superdex 200pg is used, the loading amount of Mtb antigen-NPM binding sample is controlled at about 3% to 6%; if the molecular sieve Superdex 200Increase 10 / 300 GL is used, the loading amount of Mtb antigen-NPM binding sample is controlled at 0.5 mL to 1 mL.

[0441] Chromatography procedure: Superdex 200pg or Superdex 200Increase, sterilization, equilibration buffer 12.5% sucrose TBS solution (20mM Tris-HCl, 150mM NaCl, 12.5% sucrose Mtb) equilibration column, loading, 12.5% sucrose TBS solution washing, collection of Mtb antigen-NPM components, SDS-PAGE analysis according to the method of SDS-PAGE analysis of purified antigens in Example 3.

[0442] 3. The results of Mtb antigen-NPM SDS-PAGE identification are shown in Figures 18-25:

[0443] Figure 18: SDS-PAGE identification results of 014-NPM; Figure 19A: SDS-PAGE identification results of 072-NPM; Figure 19B: SDS-PAGE identification results of 117-1-NPM; Figure 20: SDS-PAGE identification results of 076-NPM; Figure 21: SDS-PAGE identification results of 077-NPM; Figure 22: SDS-PAGE identification results of 072M7-NPM; Figure 23: SDS-PAGE identification results of 076M3-NPM; Figure 24: SDS-PAGE identification results of 099-NPM; Figure 25: SDS-PAGE identification results of 105-NPM.

[0444] The Mtb antigen-NPM purified by molecular sieve was subjected to SDS-PAGE analysis, and the results showed that the purity could reach more than 90%, and the binding rate measured by SDS-PAGE gray method was 82.5%.

[0445] 4. Particle characterization

[0446] 1) TEM detection

[0447] The Mtb antigen-NPM particles prepared in the above "purification of the binding product" were subjected to negative staining sample preparation using the floating method. A 400-mesh support film was selected, and the support film was subjected to hydrophilic treatment in advance. Deionized water and 2% uranyl formate negative staining solution were prepared. 3uL of prepared protein sample (0.1mg / ml) was directly dropped on one side of the support film; after timing for 1 minute, the excess liquid was absorbed from the edge of the support film with a clean filter paper; after drying, deionized water was dropped and quickly rinsed twice in sequence; then 5ul of negative staining solution was used for rinsing once, and finally 5ul of negative staining solution was added and timed for 1 minute. After the end, the support film was clamped with tweezers and the staining solution was absorbed with a filter paper, leaving a thin layer to dry naturally, and then detected. The detection was carried out under a transmission electron microscope (FERRITINI Tecnai Spirit) at 120kV. The overall staining of the support film was observed under a low power, and the thickness of the hole was selected for observation. Under a high power, the appropriate area was selected for photographing and saving.

[0448] 2) DLS detection

[0449] The concentration of the purified Mtb antigen-NPM particle product was diluted to 0.15mg / mL. The Zetasizer Lab instrument was used, and ≥1mL of the sample to be tested was injected into the sample cell. The instrument was run for detection, and the data were analyzed in combination with Z-Average (nm) and Polydispersity Index (PI) values, as well as Size Distribution by Intensity / Volume distribution curve, and the results were reported.

[0450] 5. Results and Analysis:

[0451] Electron micrographs of Mtb antigen-NPM particles (0.1 mg / mL, 18500×) show that the particles are uniformly distributed and there is no aggregation, as shown in Figures 26 to 33.

[0452] Figure 26: Electron microscopy results of negative staining of 014-NPM nanoparticles; Figure 27A: Electron microscopy results of negative staining of 072-NPM nanoparticles; Figure 27B: Electron microscopy results of negative staining of 117-1-NPM nanoparticles; Figure 28: Electron microscopy results of negative staining of 076-NPM nanoparticles; Figure 29: Electron microscopy results of negative staining of 077-NPM nanoparticles.

[0453] Figure 30: Electron microscopy results of negative staining of 072M7-NPM nanoparticles; Figure 31: Electron microscopy results of negative staining of 076M3-NPM nanoparticles; Figure 32: Electron microscopy results of negative staining of 099-NPM nanoparticles; Figure 33: Electron microscopy results of negative staining of 105-NPM nanoparticles.

[0454] Figures 34A-34H show the particle size analysis results of 014-NPM, 072-NPM, 076-NPM, 077-NPM, 076M3-NPM, 072M7-NPM, 099-NPM, and 117-1-NPM. The results show that the peaks of the Mtb antigen-NPM particles have good overlap and uniform particle size distribution.

[0455] In Figure 35, A represents NPM nanoparticles prepared before the RV2660c-TB10.4 mutation; B represents NPM nanoparticles prepared after the RV2660c-TB10.4 mutation. The mutation resulted in more uniform RV2660c-TB10.4-NPM nanoparticles (Z-Average and Polydispersity Index values ​​changed from 77.3 and 0.23 before the mutation to 39.8 and 0.14 after the mutation, respectively). In Figure 36, A represents NPM nanoparticles prepared before the 10⁵ molecule mutation; B represents NPM nanoparticles prepared with 10⁵ molecules. The mutation made the 105-NPM nanoparticles more uniform (Z-Average and Polydispersity Index values ​​changed from 61.35 and 0.40 before the mutation to 49.25 and 0.18 after the mutation, respectively);

[0456] DLS results show that the particle size of Mtb antigen-NPM is shown in Table 5.

[0457] Table 5: Particle antigen Z-Average (nm) and Polydispersity Index (PI) values

[0458] Example 7: Preparation of vaccines

[0459] I. Test vaccine antigens and adjuvants

[0460] 1. Test vaccine antigen stocks

[0461] Test vaccine protein stocks included Mtb antigen molecules prepared in Example 1, M72-4T control antigen prepared in Example 4, and Mtb antigen-NPM nanoparticle prepared in Example 6.

[0462] 2. Test vaccine adjuvants

[0463] Adjuvant 2 (per 0.5 ml): 25 pg Quillaja saponaria saponin QS-21, 25 pg 3D-MPL (3-0-deacylated-4'-monophosphoryl lipid A), 0.5 mg DOPC (dioleoyl phosphatidyl choline), 0.125 mg cholesterol.

[0464] Preparation method: A sterol-containing lipid suspension was prepared according to the method described in WO 96 / 33739, the lipid suspension was microfluidized until the liposome size was reduced to 100 nm; the carrier was added, or an organic solution containing 3D-MPL was added, and QS21 was added.

[0465] 3. Test vaccine preparation

[0466] 1) Mtb antigen molecule mixed vaccine

[0467] Depending on the different Mtb antigen molecule combinations, aliquots (100 pL) for injection were prepared by mixing the Mtb antigen molecule-containing buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) with 50 pL of adjuvant.

[0468] 2) Mtb antigen-NPM nanoparticle mixed vaccine

[0469] Depending on the different Mtb antigen-NPM nanoparticle combinations, aliquots (100 pL) for injection were prepared by mixing the nanoparticle-containing buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) with 50 pL of adjuvant.

[0470] 3) Single Mtb antigen-NPM nanoparticle vaccine

[0471] Depending on the different immunization groups, aliquots for injection (100 μL) were prepared by mixing the buffer containing nanoparticles (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) with 50 μL of adjuvant.

[0472] 4) M72 control vaccine

[0473] In POC-1, aliquots for injection (100 μL) were prepared by mixing the buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) of 0.8 μg, 8 μg M72-4T protein mixture with 50 μL of adjuvant 2, respectively. In POC-2, the antigen dose of M72 control was consistent with the specific experimental group antigen dose.

[0474] Example 8: Vaccine immunoprotection experiment-1 (POC-1)

[0475] 1. Experimental animals and grouping

[0476] 4-6 weeks old female C57BL / 6 mice were selected from Vantianlihua. After quarantine, the mice were metal ear tagged and randomly grouped according to body weight, and were allowed to freely eat and drink. Six candidate vaccine immunization groups (12 mice per group) were set up, including M72-4T high dose group (12 mice), M72-4T low dose group (12 mice), BCG control group (12 mice), and normal saline group (15 mice). The animals were raised in SPF standard animal rooms, and were provided with SPF animal special sterile feed and sterilized deionized water. The feeding room was provided with 12 h alternating light for day and night, temperature 21±2℃, and humidity 30-70%.

[0477] 2. Immunization and challenge

[0478] The vaccine groups (vac-1~vac-6) and M72-4T low dose group (Control-A) were single 100 μL (0.8 μg), M72-4T high dose group (Control-B) was single 100 μL (8 μg), immunized 2 times with an interval of 3 weeks, the normal saline group was immunized with the same volume of normal saline, and the BCG group (Control-C) mice were subcutaneously injected (subcutaneous injection) with 5×10 4 CFU / mouse, 100 μL / mouse, 1 time. The rest of the groups were intramuscularly injected (intramuscular injection).

[0479] Four weeks after the last immunization of the mice, aerosol challenge was performed, and four weeks after the challenge, dissection was performed, and pathological sections of the lungs were prepared. The lung grinding fluid was used for bacterial load calculation. The specific grouping and immunization scheme, challenge scheme are shown in Tables 5-7:

[0480] Table 5: Schedule of Phase 1 immunization and challenge

[0481] Table 6: Schedule of immunization detection experiments

[0482] Table 7: Phase 1 grouping and protein component, dose, adjuvant control table

[0483] 3. Detection of specific IgG after immunization

[0484] Before and after the first immunization, blood samples were collected on day 20 and day 42. The whole blood collected in centrifuge tubes was allowed to stand at room temperature for 2 hours or in the refrigerator at 4°C overnight. After the blood coagulated and the blood clot contracted, it was centrifuged at 4000 rpm for 10 minutes. The supernatant was transferred to a clean centrifuge tube and stored at -20°C.

[0485] The 96-well enzyme-labeled plates (Thermo Fisher Scientific) were coated with 014, 072, 076, and 077 proteins (1 μg / mL) at 100 ng / 50 μL / well at 4°C overnight, then washed twice with PBST (0.05% Tween 20), and then 200 μL / well of blocking solution (Thermo Fisher Scientific) was added at room temperature (25°C ± 3°C) for 1-4 hours, then washed twice, then 1:5000 diluted HRP-IgG working solution was added, 50 μL / well. After incubation at room temperature for 1 hour, wash 6 times, then add 100 μL of color developing solution to each well, develop at room temperature for 10 minutes, then add 100 μL of 1M HCL to each well to stop the reaction. Set the main wavelength to 450 nm and the reference wavelength to 620 nm on the enzyme-labeled instrument, and the sample absorbance value = OD450-OD620. The determination was completed within 5 minutes after termination, and the detection results were analyzed.

[0486] Data processing:

[0487] The data was reliable if the following conditions were met: control serum OD value ± 0.2, sample starting concentration corresponding OD value < 3.0, blank well corresponding OD value less than 0.1, and replicate well (response value) coefficient of variation less than 20%.

[0488] The raw data of the samples were moved to the "excel Endpoint ELISA template" to calculate the antibody titer. The results were analyzed using Graphpad Prism 9.1.2 software. Unpaired test or One-Way ANOVA was used to analyze the differences, and when P<0.05, the two groups of data were defined as having significant differences.

[0489] The detection results are as follows:

[0490] Figures 37A-D are, in sequence, the corresponding total IgG antibody levels detected by 072, 076, 077, 014 antigens as coating antigens for different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, saline) immunized serum, and Figure 37E is the corresponding IgG antibody level detected by M72 (0.8 μg immunized group) antigen.

[0491] The results show that the single Mtb nanoparticle antigen vaccine (vac-3 / 4 / 5 / 6) of the application, the Mtb recombinant antigen mixed vaccine (vac-2) and the Mtb nanoparticle antigen mixed vaccine (vac-1) of the application can all induce the production of IgG, and it can be seen that the vaccines of the application all have good immunogenicity. Among them, the corresponding IgG antibody levels of vac-1 and the single Mtb nanoparticle antigen vaccine group are much higher than the IgG antibody level of vac-2, which shows that the nanoparticle vaccine of the application can significantly improve the immunogenicity of Mtb antigens.

[0492] 4. Cytokine ELISPOT

[0493] 1) Commercial 96-well filter plates were coated with IFN-γ, TNFα, IL-4, IL-2 monoclonal antibodies and blocked. Three weeks after the last immunization, 6 mice per group were treated, the spleen was collected, the spleen cells were isolated, and 2.0 x 10 5 cells / well were inoculated into 96-well culture plates;

[0494] 2) Use separate culture medium and culture medium containing 014 (10 μg / mL), 072 (10 μg / mL), 076 (10 μg / mL), 077 (10 μg / mL), M72-4T (10 μg / mL), PPD (10 μg / mL), Con A (3 μg / mL), and incubate in a 37°C CO2 incubator containing 5% for 48 hours;

[0495] 3) Wash the plate wells with PBS, add biotinylated mouse IFN-γ, TNFα, IL-4, IL-2 secondary antibodies, and incubate at room temperature for 2 hours. According to the instructions of the commercial kit, the filter membrane is developed by using the substrate;

[0496] 4) After drying the plate, use an automatic ELISPOT plate reader to count the spots and analyze.

[0497] The results were analyzed using Graphpad Prism 9.1.2 software, and Unpaired test or One-Way ANOVA was used to analyze the differences. When P<0.05, the data of two groups were defined as having significant differences.

[0498] The results are as follows:

[0499] Figures 38A-D are, in turn, corresponding IFN-γ, IL-2, TNFα and IL-4 levels of the different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, saline) detected by the corresponding 072 antigen as a specific stimulating antigen; Figures 38E-H are, in turn, corresponding IFN-γ, IL-2, TNFα and IL-4 levels of the different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, saline) detected by the corresponding 076 antigen as a specific stimulating antigen.

[0500] The results show that the corresponding IFN-γ, IL-2 and TNFα levels of the vac-1 and single Mtb nanoparticle antigen vaccine groups stimulated by 072 and 076 are higher than the IFN-γ, IL-2 and TNFα levels of the vac-2, and the IFN-γ, IL-2, TNFα levels of each group are significantly higher than the IL-4 level, indicating that both 072 and 076 can induce a Th1 -type dominant cellular immune response.

[0501] Figures 39A-D are, in turn, corresponding IFN-γ, IL-2, TNFα and IL-4 levels of the different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, saline) detected by the corresponding 077 antigen as a specific stimulating antigen; Figures 39E-H are, in turn, corresponding IFN-γ, IL-2, TNFα and IL-4 levels of the different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, saline) detected by the corresponding 014 antigen as a specific stimulating antigen.

[0502] The results show that the corresponding IFN-γ, IL-2 and TNFα levels of the vac-1 and single Mtb nanoparticle antigen vaccine groups stimulated by 077 and 014 are higher than the IFN-γ, IL-2 and TNFα levels of the vac-2, and the IFN-γ, IL-2, TNFα levels of each group are significantly higher than the IL-4, indicating that both 077 and 014 can induce a Th1 -type dominant cellular immune response.

[0503] Figure 40 shows that the corresponding IFN-γ, IL-2, TNFα levels of the M72 group (0.8 μg immunization group) are significantly higher than the IL-4, indicating that the prepared M72 vaccine can induce a Th1 -type cellular immune response after immunizing mice, which is consistent with the literature reports.

[0504] 5. Challenge protection result statistics

[0505] Four weeks after the last immunization, mice were challenged with aerosol exposure to M. tuberculosis H37Rv strain, the UW-madison aerosol exposure chamber was calibrated to deliver 500-1000 CFU to the lungs, four weeks later, mice were euthanized, lung and spleen homogenates were prepared using PBS / Tween-80 (0.05%) grinding, the homogenate of single intact organ was gradient diluted and inoculated on Middlebrook 7H11 Bacto agar medium, the bacterial colonies were calculated after incubation at 37°C for 2-4 weeks under humidified, 5% CO2. The final data was expressed as the average Logio of bacteria, the Logio reduction (difference) = Logio CFU of saline treatment group - Logio CFU of vaccine treatment group.

[0506] 8. Pathological section analysis

[0507] Four weeks after the challenge, mice were euthanized, lung tissues were collected, the tissues were fixed using formaldehyde solution and HA staining, pathological sections were prepared and analyzed.

[0508] 9. Results of immune evaluation

[0509] The statistical results of challenge protection showed that, except for vac-6, vac-1-vac-5 could reduce the bacterial load in the lungs to different extents, compared with the saline group. The protection effect of vac-1 was the best, which was better than that of vac-2, and was better than that of Control-A, Control-B and Control-C (i.e. better than M72-4T and BCG), as shown in Figure 41.

[0510] Example 9: Vaccine immune protection experiment-2 (POC-2)

[0511] 1. Experimental animals and grouping

[0512] Four to six-week-old female C57BL / 6 mice were selected from Vantian Li Hua. Candidate vaccine groups (12 mice per group) were set up, M72-4T control group (Control-A, 12 mice), BCG control group (Control-B, 12 mice), and saline group (15 mice). The animals were raised in SPF standard animal rooms, provided with SPF animal special sterile feed and sterilized deionized water, the feeding room was alternately lighted for 12 h, the temperature was 21±2°C, and the humidity was 30-70%.

[0513] 2. Immunization and challenge

[0514] Vaccine group and M72-4T group (Control-A) single 100 μL (0.8 μg), immunized 2 times, interval 3 weeks, saline group immunized with same volume of saline, BCG group mice subcutaneously injected (subcutaneously) BCG (Control-B) bacteria 5 x 10 4 CFU / each, 100 μL / each, immunized 1 time, the rest of the groups were intramuscularly injected (intramuscularly).

[0515] Four weeks after the last immunization of mice, aerosol challenge was carried out, four weeks after challenge, dissection was carried out, pathological section of lung was prepared, and the grinding fluid of lung was used for bacterial load calculation, the immunodetection experiment schedule was the same as Example 8. The specific grouping and immunization scheme, challenge scheme are shown in Tables 8-11:

[0516] Table 8: Immunization and challenge schedule

[0517] Table 9: Antigen component, dose, adjuvant control table of immunization experiment 1

[0518] Table 10: Antigen component, dose, adjuvant control table of immunization experiment 2

[0519] Table 11: Antigen component, dose, adjuvant control table of immunization experiment 3

[0520] Table 12: Antigen component, dose, adjuvant control table of immunization experiment 4

[0521] 3. Detection of specific IgG after immunization

[0522] Blood was collected before and after the first immunization D20, D42, whole blood was collected in centrifuge tubes and left at room temperature for 2 h or in the refrigerator at 4°C overnight. After the blood coagulated and the blood clot contracted, it was centrifuged at 4000 rpm for 10 min. The supernatant was transferred to a clean centrifuge tube and stored at -20°C.

[0523] Respectively, the Mtb proteins (076M3, 117-1, 099, 105, 072M7) in the vaccine group, M72-4T (concentration of 1 μg / mL) were coated on 96-well enzyme-labeled plates (Thermo Fisher Scientific), 100 ng / 50 μL / well, coated at 4°C overnight, then washed twice with PBST (0.05% Tween 20), then added blocking solution (Thermo Fisher Scientific), 200 μL / well, blocked at room temperature (25°C±3°C) for 1-4 h, then washed twice, then added diluted immune serum, incubated at room temperature for 1 h, washed 4 times, then added 1:5000 diluted corresponding HRP-IgG working solution, 50 μL / well. Incubate at room temperature for 1 h, wash 6 times, then add 100 μL color developing solution to each well, develop at room temperature for 10 min, then add 100 μL 1M HCL to each well to stop. Set the main wavelength to 450 nm and the reference wavelength to 620 nm on the enzyme-labeled instrument, and the sample absorbance value = OD450-OD620. The determination is completed within 5 min after termination, and the detection results are analyzed.

[0524] Data processing:

[0525] The data is reliable if the following conditions are met:

[0526] The control serum OD value is ±0.2, the sample starting concentration corresponds to an OD value of <3.0, the blank hole corresponds to an OD value of less than 0.1, and the duplicate hole (response value) coefficient of variation should be less than 20%.

[0527] The sample raw data is moved to "excel Endpoint ELISA template" to calculate the antibody titer. When calculating, the cut-off value is 0.15. The results are analyzed using Graphpad Prism 9.1.2 software. Differences are analyzed using Unpaired test or One-Way ANOVA, and when P<0.05, two groups of data are defined as having significant differences.

[0528] 4. Cytokine ELISPOT

[0529] 1) Coated with IFN-γ, TNFα, IL-4, IL-2 monoclonal antibodies and blocked the commercial 96-well filter plate, 3 weeks after the last immunization, 6 mice per group, collect the spleen, isolate the spleen cells, inoculate 2.0×10 5 cells / well into a 96-well culture plate;

[0530] 2) Incubate the plates at 37°C in a 5% CO2 incubator for 48 hours using culture medium, culture medium containing 072M7 (10 pg / mL), 076M3 (10 pg / mL), 117-1 (10 pg / mL), 105 (10 pg / mL), 099 (10 pg / mL), M72-4T (10 pg / mL), PPD (10 pg / mL), Con A (3 pg / mL);

[0531] 3) Wash the plate wells with PBS, add biotinylated mouse IFN-g and IL-2 secondary antibodies, incubate at room temperature for 2 hours, and develop the filter membrane using the substrate according to the instructions of the commercial kit;

[0532] 4) After drying the plate, use an automatic ELISPOT plate reader to count the spots and analyze them

[0533] The results were analyzed using Graphpad Prism 9.1.2 software, and differences were analyzed using Unpaired test or One-Way ANOVA, and two groups of data were defined as having significant differences when P < 0.05.

[0534] 5, Flow cytometry detection of T cell immune response

[0535] 1) Isolation of splenocytes and antigen stimulation

[0536] At 3 weeks after the last immunization, the mouse splenocytes were isolated, the culture medium was discarded, and 1.0 x 106 cells per well were inoculated in a 96-well culture plate. The vaccine group was stimulated with the mixture of the corresponding antigens (2 pg / mL each) (e.g., vac-10 was stimulated with a mixture of 076-M3 and 117-1), to evaluate the total cytokine response; the M72 group was stimulated with M72 antigen (2 pg / mL).

[0537] 2) Dead cell staining and Fc receptor blocking

[0538] During the stimulation process, CD28 and CD49d costimulatory antibodies were added, and after 6 hours of incubation, Brefeldin A was added for continued incubation for 4 hours to block protein secretion. After incubation, the cells were washed with PBS, and Fixable Viability Stain and Fc Block were added, and incubated at room temperature for 15 minutes to exclude dead cells and block Fc receptor-mediated non-specific binding.

[0539] 3) Surface and intracellular staining

[0540] After washing the cells with PBS staining buffer containing 2% fetal bovine serum (FBS) and 0.1% sodium azide (NaN3), surface antibodies (PE-Cyanine 7-labeled anti-CD3, PE-labeled anti-CD4, PerCP-Cyanine 5.5-labeled anti-CD8) were added and incubated for 15 minutes. Subsequently, the cells were treated using a Fixation / Permeabilization Kit, and washed twice with Perm / Wash buffer, and then stained with intracellular antibodies (APC-labeled anti-IFN-γ, FITC-labeled anti-IL-2, PE-labeled anti-IL-4, R718-labeled anti-TNF-α, all diluted 1:50).

[0541] 4) Flow detection and data analysis

[0542] After staining, the cells were washed and resuspended, and detected using a flow cytometer, and the data was analyzed by CytExpert software. The experimental results were expressed as the frequency of positive cells producing IFN-γ, TNF-α or IL-2 in CD4+ and CD8+ T cells, and the corresponding background signal was subtracted.

[0543] 6, Challenge protection result statistics

[0544] Four weeks after the last immunization, the mice were challenged by exposure to a low-dose aerosol of Mycobacterium tuberculosis H37Rv strain, and the UW-madison aerosol exposure chamber was calibrated to deliver 50-100 CFU to the lungs. Four weeks later, the mice were euthanized, and lung homogenates were prepared using PBS / Tween-80 (0.05%) grinding. The homogenate of a single intact organ was gradient diluted and inoculated onto Middlebrook 7H11 Bacto agar medium, and the bacterial colonies were counted after incubation at 37°C under humidified, 5% CO2 conditions for 2-4 weeks. The final data was expressed as the average Log10 of bacteria ± SD, and the Log10 reduction (difference) of CFU = Log10 CFU of the saline treatment group - Log10 CFU of the vaccine treatment group.

[0545] 7, Pathological section analysis

[0546] Four weeks after the challenge, the mice were euthanized, and the lung tissues of the mice were collected, and the tissues were fixed using a formaldehyde solution, stained with HA, and prepared into pathological sections and analyzed.

[0547] 8, Immune evaluation results:

[0548] The results of specific IgG detection showed that the antigens in vac-7-vac-17 groups could all generate high levels of specific antibodies (Fig. 42A-B, Fig. 46A-B, Fig. 49A), and the ELISPOT results showed that the antigens covered in vac-7-vac-17 groups could all stimulate lymphocytes to produce high levels of IFN-γ and IL-2 (Fig. 43A-D, Fig. 47A-D, Fig. 50A-B), indicating that high levels of Th1 type T cell immune responses could be induced, which is consistent with the immune bias required for tuberculosis vaccines. Combined with the cytokine levels in Example 8, it can also be estimated that the total levels of IFN-γ and IL-2 of vac-17 are higher than those of M72. In addition, the ICS detection results showed that, compared with M72, vac-10 could induce more CD4 + T cells expressing IFN-γ, IL-2 and TNF-α (Fig. 44), indicating that vac-10 is superior to M72 in stimulating CD4 + T cells to prevent or treat the cytokines required by Mtb.

[0549] The statistical results of challenge protection in Fig. 45, Fig. 48 and Fig. 51 showed that, relative to the saline group, vac-7-vac-17 could all reduce the bacterial load in the lungs to different extents, and relative to the M72 and BCG groups, they could all significantly reduce the bacterial load in the lungs, and had better protective effects than M72 and BCG.

[0550] The above results showed that the VLP form antigens could enhance immune protection, and the nanoparticle antigens covered by vac-1-vac-17 could effectively stimulate the body to produce immune protection, and these vaccines could be combined in different ways as needed, and could all effectively prevent the infection of Mycobacterium tuberculosis H37Rv strain in mice, and had great potential to develop into TB subunit vaccines. The above recombinant nanoparticle Mycobacterium tuberculosis subunit vaccines had high application value.

Claims

1. One or more isolated Mycobacterium tuberculosis (Mtb) antigens selected from any one of the Mtb antigens or immunogenic fragments thereof shown in the following: Mtb32a, Mtb39a, Ag85a, RV2660c, EspC, Hrp1, Mpt64, ESAT6, CFP10, TB10.4, PE13, EsxC, EsxD, EsxV and EsxW.

2. The Mtb antigen according to claim 1, having any one or more of the following (1) to (7) features: (1) Mtb32a comprises a mutation of S177A; (2) Mtb39a lacks at least 166 amino acid residues at the N-terminus; (3) Ag85a lacks at least 42 amino acid residues at the N-terminus, optionally further comprising one or more amino acid mutations; (4) RV2660c comprises a mutation of C66A; (5) EspC comprises a mutation of C48A; (6) Hrp1 lacks at least 8 amino acid residues at the C-terminus; (7) Mpt64 mutant comprises a mutation of C16A, C17A, C29A and / or C41A.

3. The Mtb antigen according to claim 1 or 2, having any one or more of the following (1) to (15) features: (1) the amino acid sequence of Mtb32a has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 1; preferably the amino acid sequence of Mtb32a is as shown in SEQ ID NO: 1; (2) the amino acid sequence of Mtb39a has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 3; preferably the amino acid sequence of Mtb39a is as shown in SEQ ID NO: 3; (3) the amino acid sequence of Ag85a has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 5 or 7; preferably the amino acid sequence of Ag85a is as shown in SEQ ID NO: 5 or 7; (4) the amino acid sequence of RV2660c has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 9; preferably the amino acid sequence of RV2660c is as shown in SEQ ID NO: 9; (5) the amino acid sequence of EspC has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 11; preferably the amino acid sequence of EspC is as shown in SEQ ID NO: 11; (6) the amino acid sequence of Hrp1 has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 13; preferably the amino acid sequence of Hrp1 is as shown in SEQ ID NO: 13; (7) the amino acid sequence of Mpt64 has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 15; preferably the amino acid sequence of Mpt64 is as shown in SEQ ID NO: 15; (8) the amino acid sequence of ESAT6 has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 17; preferably the amino acid sequence of ESAT6 is as shown in SEQ ID NO: 17; (9) the amino acid sequence of CFP10 has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 19; preferably the amino acid sequence of CFP10 is as shown in SEQ ID NO: 19; (10) the amino acid sequence of TB10.4 has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 21; preferably the amino acid sequence of TB10.4 is as shown in SEQ ID NO: 21; (11) the amino acid sequence of PE13 has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 23; preferably the amino acid sequence of PE13 is as shown in SEQ ID NO: 23; (12) the amino acid sequence of EsxC has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 25; preferably the amino acid sequence of EsxC is as shown in SEQ ID NO: 25; (13) the amino acid sequence of EsxD has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 27; preferably the amino acid sequence of EsxD is as shown in SEQ ID NO: 27; (14) the amino acid sequence of EsxV has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 29; preferably the amino acid sequence of EsxV is as shown in SEQ ID NO: 29; and (15) the amino acid sequence of EsxW has 80% or more, 85% or more, 90% or more, 95% or more or 99% or more identity to SEQ ID NO: 31; preferably the amino acid sequence of EsxW is as shown in SEQ ID NO:

31. ​ ​ ​ ​ ​ ​ ​ (7) the amino acid sequence of Mpt64 is 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identical to SEQ ID NO: 15; preferably, the amino acid sequence of the Mpt64 mutant is set forth in SEQ ID NO: 15; (8) ESAT6 has an amino acid sequence set forth in SEQ ID NO: 17; (9) CFP10 has an amino acid sequence set forth in SEQ ID NO: 19; (10) TB10.4 has an amino acid sequence set forth in SEQ ID NO: 21; (11) PE13 has an amino acid sequence set forth in SEQ ID NO: 23; (12) EsxC has an amino acid sequence set forth in SEQ ID NO: 25; (13) EsxD has an amino acid sequence set forth in SEQ ID NO: 27; (14) EsxV has an amino acid sequence set forth in SEQ ID NO: 29; (15) EsxW has an amino acid sequence set forth in SEQ ID NO:

31.

4. The Mtb antigen of any one of claims 1-3, comprising a suitable purification tag, such as a 6xHis tag, or a PSR tag (SEQ ID NO: 45) at the N-terminus or C-terminus; or, the fusion protein comprises a signal peptide, optionally, the amino acid sequence of the signal peptide is set forth in SEQ ID NO:

46.

5. A fusion protein comprising an Mtb antigen selected from any one of claims 1-4; preferably, the fusion protein comprises two, three, or four Mtb antigens as set forth in claims 1-4.

6. The fusion protein of claim 5, comprising from N-terminus to C-terminus: (1) Ag85a-Mtb32a; (2) ESAT6-CFP10-Mtb39a; (3) EsxC-EsxD-EsxV-EsxW; (4) EspC-PE13; (5) Mpt64-Hrp1 ; or (6) RV2660c-TB10.4; optionally, the Mtb antigens in the fusion protein can be linked by a linker peptide; alternatively, the linker peptide is selected from the group consisting of (GSG) n , (GGGGS) n , (GGGGS)n, wherein n can be an integer greater than 0 and less than or equal to 5.

7. The fusion protein of any one of claims 5-6, having an amino acid sequence 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identical to SEQ ID NO: 41, 39, 35, 37, 43, or 33; preferably, the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 41, 39, 35, 37, 43, or 33.

8. The fusion protein of any one of claims 4-7, comprising a suitable purification tag, such as a 6xHis tag, or a PSR tag (SEQ ID NO: 45) at the N-terminus or C-terminus; or, the fusion protein comprises a signal peptide, optionally, the amino acid sequence of the signal peptide is set forth in SEQ ID NO:

46.

9. An immunogenic complex comprising (1) an antigen component and (2) a granulin component: (1) the antigen component comprising a: Mtb antigen of any one of claims 1-4; or b: fusion protein of any one of claims 5-8; (2) the granulin component comprising a nanogranulin; the antigen component and the granulin component form an immunogenic complex; preferably, the immunogenic complex is a virus-like particle (VLP).

10. The immunogenic complex of claim 9, wherein the antigen component and the granulin component are covalently bound via a peptide binding pair, such as Spytag-Spycatcher; SpyTag002 / SpyTag002 RG T3H-SpyCatcher; SpyTag / SpyTag002 / SpyTag002 RG T3H-SpyCatcher002; SpyTag / SpyTag002 / SpyTag002 RG T3H-SpyCatcher002(D5A, A92P, Q100D); SnoopTag / SnoopTagJr-SnoopCatcher; RrgATag / RrgATag2 / DogTag-RrgACatcher; Isopeptag-Pilin-C; Isopeptag-N-Pilin-N; PsCsTag-PsCsCatcher; SnoopTagJr-DogTag, and the like; preferably, the peptide binding pair is Spytag-Spycatcher.

11. The immunogenic complex of claim 9 or 10, wherein the nanoparticle protein displaying the antigen component comprises or consists of mi3, I3-01, Ferritin protein, I53-50, IMX313, RC_I_1, RC_I_2, AP205 VLP, Influenza HA VLP, HBsAg VLP, HBcAg VLP, HPV 18L1 VLP, HPB 16L1 VLP, HEV P239 VLP, Qβ VLP, MS2 VLP, CuMV VLP, CCMV VLP, PP7 VLP, RHDS VLP, PPV VLP, CPV VLP, Norwalk VLP, IMX313, and the like; preferably, the nanoparticle protein is mi3.

12. A method of preparing the immunogenic complex of any one of claims 9-11, comprising: (1) separately ligating the encoding genes of the antigen component and the granulin component into expression vectors, constructing expression recombinant plasmids and expression host strains, expressing the target proteins, and purifying; and / or (2) co-incubating the antigen component and the granulin component obtained in step (1) to obtain the immunogenic complex.

13. A derivative product of claims 1-11, as shown in any one of: (1) a nucleic acid sequence encoding the Mtb antigen of any one of claims 1-4, the fusion protein of any one of claims 5-8, or the immunogenic complex of any one of claims 9-11; preferably, the nucleic acid sequence is as shown in SEQ ID NO: 56-64. ​ ​ (2) a vector comprising the nucleic acid sequence of (1), operably linked to one or more control sequences suitable for directing the production of a protein of interest in a suitable host; (3) a recombinant host cell comprising the nucleic acid sequence of (1) or the vector of (2), preferably, the host cell is selected from a mammalian cell, an insect cell, a yeast cell or a bacterial cell; the mammalian cell is, for example, a HEK293 cell or a CHO cell; the bacterial cell is, for example, an E. coli cell.

14. An Mtb immunological composition comprising Mtb immunological components selected from the Mtb antigens of any one of claims 1 to 4, or the fusion proteins of any one of claims 5 to 8, or the immunogenic complexes of any one of claims 9 to 11.

15. The Mtb immunological composition of claim 14, comprising any two, three or four of the following groups: (1) Mtb32a; (2) Mtb39a; (3) Ag85a; (4) RV2660c and TB10.4; (5) EspC and PE13; (6) Mpt64 and Hrp1; (7) EsxC, EsxD, EsxV and EsxW; (8) ESAT6 and CFP10; optionally, the Mtb antigens in the above groups are present in the form of the fusion proteins of claims 5 to 8, and are also present in the form of the immunogenic complexes of claims 9 to 11; preferably, the Mtb immunological composition comprises any two, three or four of groups (1), (2), (3) and (8), and optionally further groups; more preferably, it comprises groups (1) and (3); or groups (2) and (8); or groups (1), (2), (3) and (8); or any two, three or four of groups (1), (3), (4) and (8), and optionally further groups; preferably, it comprises groups (1), (3), (4) and (8); or any two, three, four or five of groups (1), (2), (3), (4) and (8), and optionally further groups; preferably, it comprises groups (1), (2), (3), (4) and (8); or groups (5) and (6), and optionally further groups; preferably, it comprises groups (5) and (6); or groups (5) and (7), and optionally further groups; preferably, it comprises groups (5) and (7); or any two, three, four, five or six of groups (2), (3), (5), (6), (7) and (8), and optionally further groups; preferably, it comprises groups (2), (3), (5) and (8); or groups (2), (3), (6) and (8); or groups (2), (3), (7) and (8).

16. The Mtb immunological composition of claim 14 or 15, comprising components selected from the following groups: (1) Mtb32a; (2) Mtb39a; (3) Ag85a; (4) RV2660c and TB10.4; (5) EspC and PE13; (6) Mpt64 and Hrp1; (7) EsxC, EsxD, EsxV and EsxW; (8) ESAT6 and CFP10; optionally, the Mtb antigens in the above groups are present in the form of the fusion proteins of claims 5 to 8, and are also present in the form of the immunogenic complexes of claims 9 to 11; preferably, the Mtb immunological composition comprises any two, three or four of the following groups: (1) Mtb32a; (2) Mtb39a; (3) Ag85a; (4) RV2660c and TB10.4; (5) EspC and PE13; (6) Mpt64 and Hrp1; (7) EsxC, EsxD, EsxV and EsxW; (8) ESAT6 and CFP10; optionally, the Mtb antigens in the above groups are present in the form of the fusion proteins of claims 5 to 8, and are also present in the form of the immunogenic complexes of claims 9 to 11; preferably, the Mtb immunological composition comprises any two, three or four of groups (1), (2), (3) and (8), and optionally further groups; more preferably, it comprises groups (1) and (3); or groups (2) and (8); or groups (1), (2), (3) and (8); or any two, three or four of groups (1), (3), (4) and (8), and optionally further groups; preferably, it comprises groups (1), (3), (4) and (8); or any two, three, four or five of groups (1), (2), (3), (4) and (8), and optionally further groups; preferably, it comprises groups (1), (2), (3), (4) and (8); or groups (5) and (6), and optionally further groups; preferably, it comprises groups (5) and (6); or groups (5) and (7), and optionally further groups; preferably, it comprises groups (5) and (7); or any two, three, four, five or six of groups (2), (3), (5), (6), (7) and (8), and optionally further groups; preferably, it comprises groups (2), (3), (5) and (8); or groups (2), (3), (6) and (8); or groups (2), (3), (7) and (8). (1) Mtb32a, Ag85a, ESAT6-CFP10, RV2660c-TB10.4; (2) ESAT6-CFP10-Mtb39a, Ag85a-Mtb32a; (3) EsxC-EsxD-EsxV-EsxW, EspC-PE13; (4) Mpt64-Hrp1, EspC-PE13; (5) Mtb32a, Ag85a, ESAT6-CFP10-Mtb39a, RV2660c-TB10.4; preferably, the components are present in the form of an immunogenic complex comprising the antigenic components of SEQ ID NO: 2, 6, 8, 34, 36, 38, 40, 42, 44 or 54.

17. The Mtb immunological composition of any one of claims 14-16, further comprising a pharmaceutically acceptable carrier comprising a stabilizer, an excipient, a surfactant, a buffer, a pH adjuster, wherein the stabilizer comprises sucrose or arginine, the excipient comprises mannitol, the surfactant comprises Tween 80, the buffer comprises disodium hydrogen phosphate dihydrate or sodium dihydrogen phosphate dihydrate, and the pH adjuster comprises hydrochloric acid; more preferably, the pharmaceutically acceptable carrier is 20 mM Tris-HCl, pH 7.

4.

18. The Mtb immunological composition of any one of claims 14-17, wherein the immunological components are present in an amount of 10-50 μg, preferably 40 μg.

19. A vaccine for preventing or treating tuberculosis, comprising the immunological composition of any one of claims 14-18 and an adjuvant selected from the group consisting of: an aluminum salt adjuvant, Freund's complete adjuvant, propolis adjuvant, water-in-oil adjuvant, a cytokine, CpG DNA, a genetically engineered attenuated toxin, an immunostimulatory complex, and a liposome.

20. The vaccine of claim 19, wherein the water-in-oil adjuvant is a squalene adjuvant; preferably, the squalene adjuvant comprises (w / w): squalene 0.5%-5%, Span 85 0.05%-1%, Tween 80 0.05%-1%, and 10 mM citrate buffer.

21. The vaccine of claim 19, wherein, The liposome adjuvant comprises the following components (per 0.5 ml): 25-50 μg Quillaja saponaria saponins QS-21, 25-50 μg 3-O-deacylated-4'-monophosphoryl lipid A (3D-MPL), 0.5-1 mg dioleoylphosphatidylcholine (DOPC), and 0.125-0.25 mg cholesterol.

22. The vaccine of claim 19, wherein, The liposome adjuvant comprises the cationic liposome dimethylthexylammonium (DDA) and trehalose 6,6-dibehenate (TDB); optionally, the mass ratio of DDA to TDB is 5:1; preferably, the liposome adjuvant is selected from the group consisting of CAF01, CAF09b or CAF10b.

23. A Mtb nucleic acid vaccine for preventing or treating a disease caused by Mycobacterium tuberculosis infection, characterized by, a nucleic acid vaccine comprising a nucleic acid sequence encoding the Mtb immunological composition of any one of claims 14-18; preferably, the nucleic acid vaccine is an mRNA vaccine.

24. A Mtb viral vector vaccine for preventing or treating a disease caused by Mycobacterium tuberculosis infection, characterized by, a recombinant viral vector comprising a nucleic acid sequence encoding the Mtb immunological composition of any one of claims 14-18.

25. The viral vector vaccine of claim 24, wherein, the viral vector is selected from one or more of the following: an adenoviral vector, a poxvirus vector, an influenza virus vector, an adeno-associated virus vector.

26. A method of preventing or treating a disease caused by Mycobacterium tuberculosis infection, comprising the step of administering an immunologically effective amount of a vaccine comprising the Mtb antigen of claims 1-4, the Mtb fusion protein of any one of claims 5-8, the immunogenic complex of any one of claims 9-11, the immunological composition of any one of claims 14-18, the vaccine of claims 19-22, the nucleic acid vaccine of claim 23, or the viral vector vaccine of any one of claims 24-25.

27. Use of the Mtb antigen of any one of claims 1-4, the fusion protein of any one of claims 5-8, the immunogenic complex of any one of claims 9-11, the immunological composition of any one of claims 14-18, the vaccine of claims 19-22, the nucleic acid vaccine of claim 23, or the viral vector vaccine of any one of claims 24-25 in the manufacture of a medicament for preventing or treating a disease caused by Mycobacterium tuberculosis infection.

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