A nanoparticle vaccine for preventing mycobacterium tuberculosis infection and a method of preparing the same

Abstract

A vaccine for Mycobacterium tuberculosis, in particular, the vaccine comprises an immunological composition comprising an antigen component and a granulin component. The granulin component comprises a nanoparticle protein, and the antigen component is covalently bound to the granulin component through binding peptide 1 and binding peptide 2 to form an immunogenic complex. The vaccine shows excellent cellular immunogenicity and antibody immunogenicity. The preparation method of the vaccine is also involved.

Description

Nanoparticle vaccine for preventing Mycobacterium tuberculosis infection and preparation method thereof Technical Field

[0001] The present invention relates to the field of biomedicine 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 disease is primarily transmitted through respiratory droplets and can strike at any age, with those with weakened immune systems being more susceptible. Infected individuals may not experience typical symptoms for a considerable period of time, but upon onset, symptoms such as acute lung inflammation, dry cough, and fever may develop. Without prompt treatment, it can lead to serious complications and death.

[0003] Tuberculosis (TB) is a major disease in developing countries, widespread and increasingly severe. In 2021, 10.6 million new cases of TB were diagnosed worldwide, and 1.6 million people died from the disease—approximately 4,300 deaths per day. TB primarily affects people in low- and middle-income countries, with those living and working in poor conditions and experiencing malnutrition at greatest risk. Globally, up to a quarter of the population has latent TB infection. Although infection with TB bacteria produces no symptoms, it can develop into active TB.

[0004] Currently, antibiotic treatment for tuberculosis patients using drugs such as isoniazid, rifampicin, and pyrazinamide can control tuberculosis, but this treatment cannot effectively prevent the spread of the disease. Infected individuals do not develop symptoms for a period of time, but remain somewhat contagious. Furthermore, long-term antibiotic treatment poses a significant challenge to patient compliance, with some patients even unable to complete effective treatment. Furthermore, long-term antibiotic treatment can lead to drug resistance.

[0005] Effective vaccination and accurate early diagnosis are crucial for tuberculosis (TB) control. Currently, the BCG (Bacillus Calmette-Guérin) vaccine, used for infants and young children, is made from a non-toxic strain of Mycobacterium bovis. Widespread use of BCG has significantly contributed to global TB prevention and control. While BCG protects infants and young children from severe systemic TB, its effectiveness against pulmonary TB in adolescents and adults is very limited. Therefore, there is an urgent need to develop new TB vaccines that can completely replace BCG or BCG-boosted versions. Recombinant subunit TB vaccines have been proven effective in humans and have become a hot topic in the development of new TB vaccines. For example, GSK's new TB vaccine candidate, M72 / AS01E, is a recombinant fusion protein containing two Mycobacterium tuberculosis antigens (MTB32A and MTB39A). Phase II clinical data show that its protection rate is only 49.7%, barely meeting the WHO's TB vaccine requirements. While this vaccine has some immune efficacy, the M72 fusion protein is easily degraded during expression and forms inclusion bodies. The particle size of the fusion protein is significantly affected by salt ions and pH, leading to complex production processes and high costs. Therefore, developing new TB vaccines with higher protection rates and simpler production processes for population vaccination is of great significance.

[0006] Nanoparticle vaccines, a new generation of vaccine design, are not only able to elicit potent neutralizing antibodies but also enhance cellular immune responses. Examples include human HPV vaccines, hepatitis B vaccines, and veterinary PCV2 vaccines. Many natural proteins (such as ferritin, lumazine synthase, Mi3, and AP205) can self-assemble into nanoparticles. When loaded with antigens, these nanoparticles can induce strong immune responses and have been widely studied and applied. The body's resistance to Mycobacterium tuberculosis infection primarily relies on its cellular immune mechanisms. The ability to generate effective Th1 CD4+ and CD8+ T cells and generate long-lasting cellular immune memory after vaccination is a key component of new TB vaccine development. Therefore, self-assembling nanoparticles are ideal carriers and are feasible for TB vaccine research.

[0007] Summary of the Invention

[0008] The present invention provides a Mycobacterium tuberculosis vaccine and a preparation method thereof. The vaccine is a nanoparticle vaccine.

[0009] Nanoparticle vaccines: vaccines based on the formation of nanoparticle proteins, which are mainly used to display antigens.

[0010] The present invention provides an immunogenic complex comprising a protein formed by a covalent binding reaction between an antigen component and a particle protein component.

[0011] The present invention provides an immune composition comprising the immunogenic complex of the present invention and a pharmaceutically acceptable carrier, which can be in the form of a lyophilized preparation, an injection preparation, an oral preparation or a spray preparation.

[0012] The present invention provides a vaccine comprising the immune composition of the present invention and an adjuvant.

[0013] The present invention provides an immunogenic complex comprising:

[0014] (1) an antigenic component comprising a Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof;

[0015] (2) A granular protein component comprising nanoparticle protein.

[0016] The present invention provides an immunogenic complex comprising:

[0017] (1) Antigen component, which comprises Mycobacterium tuberculosis structural protein or its immunogenic fragment,

[0018] peptide 1 and binding peptide 1;

[0019] (2) a particle protein component comprising nanoparticle protein, connecting peptide 2 and binding peptide 2;

[0020] The antigen component and the granule protein component are covalently bound to each other via binding peptide 1 and binding peptide 2.

[0021] The present invention provides an immunogenic complex comprising:

[0022] (1) Antigen component, consisting of Mycobacterium tuberculosis structural protein or its immunogenic fragment, connected

[0023] Peptide 1 and binding peptide 1;

[0024] (2) granule protein component, consisting of nanoparticle protein, connecting peptide 2 and binding peptide 2;

[0025] The antigen component and the granule protein component are covalently bound to each other via binding peptide 1 and binding peptide 2.

[0026] In some embodiments, in any immunogenic complex provided by the present invention, the antigen component is formed by fusing a Mycobacterium tuberculosis structural protein with a binding peptide 1 via a connecting peptide 1 at the C-terminus.

[0027] 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.

[0028] 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 to the binding peptide 2 via the connecting peptide 2 at the N-terminus.

[0029] 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 sequentially: Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof, connecting peptide 1, and binding peptide 1; the granule protein component, from N-terminus to C-terminus, is sequentially: binding peptide 2, connecting peptide 2, and 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.

[0030] In some embodiments, in any one of the immunogenic complexes provided herein, the antigen component and / or the particle protein component comprises a histidine tag.

[0031] The present invention provides an immunogenic complex comprising:

[0032] (1) an antigen component comprising a Mycobacterium tuberculosis structural protein or an immunogenic fragment thereof, and a connecting peptide 1;

[0033] (2) The granule protein component comprises nanoparticle protein subunits.

[0034] In some embodiments, the Mycobacterium tuberculosis structural protein is linked to one subunit of the nanoparticle protein to form a fusion protein, which is then bound to another subunit of the nanoparticle protein.

[0035] In some embodiments, in any of the immunogenic complexes provided herein, the particle protein component comprises a nanoparticle protein. Preferably, the nanoparticle protein can be a virus-like particle protein, which is formed by a viral structural protein, preferably, by a bacteriophage capsid protein AP205. The particle protein component and the antigen component can be covalently bound to form a particle structure.

[0036] In some embodiments, in any immunogenic complex 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.

[0037] 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.

[0038] In some embodiments, in any immunogenic complex provided by the present invention, the binding peptide 1 comprises the amino acid sequence shown in SEQ ID NO: 1.

[0039] In some embodiments, in any one of the immunogenic complexes provided herein, the binding peptide 2 comprises the amino acid sequence shown in SEQ ID NO:24.

[0040] In some embodiments, in any one of the immunogenic complexes provided herein, the connecting peptide 1 comprises (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, in any immunogenic complex provided by the present invention, the connecting peptide 1 is preferably GSG GSG (SEQ ID NO: 2).

[0041] In some embodiments, in any one of the immunogenic complexes provided herein, the connecting peptide 2 comprises (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, in any immunogenic complex provided by the present invention, the connecting peptide 2 is preferably GGSGGSGGSGGS (SEQ ID NO: 25).

[0042] 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 the 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. 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.

[0043] Preferably, the structural protein of Mycobacterium tuberculosis of the present invention uses Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 fusion protein, 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.

[0044] 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 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.

[0045] Preferably, in any one of the immunogenic complexes provided by the present invention, the antigenic 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 antigenic component Mtb32a-4T sequence is shown in SEQ ID NO: 4, the antigenic component Ag85a-4T sequence is shown in SEQ ID NO: 6, the antigenic component ESAT6-CFP10-4T sequence is shown in SEQ ID NO: 8, and the antigenic component RV2660-TB10.4-4T sequence is shown in SEQ ID NO: 10.

[0046] In some embodiments, in any of the immunogenic complexes described above, the particle protein component is a fusion protein formed by linking peptide 2 at the N-terminus of a nanoparticle protein with binding peptide 2. Preferably, the nanoparticle protein is NPM, AP205 capsid protein 3 (AP205), or Ferritin. Specifically, in some alternative approaches, binding peptide 2 (designated "4C") is linked to the gene encoding the nanoparticle protein via linker peptide 2, inserted into a prokaryotic expression vector (e.g., pET-28a(+) or pET-30a(+)), and expressed in E. coli cells to obtain a fusion protein of binding peptide 2 and the nanoparticle protein. The fusion protein can be purified by chromatography, such as anion exchange chromatography or hydrophobic chromatography, to obtain a product. The nanoparticle protein is preferably NPM, AP205, or Ferritin; the resulting particle protein components are designated NPM-4C, AP205-4C, or Ferritin-4C.

[0047] Specifically, in some alternative embodiments, under suitable reaction conditions, any of the aforementioned antigen components is conjugated to a granule protein component, whereby the antigen component's binding peptide 1 is covalently bonded to the granule protein component's binding peptide 2 to form a coupling, thereby forming the immunogenic complex. Different immunogenic complexes can be formed using different nanoparticle proteins, and these immunogenic complexes are designated as Mtb32a-NPM, Mtb32a-AP205, or Mtb32a-Ferritin; Ag85a-NPM, Ag85a-AP205, or Ag85a-Ferritin; ESAT6-CFP10-NPM, ESAT6-CFP10-AP205, or ESAT6-CFP10-Ferritin; and RV2660-TB10.4-NPM, RV2660-TB10.4-AP205, or RV2660-TB10.4-Ferritin.

[0048] In some embodiments, the present invention provides an immunogenic complex comprising:

[0049] (1) an antigen component comprising a Mycobacterium tuberculosis structural protein, a connecting peptide 1 and a binding peptide 1;

[0050] (2) A particle protein component comprising nanoparticle protein, connecting peptide 2 and binding peptide 2.

[0051] 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 wherein n is 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 is 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.

[0052] 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.

[0053] In other embodiments, the present invention provides an immunogenic complex comprising:

[0054] (1) an antigenic component comprising a structural protein and a connecting peptide 1 of Mycobacterium tuberculosis;

[0055] (2) a granule protein component comprising nanoparticle protein subunits; preferably, the nanoparticle protein subunits are I53-50A and / or I53-50B subunits.

[0056] 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.

[0057] Specifically, in any of the immunogenic complexes provided herein, the Mycobacterium tuberculosis structural protein is linked to a subunit of the nanoparticle protein to form a fusion protein, which is then bound to another subunit of the nanoparticle protein. Preferably, the subunit of the nanoparticle protein is I53-50A or I53-50B. Furthermore, in some alternative embodiments, the Mycobacterium tuberculosis structural protein in the antigen component is linked to the nanoparticle protein I53-50A subunit at the C-terminus via linker peptide 1 to form a Mycobacterium tuberculosis structural protein-I53-50A fusion protein; this fusion protein is then bound to the nanoparticle protein I53-50B subunit.

[0058] As mentioned above, when I53-50 is selected as the nanoparticle protein, I53-50 contains 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. The coding gene for the above fusion protein is inserted into a eukaryotic cell expression vector (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 bound to form Mycobacterium tuberculosis nanoparticles, which are named Mycobacterium tuberculosis structural protein-I53-50.

[0059] Preferably, any immunogenic complex provided by the present invention comprises Mycobacterium tuberculosis structural protein-I53-50A.

[0060] In some embodiments, the present invention provides an immunogenic complex comprising any one or more of the following (1)-(7):

[0061] (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;

[0062] (2) The amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO: 2;

[0063] (3) The amino acid sequence of the binding peptide 1 is shown in SEQ ID NO: 1;

[0064] (4) the nanoparticle protein is selected from NPM, AP205 or Ferritin;

[0065] (5) the nanoparticle protein subunit is selected from I53-50A and / or I53-50B;

[0066] (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;

[0067] (7) The amino acid sequence of the binding peptide 2 is shown in SEQ ID NO: 24.

[0068] 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.

[0069] Furthermore, the present invention also provides a method for preparing any of the above-mentioned immunogenic complexes, comprising the following steps:

[0070] (1) The antigen component and granule protein component encoding genes are respectively connected into expression vectors to construct expression recombinant plasmids and expression host strains, express the target protein, and purify it;

[0071] (2) The antigen component obtained in step (1) is co-incubated with the granule protein component to obtain an immunogenic complex.

[0072] The present invention provides a method for preparing an immunogenic complex for preventing or treating Mycobacterium tuberculosis virus-related diseases:

[0073] (1) Connecting the genes encoding the Mycobacterium tuberculosis virus antigen component and the particle protein component into expression vectors to construct expression recombinant plasmids;

[0074] (2) constructing a recombinant strain capable of expressing the Mycobacterium tuberculosis virus antigen component and particle protein component in a host cell;

[0075] (3) using the recombinant strain to express the fusion protein and purify the fusion protein;

[0076] (4) The antigen component and the granule protein component are co-incubated to produce a conjugation reaction and obtain an immunogenic complex.

[0077] Preferably, the immunogenic complex obtained in the above step (4) is purified to obtain a vaccine stock solution.

[0078] 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(+).

[0079] 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 granule protein component vector is E. coli.

[0080] The immunogenic complex for preventing or treating Mycobacterium tuberculosis virus-related diseases of the present invention comprises an antigen component comprising a fusion protein formed by the Mycobacterium tuberculosis structural protein-binding peptide 1.

[0081] The immunogenic complex described herein for preventing or treating Mycobacterium tuberculosis virus-associated diseases comprises highly purified Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 antigens, obtained through molecular sieve purification, and NPM-4C, mixed at a BCA protein ratio of 6:1. A 50% sucrose stock solution is added to a final concentration of approximately 25%, and a 1M Tris-HCl stock solution is added at 10% of the total reaction volume to stabilize the pH. The binding reaction is carried out at 22°C for 48 hours. Endotoxin levels are all below 100 EU / ml, meeting the requirements for large-scale production.

[0082] 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 regulator, wherein 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 regulator is hydrochloric acid.

[0083] 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, and 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, or 50 μg / dose. The dose used in mouse experiments is 1 / 10 of the human dose.

[0084] In some embodiments, the immune composition provided by the present invention is an injection or a lyophilized preparation, preferably a lyophilized preparation.

[0085] 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 or arginine, the excipient is mannitol, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate or sodium dihydrogen phosphate dihydrate, and the pH adjuster is hydrochloric acid.

[0086] In some embodiments, the immune composition provided by the present invention is a lyophilized preparation comprising Mycobacterium tuberculosis structural protein-NPM immunogenic complex, sucrose, arginine, mannitol, Tween 80, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, and hydrochloric acid.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] In the Mycobacterium tuberculosis vaccine of the present invention, the water-oil adjuvant is a squalene adjuvant containing squalene.

[0091] 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.

[0092] 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.

[0093] 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).

[0094] 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, if the Mycobacterium tuberculosis structural protein-NPM is 5 μg / dose for mice, the human dose must be 50 μg / dose; if the adjuvant is 50 μl / dose for mice, the human dose must be 500 μl / dose (0.5 ml / dose); if the adjuvant is 25 μl / dose for mice, the human dose must be 250 μg / dose (0.25 ml / dose), and so on.

[0095] The present invention further provides a complete kit, characterized in that it comprises the Mycobacterium tuberculosis vaccine of the present invention, and the apparatus and container required for vaccinating the vaccine.

[0096] 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 prepared as a lyophilized preparation or an injectable preparation) and an adjuvant (in liquid form). The Mycobacterium tuberculosis structural protein-NPM immune composition and adjuvant are packaged in separate bottles.

[0097] The present invention provides use of a Mycobacterium tuberculosis nanoparticle immunogenic complex, immune composition or vaccine in the preparation of a medicament for preventing or treating tuberculosis.

[0098] All reagents used in the present invention can be purchased commercially.

[0099] Compared with the prior art, the present invention has the following beneficial effects:

[0100] 1. The present invention utilizes an E. coli expression system to express the structural proteins of Mycobacterium tuberculosis (Mtb32A-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T) in fusion form. These fusion proteins are all capable of achieving soluble expression. Simultaneously, NPM-4C nanoparticles are produced by expression in the E. coli expression system. The immunogenic complex of the present invention has a significant preventive effect against Mycobacterium tuberculosis (Mtb) infection. Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles prepared using Mtb 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 a tuberculosis vaccine.

[0101] 2. For the first time, Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T fusion proteins were covalently coupled to NPM-4C nanoparticles to prepare nanoparticle antigens Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM. The production process is stable, low-cost, and has great industrial potential.

[0102] 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 good cellular immunity and humoral immunity.

[0103] 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 Mycobacterium tuberculosis load in the spleen and lungs of infected mice, and has important development value.

[0104] 5. The method for preparing the nanoparticle-type tuberculosis vaccine provided by the present invention is low-cost and suitable for large-scale production. In the present invention, the granular 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. They 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 recombinant granular protein components in one batch 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.

[0105] 6. The preparation method of the recombinant granular 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 solvent used in subsequent chromatography purification; the protein product prepared using the recombinant granular 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, thereby improving safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG1 shows the expression of the recombinant protein Mtb32a-4T and its identification by Western Blot;

[0107] FIG2 shows the expression of recombinant proteins Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T by Western Blot analysis;

[0108] FIG3 shows the results of molecular sieve separation and purification of the recombinant protein Mtb32a-4T;

[0109] FIG4 shows the results of separation and purification of the recombinant protein Ag85a-4T by molecular sieve;

[0110] FIG5 shows the results of separation and purification of the recombinant protein ESAT6-CFP10-4T protein by molecular sieve;

[0111] FIG6 shows the results of molecular sieve separation and purification of the recombinant protein RV2660-TB10.4-4T;

[0112] FIG7 shows the results of separation and purification of the recombinant protein M72-4T protein by molecular sieve;

[0113] FIG8 shows the results of separation and purification of NPM-4C protein using Octyl Bestarose 4FF;

[0114] FIG9 shows the results of separation and purification of the recombinant protein Mtb32a-NPM binding product;

[0115] FIG10 shows the results of separation and purification of the recombinant protein Ag85a-NPM binding product;

[0116] FIG11 shows the results of separation and purification of the recombinant protein ESAT6-CFP10-NPM binding product;

[0117] FIG12 shows the results of separation and purification of the recombinant protein RV2660-TB10.4-NPM binding product;

[0118] Figures 13 to 16 show the negative staining electron microscopy results of recombinant protein Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles, respectively;

[0119] FIG17 shows the distribution curves of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles Distribution by Intensity / Volume;

[0120] Figure 18 shows SDS-PAGE analysis of RV2660-TB10.4T before and after mutation;

[0121] FIG19 shows the distribution curve of NPM nanoparticles prepared before and after RV2660-TB10.4T mutation by Intensity / Volume;

[0122] Figure 20 shows the total binding antibody IgG levels of the serum of the corresponding group D20 using 072, 076, 077, 014, and M72 proteins as coating antigens, respectively. Figures a and b 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.

[0123] Figure 21 shows the IgG1 and IgG2a antibody levels of the serum of the corresponding groups on D20, using 072, 076, 077, 014, and M72 proteins as coating antigens, respectively. Figures a and b 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, respectively. Figure e corresponds to the IgG1 and IgG2a antibody levels of the M72 group (0.8 μg immunization group).

[0124] Figures 22a-d show the IFN-γ, IL-2, TNFα, and IL-4 levels corresponding to the different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline) detected by using 072 antigen as a specific stimulating antigen; Figures 22e-h show the IFN-γ, IL-2, TNFα, and IL-4 levels corresponding to the different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline) detected by using 076 antigen as a specific stimulating antigen;

[0125] Figures 23a-d show the IFN-γ, IL-2, TNFα, and IL-4 levels corresponding to the different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline) detected using 077 antigen as a specific stimulating antigen; Figures 23e-h show the IFN-γ, IL-2, TNFα, and IL-4 levels corresponding to the different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and normal saline) detected using 014 antigen as a specific stimulating antigen;

[0126] FIG24 shows the specific IFN-γ, IL-2, TNFα and IL-4 levels in D20 splenocytes detected using M72 protein as a stimulation antigen. DETAILED DESCRIPTION

[0127] The principles and features of the present invention are described below with reference to examples. The examples are intended only to illustrate the present invention and are not intended 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 embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments and are not intended to limit the scope of protection of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. When numerical ranges are given in the examples, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints may be used. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the examples of the present invention may also be used to implement the present invention, based on the prior art knowledge of those skilled in the art and the description of the present invention. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

[0128] Example 1: Construction and expression of a gene encoding a Mycobacterium tuberculosis structural protein-binding peptide 1 fusion protein

[0129] 1. Construction of recombinant antigens

[0130] With reference to the sequences of the structural proteins Mtb32a, Ag85a, ESAT6, CFP10, RV2660, and TB10.4 from the Mycobacterium tuberculosis H37RV strain (GenBank: AL123456.3), Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T fusion proteins were constructed, respectively. The M72-4T fusion protein was constructed with reference to the M72 sequence from a GSK patent (Granted Publication No. CN 103249431B). The specific construction methods are as follows. The specific sequences of the constructed proteins and the reference sequences are shown in Table 1.

[0131] 1)Mtb32a-4T

[0132] The original wild-type sequence was modified from S / 177aa to A / 177aa, and a linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences were added to the C-terminus, ending with a stop codon. The resulting molecule was named 014.

[0133] 2) Ag85a-4T

[0134] The original wild-type sequence was truncated at the N-terminus to the sequence QLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAG. Furthermore, a linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences were added to the C-terminus of the protein, ending with a stop codon. The resulting molecule was named 072.

[0135] 3) ESAT6-CFP10-4T

[0136] ESAT6 was placed at the N-terminus of the fusion protein, and CFP10 was placed at the C-terminus. A linker (GSGGSG) sequence was used to connect them. A linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences were added to the C-terminus, followed by a stop codon. The resulting molecule was named 076.

[0137] 4)RV2660-TB10.4-4T

[0138] The original wild-type RV2660 sequence was mutated from C / 66aa to A / 66aa, and the mutated RV2660 was placed at the N-terminus of the fusion protein. The TB10.4 protein was placed at the C-terminus of the fusion protein, connected using a linker (GSGGSG) sequence. The C-terminus was then supplemented with a linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences, followed by a stop codon. The resulting molecule was named 077.

[0139] Through experiments, it was unexpectedly discovered that the original wild sequence of RV2660 was subjected to a C / 66aa→A / 66aa transformation, which could avoid the formation of dimers in RV2660-TB10.4-4T and enable the mutated RV2660-TB10.4-4T to maintain a soluble monomer form (SDS-PAGE analysis under both reducing and non-reducing conditions after mutation was a single band), as shown in Figure 18. The mutation also made the formed RV2660-TB10.4-NPM nanoparticles more uniform (Z-Average and Polydispersity Index values ​​changed from 77.3 and 0.23 before mutation to 39.8 and 0.14 after mutation, respectively), as shown in Figure 19.

[0140] In Figure 18, M: protein molecule marker, lane 1: RV2660-TB10.4T-4T before mutation (DTT+), lane 2: RV2660-TB10.4T-4T mutation (DTT-), lane 3: RV2660-TB10.4T-4T after mutation (DTT+), lane 4: RV2660-TB10.4T-4T after mutation (DTT-).

[0141] In Figure 19, a is the NPM nanoparticles prepared before the RV2660-TB10.4T mutation; b is the NPM nanoparticles prepared after the RV2660-TB10.4T mutation.

[0142] 5) M72-4T

[0143] 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.

[0144] 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.

[0145] Table 1: Amino acid sequences of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, RV2660-TB10.4-4T, M72-4T, binding peptide 1, linker peptide 1, and fusion proteins

[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 proteins

[0147] 2. Expression of recombinant antigens

[0148] 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 colonies were then picked and transferred to 10 ml of TB medium containing 50 μg / ml Ampicillin and cultured overnight at 37°C, 220 rpm / min.

[0149] 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.

[0150] 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 to induce expression overnight for about 16 hours. The final IPTG concentration was 500 μM.

[0151] Collect the bacterial suspension, centrifuge at 6000g, 4℃ for 15 minutes, and discard the supernatant.

[0152] Centrifuge again at 6000 g and 4°C for 3 minutes, remove the culture medium in the supernatant, and freeze the bacterial slurry in a -80°C refrigerator.

[0153] Example 2: Western blot identification of recombinant antigens

[0154] 1) Resuspend the expression bacterial slurries of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T in 20 ml of 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 buffer. After ultrasonic disruption, centrifuge at 13,000 g / min at 4°C for 30 min. Collect the supernatant and precipitate (suspend in 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 buffer).

[0155] 2) Western blot analysis was performed using LDS sample loading buffer (4x) plus the reducing agent DTT. The sample was heated at 70°C for 5 min, cooled to room temperature, centrifuged at 10,000 rpm for 20 s, and vortexed to mix. The final loading volume was 0.5 μg.

[0156] 3) Load the sample to be analyzed and prestained protein molecular weight standards onto a 4-12% Bis-Tris gel using MES running buffer. Set the voltage to 150 V and run the gel for approximately 60 minutes.

[0157] 4) Use The membrane was transferred using the Turbo instrument and corresponding reagents, incubated using the iBind instrument with Anti-his mouse monoclonal antibody and goat anti-mouse secondary antibody conjugated with AP enzyme, and then developed with colorimetric solution. The GelDoc Go was used for photography.

[0158] Western blot analysis showed that all four proteins were expressed in soluble form and inclusion bodies, and the band positions were consistent with the expected molecular weights, as shown in Figures 1 and 2.

[0159] In Figure 1 : M represents a protein molecule marker, lane 1 represents the supernatant of Mtb32a-4T, and lane 2 represents the precipitate of Mtb32a-4T;

[0160] In Figure 2: M represents a protein molecule marker, lane 1 represents the supernatant of Ag85a-4T, lane 2 represents the precipitate of Ag85a-4T, lane 3 represents the supernatant of ESAT6-CFP10-4T, lane 4 represents the precipitate of ESAT6-CFP10-4T, lane 5 represents the supernatant of RV2660-TB10.4-4T, and lane 6 represents the precipitate of RV2660-TB10.4-4T.

[0161] Example 3: Purification of recombinant antigens and SDS-PAGE analysis of purified antigens

[0162] 1. The fusion proteins Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigen components expressed in E. coli were purified by nickel column affinity chromatography and molecular sieve chromatography to obtain high-purity proteins. The specific steps are as follows:

[0163] 1) Pre-purification treatment

[0164] Resuspend the expression bacterial slurry of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T (corresponding to 200 mL of expression bacterial solution) in 20 ml of 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 buffer and perform ultrasonic disruption. After cell disruption, centrifuge at 13,000 g / min at 4°C for 30 min, discard the precipitate, and retain the supernatant.

[0165] 2) Nickel ion affinity chromatography

[0166] Affinity purification was performed using a nickel ion affinity column with a 10 mL column volume, a 5 mL / min flow rate, and a 70 mL sample load.

[0167] Chromatographic procedure: Ni-Bestarose Fast Flow, disinfection, capture buffer 20mM Trsi-HCl, 150Mm NaCl, pH7.4 equilibration column, sample 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.

[0168] 3) Molecular sieve purification

[0169] A HiLoad 16 / 600 Superdex 200 pg column was used for purification, the molecular sieve column volume was 120 mL, and the loading amount of affinity-purified samples of MTB32A, AG85A, ESAT6-CFP10, and RV2660-TB10.4 was controlled at approximately 4%.

[0170] Chromatographic procedure: Superdex 200pg; disinfection; equilibrate 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.

[0171] 2. SDS-PAGE analysis of purified antigen

[0172] Purified Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigens were analyzed by SDS-PAGE. Samples were prepared with LDS sample loading buffer (4x) plus the reducing agent DTT, heated at 70°C for 5 minutes, cooled to room temperature, centrifuged at 10,000 rpm for 20 seconds, and vortexed to mix. A final sample load of 5 μg was obtained. Samples and non-prestained protein molecular weight standards were loaded onto 4-12% Bis-Tris gels using MES running buffer. The voltage was set to 150 V and the electrophoresis lasted for approximately 60 minutes. After electrophoresis, the gel was removed and placed in a clean container. An appropriate amount of Coomassie Brilliant Blue stain was added to cover the gel and the gel was stained on a shaker for 2 hours. After staining, the staining solution was discarded and the gel was destained by immersion in purified water. Destaining was continued on a shaker until the gel background color was completely removed. The gel was then photographed using a GelDoc Go gel imager.

[0173] Results and Analysis:

[0174] Electrophoresis results showed that the four proteins, Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T, could all be purified with a purity exceeding 90%, as shown in Figures 3 to 6. Furthermore, the control protein, M72-4T, could also be successfully renatured and purified, as shown in Figure 7. In Figure 3, M represents a protein marker, and lanes 1 to 6 represent Mtb32a-4T protein; in Figure 4, M represents a protein marker, and lanes 1 to 10 represent Ag85a-4T protein; in Figure 5, M represents a protein marker, and lanes 1 to 5 represent ESAT6-CFP10-4T protein; and in Figure 6, M represents a protein marker, and lanes 1 to 7 represent RV2660-TB10.4-4T protein.

[0175] Example 4: Expression and purification of M72 fusion protein

[0176] 1) Pre-purification treatment

[0177] 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, the cells were centrifuged at 4 ° C, 13000 g / min for 30 min, the supernatant was discarded, and the precipitate was retained. The precipitate was resuspended in 20 mM Trsi-HCl, 150 mM NaCl, 8 M Urea, pH 7.4 and dissolved, and centrifuged at 4 ° C, 13000 g / min for 30 min, and the supernatant was retained.

[0178] 2) Nickel ion affinity chromatography

[0179] Affinity purification was performed using a nickel ion affinity column with a 10 mL column volume, a 5 mL / min flow rate, and a 70 mL sample load. Chromatographic procedure: Ni-Bestarose Fast Flow, disinfection, capture buffer 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH7.4 equilibration column, sample loading, 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH7.4 solution washing, 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, 2% Triton-X100, pH7.4 solution washing endotoxin, 20mM imidazole + 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH7.4 buffer washing to remove impurities, 500mM imidazole + 220mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH7.4 buffer elution M72 antigen component.

[0180] 3) Membrane package ultrafiltration refolding

[0181] The M72-4T antigen purified by nickel affinity chromatography was concentrated by tangential flow ultrafiltration using a 10 kDa ultrafiltration membrane. 20 mM Trsi-HCl, 150 mM NaCl, pH 7.4 buffer was then 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.

[0182] 4) Molecular sieve purification

[0183] 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 approximately 4%.

[0184] Chromatographic procedure: Superdex 200pg; disinfection; equilibrate the column with equilibration buffer (20mM Tris-HCl, 150mM NaCl, pH 7.4), load the sample, wash with 20mM Tris-HCl, 150mM NaCl, pH 7.4, and collect the M72-4T antigen fraction. Figure 7 shows the molecular sieve separation and purification results of the M72-4T protein (M represents protein marker, lanes 1-8 represent the M72-4T protein).

[0185] Example 5: Construction, expression and purification of the gene encoding the binding peptide 2-NPM fusion protein

[0186] 1. Construction and expression

[0187] The NPM-4C protein sequence was codon-optimized for E. coli expression, and gene synthesis and subcloning were performed. The gene encoding the fusion protein was constructed into pET30a and expressed in E. coli BL21(DE3). After harvesting, the cells were subjected to high-pressure homogenization to release the target protein and clarify the liquid to remove cell debris and impurities. The amino acid and nucleotide sequences of NPM and NPM-4C are shown in Tables 3 and 4, respectively.

[0188] 2. Pretreatment before chromatography

[0189] Clarification of the feed solution is primarily accomplished through heat treatment. A two-step heating method is used, where the supernatant from E. coli is subjected to both a first heating step and a second heating step (i.e., "two-step heating"). The effectiveness of both heating steps in removing impurities and the purity of the recombinant granular protein fraction are measured.

[0190] 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) and disrupted using a high-pressure homogenizer at 1000 bar. After centrifugation, 280 ml of supernatant was collected, of which 40 ml was subjected to a two-step heating procedure. The supernatant after disruption, the supernatant from the first heating centrifugation step, and the resuspension of the pellet from the second heating centrifugation step were analyzed by SDS-PAGE.

[0191] As shown in Table 5, in the first heating step, adjust the pH to 9.0, heat in an 80°C water bath for 1 hour, return to room temperature, and centrifuge to collect approximately 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, followed by 7 ml of 1 M Tris-HCl, and mix thoroughly. Heat in a 60°C water bath for 10 minutes, then immediately centrifuge to collect the precipitate, and reconstitute the precipitate in a buffer consisting of 20 mM Tris-HCl and 5 mM EDTA, pH 9.0.

[0192] Table 3: Amino acid sequences of NPM, NPM-4C, binding peptide 2, connecting peptide 2, and fusion protein in the examples of this application

[0193] Table 4: Nucleotide sequences of NPM, NPM-4C, binding peptide 2, connecting peptide 2, and fusion protein in the examples of this application

[0194] Table 5: Chromatographic pretreatment

[0195] After the two-step heating process, adding different concentrations of urea and sodium chloride before chromatographic purification can significantly reduce the presence of unidentified 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.

[0196] 3. Chromatographic purification

[0197] The recombinant granule protein component sample was purified using ion exchange and hydrophobic chromatography. The first chromatographic purification step was performed using a Fractogel DEAE M chromatography process. Specific steps and parameters are shown in Table 6. The Fractogel DEAE M elution pool was first diluted with buffer and stabilized with 50% (w / v) sucrose to prevent precipitation of the recombinant granule protein component during the next chromatography step. Specific parameters are shown in Table 7. The second chromatographic purification step was then performed using a hydrophobic Octyl Bestarose 4FF chromatography process. Specific steps and parameters are shown in Table 8.

[0198] First step chromatography method: chromatography medium - Fractogel DEAE M, retention time - 12.5 min.

[0199] Table 6: First step chromatography method

[0200] Table 7: Sample dilution method before the second step chromatography

[0201] Second step chromatography method: Chromatographic filler - Octyl Bestarose 4FF, retention time - 12.5min

[0202] Table 8: Second step chromatography method

[0203] Results and Analysis:

[0204] Purity testing revealed that after further purification using the above chromatographic media combination, the purity of the obtained product can reach over 99.0%. Specific SDS-PAGE analysis results of the NPM-4C protein after separation and purification using Octyl Bestarose 4FF can be seen in Figure 8 (M: protein marker; lanes 1-2: NPM-4C protein).

[0205] Example 6: Binding of Mycobacterium tuberculosis structural proteins to NPM, purification of binding products, and particle characterization

[0206] 1. Binding of antigen and NPM

[0207] High-purity Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigens obtained through molecular sieve purification were mixed with NPM-4C at a BCA protein ratio of 6:1. A 50% sucrose stock solution was added to a final concentration of approximately 25%, and a 1M Tris-HCl stock solution was added to stabilize the pH at 10% of the total reaction volume. The binding reaction was performed at 22°C for 48 hours. For example, the Mtb32a-NPM binding system might be: 6 mL of Mtb32a-4T (1 mg / mL), 1 mL of NPM-4C (1 mg / mL), 8.75 mL of 50% sucrose, and 7.4-1.75 mL of 1M Tris-HCl, for a total volume of 17.5 mL.

[0208] 2. Purification of the binding product

[0209] Use Cytiva HiLoad 16 / 600 Superdex 200 pg (column volume 120 mL) or Cytiva Superdex 200 Increase 10 / 300 GL (column volume 23 mL) to purify the Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM binding products, and separate and remove Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 antigens that are not bound to NPM-4C. If using molecular sieve HiLoad 16 / 600 Superdex 200pg, the loading amount of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM combined samples should be controlled at approximately 3% to 6%. If using molecular sieve Superdex 200Increase 10 / 300 GL, the loading amount of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM combined samples should be controlled at 0.5mL to 1mL.

[0210] Chromatographic procedure: Superdex 200 pg or Superdex 200 Increase, sterilize, equilibrate the column with 12.5% ​​sucrose TBS solution (20 mM Tris-HCl, 150 mM NaCl, 12.5% ​​sucrose Mtb), load the sample, wash with 12.5% ​​sucrose TBS solution, collect Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM fractions, and perform SDS-PAGE analysis according to the method for SDS-PAGE analysis of the purified antigen in Example 3.

[0211] Results and Analysis:

[0212] SDS-PAGE analysis of molecular sieve-purified Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles showed a purity exceeding 90%, as shown in Figures 9 to 12. In Figure 9, M represents a protein marker, lanes 1 to 3 represent Mtb32a-NPM-bound products, and lanes 4 to 10 represent unbound Mtb32a; in Figure 10, M represents a protein marker, lanes 1 to 5 represent Ag85a-NPM-bound products; in Figure 11, M represents a protein marker, lanes 1 to 5 represent ESAT6-CFP10-NPM-bound products; and in Figure 12, M represents a protein marker, lanes 1 to 5 represent RV2660-TB10.4-NPM-bound products.

[0213] According to the above method, Mtb32a, Ag85a, ESAT6-CFP10, RV2660-TB10.4 and NPM-4C were combined and reacted. The binding rate was measured by SDS-PAGE grayscale method and was 82.5%.

[0214] 3. Particle Characterization

[0215] 1) TEM detection

[0216] Using the flotation method, negatively stain the Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles prepared in the "Purification of Binding Products" section above. Prepare samples using a 400-mesh grid with a support membrane that has been pre-treated for hydrophilicity. Prepare deionized water and 2% uranyl formate negative staining solution. Place 3 μL of the prepared protein sample (0.1 mg / mL) directly onto one side of the support membrane grid. After a one-minute timer, remove excess liquid from the edge of the grid with clean filter paper. Once slightly dry, rinse quickly twice with a drop of deionized water. Rinse once with 5 μL of negative staining solution, then add 5 μL of negative staining solution for a one-minute timer. After the staining is complete, remove the grid with tweezers and remove the stain with filter paper, leaving a thin layer to air dry before testing. Check under a 120kV transmission electron microscope (FERRITINI Tecnai Spirit), observe the overall staining of the grid at low magnification, select holes of appropriate thickness for observation, and select appropriate areas under a high-power microscope to take pictures and save.

[0217] 2) DLS detection

[0218] The purified Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles were diluted to 0.15 mg / mL. Using a Zetasizer Lab instrument, ≥1 mL of the sample to be tested was injected into the sample cell. The instrument was run for detection. Data analysis was performed based on the Z-Average (nm) and Polydispersity Index (PI) values, as well as the Size Distribution by Intensity / Volume distribution curve, and the results were reported.

[0219] Results and analysis:

[0220] Electron microscopy images (0.1 mg / mL, 18500×) of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles show uniform particle distribution and no aggregation, as shown in Figures 13 to 16. The Distribution by Intensity / Volume curves analyzed by the Zetasizer Lab instrument are shown in Figure 17. The results show that the prepared Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles have good peak overlap and uniform particle size distribution. DLS results showed that the particle diameters of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM were 37.0 nm, 38.7 nm, 43.0 nm, and 39.8 nm, respectively, as shown in Table 9.

[0221] Table 9: Z-Average (nm) and Polydispersity Index (PI) values ​​of prepared particle antigens

[0222] Example 7: Preparation of vaccine

[0223] 1. Test vaccine antigens and adjuvants

[0224] 1) Test vaccine antigen solution

[0225] 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.

[0226] 2) Test vaccine adjuvant

[0227] The commercial AS01B adjuvant, QS21 (5 μg), was added to small unilamellar vesicles (SUVs) containing dioleoylphosphatidylcholine (100 μg) with cholesterol (25 μg) to prepare a dual strength AS01B (WO 96 / 33739) and monophosphoryl lipid A (MPL) (5 μg) in the membrane.

[0228] 2. Preparation method of the test vaccine

[0229] 1) Mtb recombinant antigen mixed vaccine

[0230] Aliquots for injection (50 μL) were prepared by mixing 0.8 μg of a protein mixture (0.2 μg each of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, RV2660-TB10.4-4T) in a buffer (TBS Mtb) with 50 μL of double strength AS01B.

[0231] 2) Mtb nanoparticle antigen mixed vaccine

[0232] Aliquots for injection (50 μL) were prepared by mixing 0.8 μg of a protein mix (0.2 μg each of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM) in buffer (TBS Mtb) with 50 μL of double strength AS01B.

[0233] 3) Single Mtb nanoparticle antigen vaccine

[0234] Aliquots for injection (50 μL) 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 TBS Mtb ​​buffer with 50 μL of double-strength AS01B.

[0235] 4) M72 control vaccine

[0236] Aliquots for injection (50 μL) were prepared by mixing 0.8 μg of M72-4T protein cocktail in TBS Mtb ​​buffer with 50 μL of double strength AS01B.

[0237] Example 8: Vaccine immune protection experiment

[0238] 1. Experimental animals and groups

[0239] Female C57BL / 6 mice aged 4-6 weeks were selected and purchased from Weitonglihua. After passing the quarantine, the mice were marked with metal ear tags and randomly divided into groups according to body weight. They were free to eat and drink water. Six candidate vaccine immunization groups (12 mice in each group), M72-4T group (12 mice), BCG control group (12 mice), and normal saline group (15 mice) were set up. The animals were raised in SPF standard animal rooms and supplied with sterile feed and sterilized deionized water for SPF animals. The light was alternating for 12 hours day and night in the feeding room, the temperature was 21±2°C, and the humidity was 30-70%.

[0240] 2. Immunity and anti-toxin

[0241] The vaccine groups (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 3-week intervals. 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.

[0242] Four weeks after the last immunization, mice were challenged with an aerosol at a dose of 100 bacteria per mouse. Four weeks after challenge, the mice were dissected and pathological sections of the lungs and spleens were prepared. The abrasives from the lungs and spleens were used to calculate bacterial loads. Specific groupings, immunization schedules, and challenge schedules are shown in Tables 10 and 11.

[0243] Table 10: Immunization and virus attack schedule

[0244] Table 11: Grouping and protein components, dosage, adjuvant comparison table

[0245] 3. Detection of specific IgG after immunization

[0246] 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 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.

[0247] 96-well microtiter 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, overnight at 4°C. The plates were then washed twice with PBST (0.05% Tween 20) and blocked with 200 μL / well of blocking buffer (Thermo Fisher Scientific). The plates were blocked for 1-4 hours at room temperature (25°C ± 3°C) and washed twice. Diluted immune serum was then added and incubated at room temperature for 1 hour, followed by four washes. A 1:5000 dilution of the corresponding HRP-IgG1 or HRP-IgG2a working solution was added at 50 μL / well. The plates were incubated at room temperature for 1 hour, washed six times, and 100 μL of color development solution was added to each well. The plates were developed in the dark for 10 minutes at room temperature, and then 100 μL of 1 M HCl was added to each well to terminate the reaction. The microplate reader was set to a dominant wavelength of 450 nm and a reference wavelength of 620 nm. Sample absorbance was calculated as OD450 - OD620. The assay was completed within 5 minutes of termination. Based on the test results, humoral and cellular immune patterns were analyzed.

[0248] Data processing:

[0249] Data were considered reliable if the following conditions were met: the OD value of the control serum was ±0.2, the OD value corresponding to the sample starting concentration was <3.0, the OD value corresponding to the blank well was less than 0.1, and the coefficient of variation of the replicate wells (response value) was less than 20%. The raw sample data were transferred to the "Excel Endpoint ELISA template" to calculate the antibody titer. Results were analyzed using Graphpad Prism 9.1.2 software. Differences were analyzed using the unpaired test or one-way ANOVA. Data between the two groups were considered significantly different when P < 0.05.

[0250] The test results are as follows:

[0251] Figures 20a-d show the corresponding total IgG antibody levels of immune sera from 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 20e shows the corresponding IgG antibody levels detected using the M72 (0.8 μg immunization group) antigen.

[0252] The results showed that the individual Mtb nanoparticle antigen vaccines (vac-3 / 4 / 5 / 6), the Mtb recombinant antigen mixed vaccine (vac-2), and the Mtb nanoparticle antigen mixed vaccine (vac-1) of the present invention all induced the production of IgG, demonstrating that the vaccines of the present invention possessed good immunogenicity. The IgG antibody levels corresponding to the vac-1 and individual Mtb nanoparticle antigen vaccine groups were significantly higher than those of the vac-2 group, demonstrating that the nanoparticle vaccines of the present invention significantly enhance the immunogenicity of Mtb antigens.

[0253] Figures 21a-d show 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, respectively. Figure e shows the corresponding IgG1 and IgG2a antibody levels of the M72 group (0.8 μg immunization group).

[0254] 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 all 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.

[0255] 4. Cytokine ELISA

[0256] 1) Three weeks after the last immunization, 6 mice were treated in each group, spleens were collected, splenocytes were isolated, and the cells were counted at 2.5×10 5 cells / well, seeded into 96-well culture plates;

[0257] 2) 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 Con A (3 μg / mL) was used and cultured in a 37°C 5% CO2 incubator for 72 hours;

[0258] 3) Supernatants were collected and assayed for IFN-γ, TNFα, IL-4, and IL-2 using commercially available double-antibody sandwich ELISA kits. Results were analyzed using Graphpad Prism 9.1.2 software. Unpaired test or one-way ANOVA was used to analyze differences. Data between the two groups were considered significantly different when P < 0.05.

[0259] 5. Cytokine ELISPOT

[0260] 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, and splenocytes were isolated. The cells were expressed as 2.0×10 5 cells / well, seeded into 96-well culture plates;

[0261] 2) Culture the cells in a single medium or in a 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 Con A (3 μg / mL) at 37°C in a 5% CO2 incubator for 48 hours.

[0262] 3) Wash the wells with PBS and add biotinylated mouse IFN-γ, TNFα, IL-4, and IL-2 secondary antibodies. Incubate at room temperature for 2 hours. Develop the filter membrane using substrate according to the instructions of the commercial kit.

[0263] 4) After the plate is dried, the spots are counted using an automated ELISPOT plate reader for analysis.

[0264] The results were analyzed using Graphpad Prism 9.1.2 software. Unpaired test or One-Way ANOVA was used to analyze the differences. The data between the two groups were defined as having significant differences when P < 0.05.

[0265] Figures 22a-d show 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) detected using the corresponding 072 antigen as a specific stimulation antigen; Figures 22e-h show 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) detected using the corresponding 076 antigen as a specific stimulation antigen.

[0266] The results showed that the corresponding IFN-γ, IL-2, TNFα and IL-4 levels of the 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 in each group were significantly higher than the IL-4 level, indicating that both 072 and 076 can induce a Th1-type mainly cellular immune response.

[0267] Figures 23a-d show 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) detected using the corresponding 077 antigen as a specific stimulation antigen; Figures 23e-h show 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) detected using the corresponding 014 antigen as a specific stimulation antigen.

[0268] The results showed that the corresponding IFN-γ, IL-2, TNFα and IL-4 levels of the 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 those of IL-4, indicating that 077 and 014 can both induce Th1-type cellular immune responses.

[0269] Figure 24 shows that the IFN-γ, IL-2, and TNFα levels corresponding to the M72 group (0.8 μg immunization group) were significantly higher than those of IL-4, indicating that the prepared M72 vaccine can induce a Th1 type cellular immune response after immunization of mice, which is consistent with literature reports and proves that the control vaccine was successfully prepared.

[0270] 6. Flow cytometry detection of T cell immune response

[0271] 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 were washed with staining buffer and stained for 15 minutes with a 50 μL mixture containing PE-Cyanine7 CD3 monoclonal antibody (1:50 final dilution, eBioscience), PE rat anti-mouse CD4 (1:50 final dilution, BD), and PerCP-CyTM5.5 rat anti-mouse CD8 (1:50 final dilution, BD). The cells were washed twice with 1× Perm / Wash solution, washed and resuspended in 1× Perm / Wash solution, and analyzed using DxFLEX (BECKMAN COULTER). Data were analyzed using CytExpert.

[0272] 2) ICS and flow cytometry were used to detect the expression of IFN-γ, TNFα, IL-4, and IL-2 specific CD4+ and CD8+ T cells, and spleen cells (1×10 6 Cells were re-stimulated in vitro for 6 hours using 014 (10 μg / mL), 072 (10 μg / mL), 076 (10 μg / mL), 077 (10 μg / mL), M72-4T (10 μg / mL), and a CD28 / CD49d co-stimulatory antibody (BD). For intracellular cytokine staining, cells were incubated with a protein transport inhibitor (containing Brefeldin A; BD) for 4 hours; cells were washed with PBS and stained with fixable viability stain 780 (1:1000 final dilution, BD) and mouse Fc block (BD) for 15 minutes;

[0273] 3) Cells were washed with staining buffer and stained for 15 minutes with a 50 μL 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). Cells were fixed and permeabilized using a fixation / permeabilization solution kit (BD). Cells were then washed twice with 1× Perm / Wash solution and stained 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), and R718 rat anti-mouse TNFα (1:50 final dilution, BD). Cells were then washed with 1× Perm / Wash solution, resuspended, and analyzed using DxFLEX (BECKMAN COULTER). Data were analyzed using CytExpert and expressed as the percentage of the total frequency of CD4+ T / CD8+ T cells expressing IFN-γ, TNFα, IL-4, and IL-2 after background subtraction from the mean responses of specific CD4+ and CD8+ T cells.

[0274] 7. Statistics of attack and protection results

[0275] Four weeks after the last immunization, mice were challenged by exposure to a low-dose aerosol of Mycobacterium tuberculosis H37Rv strain using a UW-Madison aerosol exposure chamber calibrated to deliver 50-100 CFU to the lungs. Four weeks later, mice were euthanized and homogenates of the lungs and spleens were prepared by grinding with PBS / Tween-80 (0.05%). Homogenates of individual whole organs were serially diluted and plated onto Middlebrook 7H11 Bacto agar medium. Bacterial colonies were counted after incubation at 37°C in a humidified, 5% CO2 environment for 2-4 weeks. Final data were expressed as the mean Log10 ± SD of bacteria, with the Log10 reduction (difference) of CFU = Log10 CFU of the saline-treated group - Log10 CFU of the vaccine-treated group.

[0276] 8. Pathological section analysis

[0277] 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.

[0278] 9. Immunity Assessment Results

[0279] The results of specific IgG detection after immunization showed that the antigens in the vac-1 to vac-6 groups can all 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 all stimulate lymphocytes to produce high levels of IFN-γ, TNFα, and IL-2. The screened antigens have a strong function of stimulating cellular immunity.

[0280] 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 thus be presented by APC to activate CTL immunity.

[0281] 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 adjuvants to improve the levels of cellular immunity and humoral immunity, and reflects the advantage of NPM nanoparticles in presenting Mycobacterium tuberculosis antigens; 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.

[0282] The above results show that VLP-form antigens enhance immune protection. The nanoparticle antigens covered by vac-1 to vac-6 can effectively stimulate the body to produce immune protection and can effectively prevent the 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.

[0283] In summary, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of protection 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 scope of protection 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 method 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 toxin-reducing agent, 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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