Nanoparticle vaccine for preventing mycobacterium tuberculosis infection and preparation method thereof

Abstract

The invention relates to a mycobacterium tuberculosis vaccine, in particular, the vaccine comprises an immune composition, and the immune composition comprises an antigen component and a granular protein component. The granular protein component comprises nano-granular protein, and the antigen component and the granular protein component are covalently bound through a binding peptide 1 and a binding peptide 2 to form an immunogenic complex. The cell immunogenicity and the antibody immunogenicity of the vaccine are excellent. The invention also discloses a preparation method of the mycobacterium tuberculosis vaccine.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an immune composition product for preventing Mycobacterium tuberculosis infection and a production method thereof. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis and other mycobacteria. The main route of transmission of the disease is droplet transmission. It can occur at any age, and people with low immunity are more susceptible to infection. The infected person may not have typical symptoms for a long time, but after the onset of the disease, it will show symptoms such as acute lung inflammation, dry cough, fever, etc. If not treated in time, it will generally lead to serious complications and death.

[0003] Currently, tuberculosis is a major disease in developing countries, widely distributed around the world, and is becoming increasingly serious. In 2021, there will be 10.6 million new cases of tuberculosis worldwide, and 1.6 million people will die from tuberculosis - equivalent to about 4,300 deaths per day. Tuberculosis mainly affects people in low- and middle-income countries. Poor people with poor living and working conditions and malnutrition are often at the highest risk of tuberculosis. Globally, up to a quarter of the population is latently infected with tuberculosis. There are no symptoms of infection with tuberculosis bacteria, but it is possible to develop active tuberculosis.

[0004] Currently, antibiotic treatment of tuberculosis patients with drugs such as isoniazid, rifampicin, and pyrazinamide can control tuberculosis, but this treatment cannot effectively prevent the spread of the disease. Infected individuals will not develop the disease for a period of time, but they are still contagious. In addition, long-term use of antibiotics for treatment poses a huge challenge to patients' medication compliance, and some patients are even unable to complete effective treatment. At the same time, long-term antibiotic treatment may lead to drug resistance.

[0005] Effective vaccination and accurate early diagnosis are important means to control tuberculosis. At present, the BCG (Bacillus Calmette-Guerin) vaccine (BCG) used for infant vaccination is prepared from a non-toxic Mycobacterium bovis. The widespread use of BCG vaccination has provided great help for the prevention and control of tuberculosis worldwide. BCG can protect infants from severe systemic tuberculosis, but its protective effect on pulmonary tuberculosis in adolescents and adults is very limited. Therefore, it is urgent to develop a new tuberculosis vaccine that can completely replace BCG or BCG booster. Recombinant subunit tuberculosis vaccines have been proven to be effective in humans and have gradually become a hot spot for the development of new tuberculosis vaccines. For example, the new tuberculosis candidate vaccine M72 / AS01E developed by GSK, M72 is a recombinant fusion protein containing two Mycobacterium tuberculosis antigens (MTB32A and MTB39A). Phase II clinical data showed that its protection rate was only 49.7%, barely meeting the WHO's TB vaccine requirements. Although the vaccine has a certain immune efficacy, the M72 fusion protein is easily degraded during the expression process and will produce inclusion bodies. The particle size of the fusion protein is greatly affected by salt ions and pH, resulting in a complex production process and high cost. Therefore, it is of great significance to develop a new TB vaccine with a higher protection rate and simpler production process for population vaccination.

[0006] At present, nanoparticle vaccines, as a new generation of vaccine design, can not only induce strong neutralizing antibodies, but also improve the level of cellular immune response. Human HPV vaccine, hepatitis B vaccine, veterinary PCV2 vaccine, etc. are all representatives. There are many natural proteins in nature (Ferritin, Lumazine Synthase, Mi3, AP205, etc.), which can self-assemble into nanoparticles. After the surface is loaded with antigens, it can induce a strong immune response. It has been widely studied and applied. The body's resistance to Mycobacterium tuberculosis infection mainly depends on the body's cellular immune mechanism. After vaccination, the effective Th1CD4+T and CD8+T levels and persistent cellular immune memory can be produced, which is the key point in the development of new tuberculosis vaccines. Therefore, self-assembled nanoparticles are very ideal carriers and are feasible in the research of tuberculosis vaccines. Summary of the invention

[0007] The invention provides a Mycobacterium tuberculosis vaccine and a preparation method thereof. The vaccine is a nanoparticle vaccine.

[0008] Nanoparticle vaccines: vaccines based on nanoparticle proteins, which are mainly used to display antigens.

[0009] The present invention provides an immunogenic complex, which comprises a protein formed by a covalent binding reaction between an antigen component and a particle protein component.

[0010] The present invention provides an immune composition, which contains the immunogenic complex of the present invention and a pharmaceutically acceptable carrier, and can be in the form of a lyophilized preparation, an injection preparation, an oral preparation or a spray preparation.

[0011] The present invention provides a vaccine comprising the immune composition of the present invention and an adjuvant.

[0012] The present invention provides an immunogenic complex comprising:

[0013] (1) an antigenic component comprising a Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof;

[0014] (2) A granular protein component, which comprises nanoparticle protein.

[0015] The present invention provides an immunogenic complex comprising:

[0016] (1) An antigen component comprising a Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof, a connecting

[0017] peptide 1 and binding peptide 1;

[0018] (2) a particle protein component, which comprises a nanoparticle protein, a connecting peptide 2 and a binding peptide 2;

[0019] The antigen component and the granule protein component are covalently bound to each other via binding peptide 1 and binding peptide 2.

[0020] The present invention provides an immunogenic complex comprising:

[0021] (1) Antigenic components, consisting of Mycobacterium tuberculosis structural proteins or immunogenic fragments thereof, linked

[0022] Peptide 1 and binding peptide 1;

[0023] (2) granule protein component, consisting of nanoparticle protein, connecting peptide 2 and binding peptide 2;

[0024] The antigen component and the granule protein component are covalently bound to each other via binding peptide 1 and binding peptide 2.

[0025] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the antigen component is formed by fusing the Mycobacterium tuberculosis structural protein with the binding peptide 1 via the connecting peptide 1 at the C-terminus.

[0026] In some embodiments, an "immunogenic fragment" refers to a portion of an oligopeptide, polypeptide, or protein that is immunogenic and elicits a protective immune response when administered to a subject.

[0027] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the particle protein component is formed by fusing the nanoparticle protein with the binding peptide 2 via the connecting peptide 2 at the N-terminus.

[0028] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the antigen component from N-terminus to C-terminus is: Mycobacterium tuberculosis structural protein or its immunogenic fragment, connecting peptide 1 and binding peptide 1; the granule protein component from N-terminus to C-terminus is: binding peptide 2, connecting peptide 2, nanoparticle protein; the antigen component and the granule protein component are covalently bound to each other through binding peptide 1 and binding peptide 2 to form an immunogenic complex.

[0029] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the antigen component and / or the particle protein component comprises a histidine tag.

[0030] The present invention provides an immunogenic complex comprising:

[0031] (1) an antigen component comprising a Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof and a connecting peptide 1;

[0032] (2) A granule protein component, which comprises nanoparticle protein subunits.

[0033] In some embodiments, the Mycobacterium tuberculosis structural protein is linked to one subunit of the nanoparticle protein to form a fusion protein, and the fusion protein is then bound to another subunit of the nanoparticle protein.

[0034] In some embodiments, in any immunogenic complex provided by the present invention, the particle protein component comprises nanoparticle protein, preferably, the nanoparticle protein can be a virus-like particle protein, and the virus-like particle protein is formed by a virus structural protein, preferably, by a bacteriophage capsid protein AP205. The particle protein component and the antigen component can form a particle structure by covalent bonding.

[0035] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the nanoparticle protein used can also be selected from: NPM particles, ferritin particles (Ferritin), I53-50 particles, Lumazine Synthase (LS) particles, etc.

[0036] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the nanoparticle protein I53-50 particles used are composed of two subunits, I53-50A and I53-50B.

[0037] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the binding peptide 1 comprises the amino acid sequence shown in SEQ ID NO:1.

[0038] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the binding peptide 2 comprises an amino acid sequence as shown in SEQ ID NO:24.

[0039] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the connecting peptide 1 comprises (GSG) n 、(GGGGS) n or (EAAAK) n The amino acid sequence of n can be an integer greater than 0 and less than or equal to 5. In some embodiments, in any immunogenic complex provided by the present invention, the connecting peptide 1 is preferably GSG GSG (SEQ ID NO: 2).

[0040] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the connecting peptide 2 comprises (GGS) n 、(SGGSGG) n or (GSGGSGGSG) n In an amino acid sequence of, n may be an integer greater than 0 and less than or equal to 10. In some embodiments, in any immunogenic complex provided by the present invention, the connecting peptide 2 is preferably GGSGGSGGSGGS (SEQ ID NO: 25).

[0041] Specifically, the structural protein of Mycobacterium tuberculosis of the present invention uses Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 fusion proteins, so that the above-mentioned proteins are connected to binding peptide 1 (the binding peptide 1 is named "4T") at the C-terminus through a specific linker peptide 1 (linker 1), and a histidine (for example, 6His, i.e., HHHHHH) purification tag can be added to the C-terminus of the fusion protein; the coding gene encoding the above-mentioned Mycobacterium tuberculosis structural protein is inserted into a prokaryotic cell expression vector (for example, pET21a), and expressed in Escherichia coli BL21 (DE3) cells to obtain a fusion protein formed by the structural protein of Mycobacterium tuberculosis-binding peptide 1, and the antigen component is subjected to nickel column affinity chromatography and molecular sieve chromatography to obtain a high-purity protein, namely, Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T.

[0042] Preferably, the structural proteins of Mycobacterium tuberculosis of the present invention use Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 fusion proteins, the Mtb32a sequence is shown in SEQ ID NO: 3, the Ag85a sequence is shown in SEQ ID NO: 5, the ESAT6-CFP10 sequence is shown in SEQ ID NO: 7, and the RV2660-TB10.4 sequence is shown in SEQ ID NO: 9.

[0043] Preferably, the structural proteins of Mycobacterium tuberculosis of the present invention use Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 fusion proteins, and the Mtb32a sequence has more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more identity with SEQ ID NO: 3, the Ag85a sequence has more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more identity with SEQ ID NO: 5, the ESAT6-CFP10 sequence has more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more identity with SEQ ID NO: 7, and the RV2660-TB10.4 sequence has more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more identity with SEQ ID NO: 9.

[0044] Preferably, in any one of the immunogenic complexes provided by the present invention, the antigen component comprises Mycobacterium tuberculosis structural protein Mtb32a (SEQ ID NO: 3), Ag85a (SEQ ID NO: 5), ESAT6-CFP10 (SEQ ID NO: 7) or RV2660-TB10.4 (SEQ ID NO: 9), connecting peptide 1-GSGGSG (SEQ ID NO: 2), binding peptide 1 (SEQ ID NO: 1), and a histidine tag; more preferably, the antigen component Mtb32a-4T sequence is shown in SEQ ID NO: 4, the antigen component Ag85a-4T sequence is shown in SEQ ID NO: 6, the antigen component ESAT6-CFP10-4T sequence is shown in SEQ ID NO: 8, and the antigen component RV2660-TB10.4-4T sequence is shown in SEQ ID NO: 10.

[0045] In some embodiments, in any of the above immunogenic complexes provided by the invention, the particle protein component is a fusion protein formed by connecting peptide 2 and binding peptide 2 at the N-terminus of the nanoparticle protein; preferably, the nanoparticle protein is NPM, AP205 capsidprotein3 (AP205) or Ferritin protein. Specifically, in some optional schemes, the binding peptide 2 (the binding peptide 2 is named "4C") is connected to the coding gene of the nanoparticle protein through the connecting peptide 2, inserted into a prokaryotic expression vector (such as pET-28a (+), pET-30a (+)), and expressed in E. coli cells to obtain a fusion protein of binding peptide 2 and nanoparticle protein, and the fusion protein can be purified by chromatography, for example, by anion exchange chromatography, hydrophobic chromatography, to obtain a product. The nanoparticle protein is preferably NPM, AP205 or Ferritin; the formed particle protein component is named NPM-4C, AP205-4C, Ferritin-4C.

[0046] Specifically, in some optional schemes, under appropriate reaction conditions, any of the above antigen components is subjected to a conjugation reaction with a particle protein component, and the immunogenic complex is formed by forming a covalent bond between the binding peptide 1 of the antigen component and the binding peptide 2 of the particle protein component. Different immunogenic complexes can be formed by using different nanoparticle proteins, and these immunogenic complexes are named Mtb32a-NPM, Mtb32a-AP205 or Mtb32a-Ferritin; Ag85a-NPM, Ag85a-AP205 or Ag85a-Ferritin; ESAT6-CFP10-NPM, ESAT6-CFP10-AP205 or ESAT6-CFP10-Ferritin; RV2660-TB10.4-NPM, RV2660-TB10.4-AP205 or RV2660-TB10.4-Ferritin.

[0047] In some embodiments, the present invention provides an immunogenic complex comprising:

[0048] (1) an antigen component comprising a Mycobacterium tuberculosis structural protein, a connecting peptide 1 and a binding peptide 1;

[0049] (2) A particle protein component, which comprises nanoparticle protein, connecting peptide 2 and binding peptide 2.

[0050] The connecting peptide 1 is any connecting peptide commonly used in the art, including but not limited to (GSG) n 、(GGGGS) n or (EAAAK) n The amino acid sequence of n can be an integer greater than 0 and less than or equal to 5, preferably GSGGSG (SEQ ID NO: 2); the connecting peptide 2 is any connecting peptide commonly used in the art, including but not limited to (GGS) n 、(SGG) n or (GSGGSGGSG) n wherein n can be an integer greater than 0 and less than or equal to 10, preferably GGSGGSGGSGGS (SEQ ID NO: 25). The nanoparticle protein is NPM, AP205 or Ferritin.

[0051] Preferably, in any immunogenic complex provided by the present invention, the particle protein component comprises NPM-4C, as shown in SEQ ID NO:27, which is a fusion protein obtained by connecting the binding peptide 2 shown in SEQ ID NO:24 to the nanoparticle protein NPM shown in SEQ ID NO:26 via the connecting peptide 2 shown in SEQ ID NO:25.

[0052] In other embodiments, the present invention provides an immunogenic complex comprising:

[0053] (1) an antigen component comprising a structural protein and a connecting peptide 1 of Mycobacterium tuberculosis;

[0054] (2) a granule protein component, which comprises nanoparticle protein subunits; preferably, the nanoparticle protein subunits are I53-50A and / or I53-50B subunits.

[0055] In some embodiments, in any one of the immunogenic complexes provided herein, the nanoparticle protein I53-50 comprises I53-50A and / or I53-50B subunits.

[0056] Specifically, in any immunogenic complex provided by the present invention, the Mycobacterium tuberculosis structural protein is connected to a subunit of the nanoparticle protein to form a fusion protein, and the fusion protein is then combined with another subunit of the nanoparticle protein. Preferably, the subunit of the nanoparticle protein is I53-50A or I53-50B. Further, in some optional schemes, the Mycobacterium tuberculosis structural protein in the antigen component is connected to the nanoparticle protein I53-50A subunit at the C-terminus through the connecting peptide 1 to form a Mycobacterium tuberculosis structural protein-I53-50A fusion protein; then, the fusion protein is combined with the nanoparticle protein I53-50B subunit.

[0057] As described above, when I53-50 is selected as the nanoparticle protein, I53-50 comprises two subunits, I53-50A and I53-50B. The Mycobacterium tuberculosis structural protein containing a specific signal peptide or not containing a signal peptide is connected to I53-50A through a connecting peptide 1, and a histidine (e.g., 6H) purification tag can be added to the C-terminus, and the coding gene encoding the above fusion protein is inserted into a eukaryotic cell expression (e.g., pcDNA3.4), expressed and purified in CHO cells, and the obtained fusion protein is named Mycobacterium tuberculosis structural protein-I53-50A; at the same time, a histidine (e.g., 6H) purification tag can be added to the C-terminus of I53-50B, and the gene encoding the above protein is inserted into a prokaryotic cell expression vector (e.g., pET-30a(+)), expressed and purified in E. coli cells, and the obtained protein is named I53-50B. Then, under appropriate reaction conditions, the Mycobacterium tuberculosis structural protein-I53-50A and I53-50B are covalently bonded to form Mycobacterium tuberculosis nanoparticles, which are named Mycobacterium tuberculosis structural protein-I53-50.

[0058] Preferably, any one of the immunogenic complexes provided by the present invention comprises Mycobacterium tuberculosis structural protein-I53-50A.

[0059] In some embodiments, the present invention provides an immunogenic complex comprising any one or more of the following (1)-(7):

[0060] (1) The amino acid sequence of the Mycobacterium tuberculosis structural protein is shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9;

[0061] (2) The amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO: 2;

[0062] (3) The amino acid sequence of the binding peptide 1 is shown in SEQ ID NO: 1;

[0063] (4) the nanoparticle protein is selected from NPM, AP205 or Ferritin;

[0064] (5) the nanoparticle protein subunit is selected from I53-50A and / or I53-50B;

[0065] (6) The connecting peptide 2 comprises an amino acid sequence of (GGS)n, (SGGSGG)n or (GSGGSGGSG)n, where n is an integer greater than 0 and less than or equal to 10; the amino acid sequence of the connecting peptide 2 is as shown in SEQ ID NO: 25;

[0066] (7) The amino acid sequence of the binding peptide 2 is shown in SEQ ID NO:24.

[0067] In some embodiments, the present invention provides an immunogenic complex consisting of an antigen component and a granule protein component, wherein the amino acid sequence of the antigen component is shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10, and the amino acid sequence of the granule protein component is shown in SEQ ID NO:27.

[0068] Furthermore, the present invention also provides a method for preparing any of the above-mentioned immunogenic complexes, comprising the following steps:

[0069] (1) connecting the antigen component and the granule protein component encoding genes into expression vectors respectively, constructing expression recombinant plasmids and expression host strains, expressing the target protein, and purifying it;

[0070] (2) The antigen component obtained in step (1) is co-incubated with the granule protein component to obtain an immunogenic complex.

[0071] The present invention provides a method for preparing an immunogenic complex for preventing or treating Mycobacterium tuberculosis virus-related diseases:

[0072] (1) connecting the genes encoding the Mycobacterium tuberculosis virus antigen component and the particle protein component into expression vectors respectively to construct expression recombinant plasmids;

[0073] (2) constructing a recombinant strain capable of expressing the Mycobacterium tuberculosis virus antigen component and particle protein component in a host cell;

[0074] (3) using the recombinant strain to express the fusion protein and purify the fusion protein;

[0075] (4) The antigen component and the granule protein component are co-incubated to produce a conjugation reaction and obtain an immunogenic complex.

[0076] Preferably, the immunogenic complex obtained in the above step (4) is purified to obtain a vaccine stock solution.

[0077] Preferably, in the method for preparing an immunogenic complex for preventing or treating Mycobacterium tuberculosis virus-related diseases, in step (1), the plasmid expressing the Mycobacterium tuberculosis virus antigen component can be pET21a, and the plasmid expressing the particle protein component can be pET-28a(+) or pET-30a(+).

[0078] In the method for preparing an immunogenic complex for preventing or treating Mycobacterium tuberculosis virus-related diseases described in the present invention, the host cell expressing the Mycobacterium tuberculosis virus antigen in step (2) is Escherichia coli, and the host cell expressing the particle protein component vector is E. coli.

[0079] The immunogenic complex for preventing or treating Mycobacterium tuberculosis virus-related diseases of the present invention comprises an antigen component of the immunogenic complex comprising a fusion protein formed by the Mycobacterium tuberculosis structural protein-binding peptide 1.

[0080] In the immunogenic complex for preventing or treating Mycobacterium tuberculosis virus-related diseases described in the present invention, the high-purity Mtb32a, Ag85a, ESAT6-CFP10, RV2660-TB10.4 antigens obtained by molecular sieve purification are mixed with NPM-4C according to a BCA protein concentration ratio of 6:1, and 50% sucrose mother solution is added to a final sucrose concentration of about 25%, and 10% of the total reaction volume of 1M Tris-HCl mother solution is added to stabilize the pH. The binding reaction is carried out at 22°C for 48 hours. Endotoxin detection is less than 100EU / ml, which meets the requirements of large-scale production.

[0081] The present invention also provides an immune composition, which comprises any one of the above-mentioned immunogenic complexes and a pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier comprises a stabilizer, an excipient, a surfactant, a buffer, and a pH adjuster, the stabilizer is sucrose and arginine, the excipient is mannitol, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate, and the pH adjuster is hydrochloric acid.

[0082] In some embodiments, the immunogenic complex of the immune composition of the present invention is contained in an amount of 0.25-100 μg / dose, preferably 0.5-50 μg / dose, more preferably 0.5 μg / dose, 1 μg / dose, 2 μg / dose, 3 μg / dose, 4 μg / dose, 5 μg / dose, 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. The dose for mouse experiments is 1 / 10 of the dose for humans.

[0083] In some embodiments, the immune composition provided by the present invention is an injection or a lyophilized preparation, preferably a lyophilized preparation.

[0084] In some embodiments, the immune composition provided by the present invention is a lyophilized preparation, which comprises a Mycobacterium tuberculosis structural protein-NPM immunogenic complex, a stabilizer, an excipient, a surfactant, a buffer, and a pH adjuster; 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, and the pH adjuster is hydrochloric acid.

[0085] In some embodiments, the immune composition provided by the present invention is a lyophilized preparation, which comprises Mycobacterium tuberculosis structural protein-NPM immunogenic complex, sucrose, arginine, mannitol, Tween 80, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, and hydrochloric acid.

[0086] In some embodiments, the immune composition provided by the present invention is an injection, which comprises a Mycobacterium tuberculosis structural protein-NPM immunogenic complex, a stabilizer, a surfactant, a buffer, and a pH adjuster; preferably, the stabilizer is sucrose, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, and the pH adjuster is hydrochloric acid.

[0087] In some embodiments, the immune composition provided by the present invention is an injection solution, which comprises Mycobacterium tuberculosis structural protein-NPM immunogenic complex, sucrose, Tween 80, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, and hydrochloric acid.

[0088] The present invention further provides a Mycobacterium tuberculosis vaccine, which comprises any one of the above-mentioned immune compositions and an adjuvant, wherein the adjuvant is selected from at least one of: aluminum salt adjuvants, Freund's complete adjuvant, propolis adjuvant, water-oil adjuvant, cytokine, CpG DNA, genetically engineered attenuated toxin, immunostimulatory complex, and liposome.

[0089] In the Mycobacterium tuberculosis vaccine described in the present invention, the water-oil adjuvant is a squalene adjuvant containing squalene.

[0090] The Mycobacterium tuberculosis vaccine of the present invention contains the immunogenic complex in an amount of 5-50 μg / dose per unit dose of the vaccine for human use, preferably 5 μg, 25 μg or 50 μg.

[0091] The squalene adjuvant of the present invention contains: (w / w) squalene 0.5%-5%, Span 85 0.05%-1%, Tween 80 0.05%-1%, and 10mM citrate buffer.

[0092] The squalene adjuvant of the present invention preferably contains: (w / w) squalene 2%-4.5%, Span 85 0.2%-0.5%, Tween 80 0.2%-0.5%, and 10 mM citrate buffer. Among them, the more preferred amount of squalene is 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 more preferred amount of Span 85 is 0.3%-0.4% (w / w), and the more preferred amount of Tween 80 is 0.3%-0.4% (w / w).

[0093] As mentioned above, the dosages of the immunogenic complex, Mycobacterium tuberculosis structural protein-NPM, and adjuvant are different for humans and mice, and the corresponding relationship is: when used as human doses, the dosages of Mycobacterium tuberculosis structural protein-NPM and adjuvant are 10 times the dosages used in mice, for example: the Mycobacterium tuberculosis structural protein-NPM for mice is 5 μg / dose, and the human dose needs to be 50 μg / dose; the adjuvant for mice is 50 μl / dose, and the human dose needs to be 500 μl / dose (0.5 ml / dose); the adjuvant for mice is 25 μl / dose, and the human dose needs to be 250 μg / dose (0.25 ml / dose), and so on.

[0094] The present invention further provides a complete kit, characterized in that it comprises the Mycobacterium tuberculosis vaccine described in the present invention, and instruments and containers required for inoculating the vaccine.

[0095] The present invention provides a Mycobacterium tuberculosis vaccine, comprising a Mycobacterium tuberculosis structural protein-NPM immune composition (i.e., an immune combination containing Mycobacterium tuberculosis structural protein-NPM, which can be made into a lyophilized preparation or an injection preparation) and an adjuvant (liquid). The Mycobacterium tuberculosis structural protein-NPM immune composition and the adjuvant are packaged in separate bottles.

[0096] The present invention provides use of a Mycobacterium tuberculosis nanoparticle immunogenic complex, immune composition or vaccine in the preparation of a medicine for preventing or treating tuberculosis.

[0097] All reagents used in the present invention can be purchased commercially.

[0098] Compared with the prior art, the present invention has the following beneficial effects:

[0099] 1. The present invention uses an E. coli expression system to express the structural proteins MTB32A-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T of Mycobacterium tuberculosis in the form of fusion expression. These fusion proteins can all be expressed in a soluble form. At the same time, NPM-4C nanoparticles are prepared by expression with the aid of the E. coli expression system. The immunogenic complex of the present invention has a significant preventive effect on Mycobacterium tuberculosis (Mtb) infection. Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles prepared using Mtb's structural proteins Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 have uniform particle size, even distribution, and no aggregation. The product performance is stable and the endotoxin level is qualified, making them suitable for non-clinical development and antibody immunogenicity testing, and thus suitable as tuberculosis vaccines.

[0100] 2. For the first time, Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T fusion proteins were covalently coupled with NPM-4C nanoparticles to prepare nanoparticle antigens Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM. The production process is stable, the cost is low, and it has great industrial potential.

[0101] 3. The results of animal immunization experiments showed that Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM were able to produce high levels of specific IgG1 and IgG2a antibodies after immunizing mice, indicating that Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles have good immunogenicity and can produce better cellular immunity and humoral immunity.

[0102] 4. The mixture of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T fusion proteins can significantly reduce the infection of Mycobacterium tuberculosis. The mixture of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM can significantly reduce the load of Mycobacterium tuberculosis in the spleen and lungs of infected mice, and has important development value.

[0103] 5. The method for preparing the tuberculosis vaccine of the nanoparticle type provided by the present invention is low in cost and suitable for large-scale production. In the present invention, the granule protein component is prepared by Escherichia coli fermentation and chromatography purification, and the Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigen components can be prepared by cell reactor culture and chromatography purification, which are all suitable for industrial large-scale production, and have the advantages of high expression, stable process and yield, and simple operation. The amount of a batch of recombinant granule protein components can be combined with multiple batches of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigen components to improve production efficiency and save the cost of large-scale production.

[0104] 6. The method for preparing the recombinant granule protein component provided by the present 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 solvents used in subsequent chromatography purification; the protein product prepared using the recombinant granule protein component method provided by the present invention effectively reduces the side effects caused by the residues of impurities, host proteins, organic solvents, exogenous DNA, antibiotics, bacterial endotoxins and other substances in the particles, and improves safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 Shows the expression of recombinant protein Mtb32a-4T identified by Western Blot;

[0106] Figure 2Shows the expression of recombinant proteins Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T by Western Blot;

[0107] Figure 3 The results of molecular sieve separation and purification of the recombinant protein Mtb32a-4T are shown;

[0108] Figure 4 The results of molecular sieve separation and purification of the recombinant protein Ag85a-4T are shown;

[0109] Figure 5 The results of molecular sieve separation and purification of the recombinant protein ESAT6-CFP10-4T are shown;

[0110] Figure 6 The results of molecular sieve separation and purification of the recombinant protein RV2660-TB10.4-4T are shown;

[0111] Figure 7 The results of molecular sieve separation and purification of the recombinant protein M72-4T are shown;

[0112] Figure 8 The results of separation and purification of NPM-4C protein using Octyl Bestarose 4FF are shown;

[0113] Fig. 9 The results of separation and purification of the recombinant protein Mtb32a-NPM binding product are shown;

[0114] Fig.10 The results of separation and purification of the recombinant protein Ag85a-NPM binding product are shown;

[0115] Fig.11 The results of separation and purification of the recombinant protein ESAT6-CFP10-NPM binding product are shown;

[0116] Fig.12 The results of separation and purification of the recombinant protein RV2660-TB10.4-NPM binding product are shown;

[0117] Figures 13 to 16 The negative staining electron microscopy results of recombinant protein Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles are shown in sequence;

[0118] Fig.17The distribution curves of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles Distribution by Intensity / Volume are shown;

[0119] Fig.18 SDS-PAGE analysis of RV2660-TB10.4T before and after mutation is shown;

[0120] Fig.19 The distribution curve of NPM nanoparticles prepared before and after RV2660-TB10.4T mutation Distribution by Intensity / Volume is shown;

[0121] Fig. 20 The total binding antibody IgG levels of the serum of the corresponding groups of D20 were detected using 072, 076, 077, 014, and M72 proteins as coating antigens, respectively. Figures a to e are the corresponding IgG antibody levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline) detected using 072, 076, 077, and 014 antigens as coating antigens, respectively. Figure e corresponds to the IgG antibody level detected by M72 antigen.

[0122] Fig.21 It shows the IgG1 and IgG2a antibody levels of the serum of the corresponding groups of D20 detected using 072, 076, 077, 014, and M72 proteins as coating antigens, respectively. Figures a to e are the corresponding IgG1 and IgG2a antibody levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline) detected using 072, 076, 077, and 014 antigens as coating antigens, respectively. Figure e corresponds to the IgG1 and IgG2a antibody levels of the M72 group (0.8 μg immunization group);

[0123] Fig. 22 ad, in order, 072 antigen was used as the specific stimulation antigen to detect the corresponding IFN-γ, IL-2, TNFα, and IL-4 levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline); Fig. 22 eh, in turn, 076 antigen was used as the specific stimulation antigen to detect the corresponding IFN-γ, IL-2, TNFα, and IL-4 levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline);

[0124] Fig.23ad, in order, 077 antigen was used as the specific stimulation antigen to detect the corresponding IFN-γ, IL-2, TNFα, and IL-4 levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline); Fig.23 eh, in turn, 014 antigen was used as the specific stimulation antigen to detect the corresponding IFN-γ, IL-2, TNFα, and IL-4 levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline);

[0125] Fig.24 The results show that the specific IFN-γ, IL-2, TNFα and IL-4 levels in D20 splenocytes were detected using M72 protein as a stimulating antigen. DETAILED DESCRIPTION

[0126] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, rather than to limit the scope of protection of the present invention. The test methods for which specific conditions are not indicated in the following examples are usually carried out under conventional conditions or under conditions recommended by various manufacturers. When the examples give a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, according to the technical personnel in the art's grasp of the prior art and the records of the present invention, any methods, equipment, and materials of the prior art similar to or equivalent to the methods, equipment, and materials described in the examples of the present invention can also be used to implement the present invention. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

[0127] Example 1: Construction and expression of the gene encoding the Mycobacterium tuberculosis structural protein-binding peptide 1 fusion protein 1. Construction of recombinant antigen

[0128] Referring to the sequences of the structural proteins Mtb32a, Ag85a, ESAT6, CFP10, RV2660, and TB10.4 of Mycobacterium tuberculosis H37RV strain (GenBank: AL123456.3), Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T fusion proteins were constructed respectively; referring to the M72 sequence in the patent published by GSK (Grant Announcement No. CN 103249431B), the M72-4T fusion protein was constructed; the specific construction method is as follows. The specific sequence of the constructed protein and the above reference sequence are shown in Table 1.

[0129] 1)Mtb32a-4T

[0130] The original wild-type sequence was modified from S / 177aa to A / 177aa, and the linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences were added to the C-terminus, and the end was terminated with a stop codon. The molecule was named 014.

[0131] 2) Ag85a-4T

[0132] The original wild sequence was truncated at the N-terminus, and the specific sequence truncated at the N-terminus was QLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAG. In addition, linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences were added to the C-terminus of the protein in sequence, and the end was terminated with a stop codon. Named as 072 molecule.

[0133] 3)ESAT6-CFP10-4T

[0134] ESAT6 was placed at the N-terminus of the fusion protein, CFP10 protein was placed at the C-terminus of the fusion protein, and the linker (GSGGSG) sequence was used for connection in the middle. The linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences were added to the C-terminus in sequence, and the end was terminated with a stop codon. Named as 076 molecule.

[0135] 4)RV2660-TB10.4-4T

[0136] The original wild sequence of RV2660 was mutated from C / 66aa to A / 66aa, and the mutated RV2660 was placed at the N-terminus of the fusion protein, and the TB10.4 protein was placed at the C-terminus of the fusion protein, and the linker (GSGGSG) sequence was used to connect them in the middle. The linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences were added to the C-terminus in sequence, and the end was terminated with a stop codon. Named as 077 molecule.

[0137] It was unexpectedly discovered through experiments that the original wild-type sequence of RV2660 was modified from C / 66aa to A / 66aa, which could avoid dimer formation in RV2660-TB10.4-4T and keep RV2660-TB10.4-4T in a soluble monomer form after mutation (SDS-PAGE analysis showed a single band in both reduced and non-reduced states after mutation). Fig.18 As shown in Figure 2, the mutation makes the formed RV2660-TB10.4-NPM nanoparticles more uniform (Z-Average and Polydispersity Index values ​​change from 77.3 and 0.23 before mutation to 39.8 and 0.14 after mutation, respectively). Fig.19 shown.

[0138] Fig.18 In the figure, M: protein molecule marker, lane 1: before RV2660-TB10.4T-4T mutation (DTT+), lane 2: after RV2660-TB10.4T-4T mutation (DTT-), lane 3: after RV2660-TB10.4T-4T mutation (DTT+), lane 4: after RV2660-TB10.4T-4T mutation (DTT-).

[0139] Fig.19 In the figure, a is the NPM nanoparticles prepared before the RV2660-TB10.4T mutation; b is the NPM nanoparticles prepared after the RV2660-TB10.4T mutation.

[0140] 5)M72-4T

[0141] Referring to the sequence of the M72 fusion protein in the patent published by GSK (authorization announcement number CN 103249431B), 6His (HHHHHH), 4T (AHIVMVDAYKPTK), and linker (GSGGSG) sequences were added to the N-terminus of the original sequence in sequence, and ended with a stop codon.

[0142] The protein sequence designed above (sequence shown in Table 1) was codon optimized and gene synthesized in E. coli (sequence shown in Table 2 below), cloned into the restriction sites (5'NdeI, 3'HindⅢ) in the pET21a vector, and transformed into BL21 (DE3) for downstream expression.

[0143] Table 1: Amino acid sequences of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, RV2660-TB10.4-4T, M72-4T, binding peptide 1, connecting peptide 1 and fusion protein

[0144]

[0145]

[0146] Table 2: Nucleotide sequences of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, RV2660-TB10.4-4T, M72-4T, binding peptide 1, connecting peptide 1 and fusion protein

[0147]

[0148]

[0149]

[0150]

[0151]

[0152] 2. Expression of recombinant antigens

[0153] The BL21 (DE3) expression bacteria of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T were streaked onto LB agar plates (containing 50 μg / ml Ampicillin) and cultured overnight at 37°C. Single clones were picked and transferred to 10 ml TB medium containing 50 μg / ml Ampicillin and cultured overnight at 37°C, 220 rpm / min.

[0154] The bacterial liquid was inoculated into TB medium containing 50 μg / ml Ampicillin at a ratio of 1 / 100 and cultured at 37°C, 220 rpm / min for 2 to 3 hours.

[0155] The OD600 of the bacterial solution was measured using Nanodrop. When the OD value reached 0.6-0.8, the bacterial solution was transferred to an 18°C ​​shaker for cooling. IPTG was added overnight to induce expression for about 16 hours. The final concentration of IPTG was 500 μM.

[0156] Collect the bacterial suspension, centrifuge at 6000g, 4°C for 15 min, and discard the supernatant.

[0157] Centrifuge again at 6000g, 4°C, for 3 min, remove the culture medium in the supernatant, and freeze the bacterial sludge in a -80°C refrigerator.

[0158] Example 2: Western blot identification of recombinant antigens

[0159] 1) Resuspend the expression bacterial sludge of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T and RV2660-TB10.4-4T in 20 ml of 20 mM Trsi-HCl, 150 mM NaCl, pH 7.4 buffer, and after ultrasonic disruption, centrifuge at 4°C, 13000 g / min for 30 min, take the supernatant and precipitate (20 mM Trsi-HCl, 150 mM NaCl, pH 7.4 buffer suspension).

[0160] 2) Western blot was performed using LDS sample loading buffer (4x) plus reducing agent DTT, heated at 70°C for 5 min, cooled to room temperature, centrifuged at 10,000 rpm for 20 s, vortexed to mix, and the final loading amount was 0.5 μg.

[0161] 3) The samples to be analyzed and the prestained protein molecular weight standards are loaded onto a 4-12% Bis-Tris gel, with MES electrophoresis buffer, the voltage is set to 150 V, and the electrophoresis lasts for about 60 minutes.

[0162] 4) Use The membrane was transferred using the Turbo instrument and the corresponding reagents, and then incubated using the iBind instrument with Anti-his mouse monoclonal antibody and goat anti-mouse secondary antibody coupled to AP enzyme, and then developed with colorimetric solution, and photographed with GelDoc Go. Western blot identification results showed that all four proteins could be expressed in soluble form and inclusion bodies, and the band positions were consistent with the expected molecular weight, as shown in the following figure. Figure 1 and Figure 2 shown.

[0163] Figure 1 Middle: M indicates protein molecule marker, lane 1 indicates Mtb32a-4T supernatant, and lane 2 indicates Mtb32a-4T precipitate;

[0164] Figure 2Middle: M represents protein molecule marker, lane 1 represents Ag85a-4T supernatant, lane 2 represents Ag85a-4T precipitate, lane 3 represents ESAT6-CFP10-4T supernatant, lane 4 represents ESAT6-CFP10-4T precipitate, lane 5 represents RV2660-TB10.4-4T supernatant, and lane 6 represents RV2660-TB10.4-4T precipitate.

[0165] Example 3: Purification of recombinant antigens and SDS-PAGE analysis of purified antigens

[0166] 1. The fusion proteins Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigen components obtained by E. coli expression were purified by nickel column affinity chromatography and molecular sieve chromatography to obtain high-purity proteins. The specific steps are as follows:

[0167] 1) Pre-purification treatment

[0168] Use 20 ml of 20 mM Trsi-HCl, 150 mM NaCl, pH 7.4 buffer to resuspend the expression bacterial sludge of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T (corresponding to 200 mL of expression bacterial solution) and perform ultrasonic disruption; after cell disruption, centrifuge at 4°C, 13000 g / min for 30 min, discard the precipitate, and retain the supernatant.

[0169] 2) Nickel ion affinity chromatography

[0170] Affinity purification was performed using a nickel ion affinity column with a column volume of 10 mL, a flow rate of 5 mL / min, and a sample load of 70 mL.

[0171] Chromatographic procedure: Ni-Bestarose Fast Flow, disinfection, capture buffer 20mM Trsi-HCl, 150Mm NaCl, pH7.4 equilibration column, loading, 20mM Trsi-HCl, 150Mm NaCl, pH7.4 solution washing, 20mM Trsi-HCl, 150Mm NaCl, 2% Triton-X100, pH7.4 solution washing endotoxin, 20mM imidazole + 20mM Trsi-HCl, 150Mm NaCl, pH7.4 buffer washing away impurities, 500mM imidazole + 20mM Trsi-HCl, 150Mm NaCl, pH7.4 buffer elution Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, RV2660-TB10.4-4T antigen components.

[0172] 3) Molecular sieve purification

[0173] HiLoad 16 / 600 Superdex 200 pg was used for purification, the molecular sieve column volume was 120 mL, and the loading amount of MTB32A, AG85A, ESAT6-CFP10, and RV2660-TB10.4 affinity purified samples was controlled at about 4%.

[0174] Chromatographic procedure: Superdex 200pg; disinfection; balance the column with equilibration buffer (20mM Trsi-HCl, 150Mm NaCl, pH7.4), load the sample, wash with TBS solution, and collect Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigen components.

[0175] 2. SDS-PAGE analysis of purified antigen

[0176] The purified Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigens were subjected to SDS-PAGE analysis. The samples to be analyzed were prepared with LDS sample loading buffer (4x) plus reducing agent DTT, heated at 70°C for 5min, cooled to room temperature, centrifuged at 10000rpm for 20s, vortexed and mixed, and the final loading amount was 5μg. The samples to be analyzed and the non-prestained protein molecular weight standards were loaded onto 4-12% Bis-Tris gels, matched with MES electrophoresis buffer, set the voltage to 150V, and the electrophoresis lasted for about 60 minutes. After the electrophoresis, the gel was removed, placed in a clean container, and an appropriate amount of Coomassie brilliant blue staining solution was added to cover the gel, and stained on a shaker for 2h. After the staining was completed, the staining solution was poured out, and the gel was soaked in purified water for decolorization. The decolorization was continued on a shaker until the gel background color was completely removed, and the gel was photographed using a GelDoc Go gel imager.

[0177] Results and analysis:

[0178] The electrophoresis results showed that the four proteins Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T could be purified with a purity of more than 90%. Figure 3 to Figure 6 As shown, in addition, the control protein M72-4T can also be successfully renatured and purified, as shown in Figure 7 shown. Figure 3 In the figure, M indicates a protein molecule marker, and lanes 1 to 6 indicate Mtb32a-4T protein; Figure 4 In the figure, M indicates protein molecule marker, and lanes 1 to 10 indicate Ag85a-4T protein; Figure 5 In the figure, M indicates protein molecule marker, and lanes 1 to 5 indicate ESAT6-CFP10-4T protein; Figure 6 In the figure, M represents a protein molecule marker, and lanes 1 to 7 represent RV2660-TB10.4-4T protein.

[0179] Example 4: Expression and purification of M72 fusion protein

[0180] 1) Pre-purification treatment

[0181] According to the method in Example 1 "Expression of recombinant protein", 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 with 20 ml of 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 buffer and ultrasonically disrupted. After cell disruption, centrifuge at 4 ° C, 13000 g / min for 30 min, discard the supernatant, retain the precipitate, resuspend the precipitate in 20 mM Trsi-HCl, 150 Mm NaCl, 8 M Urea, pH 7.4 and dissolve it, centrifuge at 4 ° C, 13000 g / min for 30 min, and retain the supernatant.

[0182] 2) Nickel ion affinity chromatography

[0183] Affinity purification was performed using a nickel ion affinity column with a column volume of 10 mL, a flow rate of 5 mL / min, and a sample load of 70 mL. Chromatographic procedure: Ni-Bestarose Fast Flow, disinfection, capture buffer 20mM Trsi-HCl, 150MmNaCl, 8M Urea, pH7.4 equilibration column, loading, 20mM Trsi-HCl, 150MmNaCl, 8M Urea, pH7.4 solution washing, 20mM Trsi-HCl, 150MmNaCl, 8M Urea, 2% Triton-X100, pH7.4 solution washing endotoxin, 20mM imidazole + 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH7.4 buffer washing impurities, 500mM imidazole + 220mMTrsi-HCl, 150Mm NaCl, 8M Urea, pH7.4 buffer elution M72 antigen component.

[0184] 3) Membrane package ultrafiltration refolding

[0185] The M72-4T antigen purified by nickel ion affinity chromatography was concentrated by tangential flow ultrafiltration using a 10 kDa ultrafiltration membrane package, and then 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 buffer was continuously added to the concentrated protein solution to gradually remove Urea from the M72-4T antigen and completely renature the M72 antigen into 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 buffer.

[0186] 4) Molecular sieve purification

[0187] The renatured M72-4T antigen was purified using HiLoad 16 / 600 Superdex 200 pg, the molecular sieve column volume was 120 mL, and the loading amount of the M72-4T affinity purified sample was controlled at about 4%.

[0188] Chromatographic procedure: Superdex 200 pg; disinfection; balance the column with equilibration buffer (20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4), load the sample, wash with 20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 solution, and collect the M72-4T antigen component. Figure 7 It represents the results of molecular sieve separation and purification of M72-4T protein (M represents protein molecule marker, lanes 1-8 represent M72-4T protein).

[0189] Example 5: Construction, expression and purification of the gene encoding the binding peptide 2-NPM fusion protein

[0190] 1. Construction and expression

[0191] The NPM-4C protein sequence was codon optimized for E. coli expression, and gene synthesis and subcloning were performed. The coding gene of the fusion protein was constructed into pET30a and expressed in E. coli BL21 (DE3). After the bacteria were harvested, high-pressure homogenization was required to release the target protein and clarify the liquid, the main purpose of which was to remove bacterial fragments and impurity proteins. The amino acid and nucleotide sequences of NPM and NPM-4C are shown in Tables 3 and 4, respectively.

[0192] 2. Pretreatment before chromatography

[0193] The clarification of the feed liquid is mainly completed by heating treatment. The heating treatment is carried out using a two-step heating method, in which the supernatant after E. coli crushing is subjected to the first step heating and the second step heating (i.e., "two-step heating"), and the impurity removal effect of the two-step heating steps and the purity of the recombinant particle protein component are measured.

[0194] 60 g of wet E. coli cells collected by centrifugation were resuspended in 240 ml of buffer (20 mM Tris-HCl, 2 mM PMSF, pH = 9.0), crushed using a high-pressure homogenizer at a pressure of 1000 bar, and 280 ml of supernatant was collected after centrifugation, of which 40 ml was taken for two-step heating operation. SDS-PAGE analysis was performed on the supernatant after crushing, the supernatant of the first step of heating centrifugation, and the resuspended precipitate of the second step of heating centrifugation.

[0195] As shown in Table 5, in the first heating step, adjust pH=9.0, 80°C, heat in a water bath for 1 hour, return to room temperature and centrifuge to collect about 35 ml of supernatant. In the second heating step, add 35 ml of 100 mM Tris-HCl, 5 mM EDTA, 4% Triton, pH 7.4 buffer, then add 7 ml of 1 M Tris-HCl and mix. Heat in a 60°C water bath for 10 min, immediately centrifuge to collect the precipitate, and use 20 mM Tris-HCl 5 mM EDTA pH=9.0 buffer to re-dissolve the precipitate.

[0196] Table 3: Amino acid sequences of NPM, NPM-4C, binding peptide 2, connecting peptide 2 and fusion protein in the examples of the present application

[0197]

[0198] Table 4: Nucleotide sequences of NPM, NPM-4C, binding peptide 2, connecting peptide 2 and fusion protein in the examples of the present application

[0199]

[0200]

[0201] Table 5: Chromatographic pretreatment

[0202]

[0203]

[0204] After the two-step heating process, adding different concentrations of urea and sodium chloride before chromatography purification can significantly reduce the presence of unknown substances near the target recombinant granule protein band. The optimal process conditions for pretreatment of recombinant granule protein component samples before Fractogel DEAE M chromatography are soaking in 8M urea and 50-200mM sodium chloride.

[0205] 3. Chromatographic purification

[0206] The above-mentioned recombinant granule protein component sample solution was refined by ion exchange and hydrophobic chromatography. The first step of chromatographic purification was performed by Fractogel DEAE M chromatography process. The specific steps and parameters are shown in Table 6. The Fractogel DEAEM elution collection solution sample was first diluted with buffer, and 50% (w / v) sucrose stabilizer was added to prevent precipitation of the recombinant granule protein component during the next step of chromatography. The specific parameters are shown in Table 7. Then, the hydrophobic chromatography Octyl Bestarose 4FF chromatography process was used for purification (second step of chromatographic purification). The specific steps and parameters are shown in Table 8.

[0207] First step chromatography method: Chromatographic filler - Fractogel DEAE M, retention time - 12.5 min.

[0208] Table 6: First step chromatography method

[0209] Chromatography steps Chromatography buffer / conditions parameter Equilibration buffer 20mMTris-HCl,5mMEDTA,8MUrea,50mMNaCl,pH9.0 6CV pH after equilibrium 8.8±0.05 8.80 Wash 1 Buffer 20mMTris-HCl,5mMEDTA,8MUrea,50mMNaCl,pH9.0 1.5CV Wash 2 Buffer 20mM Tris-HCl, 5mMEDTA, 8MUrea, 2% Triton, pH9.0 5CV Wash 3 Buffer 20mM Tris-HCl, 8MUrea, pH 9.0 5CV Wash 4 Buffer 20mM Tris-HCl, 4M Urea, pH 9.0 5CV Elution buffer 20mM Tris-HCl, 4MUrea, 150mM, pH9.0 2CV Collection interval 50mAU-50mAU Light diameter 2mm

[0210] Table 7: Sample dilution method before the second step chromatography

[0211] Process steps Dilution buffer Dilution volume First step chromatography eluate collection N / A N / A Buffer dilution 20 mM Tris-HCl, 1 M NaCl, 50% (w / v) sucrose, pH 9.0 2 times the volume of elution buffer Buffer dilution 20mM Tris-HCl, 2M NaCl, pH 9.0 1 times the volume of elution buffer

[0212] Second step chromatography method: Chromatographic filler-OctylBestarose4FF, retention time-12.5min

[0213] Table 8: Second step chromatography method

[0214] Chromatography steps Chromatography buffer / conditions parameter Equilibration buffer 20 mM Tris-HCl, 1 M NaCl, 25% (w / v) sucrose, pH 9.0 2CV Wash buffer 20 mM Tris-HCl, 1 M NaCl, 25% (w / v) sucrose, pH 9.0 1.5CV Elution buffer 20 mM Tris-HCl, 25% (w / v) sucrose, pH 9.0 3CV Collection interval 50mAU-50mAU Light diameter 2mm

[0215] Results and analysis:

[0216] Through purity detection, it was found that after further purification by the above chromatographic medium combination, the purity of the obtained product can reach more than 99.0%. The specific SDS-PAGE analysis results of NPM-4C protein after separation and purification by Octyl Bestarose 4FF can be seen Figure 8 (M: protein molecule marker; lanes 1-2: NPM-4C protein).

[0217] Example 6: Binding of Mycobacterium tuberculosis structural proteins and NPM, purification of binding products, and particle characterization 1. Binding of antigens and NPM

[0218] The high-purity Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, RV2660-TB10.4-4T antigens obtained by molecular sieve purification were mixed with NPM-4C at a BCA protein concentration ratio of 6:1, and 50% sucrose mother solution was added to a final concentration of about 25% sucrose, and 10% of the total reaction volume of 1M Tris-HCl mother solution was added to stabilize the pH. The binding reaction was carried out at 22°C for 48 hours. As an example, the Mtb32a-NPM binding system can be specifically: Mtb32a-4T (1 mg / mL) 6mL, NPM-4C (1 mg / mL) 1mL, 50% sucrose 8.75mL, 1M Tris-HCl 7.41.75mL, total volume 17.5mL.

[0219] 2. Purification of the binding product

[0220] Use Cytiva HiLoad 16 / 600Superdex 200pg (column volume 120mL) or CytivaSuperdex 200 Increase 10 / 300GL (column volume 23mL) to purify the binding products of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM, and separate and remove Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 antigens that are not bound to NPM-4C. If the molecular sieve HiLoad 16 / 600 Superdex200pg is used, the loading amount of the combined samples of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM should be controlled at about 3% to 6%; if the molecular sieve Superdex 200Increase 10 / 300 GL is used, the loading amount of the combined samples of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM should be controlled at 0.5mL to 1mL.

[0221] Chromatographic procedure: Superdex 200pg or Superdex 200Increase, disinfection, equilibration buffer 12.5% ​​sucrose TBS solution (20mM Tris-HCl, 150mM NaCl, 12.5% ​​sucrose Mtb) equilibration column, loading, washing with 12.5% ​​sucrose TBS solution, collecting Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, RV2660-TB10.4-NPM components, and performing SDS-PAGE analysis according to the method of SDS-PAGE analysis of purified antigens in Example 3.

[0222] Results and analysis:

[0223] The molecular sieve-purified Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles were subjected to SDS-PAGE analysis, and the results showed that the purity could reach more than 90%. Figures 9 to 12 shown. Fig. 9 In the figure, M represents a protein molecule marker, lanes 1 to 3 represent Mtb32a-NPM binding products, and lanes 4 to 10 represent unbound Mtb32a; Fig.10 In the figure, M represents protein molecule marker, and lanes 1 to 5 represent Ag85a-NPM binding products; Fig.11 In the figure, M represents a protein molecule marker, and lanes 1 to 5 represent the ESAT6-CFP10-NPM binding products; Fig.12 In the figure, M represents a protein molecule marker, and lanes 1 to 5 represent the RV2660-TB10.4-NPM binding products.

[0224] According to the above method, Mtb32a, Ag85a, ESAT6-CFP10, RV2660-TB10.4 and NPM-4C were subjected to binding reaction, and the binding rate was measured by SDS-PAGE grayscale method to be 82.5%.

[0225] 3. Particle characterization

[0226] 1) TEM detection

[0227] The Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles prepared in the above "Purification of Binding Products" were negatively stained using the floating method to prepare samples. A 400-mesh grid with a support membrane was selected, which was pre-treated for hydrophilicity, and deionized water and 2% uranium formate negative staining solution were prepared. Take the prepared 3uL protein sample (0.1mg / ml) and drop it directly on one side of the grid of the support membrane; after timing for 1 minute, use a clean filter paper to absorb the excess liquid from the edge of the grid; after drying slightly, rinse it twice quickly with deionized water droplets; then take 5ul of negative staining solution and rinse once, and finally add 5ul of negative staining solution for 1 minute. After the end, use tweezers to pick up the grid and use filter paper to absorb the staining solution, leaving a thin layer to let it dry naturally before testing. Check under a 120 kV transmission electron microscope (FERRITINI Tecnai Spirit), observe the overall staining of the grid at low magnification, select holes with appropriate thickness for observation, and select appropriate areas under a high magnification microscope to take pictures and save.

[0228] 2) DLS detection

[0229] The purified Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles were diluted to 0.15 mg / mL. Using the Zetasizer Lab instrument, ≥1 mL of the sample to be tested was injected into the sample pool. The instrument was run for detection. The data were analyzed based on the Z-Average (nm) and Polydispersity Index (PI) values, as well as the SizeDistribution by Intensity / Volume distribution curve, and the results were reported.

[0230] Results and analysis:

[0231] Electron microscopic examination results of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles (0.1 mg / mL, 18500×). The images show that the particles are evenly distributed without aggregation. Figures 13 to 16 The distribution curve of Distribution by Intensity / Volume analyzed by Zetasizer Lab is shown in Fig.17As shown in Table 9, the peaks of the prepared Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles overlap well and have uniform particle size distribution. DLS results show that the particle diameters of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM are 37.0 nm, 38.7 nm, 43.0 nm, and 39.8 nm, respectively, as shown in Table 9.

[0232] Table 9: Z-Average (nm) and Polydispersity Index (PI) values ​​of prepared particle antigens

[0233] Sample name Z-Average(nm) Polydispersity(PI) Mtb32a-NPM 37.0 0.17 Ag85a-NPM 38.7 0.13 ESAT6-CFP10-NPM 43.0 0.14 RV2660-TB10.4-NPM 39.8 0.14

[0234] Example 7: Preparation of vaccine

[0235] 1. Test vaccine antigens and adjuvants

[0236] 1) Test vaccine antigen solution

[0237] The test vaccine protein stock solution was prepared by Guangzhou Painuo Biotechnology Co., Ltd., including Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, RV2660-TB10.4-NPM particles, Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, RV2660-TB10.4-4T antigens, and M72-4T control antigen.

[0238] 2) Test vaccine adjuvant

[0239] Commercial AS01B adjuvant, dual strength AS01B (WO 96 / 33739) was prepared by adding QS21 (5 μg) to small unilamellar vesicles (SUV) containing dioleoylphosphatidylcholine (100 μg) with cholesterol (25 μg) and monophosphoryl lipid A (MPL) (5 μg) in the membrane.

[0240] 2. Preparation method of the test vaccine

[0241] 1) Mtb recombinant antigen mixed vaccine

[0242] Aliquots (50 μL) for injection were prepared by mixing 0.8 μg of protein mixture (0.2 μg each of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T) in a buffer (TBS Mtb) with 50 μL of double strength AS01B. 2) Mtb nanoparticle antigen mixed vaccine

[0243] Aliquots for injection (50 μL) were prepared by mixing 0.8 μg of the protein mixture (0.2 μg each of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, RV2660-TB10.4-NPM) in buffer (TBS Mtb) with 50 μL of double strength AS01B.

[0244] 3) Single Mtb nanoparticle antigen vaccine

[0245] Aliquots (50 μL) for injection were prepared by mixing 0.8 μg of a protein mixture of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM in a buffer solution (TBS Mtb) with 50 μL of double strength AS01B.

[0246] 4) M72 control vaccine

[0247] Aliquots (50 μL) for injection were prepared by mixing 0.8 μg of M72-4T protein cocktail in TBS Mtb ​​with 50 μL of double strength AS01B.

[0248] Example 8: Vaccine immune protection experiment

[0249] 1. Experimental animals and groups

[0250] Select 4-6 week old female C57BL / 6, female, purchased from Vital River. After quarantine, mice were marked with metal ear tags, randomly divided into groups according to body weight, and allowed to eat and drink freely. Set up 6 candidate vaccine immunization groups (12 mice in each group), M72-4T group (12 mice), BCG control group (12 mice), and saline group (15 mice). Animals were raised in SPF standard animal rooms, supplied with sterile feed and sterilized deionized water for SPF animals, and the light was alternated for 12 hours day and night in the feeding room, the temperature was 21±2°C, and the humidity was 30-70%.

[0251] 2. Immunity and anti-virus

[0252] The vaccine group (vac-1 to vac-6) and the M72-4T group (Control-A) were immunized twice with a single dose of 100 μL (0.8 μg) at an interval of 3 weeks. The saline group was immunized with the same volume of saline. The BCG group (Control-B) was subcutaneously injected with 5×10 BCG bacteria. 4 CFU / mouse, 100 μL / mouse, immunization once. The other groups were injected intramuscularly.

[0253] Four weeks after the last immunization, mice were challenged with aerosols at a dose of 100 bacteria per mouse. Four weeks after the challenge, the mice were dissected and pathological sections of the lungs and spleens were prepared. The grinding fluid of the lungs and spleens was used to calculate the bacterial load. The specific grouping, immunization scheme, and challenge scheme are shown in Tables 10 and 11:

[0254] Table 10: Immunization and virus attack schedule

[0255]

[0256]

[0257] Table 11: Grouping and protein components, dosages, and adjuvant comparison table

[0258]

[0259] 3. Detection of specific IgG after immunization

[0260] Blood was collected on D20 after the first immunization. The whole blood collected in the centrifuge tube was placed at room temperature for 2 hours or in a 4°C refrigerator overnight. After the blood coagulated and the blood clot shrank, it was centrifuged at 4000 rpm for 10 minutes. The supernatant was collected and placed in a clean centrifuge tube and stored at -20°C.

[0261] 96-well ELISA plates (Thermo Fisher Scientific) were coated with 014, 072, 076, and 077 proteins (at a concentration of 1 μg / mL) at 100 ng / 50 μL / well at 4°C overnight, then washed twice with PBST (0.05% Tween20) and added with blocking solution (Thermo Fisher Scientific) at 200 μL / well. After blocking at room temperature (25°C ± 3°C) for 1-4 hours, the plates were washed twice, and then diluted immune serum was added and incubated at room temperature for 1 hour, followed by washing 4 times. The corresponding HRP-IgG1 and HRP-IgG2a working solutions at a dilution of 1:5000 were added at 50 μL / well. After incubation at room temperature for 1 hour, the plates were washed 6 times, and then 100 μL of color development solution was added to each well. After color development at room temperature in the dark for 10 minutes, 100 μL of 1M HCL was added to each well to terminate the reaction. The main wavelength of the microplate reader is set to 450nm, the reference wavelength is set to 620nm, and the sample absorbance value = OD450-OD620. The determination is completed within 5 minutes after termination, and the body fluid and cellular immune types are analyzed according to the test results.

[0262] Data processing:

[0263] The data are reliable if the following conditions are met: the control serum OD value is ± 0.2, the sample initial concentration corresponds to an OD value of < 3.0, the blank well corresponds to an OD value of less than 0.1, and the coefficient of variation of the duplicate wells (response value) should be less than 20%. The original sample data was transferred to the "excelEndpoint 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 the two groups of data were defined as having significant differences when P < 0.05.

[0264] The test results are as follows:

[0265] Fig. 20 ad is 072, 076, 077, 014 antigens as coating antigens to detect the corresponding total IgG antibody levels of immune sera in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, normal saline), Fig. 20 e corresponds to the corresponding IgG antibody level detected by M72 (0.8 μg immunization group) antigen.

[0266] The results showed that the single Mtb nanoparticle antigen vaccine (vac-3 / 4 / 5 / 6), Mtb recombinant antigen mixed vaccine (vac-2) and Mtb nanoparticle antigen mixed vaccine (vac-1) of the present invention can induce the production of IgG, which shows that the vaccines of the present invention have good immunogenicity. Among them, the IgG antibody levels corresponding to the vac-1 and single Mtb nanoparticle antigen vaccine groups are much higher than the IgG antibody level of vac-2, indicating that the nanoparticle vaccine of the present invention can significantly improve the immunogenicity of Mtb antigens.

[0267] Fig.21 Figures ad are the corresponding IgG1 and IgG2a antibody levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline) detected using 072, 076, 077, and 014 antigens as coating antigens, and Figure e corresponds to the IgG1 and IgG2a antibody levels of the M72 group (0.8 μg immunization group).

[0268] The results showed that the IgG1 and IgG2a levels of the vac-1 and single Mtb nanoparticle antigen vaccine groups were much higher than those of the vac-2. The IgG2a / IgG1 ratios of the vac-1 and single Mtb nanoparticle antigen vaccine groups were greater than 1.0, indicating that 072, 076, 077, and 014 can all induce Th1 type cellular immune responses. The IgG2a / IgG1 ratio of the M72 group was greater than 1.0, indicating that it can induce Th1 type cellular immune responses, which is consistent with literature reports and proves that the control vaccine was successfully prepared.

[0269] 4. Cytokine ELISA

[0270] 1) Three weeks after the last immunization, 6 mice were treated in each group, spleens were collected, splenocytes were isolated, and 2.5×10 5 cells / well, seeded into 96-well culture plates;

[0271] 2) using a 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), and ConA (3 μg / mL), and culturing in a 37°C incubator containing 5% CO2 for 72 hours;

[0272] 3) The supernatant was collected and IFN-γ, TNFα, IL-4, and IL-2 were detected by double antibody sandwich ELISA using commercial kits; the results were analyzed using Graphpad Prism 9.1.2 software. Unpaired test or One-Way ANOVA was used to analyze the differences, and the two groups of data were defined as having significant differences when P < 0.05.

[0273] 5. Cytokine ELISPOT

[0274] 1) A commercial 96-well filter plate was coated with IFN-γ, TNFα, IL-4, and IL-2 monoclonal antibodies and blocked. Three weeks after the last immunization, 6 mice were treated in each group, spleens were collected, spleen cells were isolated, and the number of cells was 2.0×10 5 1) The cells were inoculated into 96-well culture plates; 2) the culture medium alone and the 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), and ConA (3 μg / mL) were used and cultured in a 37°C incubator containing 5% CO2 for 48 hours;

[0275] 3) Wash the wells with PBS, add biotinylated mouse IFN-γ, TNFα, IL-4, and IL-2 secondary antibodies, incubate at room temperature for 2 hours, and develop the filter membrane using substrate according to the instructions of the commercial kit;

[0276] 4) After the plate is dried, the spots are counted using an automated ELISPOT plate reader for analysis.

[0277] The results were analyzed using Graphpad Prism 9.1.2 software. Unpaired test or One-Way ANOVA was used to analyze the differences. The two groups of data were defined as having significant differences when P < 0.05.

[0278] Fig. 22 ad are the corresponding 072 antigens as specific stimulation antigens to detect the corresponding IFN-γ, IL-2, TNFα, and IL-4 levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline); Fig. 22 eh are the corresponding 076 antigens used as specific stimulation antigens to detect the corresponding IFN-γ, IL-2, TNFα and IL-4 levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline).

[0279] The results showed that the corresponding IFN-γ, IL-2, TNFα and IL-4 levels of vac-1 and single Mtb nanoparticle antigen vaccine groups stimulated by 072 and 076 were higher than those of vac-2, and the IFN-γ, IL-2 and TNFα levels of each group were significantly higher than the IL-4 level, indicating that 072 and 076 can both induce Th1-type cellular immune responses.

[0280] Fig.23 ad are the corresponding 077 antigens as specific stimulation antigens to detect the corresponding IFN-γ, IL-2, TNFα, and IL-4 levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline); Fig.23 eh is the corresponding 014 antigen as the specific stimulation antigen to detect the corresponding IFN-γ, IL-2, TNFα and IL-4 levels of different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, normal saline).

[0281] The results showed that the corresponding IFN-γ, IL-2, TNFα and IL-4 levels of vac-1 and single Mtb nanoparticle antigen vaccine groups stimulated by 077 and 014 were higher than those of vac-2, and the IFN-γ, IL-2 and TNFα levels in each group were significantly higher than IL-4, indicating that 077 and 014 can both induce Th1-type cellular immune responses.

[0282] Fig.24The results showed that the IFN-γ, IL-2, and TNFα levels in the M72 group (0.8 μg immunization group) were significantly higher than those in IL-4, indicating that the prepared M72 vaccine can induce Th1 type cellular immune response after immunization of mice, which is consistent with literature reports and proves that the control vaccine was successfully prepared.

[0283] 6. Flow cytometry detection of T cell immune response

[0284] 1) Flow cytometry detection of CD4+ and CD8+ T cells: Three weeks after the last immunization, 6 mice were treated in each group, spleens were collected, and spleen cells (1×10 6 cells), washed with staining buffer and stained with 50 μL of a total volume of PE-Cyanine7 CD3 monoclonal antibody (1:50 final dilution, eBioscience), PE rat anti-mouse CD4 (1:50 final dilution, BD), and PerCP-Cy TM 5.5 Rat anti-mouse CD8 (1:50 final dilution, BD) mixture staining for 15 minutes. Wash twice with 1× Perm / Wash solution, wash cells with 1× Perm / Wash solution and resuspend, then analyze using DxFLEX (BECKMANCOULTER), and analyze data using CytExpert.

[0285] 2) ICS and flow cytometry were used to detect the expression of IFN-γ, TNFα, IL-4, IL-2 specific CD4+ and CD8+ T cells, and spleen cells (1×10 6 cells) and restimulated in vitro for 6 h with 014 (10 μg / mL), 072 (10 μg / mL), 076 (10 μg / mL), 077 (10 μg / mL), M72-4T (10 μg / mL), and CD28 / CD49d co-stimulatory antibody (BD). For intracellular cytokine staining, cells were incubated with protein transport inhibitor (containing Brefeldin A; BD) for 4 h; cells were washed with PBS and stained with fixable viability stain 780 (1:1000 final dilution, BD) and mouse Fc block (BD) for 15 min;

[0286] 3) Wash the cells with staining buffer and stain them with a total of 50 μL of a mixture containing PE-Cyanine7 CD3 monoclonal antibody (1:50 final dilution, eBioscience), PE rat anti-mouse CD4 (1:50 final dilution, BD) and PerCP-Cynce rat anti-mouse CD8 (1:50 final dilution, BD) for 15 minutes. Fix and permeabilize the cells using a fixation / permeabilization solution kit (BD). Then wash twice with 1× Perm / Wash solution and stain with APC rat anti-mouse IFN-γ (1:50 final dilution, BD), FITC rat anti-mouse IL-2 (1:50 final dilution, BD), PE rat anti-mouse IL-4 (1:50 final dilution, BD), R718 rat anti-mouse TNFα (1:50 final dilution, BD). Wash the cells with 1× Perm / Wash solution and resuspend them, and then analyze them using DxFLEX (BECKMAN COULTER). Data were analyzed using CytExpert and are presented as background subtraction from the mean responses of specific CD4+ and CD8+ T cells and expressed as a percentage of the total frequency of CD4+ T / CD8+ T cells expressing IFN-γ, TNFα, IL-4, and IL-2.

[0287] 7. Statistics of attack and protection results

[0288] 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 used was calibrated to deliver 50-100 CFU to the lungs. Four weeks later, the mice were euthanized, and the homogenates of the lungs and spleens were prepared by grinding with PBS / Tween-80 (0.05%). The homogenates of the individual whole organs were diluted and inoculated on Middlebrook 7H11 Bacto agar medium, and the bacterial colonies were counted after incubation at 37°C for 2-4 weeks under humidified, 5% CO2 conditions. The final data are expressed as the average Log10±SD of bacteria, and the Log10 reduction (difference) of CFU = Log10 CFU of the saline-treated group-Log10 CFU of the vaccine-treated group.

[0289] 8. Pathological section analysis

[0290] Four weeks after infection, all groups of mice were euthanized, and their spleen and lung tissues were collected. The tissues were fixed with formaldehyde solution and sent to the company for HA staining, pathological sections were prepared, and analysis was performed.

[0291] 9. Immunity Assessment Results

[0292] The results of specific IgG detection after immunization showed that the antigens in the vac-1 to vac-6 groups can produce good antibodies, and both the single antigen and mixed antigen groups have good immunogenicity. The cytokine ELISPOT results showed that the antigens covered in the vac-1 to vac-6 groups can stimulate lymphocytes to produce high levels of IFN-γ, TNFα, and IL-2. The screened antigens have a strong function of stimulating cellular immunity.

[0293] Mycobacterium tuberculosis mainly causes disease by infecting human macrophages. It is an intracellular parasite. Tuberculosis prevention and control requires intracellular anti-infection. This vaccine uses NPM nanoparticles to display Mycobacterium tuberculosis antigens 072, 076, 077 and 014, which can effectively stimulate the body to produce high levels of IFN-γ, TNFα, and IL-2. Among them, IFN-γ can activate macrophages to kill Mycobacterium tuberculosis, while enhancing the killing effect of NK cells, and IL-2 can also enhance the killing power of NK cells. TNFα, as an important cytokine, plays an important role in the anti-Mycobacterium tuberculosis infection process. Its main function is to promote the apoptosis of infected macrophages, expose the hidden Mtb, and then be presented by APC to activate CTL immunity.

[0294] The present application found that the IgG of the mixed NPM group (vac-1) and single NPM group of 072, 076, 077 and 014 prepared was higher than that of the mixed recombinant protein group (vac-2), and the IFN-γ, TNFα, IL-4 and IL-2 of the mixed NPM group and single NPM group were also higher than those of the mixed recombinant protein group, which shows that the Mycobacterium tuberculosis nanoparticle antigen after NPM display can better cooperate with adjuvant to improve the level of cellular immunity and humoral immunity, and reflects the advantage of NPM nanoparticles in presenting Mycobacterium tuberculosis antigen; at the same time, the vaccine of the present invention can produce a significant Th1 type immune response. TB vaccine mainly relies on cellular immune response, and this vaccine type with a Th1 type T cell immune response is in line with the immune bias of TB vaccine development.

[0295] The above results indicate that VLP-form antigens enhance immune protection. Nanoparticle antigens covered by vac-1 to vac-6 can effectively stimulate the body to produce immune protection and effectively prevent infection of Mycobacterium tuberculosis H37Rv strain in mice. They have great potential to be developed into TB subunit vaccines. The above recombinant nanoparticle Mycobacterium tuberculosis subunit vaccines have high application value.

[0296] To sum up, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An immunogenic complex, characterized in that It contains: (1) an antigen component, which comprises a Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof and a binding peptide 1, wherein the Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof and the binding peptide 1 form a fusion protein; (2) a particle protein component, which comprises a nanoparticle protein and a binding peptide 2; the nanoparticle protein and the binding peptide 2 form a fusion protein; The Mycobacterium tuberculosis structural protein or immunogenic fragment thereof is selected from Mtb32a, Ag85a, ESAT6, CFP10, RV2660 or TB10.4, and a fusion protein formed by two or more of the structural proteins or immunogenic fragments thereof; The antigen component and the granule protein component are covalently bound to each other via binding peptide 1 and binding peptide 2 to form an immunogenic complex.

2. The immunogenic complex according to claim 1, characterized in that: The binding peptide 1 contains the amino acid sequence shown in SEQ ID NO: 1, and the binding peptide 2 contains the amino acid sequence shown in SEQ ID NO: 24; Preferably, the antigen component further comprises a connecting peptide 1, and the particle protein component further comprises a connecting peptide 2; the antigen component is formed by fusing the Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof at the C-terminus via a connecting peptide 1 and a binding peptide 1; the particle protein component is formed by fusing the nanoparticle protein at the N-terminus via a connecting peptide 2 and a binding peptide 2; Optionally, the connecting peptide 1 is selected from (GSG) n 、(GGGGS) n or (EAAAK) n The amino acid sequence of n can be an integer greater than 0 and less than or equal to 5; the connecting peptide 2 is selected from (GGS) n 、(SGGSGG) n or (GSGGSGGSG) n An amino acid sequence of, n may be an integer greater than 0 and less than or equal to 10; Preferably, the connecting peptide 1 contains the amino acid sequence shown in SEQ ID NO: 2, and the connecting peptide 2 contains the amino acid sequence shown in SEQ ID NO: 25; Optionally, both the antigen component and the particle protein component comprise a histidine tag.

3. The immunogenic complex according to claim 1 or 2, characterized in that: The Mycobacterium tuberculosis structural protein or its immunogenic fragment or the fusion protein formed by the structural protein or immunogenic fragment is selected from the following sequences: (1) Mtb32a: whose sequence is 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more identical to SEQ ID NO: 3; (2) Ag85a: whose sequence is 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more identical to SEQ ID NO:5; (3) ESAT6-CFP10: whose sequence is 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more identical to SEQ ID NO:7; (4) RV2660-TB10.4: Its sequence has 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more identity with SEQ ID NO:9; Preferably, the Mycobacterium tuberculosis structural protein or its immunogenic fragment or the fusion protein formed by the structural protein or the immunogenic fragment is selected from: Mtb32a as shown in SEQ ID NO:3, Ag85a as shown in SEQ ID NO:5, ESAT6-CFP10 as shown in SEQ ID NO:7, and RV2660-TB10.4 as shown in SEQ ID NO:

9.

4. The immunogenic complex according to any one of claims 1 to 3, characterized in that: The nanoparticle protein is NPM, AP205 or Ferritin protein; the amino acid sequence of NPM is shown in SEQ ID NO:

26.

5. The immunogenic complex according to any one of claims 1 to 4, characterized in that Any one or more of the following (1)-(8): (1) the amino acid sequence of a Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof is shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9; (2) the amino acid sequence of binding peptide 1 is shown in SEQ ID NO: 1; (3) the amino acid sequence of connecting peptide 1 is shown in SEQ ID NO: 2; (4) The nanoparticle protein is selected from NPM, AP205 or Ferritin; wherein the amino acid sequence of NPM is as shown in SEQ ID NO: 26; (5) the amino acid sequence of binding peptide 2 is shown in SEQ ID NO: 24; (6) the amino acid sequence of connecting peptide 2 is shown in SEQ ID NO: 25; (7) the amino acid sequence of the antigen component is selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10; (8) The amino acid sequence of the granule protein component is shown in SEQ ID NO:

27.

6. A recombinant antigen protein comprising the Mycobacterium tuberculosis structural protein or its immunogenic fragment according to claim 3 or 5, or a fusion protein formed by the structural protein or immunogenic fragment.

7. The method for preparing the immunogenic complex according to any one of claims 1 to 5, characterized in that: include: (1) connecting the antigen component and the granule protein component encoding genes into expression vectors respectively, constructing expression recombinant plasmids and expression host strains, expressing the target protein, and purifying it; (2) The antigen component obtained in step (1) is co-incubated with the granule protein component to obtain an immunogenic complex.

8. An immune composition, characterized in that The invention comprises the immunogenic complex of any one of claims 1 to 5 or the recombinant antigen protein of claim 6, and further comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a stabilizer, an excipient, a surfactant, a buffer, and 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.

9. A Mycobacterium tuberculosis vaccine, characterized in that: The invention comprises the immune composition according to claim 8 and an adjuvant, wherein the adjuvant is selected from at least one of aluminum salt adjuvants, Freund's complete adjuvant, propolis adjuvant, water-oil adjuvant, cytokine, CpG DNA, genetically engineered attenuated toxin, immunostimulatory complex, and liposome.

10. Use of the immunogenic complex according to any one of claims 1 to 5, the recombinant antigen protein according to claim 6, the immune composition according to claim 8, or the Mycobacterium tuberculosis vaccine according to claim 9 in the preparation of a medicament for preventing or treating a disease caused by Mycobacterium tuberculosis infection.

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