A nanoparticle vaccine for the prevention of Mycobacterium tuberculosis infection and its preparation method

Abstract

This invention relates to a Mycobacterium tuberculosis vaccine. Specifically, the vaccine comprises an immune composition containing an antigen component and a particulate protein component. The particulate protein component comprises nanoparticle proteins, and the antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2 to form an immunogenic complex. This vaccine exhibits excellent cellular immunogenicity and antibody immunogenicity. The invention also discloses a method for preparing the Mycobacterium tuberculosis vaccine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an immune composition product for the prevention of Mycobacterium tuberculosis infection and its production preparation method. Background Technology

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis and other mycobacteria. The main route of transmission is through droplets. It can occur at any age, but those with weakened immune systems are more susceptible. Infected individuals may not experience typical symptoms for a considerable period, but once symptoms appear, they manifest as acute lung inflammation, dry cough, fever, and other symptoms. Without timely treatment, it generally leads to serious complications and death.

[0003] Currently, tuberculosis (TB) is a major disease in developing countries, widely distributed globally, and its severity is increasing. In 2021, there were 10.6 million new TB cases worldwide, and 1.6 million people died from TB—equivalent to approximately 4,300 deaths per day. TB primarily affects populations in low- and middle-income countries, with those living and working in poor conditions and suffering from malnutrition often at the highest risk. Globally, up to a quarter of the population is latently infected with TB. Infection with Mycobacterium tuberculosis may not cause any symptoms, but it can potentially develop into active TB.

[0004] Currently, antibiotic treatment with drugs such as isoniazid, rifampin, and pyrazinamide can control tuberculosis, but this treatment cannot effectively prevent the spread of the disease. Infected individuals may not develop symptoms for a period of time, but they are still infectious. Furthermore, long-term antibiotic use poses a significant challenge to patient adherence, with some patients failing to complete effective treatment. Additionally, long-term antibiotic treatment may lead to drug resistance.

[0005] Effective vaccination and accurate early diagnosis are crucial for controlling tuberculosis (TB). Currently, the BCG (Bacillus Calmette-Guerin) vaccine used for infants and young children is prepared from a non-toxic Mycobacterium bovis. The widespread use of BCG has greatly aided global TB prevention and control. While BCG protects infants and young children from severe systemic TB, its protective effect 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 booster versions. Recombinant subunit TB vaccines have been proven effective in humans and are gradually becoming a hot topic in new TB vaccine development. For example, GSK's novel TB candidate vaccine M72 / AS01E contains a recombinant fusion protein with two Mycobacterium tuberculosis antigens (MTB32A and MTB39A). Phase II clinical data shows that its protection rate is only 49.7%, barely meeting the WHO's requirements for TB vaccines. While this vaccine possesses some immunogenicity, the M72 fusion protein is easily degraded during expression and produces inclusion bodies. Furthermore, the particle size of the fusion protein is significantly affected by salt ions and pH, leading to a complex production process and high costs. Therefore, developing novel TB vaccines with higher protection rates and simpler production processes for population vaccination is of great significance.

[0006] Currently, nanoparticle vaccines, as a new generation of vaccine design, can not only induce strong neutralizing antibodies but also enhance cellular immune responses. Human HPV vaccines, hepatitis B vaccines, and veterinary PCV2 vaccines are representative examples. Many natural proteins (ferritin, lumazine synthase, Mi3, AP205, etc.) exist in nature and can self-assemble into nanoparticles. After loading antigens onto their surface, they can induce strong immune responses and are currently widely studied and applied. The body's resistance to Mycobacterium tuberculosis infection mainly relies on the body's cellular immune mechanisms. Vaccine immunization can produce effective Th1CD4+T and CD8+T levels and generate lasting cellular immune memory, which is a key point in the development of novel tuberculosis vaccines. Therefore, self-assembled nanoparticles are an ideal carrier and are feasible in tuberculosis vaccine research. Summary of the Invention

[0007] This invention provides a Mycobacterium tuberculosis vaccine and its preparation method, wherein 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 comprising a protein formed by a covalent binding reaction of an antigenic component and a particulate protein component.

[0010] The present invention provides an immune composition comprising the immunogenic complex of the present invention and a pharmaceutically acceptable carrier, which may be a lyophilized dosage form, an injectable dosage form, an oral dosage form or a spray dosage form.

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

[0012] This invention provides an immunogenic complex comprising:

[0013] (1) Antigen components, which contain Mycobacterium tuberculosis structural proteins or their immunogenic fragments;

[0014] (2) Particulate protein component, which includes nanoparticle protein.

[0015] This invention provides an immunogenic complex comprising:

[0016] (1) Antigen components, which contain Mycobacterium tuberculosis structural proteins or their immunogenic fragments, linkers

[0017] Peptide 1 and binding peptide 1;

[0018] (2) Particulate protein component, which includes nanoparticle protein, linker peptide 2 and binding peptide 2;

[0019] The antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2.

[0020] This invention provides an immunogenic complex comprising:

[0021] (1) Antigen components, consisting of Mycobacterium tuberculosis structural proteins or their immunogenic fragments, linkers

[0022] It consists of peptide 1 and binding peptide 1;

[0023] (2) The particulate protein component consists of nanoparticle protein, linker peptide 2 and binding peptide 2;

[0024] The antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2.

[0025] In some embodiments, in any of the immunogenic complexes provided by the present invention, the antigenic component is formed by fusing a Mycobacterium tuberculosis structural protein at its C-terminus with a linker peptide 1 and a binding peptide 1.

[0026] In some implementations, 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 of the immunogenic complexes provided by the present invention, the particulate protein component is formed by fusing nanoparticle protein at the N-terminus with binding peptide 2 via linker peptide 2.

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

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

[0030] This invention provides an immunogenic complex comprising:

[0031] (1) Antigen components, which include Mycobacterium tuberculosis structural proteins or their immunogenic fragments and linker peptide 1;

[0032] (2) Particulate protein component, which contains nanoparticle protein subunits.

[0033] In some implementations, a Mycobacterium tuberculosis structural protein is linked to one subunit of the nanoparticle protein to form a fusion protein, which in turn binds to another subunit of the nanoparticle protein.

[0034] In some embodiments, in any of the immunogenic complexes provided by the present invention, the particulate protein component comprises nanoparticle proteins. Preferably, the nanoparticle proteins can be virus-like particulate proteins formed from viral structural proteins, and more preferably, from bacterial phage capsid protein AP205. The particulate protein component and the antigen component can form a particulate structure through covalent bonding.

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

[0036] In some embodiments, in any 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 of the immunogenic complexes provided by the present invention, the binding peptide 1 contains an amino acid sequence as shown in SEQ ID NO:1.

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

[0039] In some embodiments, in any of the immunogenic complexes provided by the present invention, the linker peptide 1 comprises (GSG). n (GGGGS) n Or (EAAAK) n The amino acid sequence, where 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 linker peptide 1 is preferably GSG GSG (SEQ ID NO:2).

[0040] In some embodiments, in any of the immunogenic complexes provided by the present invention, the linker peptide 2 comprises (GGS). n (SGGSGG) n Or (GSGGSGGSG) n The amino acid sequence, where n can 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 linker peptide 2 is preferably GGSGGSGGSGGS (SEQ ID NO:25).

[0041] Specifically, the structural protein of Mycobacterium tuberculosis in this invention uses a fusion protein of Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4, such that the above proteins are linked to a binding peptide 1 (named "4T") at the C-terminus via a specific linker 1. Simultaneously, a histidine residue (e.g., 6His, i.e., HHHHHH) purification tag can be added to the C-terminus of the fusion protein. The coding gene for the above Mycobacterium tuberculosis structural protein is inserted into a prokaryotic cell expression vector (e.g., pET21a) and expressed in Escherichia coli BL21(DE3) cells to obtain the fusion protein formed by the Mycobacterium tuberculosis structural protein and binding peptide 1. This antigenic component is purified to high purity by nickel column affinity chromatography and molecular sieve chromatography. This antigenic component is 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 a fusion protein of Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4, 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 the fusion proteins Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4. The Mtb32a sequence has more than 40%, 50%, 60%, 70%, 80%, or 90% identity with SEQ ID NO: 3; the Ag85a sequence has more than 40%, 50%, 60%, 70%, 80%, or 90% identity with SEQ ID NO: 5; the ESAT6-CFP10 sequence has more than 40%, 50%, 60%, 70%, 80%, or 90% identity with SEQ ID NO: 7; and the RV2660-TB10.4 sequence has more than 40%, 50%, 60%, 70%, 80%, or 90% identity with SEQ ID NO: 9.

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

[0045] In some embodiments, in any of the immunogenic complexes provided by the invention, the particulate protein component is a fusion protein formed by linker peptide 2 and binding peptide 2 at the N-terminus of the nanoparticle protein; preferably, the nanoparticle protein is NPM, AP205 capsid protein 3 (AP205), or Ferritin protein. Specifically, in some alternative embodiments, binding peptide 2 (named "4C") is linked to the encoding gene of the nanoparticle protein via linker peptide 2, inserted into a prokaryotic expression vector (e.g., pET-28a(+), pET-30a(+)), and expressed in E. coli cells to obtain a fusion protein of binding peptide 2 and nanoparticle protein. The fusion protein can be purified by chromatography, such as by anion exchange chromatography or hydrophobic chromatography, to obtain the product. The nanoparticle protein is preferably NPM, AP205, or Ferritin; the formed particulate protein component is named NPM-4C, AP205-4C, or Ferritin-4C.

[0046] Specifically, in some alternative approaches, under suitable reaction conditions, any of the above-mentioned antigen components and particulate protein components undergo a conjugated binding reaction. Coupling occurs through a covalent bond formed between the binding peptide 1 of the antigen component and the binding peptide 2 of the particulate protein component, thereby forming the immunogenic complex. Different nanoparticle proteins can be used to form different immunogenic complexes, which 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) Antigen components, which include Mycobacterium tuberculosis structural proteins, linker peptide 1 and binding peptide 1;

[0049] (2) Particulate protein component, which includes nanoparticle protein, linker peptide 2 and binding peptide 2.

[0050] The linker peptide 1 is any linker peptide commonly used in the art, including but not limited to (GSG). n (GGGGS) n Or (EAAAK) n The amino acid sequence, n can be an integer greater than 0 and less than or equal to 5, preferably GSGGSG (SEQ ID NO:2); the linker peptide 2 is any linker peptide commonly used in the art, including but not limited to (GGS). n (SGG) n Or (GSGGSGGSG) n The amino acid sequence is specified, where 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 of the immunogenic complexes provided by the present invention, the particulate protein component comprises NPM-4C, as shown in SEQ ID NO:27, which is a fusion protein obtained by linking binding peptide 2 as shown in SEQ ID NO:24 to nanoparticle protein NPM as shown in SEQ ID NO:25 via linking peptide 2 as shown in SEQ ID NO:26.

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

[0053] (1) Antigen components, which contain structural proteins and linker peptide 1 of Mycobacterium tuberculosis;

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

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

[0056] Specifically, in any of the immunogenic complexes provided by this invention, the Mycobacterium tuberculosis structural protein is linked to one subunit of the nanoparticle protein to form a fusion protein, and the fusion protein then binds to another subunit of the nanoparticle protein. Preferably, the subunit of the nanoparticle protein is I53-50A or I53-50B. Further, in some alternative embodiments, the Mycobacterium tuberculosis structural protein in the antigen component forms a Mycobacterium tuberculosis structural protein-I53-50A fusion protein by linking peptide 1 at the C-terminus of the nanoparticle protein; then, the fusion protein binds to the I53-50B subunit of the nanoparticle protein.

[0057] As described above, when the nanoparticle protein I53-50 is selected, I53-50 contains two subunits, I53-50A and I53-50B. The aforementioned Mycobacterium tuberculosis structural protein, which may or may not contain a specific signal peptide, is linked to I53-50A via linker peptide 1. A purification tag (e.g., 6H) can be added to the C-terminus. The gene encoding the aforementioned fusion protein is inserted into a eukaryotic cell expression vector (e.g., pcDNA3.4), expressed and purified in CHO cells, and the resulting fusion protein is named Mycobacterium tuberculosis structural protein-I53-50A. Simultaneously, a purification tag (e.g., 6H) can be added to the C-terminus of I53-50B, and the gene encoding the aforementioned protein is inserted into a prokaryotic cell expression vector (e.g., pET-30a(+)), expressed and purified in E. coli cells, and the resulting protein is named I53-50B. Then, under suitable reaction conditions, Mycobacterium tuberculosis structural protein-I53-50A and I53-50B were covalently bound to form Mycobacterium tuberculosis nanoparticles, which were named Mycobacterium tuberculosis structural protein-I53-50.

[0058] Preferably, any immunogenic complex provided by the present invention contains 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 items (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 linker 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 protein subunits of the nanoparticles are selected from I53-50A and / or I53-50B;

[0065] (6) The linker peptide 2 comprises an amino acid sequence of (GGS)n, (SGGSGG)n or (GSGGSGGSG)n, where n can be an integer greater than 0 and less than or equal to 10; the amino acid sequence of the linker peptide 2 is 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 composed of an antigenic component and a particulate protein component, wherein the amino acid sequence of the antigenic 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 particulate 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) The antigen component and the particulate protein component encoding genes were respectively ligated into expression vectors to construct recombinant expression plasmids and expression host strains, express the target protein, and then purified.

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

[0071] This invention provides a method for preparing an immunogenic complex for the prevention or treatment of Mycobacterium tuberculosis virus-related diseases:

[0072] (1) The genes encoding the antigenic components and particulate protein components of Mycobacterium tuberculosis virus were respectively ligated into expression vectors to construct recombinant expression plasmids;

[0073] (2) Construct a recombinant strain capable of expressing the Mycobacterium tuberculosis virus antigen components and particulate protein components in host cells;

[0074] (3) The recombinant strain was used to express the fusion protein, and the fusion protein was purified.

[0075] (4) The above antigen components and particulate protein components are co-incubated to undergo a conjugation reaction and obtain an immunogenic complex.

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

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

[0078] In step (2) of the method for preparing an immunogenic complex for the prevention or treatment of Mycobacterium tuberculosis virus-related diseases according to the present invention, the host cell expressing the Mycobacterium tuberculosis virus antigen is Escherichia coli, and the host cell expressing the particle protein component carrier is E. coli.

[0079] The present invention discloses an immunogenic complex for the prevention or treatment of Mycobacterium tuberculosis virus-related diseases, wherein the antigenic component of the immunogenic complex comprises a fusion protein formed by the above-mentioned Mycobacterium tuberculosis structural protein-binding peptide 1.

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

[0081] The present invention also provides an immune composition comprising any of the above-described 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, wherein the stabilizer is sucrose or arginine, the excipient is mannitol, the surfactant is Tween 80, the buffer is disodium hydrogen phosphate dihydrate or disodium dihydrogen phosphate dihydrate, and the pH adjuster is hydrochloric acid.

[0082] In some embodiments, the immunogenic composition of the present invention contains an immunogenic complex at 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 dosage used in mouse experiments is 1 / 10 of the human dosage.

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

[0084] In some embodiments, the immune composition provided by the present invention is a lyophilized preparation comprising 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 disodium 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 comprising Mycobacterium tuberculosis structural protein-NPM immunogenic complex, sucrose, arginine, mannitol, Tween 80, disodium hydrogen phosphate dihydrate, disodium dihydrogen phosphate dihydrate, and hydrochloric acid.

[0086] In some embodiments, the immune composition provided by the present invention is an injection solution comprising 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 or 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 comprising 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 comprising any of the above-described immune compositions and an adjuvant, wherein the adjuvant is selected from at least one of the following: aluminum salt adjuvants, Freund's complete adjuvant, propolis adjuvant, water-oil adjuvant, cytokines, CpG DNA, genetically engineered attenuated toxins, immunostimulatory complexes, and liposomes.

[0089] The present invention discloses a Mycobacterium tuberculosis vaccine, wherein the water-oil adjuvant is a squalene adjuvant containing squalene.

[0090] The Mycobacterium tuberculosis vaccine of the present invention contains, in human use, 5-50 μg of the immunogenic complex per unit dose, 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) 2%-4.5% squalene, 0.2%-0.5% Span 85, 0.2%-0.5% Tween 80, and 10mM citrate buffer. The preferred amounts of squalene are 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%, and 4.4% (w / w), with Span 85 more preferably at 0.3%-0.4% (w / w) and Tween 80 more preferably at 0.3%-0.4% (w / w).

[0093] As mentioned above, the dosages of the immunogenic complex, Mycobacterium tuberculosis structural protein-NPM, and adjuvant differ for humans and mice. The corresponding relationships are as follows: When used for human purposes, the dosages of Mycobacterium tuberculosis structural protein-NPM and adjuvant are 10 times the dosages used in mice. For example, the dosage of Mycobacterium tuberculosis structural protein-NPM in mice is 5 μg / dose, while the dosage in humans is 50 μg / dose; the dosage of adjuvant in mice is 50 μl / dose, while the dosage in humans is 500 μl / dose (0.5 ml / dose); the dosage of adjuvant in mice is 25 μl / dose, while the dosage in humans is 250 μg / dose (0.25 ml / dose), and so on.

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

[0095] This invention provides a Mycobacterium tuberculosis vaccine comprising a Mycobacterium tuberculosis structural protein-NPM immune composition (i.e., an immune composition containing Mycobacterium tuberculosis structural protein-NPM, which can be formulated as a lyophilized preparation or an injectable preparation) and an adjuvant (in liquid form). The Mycobacterium tuberculosis structural protein-NPM immune composition and the adjuvant are packaged separately in vials.

[0096] This invention provides the use of Mycobacterium tuberculosis nanoparticle immunogenic complex, immune composition, or vaccine in the preparation of medicaments for the prevention or treatment of tuberculosis.

[0097] All reagents used in this invention are commercially available.

[0098] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0099] 1. This invention utilizes 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 fusion expression form. These fusion proteins can all be expressed in a soluble manner. Simultaneously, NPM-4C nanoparticles are prepared using the *E. coli* expression system. The immunogenic complex of this invention has a significant preventive effect against *Mycobacterium tuberculosis* (Mtb) infection. The Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles prepared using the structural proteins Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 exhibit uniform particle size, even distribution, and no aggregation. The products demonstrate stable performance and meet endotoxin standards, making them suitable for non-clinical development and antibody immunogenicity testing, thus qualifying them 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, low-cost, and has great potential for industrialization.

[0101] 3. Animal immunization experiments showed that Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM all produced high levels of specific IgG1 and IgG2a antibodies in mice after immunization, indicating that Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles have good immunogenicity and can produce good cellular and humoral immunity.

[0102] 4. A mixture of fusion proteins Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T can significantly reduce Mycobacterium tuberculosis infection. A 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.

[0103] 5. The method for preparing a tuberculosis vaccine using nanoparticles provided by this invention is low-cost and suitable for large-scale production. In this invention, the particle protein component is prepared using E. coli fermentation and chromatographic purification. The Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigen components can be prepared using cell reactor culture and chromatographic purification, all suitable for large-scale industrial production. Furthermore, it possesses advantages such as high expression levels, stable process and yield, and simple operation. The amount of recombinant particle protein component in one batch can correspond to multiple batches of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigen components for binding, improving production efficiency and saving costs associated with large-scale production.

[0104] 6. The method for preparing recombinant particulate protein components provided by this invention is suitable for industrial production, can reduce the cost of large-scale industrial production, is simple to operate, and reduces the amount of organic solvent used in subsequent chromatography purification; the protein products prepared using the method for preparing recombinant particulate protein components provided by this invention effectively reduce the side effects caused by the residues of impurities, host proteins, organic solvents, exogenous DNA, antibiotics, bacterial endotoxins, etc. in the particles, and improve safety. Attached Figure Description

[0105] Figure 1 The expression of recombinant protein Mtb32a-4T was shown by Western blot identification.

[0106] Figure 2The expression of recombinant proteins Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T was shown by Western Blot.

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

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

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

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

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

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

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

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

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

[0116] Figure 12 The results of the isolation 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] Figure 17The distribution curves of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM nanoparticles by intensity / volume are shown.

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

[0120] Figure 19 The distribution curves by intensity / volume of NPM nanoparticles prepared before and after the RV2660-TB10.4T mutation are shown.

[0121] Figure 20 The figures show the total binding antibody IgG levels in the serum of the D20 group detected using proteins 072, 076, 077, 014, and M72 as coating antigens, respectively. Figures a and b show the corresponding IgG antibody levels detected in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline) using antigens 072, 076, 077, and 014 as coating antigens, respectively. Figure e shows the IgG antibody levels detected by the M72 antigen.

[0122] Figure 21 The figures show the detection of IgG1 and IgG2a antibody levels in serum of the D20 group using proteins 072, 076, 077, 014, and M72 as coating antigens, respectively. Figures a and b show the detection of IgG1 and IgG2a antibody levels in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline) using antigens 072, 076, 077, and 014 as coating antigens, respectively. Figure e shows the IgG1 and IgG2a antibody levels in the M72 group (0.8 μg immunization group).

[0123] Figure 22 The ad group, in turn, used the 072 antigen as a specific stimulating antigen to detect the levels of IFN-γ, IL-2, TNFα, and IL-4 in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline). Figure 22 EH1C uses the 076 antigen as a specific stimulating antigen to detect the levels of IFN-γ, IL-2, TNFα, and IL-4 in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline).

[0124] Figure 23The ad group, in turn, used the 077 antigen as a specific stimulating antigen to detect the levels of IFN-γ, IL-2, TNFα, and IL-4 in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline). Figure 23 EH14 was used as a specific stimulating antigen to detect the levels of IFN-γ, IL-2, TNFα and IL-4 in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline).

[0125] Figure 24 The study demonstrates the detection of specific IFN-γ, IL-2, TNFα, and IL-4 levels in D20 spleen cells using M72 protein as a stimulating antigen. Detailed Implementation

[0126] The principles and features of the present invention are described below with reference to examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. Before further describing specific embodiments of the invention, it should be understood that the scope of protection of the invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the invention is for describing specific embodiments and not for limiting the scope of protection of the invention. Test methods in the following embodiments without specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the 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 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 embodiments, based on the knowledge of the prior art by those skilled in the art and the description of the invention, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the embodiments of the invention can be used to implement the invention. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional reagent companies.

[0127] Example 1: Construction and expression of the gene encoding the structural protein-binding peptide 1 fusion protein of Mycobacterium tuberculosis 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), fusion proteins Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T were constructed, respectively. Referring to the M72 sequence in the GSK patent (authorization announcement number CN 103249431B), the M72-4T fusion protein was constructed. The specific construction methods are as follows. The specific sequences of the constructed proteins and the above-mentioned reference sequences are shown in Table 1.

[0129] 1) Mtb32a-4T

[0130] The original wild-type sequence was modified by S / 177aa → A / 177aa, and the C-terminus was sequentially modified with a linker (GSGGSG), a 4T (AHIVMVDAYKPTK), and a 6His (HHHHHH) sequence, ending with a stop codon. The molecule was named 014.

[0131] 2)Ag85a-4T

[0132] The original wild-type sequence was truncated at the N-terminus. The specific N-terminal truncated sequence is QLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAG. Additionally, linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences were sequentially added to the C-terminus of the protein, and the protein was terminated with a stop codon. It was named molecule 072.

[0133] 3) ESAT6-CFP10-4T

[0134] The ESAT6 protein was placed at the N-terminus of the fusion protein, and the CFP10 protein was placed at the C-terminus. A linker (GSGGSG) sequence was used to link them together. The C-terminus was then appended with the linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences, ending with a stop codon. This molecule was named 076.

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

[0136] The original wild-type sequence of RV2660 was modified from C / 66aa to A / 66aa. The mutated RV2660 was placed at the N-terminus of the fusion protein, and the TB10.4 protein was placed at the C-terminus. The two sequences were linked together using a linker (GSGGSG) sequence. The linker (GSGGSG), 4T (AHIVMVDAYKPTK), and 6His (HHHHHH) sequences were added sequentially to the C-terminus, and the fusion protein ended with a stop codon. This molecule was named 077.

[0137] An unexpected discovery during the experiment was that modifying the original wild-type sequence of RV2660 from C / 66aa to A / 66aa could prevent the formation of dimers in RV2660-TB10.4-4T, thus maintaining the mutated RV2660-TB10.4-4T in a soluble monomeric form (SDS-PAGE analysis in both reduced and unreduced states showed a single band after mutation). See details... Figure 18 As shown, the mutation resulted in more uniform RV2660-TB10.4-NPM nanoparticles (Z-Average and Polydispersity Index values ​​changed from 77.3 and 0.23 before the mutation to 39.8 and 0.14 after the mutation, respectively). See details in [link to documentation]. Figure 19 As shown.

[0138] Figure 18 In the diagram, M represents the protein marker. Lane 1: RV2660-TB10.4T-4T before mutation (DTT+), Lane 2: RV2660-TB10.4T-4T after mutation (DTT-), Lane 3: RV2660-TB10.4T-4T after mutation (DTT+), and Lane 4: RV2660-TB10.4T-4T after mutation (DTT-).

[0139] Figure 19 In the figure, a represents NPM nanoparticles prepared before the RV2660-TB10.4T mutation; b represents 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), the sequences 6His (HHHHHH), 4T (AHIVMVDAYKPTK), and linker (GSGGSG) were added sequentially to the N-terminus of the original sequence, and the sequence ended with a stop codon.

[0142] The designed protein sequences (sequences shown in Table 1) were codon optimized and gene synthesized in E. coli (sequences shown in Table 2 below), cloned into the pET21a vector at restriction sites (5'NdeI, 3'HindⅢ), 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, linker 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, linker peptide 1, and fusion protein.

[0147]

[0148]

[0149]

[0150]

[0151]

[0152] 2. Expression of recombinant antigens

[0153] 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 cultured overnight at 37°C and 220 rpm / min in 10 ml of TB medium containing 50 μg / ml Ampicillin.

[0154] Inoculate the bacterial culture at a ratio of 1 / 100 into TB medium containing 50 μg / ml Ampicillin, and incubate at 37°C and 220 rpm / min for 2–3 hours.

[0155] The OD600 of the bacterial culture was measured using Nanodrop. When the OD value reached 0.6–0.8, the culture was transferred to a shaker at 18°C ​​to cool down. IPTG was added overnight to induce expression for about 16 hours, with a final IPTG concentration of 500 μM.

[0156] Collect 6000g of bacterial culture, centrifuge at 4℃ for 15 minutes, and discard the supernatant.

[0157] Centrifuge again at 6000g, 4℃ for 3 minutes to remove the culture medium from the supernatant. Store the bacterial sludge frozen at -80℃.

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

[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, and pH 7.4 buffer. After sonication, centrifuge at 13000 g / min for 30 min at 4 °C. Collect the supernatant and precipitate (20 mM Trsi-HCl, 150 Mm NaCl, pH 7.4 buffer).

[0160] 2) Perform Western blot identification. Prepare LDS sample loading buffer (4x) with reducing agent DTT, heat at 70℃ for 5 min, cool to room temperature, centrifuge at 10000 rpm for 20 s, vortex to mix, and finally load 0.5 μg.

[0161] 3) Load the sample to be analyzed and the pre-stained protein molecular weight standard into a 4-12% Bis-Tris gel, add MES electrophoresis buffer, set the voltage to 150V, and electrophoresis for about 60 minutes.

[0162] 4) Use Transfer was performed using a Turbo instrument and corresponding reagents. Incubation was performed using an iBind instrument with anti-his mouse monoclonal antibody and goat anti-mouse secondary antibody conjugated with AP enzyme. Color development was then performed, and images were taken using GelDoc Go. Western blot results showed that all four proteins exhibited soluble expression and inclusion body expression, with band positions consistent with expected molecular weights. (Specific details are as follows...) Figure 1 and Figure 2 As shown.

[0163] Figure 1 In the text: M represents the protein marker, lane 1 represents the Mtb32a-4T supernatant, and lane 2 represents the Mtb32a-4T precipitate;

[0164] Figure 2In the diagram: M represents the protein marker; lane 1 represents the Ag85a-4T supernatant; lane 2 represents the Ag85a-4T precipitate; lane 3 represents the ESAT6-CFP10-4T supernatant; lane 4 represents the ESAT6-CFP10-4T precipitate; lane 5 represents the RV2660-TB10.4-4T supernatant; and lane 6 represents the RV2660-TB10.4-4T precipitate.

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

[0166] 1. The antigenic fractions of the fusion proteins Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T obtained through expression 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:

[0167] 1) Pretreatment before purification

[0168] Resuspend the expression bacterial sludge (corresponding to 200 mL of expression bacterial culture) of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T in 20 mL of 20 mM Trsi-HCl, 150 Mm NaCl, and pH 7.4 buffer, and sonicate to disrupt the cells. After cell disruption, centrifuge at 13000 g / min for 30 min at 4 °C, discard the precipitate, and retain the supernatant.

[0169] 2) Nickel ion affinity chromatography

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

[0171] Chromatographic procedure: Ni-Bestarose Fast Flow, sterilization, column equilibration with capture buffer 20mM Trsi-HCl, 150Mm NaCl, pH 7.4, sample loading, washing with 20mM Trsi-HCl, 150Mm NaCl, pH 7.4 solution, washing with 20mM Trsi-HCl, 150Mm NaCl, 2% Triton-X100, pH 7.4 solution to remove endotoxins, washing with 20mM imidazole + 20mM Trsi-HCl, 150Mm NaCl, pH 7.4 buffer to remove contaminating proteins, eluting Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigen components with 500mM imidazole + 20mM Trsi-HCl, 150Mm NaCl, pH 7.4 buffer.

[0172] 3) Molecular sieve purification

[0173] Purification was performed using a HiLoad 16 / 600 Superdex 200pg filter with a molecular sieve column volume of 120mL. The loading volume of the sample for affinity purification using MTB32A, AG85A, ESAT6-CFP10, and RV2660-TB10.4 was controlled at approximately 4%.

[0174] Chromatographic procedure: Superdex 200pg; sterilization; equilibration of column with equilibration buffer (20mM Trsi-HCl, 150Mm NaCl, pH 7.4), sample loading, washing with TBS solution, collection of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T antigen fractions.

[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 analyzed by SDS-PAGE. The samples to be analyzed were prepared with LDS sample loading buffer (4x) and DTT reducing agent, heated at 70°C for 5 min, cooled to room temperature, centrifuged at 10000 rpm for 20 s, vortexed to mix, and the final loading volume was 5 μg. The samples to be analyzed and non-prestained protein molecular weight standards were loaded onto a 4-12% Bis-Tris gel, and MES electrophoresis buffer was used. Electrophoresis was performed at 150V for approximately 60 minutes. After electrophoresis, the gel was removed and placed in a clean container. An appropriate amount of Coomassie Brilliant Blue staining solution was added to cover the gel, and staining was performed on a shaker for 2 h. After staining, the staining solution was discarded, and the gel was soaked in purified water for destaining. Destaining was continued on a shaker until the gel background color was completely removed. The gel was then photographed using a GelDoc Go gel imaging system.

[0177] Results and Analysis:

[0178] 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 of over 90%. (Specific details are as follows...) Figures 3-6 As shown, the control protein M72-4T can also be successfully refolded and purified, as shown in the figure. Figure 7 As shown. Figure 3 In the text, M represents the protein molecule marker, and lanes 1-6 represent the Mtb32a-4T protein; Figure 4 In the text, M represents the protein molecule marker, and lanes 1-10 represent the Ag85a-4T protein; Figure 5 In the text, M represents the protein molecule marker, and lanes 1-5 represent the ESAT6-CFP10-4T protein; Figure 6 In the text, M represents the protein marker, and lanes 1-7 represent the RV2660-TB10.4-4T protein.

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

[0180] 1) Pretreatment before purification

[0181] Following the method described 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 in 20 mL of 20 mM Trsi-HCl, 150 Mm NaCl, and pH 7.4 buffer and sonicated. After cell disruption, the cells were centrifuged at 13000 g / min for 30 min at 4 °C, the supernatant was discarded, and the precipitate was retained. The precipitate was resuspended and dissolved in 20 mM Trsi-HCl, 150 Mm NaCl, 8 M Urea, and pH 7.4, and centrifuged at 13000 g / min for 30 min at 4 °C, and the supernatant was retained.

[0182] 2) Nickel ion affinity chromatography

[0183] Affinity purification was performed using a nickel ion affinity chromatography column. The column volume was 10 mL, the flow rate was 5 mL / min, and 70 mL of sample was loaded. Chromatographic procedure: Ni-Bestarose Fast Flow, sterilization, column equilibration with capture buffer 20mM Trsi-HCl, 150MmNaCl, 8M Urea, pH 7.4, sample loading, washing with 20mM Trsi-HCl, 150MmNaCl, 8M Urea, pH 7.4 solution, washing with endotoxin with 20mM Trsi-HCl, 150MmNaCl, 8M Urea, 2% Triton-X100, pH 7.4 solution, washing away contaminating proteins with 20mM imidazole + 20mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH 7.4 buffer, eluting M72 antigen components with 500mM imidazole + 220mM Trsi-HCl, 150Mm NaCl, 8M Urea, pH 7.4 buffer.

[0184] 3) Membrane-encapsulated ultrafiltration refolding

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

[0186] 4) Molecular sieve purification

[0187] The refolded M72-4T antigen was purified using a HiLoad 16 / 600 Superdex 200pg filter with a molecular sieve column volume of 120mL. The loading volume of the M72-4T affinity purified sample was controlled at approximately 4%.

[0188] Chromatographic procedure: Superdex 200pg; sterilization; equilibration buffer (20mM Trsi-HCl, 150Mm NaCl, pH 7.4) to equilibrate the column, loading the sample, washing with 20mM Trsi-HCl, 150Mm NaCl, pH 7.4 solution, and collecting the M72-4T antigen fraction. Figure 7 The results of molecular sieve separation and purification of M72-4T protein are shown (M represents protein molecular marker, lanes 1-8 represent M72-4T protein).

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

[0190] 1. Construction and Expression

[0191] The NPM-4C protein sequence was optimized using host codons in *E. coli*, followed by gene synthesis and subcloning. The encoding gene of the fusion protein was constructed into pET30a and expressed in *E. coli* BL21(DE3). After harvesting the cells, the target protein was released by high-pressure homogenization, and the liquid was clarified to remove cell debris 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] Clarification of the feed solution was mainly achieved through heat treatment. A two-step heating method was used, with the supernatant after E. coli disruption subjected to a first heating step and a second heating step (i.e., "two-step heating"). The impurity removal effect of the two heating steps and the purity of the recombinant particle protein components were calculated.

[0194] 60 g of E. coli wet cells collected by centrifugation were resuspended in 240 ml of buffer (20 mM Tris-HCl, 2 mM MPMSF, pH 9.0), and homogenized using a high-pressure homogenizer at 1000 bar. After centrifugation, 280 ml of supernatant was collected, and 40 ml of this supernatant was subjected to a two-step heating process. SDS-PAGE analysis was performed on the homogenized supernatant, the supernatant from the first heating and centrifugation step, and the resuspended precipitate from the second heating and centrifugation step.

[0195] As shown in Table 5, in the first heating step, the pH was adjusted to 9.0, and the mixture was heated in a water bath at 80°C for 1 hour. After returning to room temperature, approximately 35 ml of supernatant was collected by centrifugation. In the second heating step, 35 ml of 100 mM Tris-HCl, 5 mM EDTA, 4% Triton, and pH 7.4 buffer were added, followed by 7 ml of 1 M Tris-HCl, and the mixture was stirred well. The mixture was then heated in a 60°C water bath for 10 min, and the precipitate was immediately collected by centrifugation. The precipitate was reconstituted using a buffer solution of 20 mM Tris-HCl, 5 mM EDTA, and pH 9.0.

[0196] Table 3: Amino acid sequences of NPM, NPM-4C, binding peptide 2, linker peptide 2, and fusion protein in the embodiments of this application.

[0197]

[0198] Table 4: Nucleotide sequences of NPM, NPM-4C, binding peptide 2, linker peptide 2, and fusion protein in the embodiments of this application.

[0199]

[0200]

[0201] Table 5: Pretreatment before chromatography

[0202]

[0203]

[0204] Adding different concentrations of urea and sodium chloride after the two-step heating process and before chromatographic purification can significantly reduce the presence of unknown substances near the target recombinant particle protein bands. The preferred process conditions for pretreatment of recombinant particle protein component samples before Fractogel DEAE M chromatography are soaking in 8M urea and 50-200mM sodium chloride.

[0205] 3. Chromatographic purification

[0206] The recombinant particle protein fraction sample solution was purified using ion exchange and hydrophobic chromatography. The first step of chromatographic purification was performed using Fractogel DEAEM chromatography; specific steps and parameters are shown in Table 6. The Fractogel DEAEM elution sample was first diluted with buffer, and 50% (w / v) sucrose stabilizer was added to prevent precipitation of recombinant particle protein fractions during the next chromatographic step; specific parameters are shown in Table 7. Then, hydrophobic chromatography using Octyl Bestarose 4FF chromatography was performed for further purification (second step of chromatographic purification); specific steps and parameters are shown in Table 8.

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

[0208] Table 6: First-Step Chromatography Method

[0209] Chromatography steps Chromatography buffer / conditions parameter Balanced 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 20 mM Tris-HCl, 8 M Urea, pH 9.0 5CV Wash 4 buffer 20 mM Tris-HCl, 4 M Urea, pH 9.0 5CV Elution buffer 20mM Tris-HCl, 4MUrea, 150mM, pH9.0 2CV Collection range 50mAU-50mAU 2mm optical path

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

[0211] Process steps Dilution buffer Dilution volume Step 1: Collection of eluent from chromatography 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 eluent Buffer dilution 20 mM Tris-HCl, 2 M NaCl, pH 9.0 1 volume of eluent

[0212] Second step chromatography method: chromatography packing material - Octyl Bestarose 4FF, retention time - 12.5 min

[0213] Table 8: Second-Step Chromatography Method

[0214] Chromatography steps Chromatography buffer / conditions parameter Balanced 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 range 50mAU-50mAU 2mm optical path

[0215] Results and Analysis:

[0216] Purity testing revealed that after further purification using the above-mentioned combination of chromatographic media, the purity of the product reached over 99.0%. Specific SDS-PAGE analysis results of the purified NPM-4C protein using Octyl Bestarose 4FF are shown below. Figure 8 (M: protein marker; lanes 1-2: NPM-4C protein).

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

[0218] 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 concentration ratio of 6:1. A 50% sucrose stock solution was added to approximately a final sucrose concentration of 25%, and 10% of the total reaction volume of 1M Tris-HCl stock 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 could specifically 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.

[0219] 2. Purification of the combined product

[0220] Purification of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM binding products was performed using Cytiva HiLoad 16 / 600 Superdex 200pg (column volume 120mL) or Cytiva Superdex 200 Increase 10 / 300GL (column volume 23mL), and unbound Mtb32a, Ag85a, ESAT6-CFP10, and RV2660-TB10.4 antigens was separated and removed. If using HiLoad 16 / 600 Superdex 200pg molecular sieves, 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 Superdex 200Increase 10 / 300 GL molecular sieves, 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.

[0221] 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 the 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 purified antigen in Example 3.

[0222] Results and Analysis:

[0223] Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles purified by molecular sieve were analyzed by SDS-PAGE. The results showed that the purity could reach over 90%. (See details...) Figures 9-12 As shown. Figure 9 In the text, M represents the protein molecule marker, lanes 1-3 represent the Mtb32a-NPM binding product, and lanes 4-10 represent unbound Mtb32a. Figure 10 In the text, M represents the protein molecule marker, and lanes 1-5 represent the Ag85a-NPM binding products; Figure 11 In the text, M represents the protein marker, and lanes 1-5 represent the ESAT6-CFP10-NPM binding product; Figure 12 In the text, M represents the protein marker, and lanes 1-5 represent the RV2660-TB10.4-NPM binding products.

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

[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 "Purification of Binding Products" section above were negatively stained using the flotation method. A 400-mesh screen with a supporting membrane was selected and pre-treated for hydrophilicity. Deionized water and 2% uranium formate negative staining solution were prepared. 3 μL of the prepared protein sample (0.1 mg / ml) was dropped directly onto one side of the screen with the supporting membrane. After timing for 1 minute, excess liquid was absorbed from the edge of the screen with clean filter paper. After slightly drying, the sample was rinsed twice quickly with deionized water. Then, 5 μL of negative staining solution was added for rinsing once. Finally, 5 μL of negative staining solution was added and timing for 1 minute. After the rinsing time, the screen was removed with tweezers, and the staining solution was absorbed with filter paper, leaving a thin layer to air dry naturally for analysis. The examination was conducted under a 120kV transmission electron microscope (FERRITINI Tecnai Spirit). The overall staining of the screen was observed under low magnification. Wells of appropriate thickness were selected for observation. Under high magnification, suitable areas were photographed and preserved.

[0228] 2) DLS detection

[0229] The purified Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles were diluted to a concentration of 0.15 mg / mL. Using a Zetasizer Lab instrument, ≥1 mL of the sample was injected into the sample cell, and the instrument was run for detection. Data analysis was performed using Z-Average (nm) and Polydispersity Index (PI) values, as well as the Size Distribution by Intensity / Volume distribution curve, and the results were reported.

[0230] Results and analysis:

[0231] Electron microscopy images of Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles (0.1 mg / mL, 18500×) show uniform particle distribution and no aggregation. (Details are as follows...) Figures 13 to 16 As shown. The distribution curve of Distribution by Intensity / Volume analyzed by Zetasizer Lab instruments is shown in the figure. Figure 17As shown in the figure, the results indicate that the prepared Mtb32a-NPM, Ag85a-NPM, ESAT6-CFP10-NPM, and RV2660-TB10.4-NPM particles exhibit good peak overlap and 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 detailed in Table 9.

[0232] Table 9: Z-Average (nm) and Polydispersity Index (PI) values ​​of particulate 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: Vaccine Preparation

[0235] 1. Test vaccine antigens and adjuvants

[0236] 1) Test vaccine antigen solution

[0237] The test vaccine protein stock solution was prepared by Guangzhou Pano 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) Adjuvants to the test vaccine

[0239] Commercial AS01B adjuvant was prepared by adding QS21 (5 μg) to small monolayer vesicles (SUVs) containing cholesterol (25 μg) and dioleoylphosphatidylcholine (100 μg) to prepare dual-strength AS01B (WO 96 / 33739) and monophosphatidyllipid 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 a protein mixture (0.2 μg each of Mtb32a-4T, Ag85a-4T, ESAT6-CFP10-4T, and RV2660-TB10.4-4T) in TBS Mtb ​​buffer with 50 μL of dual-strength AS01B. 2) Mtb nanoparticle antigen-mixed vaccine

[0243] Aliquots (50 μL) for injection were prepared by mixing 0.8 μg of protein mixture (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 dual-strength AS01B.

[0244] 3) Single Mtb nanoparticle antigen vaccine

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

[0246] 4) M72 control vaccine

[0247] Aliquots (50 μL) for injection were prepared by mixing 0.8 μg of the M72-4T protein mixture buffer (TBS Mtb) with 50 μL of dual-strength AS01B.

[0248] Example 8: Vaccine Immunoprotection Experiment

[0249] 1. Laboratory animals and grouping

[0250] Female C57BL / 6 mice aged 4-6 weeks were selected and purchased from Vital River. After quarantine, the mice were ear-tagged with metal tags and randomly grouped according to body weight, with free access to food and water. Six candidate vaccine immunization groups were established (n=12 per group): the M72-4T group (n=12), the BCG control group (n=12), and the saline group (n=15). Animals were housed in an SPF-standard animal facility, provided with SPF-specific sterile feed and sterile deionized water. The housing was maintained with alternating 12-hour light and dark cycles, a temperature of 21±2℃, and humidity of 30-70%.

[0251] 2. Immunization and virus attack

[0252] Mice in the vaccine group (vac-1 to vac-6) and the M72-4T group (Control-A) received a single 100 μL (0.8 μg) immunization, administered twice at a 3-week interval. The saline group received the same volume of saline immunization. Mice in the BCG group (Control-B) received a subcutaneous injection of 5 × 10⁶ BCG bacteria. 4 CFU / animal, 100μL / animal, immunized once. The remaining groups were immunized intramuscularly.

[0253] Four weeks after the last immunization, mice were challenged with aerosol virus at a dose of 100 bacteria per mouse. Four weeks post-challenge, the lungs and spleen were dissected, and pathological sections were prepared. The homogenates from the lungs and spleen were used to calculate the bacterial load. Specific grouping, immunization protocols, and challenge protocols are shown in Tables 10 and 11.

[0254] Table 10: Immunization and Challenge Plan

[0255]

[0256]

[0257] Table 11: Grouping and Protein Components, Dosage, and Adjuvant Comparison Table

[0258]

[0259] 3. Detection of specific IgG after immunization

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

[0261] 96-well microplates (Thermo Fisher Scientific) were coated with proteins 014, 072, 076, and 077 (1 μg / mL), respectively, at 100 ng / 50 μL / well, and incubated overnight at 4°C. The plates were then washed twice with PBST (0.05% Tween 20), followed by blocking buffer (Thermo Fisher Scientific), 200 μL / well, and incubated at room temperature (25°C ± 3°C) for 1-4 h. After washing twice, diluted immune serum was added, and the plates were incubated at room temperature for 1 h, followed by 4 washes. Then, 50 μL / well of the corresponding 1:5000 diluted HRP-IgG1 and HRP-IgG2a working solutions were added, respectively. After incubation at room temperature for 1 h, the plates were washed 6 times. Finally, 100 μL of chromogenic buffer was added to each well, and the plates were incubated at room temperature in the dark for 10 min. The incubation was terminated by adding 100 μL of 1M HCl to each well. The microplate reader was set to a master wavelength of 450 nm and a reference wavelength of 620 nm. The sample absorbance was calculated as OD450 - OD620. The assay was completed within 5 minutes after termination. Based on the results, the humoral and cellular immune types were analyzed.

[0262] Data processing:

[0263] Data are considered reliable if the following conditions are met: control serum OD value ±0.2, initial sample concentration corresponding to OD value <3.0, blank well corresponding to OD value <0.1, and replicate well (response value) coefficient of variation <20%. Import the raw sample data into the "Excel Endpoint ELISA template" to calculate the antibody titer. Analyze the results using Graphpad Prism 9.1.2 software. Analyze differences using the Unpaired Test or One-Way ANOVA; a p-value <0.05 is defined as statistically significant between the two groups.

[0264] The test results are as follows:

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

[0266] The results showed that the single Mtb nanoparticle antigen vaccines (vac-3 / 4 / 5 / 6), Mtb recombinant antigen mixed vaccines (vac-2), and Mtb nanoparticle antigen mixed vaccines (vac-1) of the present invention could all induce IgG production, indicating that the vaccines of the present invention all have good immunogenicity. In particular, the IgG antibody levels corresponding to vac-1 and the single Mtb nanoparticle antigen vaccine groups were significantly higher than the IgG antibody levels of vac-2, indicating that the nanoparticle vaccines of the present invention can significantly improve the immunogenicity of Mtb antigen.

[0267] Figure 21 Figures a, b, and c show the levels of IgG1 and IgG2a antibodies in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline) using antigens 072, 076, 077, and 014 as coating antigens, respectively. Figure e shows the levels of IgG1 and IgG2a antibodies in the M72 group (0.8 μg immunization group).

[0268] The results showed that the IgG1 and IgG2a levels in the vac-1 and single Mtb nanoparticle antigen vaccine groups were significantly higher than those in the vac-2 group. The IgG2a / IgG1 ratio in both the vac-1 and single Mtb nanoparticle antigen vaccine groups was greater than 1.0, indicating that 072, 076, 077, and 014 could all induce Th1-type cellular immune responses. The IgG2a / IgG1 ratio in the M72 group was greater than 1.0, indicating that it could 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, six mice in each group were treated, spleens were collected, spleen cells were isolated, and the cells were processed at a rate of 2.5 × 10⁻⁶. 5 10 cells / well, seeded into a 96-well culture plate;

[0271] 2) Use 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 incubate at 37°C in a 5% CO2 incubator for 72 hours;

[0272] 3) Collect the supernatant and use a commercially available kit to detect IFN-γ, TNFα, IL-4, and IL-2 using a double-antibody sandwich ELISA. Analyze the results using Graphpad Prism 9.1.2 software. Analyze the differences using the unpaired test or one-way ANOVA. A p-value < 0.05 was defined as statistically significant between the two groups.

[0273] 5. Cytokine ELISPOT

[0274] 1) Commercially available 96-well filter plates were coated with monoclonal antibodies against IFN-γ, TNFα, IL-4, and IL-2 and then blocked. Three weeks after the last immunization, six mice were treated in each group, spleens were collected, spleen cells were isolated, and the cells were processed at a concentration of 2.0 × 10⁻⁶. 5 1) Seed cells / well into a 96-well culture plate; 2) Use separate culture medium and 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 incubate at 37°C in a 5% CO2 incubator 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 the substrate according to the instructions of the commercial kit.

[0276] 4) After drying the plates, use an automated ELISPOT plate reader to count and analyze the spots.

[0277] The results were analyzed using Graphpad Prism 9.1.2 software. Differences were analyzed using the Unpaired Test or One-Way ANOVA. Two groups of data were defined as having a significant difference when P < 0.05.

[0278] Figure 22 The ad refers to the corresponding 072 antigen as a specific stimulating antigen to detect the corresponding IFN-γ, IL-2, TNFα and IL-4 levels in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline); Figure 22 EH represents the corresponding 076 antigen as a specific stimulating antigen to detect the corresponding IFN-γ, IL-2, TNFα and IL-4 levels in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline).

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

[0280] Figure 23 The ad groups, in turn, use the corresponding 077 antigen as specific stimulating antigens to detect the corresponding IFN-γ, IL-2, TNFα, and IL-4 levels in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline). Figure 23 Eh represents the corresponding 014 antigen as a specific stimulating antigen to detect the corresponding IFN-γ, IL-2, TNFα and IL-4 levels in different groups (vac-1, vac-2, single Mtb nanoparticle antigen vaccine, and saline).

[0281] The results showed that the levels of IFN-γ, IL-2, TNFα, and IL-4 in vac-1 stimulated by 077 and 014 and in the single Mtb nanoparticle antigen vaccine group were higher than those in vac-2. Furthermore, the levels of IFN-γ, IL-2, and TNFα in each group were significantly higher than those in IL-4, indicating that both 077 and 014 can induce a th1-type-dominant cellular immune response.

[0282] Figure 24The results showed that the levels of IFN-γ, IL-2, and TNFα 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 responses in mice after immunization, which is consistent with the 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 in each group were treated, spleens were collected, and spleen cells were harvested (1×10⁻⁶). 6 Cells were washed with staining buffer and then stained with a total volume of 50 μL containing 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 Stain with a mixture of rat anti-mouse CD8 (1:50 final dilution, BD) 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 CD4+ and CD8+ T cells expressing IFN-γ, TNFα, IL-4, and IL-2. Spleen cells were isolated and harvested (1×10⁻⁶ cells). 6 Cells were in vitro stimulated for 6 hours 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 co-incubated with a protein transport inhibitor (containing Brefeldin A; BD) for 4 hours; cells were washed with PBS and stained with fixative viability stain 780 (1:1000 final dilution, BD) and mouse Fc blocks (BD) for 15 minutes.

[0286] 3) Wash cells with staining buffer and stain for 15 minutes with a mixture of 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). Fix and permeabilize cells using a fixation / permeabilization kit (BD). Wash twice with 1×Perm / Wash solution, 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), and R718 rat anti-mouse TNFα (1:50 final dilution, BD), then wash cells with 1×Perm / Wash solution and resuspend. Analyze using DxFLEX (BECKMAN COULTER). Data were analyzed using CytExpert, and the data are expressed as the percentage of the total frequency of CD4+ T / CD8+ T cells expressing IFN-γ, TNFα, IL-4, and IL-2, after subtracting background from the mean response of specific CD4+ and CD8+ T cells.

[0287] 7. Statistics on the results of virus challenge protection

[0288] Four weeks after the final immunization, mice were challenged with a low-dose aerosol of Mycobacterium tuberculosis H37Rv. The UW-madison aerosol contact chamber was calibrated to deliver 50-100 CFU to the lungs. Four weeks later, mice were euthanized, and lung and spleen homogenates were prepared using PBS / Tween-80 (0.05%). The homogenates of individual intact organs were serially diluted and inoculated onto Middlebrook 7H11 Bacto agar. After incubation at 37°C under humid, 5% CO2 conditions for 2-4 weeks, bacterial colonies were counted. The final data are expressed as mean Log10 ± SD. The Log10 reduction (difference) of CFUs = Log10 CFUs in the saline-treated group - Log10 CFUs in the vaccine-treated group.

[0289] 8. Pathological section analysis

[0290] Four weeks after challenge with the virus, all mice were euthanized, and spleen and lung tissues were collected. The tissues were fixed with formaldehyde solution, sent to the company for HA staining, pathological sections were prepared, and analyzed.

[0291] 9. Immunological assessment results

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

[0293] Mycobacterium tuberculosis primarily infects human macrophages, acting as an intracellular parasite. Controlling tuberculosis requires intracellular anti-infection agents. This vaccine, utilizing NPM nanoparticles to display Mycobacterium tuberculosis antigens 072, 076, 077, and 014, effectively stimulates the body to produce high levels of IFN-γ, TNFα, and IL-2. IFN-γ activates macrophages to kill Mycobacterium tuberculosis and enhances the killing effect of NK cells, while IL-2 also enhances the killing power of NK cells. TNFα, as an important cytokine, plays a crucial role in combating Mycobacterium tuberculosis infection; its main function is to promote apoptosis of infected macrophages, exposing occult Mtb, which is then presented by APCs and activates CTL immunity.

[0294] This application found that the IgG levels in the mixed NPM group (vac-1) and the single NPM group (072, 076, 077, and 014) were higher than those in the mixed recombinant protein group (vac-2). Furthermore, the IFN-γ, TNFα, IL-4, and IL-2 levels in the mixed NPM group and the single NPM group were also higher than those in the mixed recombinant protein group. This demonstrates that the Mycobacterium tuberculosis nanoparticle antigen presented by NPM can better cooperate with adjuvants to enhance cellular and humoral immunity levels, showcasing the advantages of NPM nanoparticles in presenting Mycobacterium tuberculosis antigen. Simultaneously, the vaccine of this invention can generate a significant Th1-type immune response. TB vaccines mainly rely on cellular immune responses, while this type of vaccine, which favors Th1-type T-cell immune responses, aligns with the immunogenic bias in TB vaccine development.

[0295] The above results indicate that VLP-form antigens enhance immune protection, and the nanoparticle antigens covered by vac-1 to vac-6 can effectively stimulate the body to produce immune protection, effectively preventing 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] In summary, the above embodiments and accompanying drawings are merely 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 within the scope of protection of the present invention.

Claims

1. An immune composition, characterized in that, It comprises the following four immunogenic complexes, wherein the immunogenic complexes comprise: (1) Antigen component, which is formed by the fusion of Mycobacterium tuberculosis structural protein at the C-terminus with binding peptide 1 via linker peptide 1; (2) Particulate protein component, which is formed by the fusion of nanoparticle protein with linking peptide 2 and binding peptide 2 at the N-terminus; The antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2 to form an immunogenic complex. The Mycobacterium tuberculosis structural proteins are 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; the amino acid sequence of the nanoparticle protein is shown in SEQ ID NO:

26. The amino acid sequence of the binding peptide 1 is shown in SEQ ID NO:1, and the amino acid sequence of the binding peptide 2 is shown in SEQ ID NO:

24. The amino acid sequence of linker peptide 1 is shown in SEQ ID NO:2, and the amino acid sequence of linker peptide 2 is shown in SEQ ID NO:

25.

2. The immune composition according to claim 1, characterized in that: The antigen component and / or the particulate protein component contain a histidine tag.

3. The immune composition according to claim 1 or 2, wherein the immune composition further comprises a pharmaceutically acceptable carrier.

4. The immunomodulatory composition according to claim 3, 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 disodium dihydrogen phosphate dihydrate, and the pH adjuster comprises hydrochloric acid.

5. A method for preparing the immune composition according to any one of claims 1-4, characterized in that, include: (1) The antigen component and the particulate protein component encoding genes were respectively ligated into expression vectors to construct recombinant expression plasmids and expression host strains, express the target protein, and then purified. (2) The antigen component obtained in step (1) is co-incubated with the particulate protein component to obtain an immunogenic complex.

6. A Mycobacterium tuberculosis vaccine, characterized in that, The invention comprises the immune composition and adjuvant according to any one of claims 1-4, wherein the adjuvant is selected from at least one of: aluminum salt adjuvants, Freund's complete adjuvants, propolis adjuvants, water-oil adjuvants, cytokines, CpG DNA, genetically engineered attenuated toxins, immunostimulatory complexes, and liposomes.

7. The use of the immune composition according to any one of claims 1-4 or the Mycobacterium tuberculosis vaccine according to claim 6 in the preparation of a medicament for the prevention or treatment of diseases caused by Mycobacterium tuberculosis infection.

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