Novel tuberculosis subunit vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a novel tuberculosis subunit vaccine as well as a preparation method and application thereof, and the preparation method comprises the following steps: S1, culturing a mycobacterium bovis attenuated strain; s2, extracting the cell wall of the attenuated strain of mycobacterium bovis; s3, identifying cell walls of the attenuated strains of the mycobacterium bovis; s4, preparing a cell wall and manganese adjuvant subunit vaccine; s5, subunit vaccine intranasal immunization of mice; and S6, performing a mouse challenge protection experiment. Two novel tuberculosis subunit vaccines named as CW-MnJ (alpha) and CW-MnJ (beta) are prepared by extracting cell wall components of a mycobacterium bovis attenuated strain and using the cell wall components as antigens and combining the cell wall components with manganese adjuvants (MnJ (alpha) and MnJ (beta)). A mouse immune protection experiment proves that the lung and spleen bacterium load can be remarkably reduced by the mouse intranasal immunization CW-MnJ (alpha) and CW-MnJ (beta), and the spleen bacterium load of the mouse immunized with the CW-MnJ (alpha) is remarkably reduced compared with a positive control group (BCG immunization group), so that the CW-MnJ (alpha) and CW-MnJ (beta) subunit vaccines prepared by the invention have very good immune protection effects.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of medical technology, and in particular relates to a novel tuberculosis subunit vaccine and a preparation method and application thereof. Background Art

[0002] Tuberculosis is a zoonosis with a significant impact worldwide. It is caused by the Mycobacterium tuberculosis complex, including Mycobacterium tuberculosis and Mycobacterium bovis. Tuberculosis is mainly transmitted through the respiratory tract, often in the lungs. The intestines and spine can also be infected with Mycobacterium tuberculosis, which is called extrapulmonary tuberculosis. The general clinical symptoms and signs of tuberculosis mainly include cough, chest pain, fatigue, fever, loss of appetite, weight loss and night sweats.

[0003] Bacillus Calmette-Guérin (BCG) is the only approved tuberculosis vaccine in the world, but its protective effect in adults is limited. Data released by the Communicable Disease Surveillance Information System of the Chinese Center for Disease Control and Prevention show that the incidence rate of children over 10 years old has increased significantly. There are also research reports that after BCG immunization, usually 10 to 15 years later, that is, in adolescence, the immune protection will basically disappear. Therefore, the development of new tuberculosis vaccines is imminent. At present, there are also many new tuberculosis vaccines entering the clinical trial stage in the world, including recombinant BCG vaccine, live attenuated vaccine, viral vector vaccine, recombinant protein subunit vaccine, DNA vaccine and mRNA vaccine, but its protective effect in the population is still unclear. However, it is worth mentioning that the Phase II clinical trial of the M72 / AS01E recombinant protein vaccine developed by GlaxoSmithKline showed that its protection rate reached 54%, which is the only tuberculosis vaccine with a population protection efficiency of more than 50% in the past few decades. The M72 / AS01E vaccine is a subunit vaccine composed of Mycobacterium tuberculosis PPE18 and pepA fusion proteins as antigens and AS01E as adjuvant. It is currently undergoing Phase III clinical trials, which suggests that the research strategy of recombinant protein subunit vaccines is promising for the development of a successful and highly effective new tuberculosis vaccine.

[0004] Mycobacterium tuberculosis has a very complex and special cell wall (CW) structure, which is mainly composed of lipids and carbohydrates, providing a permeability barrier against hydrophilic drugs for mycobacteria, which is essential for its survival and virulence. The inner layer of the mycobacterial cell wall is the plasma membrane, the middle is the periplasmic space, and the outer layer is a cross-linked network of highly branched arabinogalactan (AG) and peptidoglycan (PG), namely the arabinogalactan-peptidoglycan complex (AGP). Arabinogalactan residues are connected to mycolic acids (MA) and are located in the outermost layer, forming a waxy shell unique to mycobacteria, also known as the mycobacterial outer membrane. The mycolic acid-arabinogalactan-peptidoglycan complex (mAGP) is the core structure of the mycobacterial cell wall. In addition, mycobacterial cell walls also contain lipomannan (LM), lipoarabinomannan (LAM) and various free lipids, such as glycolipids, thiosterol bifidose, cord factors or bifidose acyltrehalose, sulfolipids and phosphatidylinositol mannose. Many of the mycobacterial cell wall components have been shown to be virulence factors, and some components have immunomodulatory properties (such as lipoarabinomannan). Mannose-capped lipoarabinomannan (ManLAM) is a high-molecular-weight amphoteric lipopolysaccharide that can bind to the macrophage mannose receptor (MR) to induce the production of the anti-inflammatory factor IL-10, impair the polarization of M1 macrophages, participate in the inhibition of phagosome maturation, and inhibit the fusion of phagosomes and lysosomes, thereby facilitating the survival of mycobacteria in macrophages. In addition to affecting the function of macrophages against tuberculosis infection, ManLAM can also affect the maturation and activation of dendritic cells, T cell function, and antibody production.

[0005] In summary, the inventors of the present invention prepared two new tuberculosis subunit vaccines (CW-MnJ(α) and CW-MnJ(β)) by extracting cell wall components of attenuated strains of Mycobacterium bovis and using them as antigens in combination with novel adjuvants manganese adjuvants (MnJ(α) and MnJ(β)). The results of the mouse immune protection experiment showed that intranasal immunization of mice with CW-MnJ(α) and CW-MnJ(β) could significantly reduce the bacterial load in the lungs and spleen, and the bacterial load in the spleen of mice immunized with CW-MnJ(α) was significantly reduced compared with that of the BCG immunization group, indicating that the two subunit vaccines CW-MnJ(α) and CW-MnJ(β) have very good immune protection effects. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for preparing a novel tuberculosis subunit vaccine and a method for evaluating the immune effect on mice.

[0007] The present invention is achieved by a method for preparing a novel tuberculosis subunit vaccine, comprising the following steps:

[0008] S1, culture the attenuated strain of Mycobacterium bovis to obtain the bacteria;

[0009] S2, extraction of cell wall components of attenuated strains of Mycobacterium bovis;

[0010] S3, identifying whether the extracted cell wall contains polysaccharides and determining their concentration;

[0011] S4, cell wall components were formulated into subunit vaccines using manganese adjuvants;

[0012] S5, mice were immunized with subunit vaccine by intranasal administration;

[0013] S6, challenge and protection experiments were performed on immunized mice to evaluate the immune effect of the vaccine.

[0014] Further, the S1 step specifically includes:

[0015] Take 100 μl of the attenuated strain of Mycobacterium bovis preserved in the laboratory and spread it on 7H10 solid medium;

[0016] The coated culture medium was placed in a 37°C water-tight constant temperature incubator and cultured for 14-30 days to obtain an appropriate amount of bacteria.

[0017] Further, the S4 step includes the preparation of the following two subunit vaccines:

[0018] CW-MnJ(α): Mix 10 μg of polysaccharide-containing cell wall components with 10 μg (100 μL) of MnJ(α) adjuvant and emulsify for 5 minutes for mouse immunization. The antigen and adjuvant must be prepared and used immediately.

[0019] CW-MnJ(β): Mix 10 μg of cell wall components with polysaccharide content and 50 μg of MnJ(β) adjuvant with a concentration of 5 mg / mL at a ratio of 1:5. Mix well on a vortexer and let stand for 3 minutes. If no stratification is performed, it can be used for immunization.

[0020] Further, the S6 step specifically includes:

[0021] Mice immunized three times were infected intranasally with a virulent strain of Mycobacterium bovis;

[0022] The mice were autopsied 4 weeks after infection, and lung and spleen samples were collected to detect bacterial loads to evaluate the protective effect of the vaccine.

[0023] The present invention also provides a method for evaluating the immune effect of a novel tuberculosis subunit vaccine on mice, comprising the following steps:

[0024] The prepared subunit vaccine was used to immunize mice by intranasal drops;

[0025] After immunization, a challenge experiment was performed to infect mice to evaluate the protective effect of the vaccine;

[0026] Lung and spleen tissue samples were collected from mice at set times after infection;

[0027] The bacterial load in the lungs and spleen was measured and the immune effect was analyzed.

[0028] Furthermore, the mouse immunization procedure includes three nasal drop immunizations, with one week between each immunization to enhance the specific immune response of the mice and ensure the effectiveness of the protective effect.

[0029] Furthermore, the challenge experiment specifically involved infecting mice after three immunizations with a virulent strain of Mycobacterium bovis through nasal drops, and dissecting the mice 4 weeks after infection to collect lung and spleen samples for bacterial load testing.

[0030] Furthermore, the bacterial load in the lungs and spleens of mice was quantitatively analyzed through the data acquisition and analysis system to generate an immune effect evaluation report to facilitate scientific verification and comparative analysis of the protective effect of the vaccine.

[0031] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0032] First, the present invention relates to a method for preparing a novel tuberculosis subunit vaccine, which uses the cell wall of an attenuated strain of Mycobacterium bovis as an immunogen and uses a manganese adjuvant to improve the immune effect. The present invention verifies its effect through an immune experiment on a mouse model. Its vaccine preparation and immune process are closely combined with experimental results and data, bringing significant technological progress and innovative effects:

[0033] High-efficiency cell wall extraction method: Through the optimized culture and cell wall extraction method of attenuated Mycobacterium bovis, the effective extraction and high purity of polysaccharides in the cell wall are ensured, and the stability and activity of the vaccine components are guaranteed. This extraction method lays the foundation for the preparation of high-efficiency vaccines. The addition of Tween 80 to the attenuated Mycobacterium bovis strain cultured in liquid will destroy the complete structure of the cell wall, and the bacteria cultured without Tween 80 are prone to form bacterial agglomerates. The present invention uses the attenuated Mycobacterium bovis strain cultured in solid culture to extract the cell wall, which saves costs while also ensuring the integrity of the Mycobacterium bovis cell wall.

[0034] Design of new subunit vaccines: The subunit vaccines CW-MnJ (α) and CW-MnJ (β) developed by the present invention combine the cell wall components of Mycobacterium bovis with different types of manganese adjuvants to improve the immunogenicity of the vaccine. Its immune effect is more targeted than that of the traditional BCG vaccine.

[0035] Significant immune protection effect: By immunizing mice with nasal drops and conducting virus challenge experiments, the present invention verified the good protective effect of the vaccine against virulent strains of Mycobacterium bovis, especially showing significant advantages in reducing the pathogen load in the lungs and spleen. This effect is highly innovative and solves the problem that traditional subunit vaccines are not effective in mouse virus challenge experiments.

[0036] Second, from the perspective of the overall product, the technical solution of the present invention has the following technical effects and advantages:

[0037] The preparation method is efficient and stable: The present invention provides an optimized method for extracting the cell wall of Mycobacterium bovis and preparing a vaccine, which ensures the high purity and stability of the cell wall components, especially the stability of the polysaccharide content verified by PAS staining and phenol-sulfuric acid method. This lays a technical foundation for the efficient preparation of subunit vaccines.

[0038] Adjuvant combination enhances immune response: The present invention combines a novel manganese adjuvant with the cell wall of Mycobacterium bovis to form two different types of subunit vaccines, CW-MnJ (α) and CW-MnJ (β). The immunogenicity and protective effect of the vaccine are enhanced through a reasonable adjuvant ratio and preparation method.

[0039] Verification of the mouse challenge experiment: In the nasal challenge experiment of immunized mice, the subunit vaccine of the present invention significantly reduced the bacterial load in the lungs and spleens of infected mice, verifying the superiority of its immune protection effect.

[0040] Innovative method to improve immune effect: Compared with the traditional BCG vaccine, the subunit vaccine of the present invention enhances the immune effect while reducing the side effects during use. By using manganese adjuvant, the vaccine of the present invention is expected to solve the technical problem of the limited effect of the existing BCG vaccine.

[0041] Third, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0042] Expanded technical means: The subunit vaccine of the present invention expands the technical means for tuberculosis vaccine research through the use of an innovative cell wall extraction method and a new manganese adjuvant.

[0043] Overcoming technical prejudice: The combination of manganese adjuvant and cell wall components adopted in the present invention breaks the limitation of adjuvant selection in traditional vaccine preparation, and provides a new and efficient adjuvant combination scheme, which is easy to scale up production and popularize and apply.

[0044] In summary, the present invention provides a highly effective subunit vaccine for tuberculosis through innovative technical means, which not only overcomes the problems in the prior art, but also brings new ideas and breakthroughs to the research and development and production of tuberculosis vaccines.

[0045] Fourth, the technical solution of the present invention effectively solves the following key problems in the prior art in industrial applications and achieves significant technical progress:

[0046] 1. Improve the safety and immune effect of tuberculosis subunit vaccines

[0047] Traditional tuberculosis vaccines use live bacteria or attenuated vaccines, which pose potential safety risks, especially in immunocompromised populations. The present invention uses cell wall components of attenuated Mycobacterium bovis strains and combines them with manganese adjuvants to make subunit vaccines, avoiding the use of live bacteria. At the same time, scientific immune adjuvants are used to enhance the immune effect of the vaccine, thereby ensuring safety while improving the immune effect.

[0048] 2. The local immunization method of the vaccine is realized to enhance respiratory immunity

[0049] Traditional vaccines are mostly injected, and tuberculosis is mainly transmitted through the respiratory tract. The present invention uses nasal drops to directly act on the respiratory mucosa, enhance local immune response, effectively simulate the natural infection route, and improve the protective effect against tuberculosis pathogens. This local immunization method has a high promotion value in the application of respiratory disease vaccines.

[0050] 3. Established an efficient immune effect evaluation system

[0051] The present invention evaluates the protective effect of the vaccine against tuberculosis through a mouse challenge experiment, clearly determines the bacterial load in the lungs and spleen, and realizes the scientific quantification of the vaccine effect. This evaluation method provides an effective experimental basis for vaccine research and development, making the vaccine research and development and evaluation process more efficient and reliable, and helping to promote the application of vaccines in industrialization.

[0052] 4. Significantly improved the industrial production efficiency of tuberculosis vaccines

[0053] The cell wall extraction and immune adjuvant ratio process used in the present invention is simple and controllable, and is convenient for large-scale production. By establishing a standardized preparation process and immune effect evaluation method, the technical solution can be quickly promoted to industrial application, improving the production efficiency and product quality of tuberculosis vaccines. This technological advancement not only optimizes the vaccine research and development process, but also provides the market with a safer and more effective means of tuberculosis prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a colony diagram of the attenuated Mycobacterium bovis (BCG) strain provided in an embodiment of the present invention cultured on a 7H10 solid culture medium;

[0055] Figure 2 This is a diagram showing the results of PAS staining of the cell wall of Mycobacterium bovis provided in an embodiment of the present invention;

[0056] Figure 3 This is a result diagram of measuring the polysaccharide concentration of the cell wall using the phenol-sulfuric acid method provided in an embodiment of the present invention;

[0057] Figure 4 It is a schematic diagram of animal immunization program and grouping provided by an embodiment of the present invention;

[0058] Figure 5 It is a schematic diagram of the gross pathological observation results of the lungs and spleen of mice after immunization and before challenge with a virus provided in an embodiment of the present invention;

[0059] Figure 6 This is a schematic diagram of the gross pathological observation results of the lungs and spleens of mice immunized with the subunit vaccine provided in an embodiment of the present invention after infection with Mycobacterium bovis;

[0060] Figure 7 It is a schematic diagram of the bacterial load in the lungs and spleen of mice immunized with the subunit vaccine provided in an embodiment of the present invention after infection with Mycobacterium bovis;

[0061] Figure 8 It is a flow chart of the method for preparing a novel tuberculosis subunit vaccine and evaluating the immune effect on mice provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0063] In view of the limited protective effect of BCG vaccine in adults, the present invention aims to provide the preparation of two new tuberculosis subunit vaccines using Mycobacterium bovis cell wall as immunogen and the evaluation of their immune effect in mice. The present invention is described in detail below in conjunction with the accompanying drawings.

[0064] 1. Cultivation of attenuated strains of Mycobacterium bovis

[0065] The attenuated Mycobacterium bovis strain used in the present invention is the Pasteur strain of Bacillus Calmette-Guerin (BCG), which is stored in the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The recovery culture process of the strain is to take 1 ml of glycerol bacteria, add it to a 50 ml centrifuge tube containing 9 ml of 7H9 liquid culture medium, and then put it into a 37°C constant temperature incubator for static culture or put it in a 37°C shaking table for culture. After 14 days of culture, take 100 μl of the attenuated Mycobacterium bovis strain stored in this laboratory and spread it on the 7H10 solid culture medium, and place it in a 37°C water-insulated constant temperature incubator for culture for 14-30 days. After the colony is full, it is used for use (the colony morphology is as shown in the attached Figure 1 shown).

[0066] Preparation of 7H9 liquid culture medium (200 ml): 7H9 Agar 0.94 g; sodium pyruvate 0.4 g; Tween 80 100 μl; distilled water 180 mL; autoclave at 121 °C for 15 min, add 20 mL OADC when the temperature cools to 55 °C-60 °C, shake quickly, seal and store at 4 °C away from light.

[0067] Preparation of 7H10 solid culture medium (200ml): 3.8g 7H10 Agar; 0.4g sodium pyruvate; 1mL glycerol; 180mL distilled water. Autoclave at 121℃ for 15min, add 20mL OADC, polymyxin and amphotericin B when the temperature cools to 55℃~60℃, mix quickly, pour into a plate, cool and solidify, seal and store at 4℃.

[0068] (II) Extraction of cell wall of attenuated Mycobacterium bovis strain

[0069] 1. Collect a large number of colonies on 7H10 medium and place them in sterile PBS;

[0070] 2. Centrifuge the collected bacterial solution at 4°C and 11000 rpm for 10 min and collect the precipitate (about 300-500 mg);

[0071] 3. Wash the precipitate three times with PBS and then inactivate it in a 80°C water bath for 30 min;

[0072] 4. The inactivated bacterial precipitate was resuspended in 20 mL of pre-cooled PBS (if there are too many bacterial precipitates, the amount of PBS can be increased), and the resuspended liquid was sonicated in an ice bath (ultrasonication conditions: sonication for 2 seconds, stop for 3 seconds, power of 50%), placed on ice for 15 minutes and then sonicated again until the bacterial solution becomes clear and transparent;

[0073] 5. Centrifuge the ultrasonic product at 4°C, 10,000 rpm for 10 min and collect the supernatant;

[0074] 6. Add nuclease to the supernatant (add 2 μL of 5 KU nuclease to 20 mL supernatant) and incubate at 4°C for 1 hour.

[0075] 7. Add 4 μL of proteinase K (10 mg / mL) and allow to react at room temperature for 15 min;

[0076] 8. Centrifuge the treated supernatant at 4°C, 27,000 g for 1 h. The precipitate is the cell wall. Resuspend it in PBS and store it at -20°C.

[0077] 3. Identification of the cell wall of attenuated Mycobacterium bovis strains

[0078] The extracted cell walls were stained with Periodic Acid-Schiffstain (PAS) to identify whether the extracted cell walls contained polysaccharides and to determine the polysaccharide concentration in the cell walls.

[0079] 1.PAS staining method is as follows:

[0080] (1) Place the protein gel after electrophoresis into the pre-prepared destaining solution for overnight destaining;

[0081] (2) Discard the decolorizing solution, add 7% acetic acid solution, and equilibrate at 4°C for 1 h;

[0082] (3) Discard the equilibrated solution, add 7% acetic acid solution containing 1% sodium periodate, and incubate at 4°C in the dark for 1 h;

[0083] (4) Discard the liquid, wash with deionized water for 5 minutes, then add Schiff reagent and incubate in the dark to complete color development. Check every 5 minutes to avoid overexposure.

[0084] (5) The results of cell wall PAS staining are shown in the attached figure. Figure 2 shown.

[0085] 2. The polysaccharide concentration was determined using the phenol-sulfuric acid method. The phenol-sulfuric acid method is as follows:

[0086] (1) Prepare glucose standard 0.1 mg / mL;

[0087] (2) Prepare the color developing solution: Add concentrated sulfuric acid to 5% phenol solution on ice.

[0088] (3) Prepare a standard curve: aspirate 0 μL, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, and 60 μL of 0.1 mg / mL glucose solution, respectively, and then make up to 100 μL each with ultrapure water.

[0089] (4) Add 300 μL of the prepared colorimetric solution to each of the standard and sample and mix thoroughly by pipetting;

[0090] (5) Metal bath cooking: 100°C, 25 min;

[0091] (6) Ice bath: Place the cooked sample on ice to cool down;

[0092] (7) Reading: Use an ELISA reader to read the OD 490 The concentration of polysaccharides in the extracted cell wall fraction was then determined using the phenol-sulfuric acid method, and the OD of the cell wall polysaccharide was 0.445.

[0093] (8) Then substitute the standard curve linear equation y = 0.5104x-0.0288, R2 = 0.9815 (see Appendix Figure 3 As shown), after calculation, the polysaccharide concentration of the cell wall was 2 mg / mL.

[0094] (IV) Preparation of cell wall and manganese adjuvant subunit vaccines

[0095] 1. Preparation of CW-MnJ(α): Mix the cell wall containing 10μg polysaccharide with 10μg (100μL) MnJ(α) adjuvant and mix or emulsify (for best effect) for 5 minutes, then immunize mice by intranasal drip. The mixture of antigen and adjuvant must be prepared and used immediately, and the best effect is to complete the immunization within 30 minutes. MnJ(α) manganese adjuvant is an oil-in-water adjuvant with a manganese content of 100μg / mL. The core principle is that Mn 2+ Activate the cGAS-STING signaling pathway, efficiently and rapidly induce dendritic cells to mature and present antigens, thereby inducing adaptive immune responses and generating immune memory.

[0096] 2. Preparation of CW-MnJ(β): According to the ratio of antigen to adjuvant = 1:5, take 10μg of polysaccharide cell wall and 50μg of 5mg / mL MnJ(β) adjuvant and mix them evenly on a vortexer. Let it stand at room temperature for 3 minutes. If there is no stratification, the mice can be immunized by intranasal drops. MnJ(β) colloidal manganese adjuvant is a manganese nanoparticle aqueous adjuvant with a manganese content of 5mg / mL. Its core principle is that Mn 2+ Activate the cGAS-STING signaling pathway, efficiently and rapidly induce dendritic cells to mature and present antigens, thereby inducing adaptive immune responses and generating immune memory.

[0097] (V) Immunization of mice with subunit vaccine by intranasal drops

[0098] The present invention also sets up a control group with Mycobacterium tuberculosis Ag85A as an immunogen, and chitosan nanoparticles (CSNPs) as an adjuvant as a control. Therefore, the experimental grouping of the present invention is divided into seven groups, namely: Ag85A-CW-CSNPs, Ag85A-CSNPs, Ag85A-MnJα, Ag85A-MnJβ, CW-CSNPs, CW-MnJα and CW-MnJβ; PBS is used as a negative control group, and BCG is used as a positive control group. A total of 9 groups, each with 12 mice, are grouped as shown in the attached Figure 4 As shown in A.

[0099] The prepared vaccine was administered by intranasal immunization. The mouse immunization procedure is as shown in the attached Figure 4 As shown in B. The PBS immunization dose is 20 μL, and the BCG immunization dose is 5×10 6 CFU, the immunization dose of the seven test groups was 20 μg / mouse. The mice were weighed and immunized at 0, 2 and 4 weeks, and weighed and blood was collected at 9 weeks. Six mice were killed in each group, and the gross lesions of their lungs and spleens were observed (see Appendix). Figure 5 Next, the remaining 6 mice in each group were transferred to the IVC cages in the animal biosafety level 3 laboratory and intranasally infected with a virulent strain of Mycobacterium bovis (clinical isolates preserved in this laboratory).

[0100] The process of intranasal immunization of mice: Before intranasal drops, the mice were anesthetized with intraperitoneal injection of Zotai 50. After anesthesia, the mice were immunized with intranasal drops using a 100μL pipette. The intranasal drop process needed to be carried out slowly in coordination with the breathing of the mice. After confirming that the mouse had inhaled, another drop was dripped to ensure that the antigen for intranasal immunization was effectively inhaled. (VI) Mouse challenge and protection experiment

[0101] 1. Establishment of mouse infection model

[0102] (1) 54 immunized C57BL / 6 mice were transferred into IVC cages in an animal biosafety level 3 laboratory;

[0103] (2) After anesthesia with Zota 50, mice in nine groups were infected nasally with Mycobacterium bovis (200 CFU);

[0104] (3) 24 h after infection with Mycobacterium bovis, three infected mice were randomly killed, the mouse lungs were homogenized, the homogenized tissues were graded diluted, and the plates were plated on 7H10 agar plates. After about two weeks, the CFU were counted to confirm the actual infection dose;

[0105] (4) After infection, mice in each group were weighed weekly;

[0106] (5) Blood was collected and mice were killed on the 28th day after infection to observe the gross organ lesions of mice in each group. Lungs, spleen and other organs were collected aseptically. The lungs and spleen were weighed and photographed. The left lobe of the lung, 1 / 2 of the spleen, part of the liver and other organs were fixed with 10% neutral formalin solution for subsequent histopathological observation; unfixed organs were frozen in time for subsequent experiments;

[0107] (6) All the above operations were carried out in an ABSL-3 level laboratory.

[0108] (7) The gross lesions of the lungs and spleens of the 9 groups of mice after infection are shown in the attached figure. Figure 6 shown.

[0109] 2. Detection of bacterial load (CFU) in mouse lungs and spleen

[0110] (1) Aseptically collect the lungs and spleen, weigh the right upper lobe of the lung and spleen (30 mg), and place them in a sterile 2 mL homogenate tube (containing 2-3 sterile small magnetic beads);

[0111] (2) Place the homogenization tube into the tissue homogenizer and operate according to the instrument instructions. Homogenize for a total of 5 minutes, with the homogenization program running for 60 seconds and stopping for 60 seconds;

[0112] (3) After homogenization, remove the homogenization tube and observe whether the lung and spleen tissues are thoroughly homogenized (generally, the homogenization time is set to 5 minutes according to the program);

[0113] (4) In the biosafety cabinet of the BSL-3 laboratory, the tissue homogenate was diluted with sterile PBS in a continuous gradient. 3 Times and 10 4 The diluted solution was spread on the 7H10 solid culture plate containing OADC, and each concentration gradient was repeated 3 plates; Note: The gradient dilution process is best to use a 1mL sterile syringe to blow and aspirate 10-15 times to blow away the bacterial clumps in the lung tissue homogenate;

[0114] (5) Seal the 7H10 plate coated with lung tissue homogenate dilution with sealing film and put it into No. 9 ziplock bags according to the groups. Mark the group, date and dilution multiple, seal it and place it in a 37°C constant temperature bacterial incubator for culture;

[0115] (6) After 2-3 weeks of culture, the number of colonies was counted. The bacterial load in the lungs and spleen was as shown in the attached Figure 7 As shown;

[0116] (7) All the above operations were carried out in an ABSL-3 level laboratory.

[0117] like Figure 8 As shown, the method for preparing a novel tuberculosis subunit vaccine and evaluating the immune effect on mice provided by the embodiment of the present invention comprises:

[0118] S1, culture of attenuated strain of Mycobacterium bovis;

[0119] S2, extraction of cell wall of attenuated strain of Mycobacterium bovis;

[0120] S3, Characterization of the cell wall of attenuated strains of Mycobacterium bovis;

[0121] S4, preparation of cell wall and manganese adjuvanted subunit vaccines;

[0122] S5, mice immunized with subunit vaccine by intranasal drip;

[0123] S6, mouse challenge and protection experiment.

[0124] The preparation method of this new tuberculosis subunit vaccine uses cell wall components of attenuated Mycobacterium bovis strains and combines them with manganese adjuvants to prepare subunit vaccines to stimulate host immune responses and provide protection against tuberculosis infection. First, the required bacteria are obtained by culturing the attenuated Mycobacterium bovis strain. This step is completed on a special 7H10 solid culture medium to ensure that the strain can grow for 14-30 days in a stable 37°C environment to produce a large number of bacteria for subsequent extraction. This specific culture condition ensures the growth activity of the strain, so that the cell wall components finally extracted maintain effective immune activity.

[0125] Secondly, the cell wall extraction step uses a combination of physical and chemical methods to purify the immune active ingredients to the maximum extent. After collecting the bacteria with PBS solution under sterile conditions, the bacteria are inactivated at high temperature to ensure their biosafety, and then subjected to ice bath ultrasonic treatment to break up the bacterial structure and release internal components. The extraction process includes a series of centrifugation, nuclease and protease steps to remove other impurities and finally obtain purified cell wall components. These components are recognized as antigens in the immune response and can effectively stimulate the host's immune system.

[0126] Next, the extracted cell wall was combined with a manganese adjuvant to form a subunit vaccine. Manganese adjuvants can enhance the immunogenicity of vaccines as immune adjuvants. Their mechanism of action is to activate the host's innate immune cells, thereby enhancing the recognition and response to antigens. Combining cell wall components with manganese adjuvants can form a stable subunit vaccine formula, which is immunized into mice by nasal drops to locally stimulate the respiratory immune system and simulate the natural transmission route of tuberculosis infection.

[0127] Finally, in order to evaluate the effectiveness of the vaccine, a mouse challenge and protection experiment was conducted to observe the immune protection effect of mice after vaccination. The protective effect of the vaccine was verified by detecting the immune response indicators and the number of infected pathogens in the mice. The mouse challenge experiment provides an evaluation of the protective efficacy of the vaccine under actual infection conditions, providing data support for the subsequent improvement and application of the vaccine.

[0128] In order to prove the creativity and technical value of the technical solution of the present invention, this section provides application examples of the claimed technical solution on specific products or related technologies.

[0129] Compared with the approved BCG, the novel subunit vaccines (CW-MnJ (α) and CW-MnJ (β)) prepared by the present invention achieved the same immune protection as BCG after intranasal immunization of mice, indicating that the tuberculosis subunit vaccine can greatly enhance the body's ability to resist Mycobacterium bovis and can effectively prevent and treat tuberculosis.

[0130] The following is an explanation of the evidence related to the technical effects obtained by the embodiments of the present invention.

[0131] 1. This invention is the first to identify the cell wall of extracted Mycobacterium bovis and accurately detect the polysaccharide concentration

[0132] The cell wall components of Mycobacterium bovis obtained in the present invention were identified by PAS staining. The PAS staining results showed a diffuse band at 35 kDa, which was consistent with the size of ManLAM in the cell wall (see attached Figure 2 Further, the present invention uses the phenol-sulfuric acid method to determine the concentration of cell wall polysaccharides, and the results show that the concentration of cell wall polysaccharides is 2 mg / mL (as shown in the attached Figure 3 As shown), the above technical effects prove the stability of the polysaccharide content in the vaccine components used and ensure the reliability of the immune effect.

[0133] 2. No obvious gross lesions were observed in the organs of mice after intranasal immunization with the novel subunit vaccine prepared by the present invention

[0134] After 9 weeks of intranasal immunization, 6 mice in each group were autopsied and their lung and spleen lesions were observed. The results of the observation of gross lesions showed that after intranasal immunization with the subunit vaccines CW-MnJ (α) and CW-MnJ (β) prepared by the present invention, no obvious lesions were observed in the lungs and spleens of the mice compared with the PBS group (such as the attached Figure 5 As shown), this indicates that the CW-MnJ (α) and CW-MnJ (β) subunit vaccines prepared by the present invention are safe after nasal immunization and will not cause damage to the lungs and spleen.

[0135] 3. The bacterial load of mouse organs significantly decreased after immunization with the novel subunit vaccine prepared by the present invention

[0136] Nine weeks after immunization with the CW-MnJ (α) and CW-MnJ (β) subunit vaccines prepared by the present invention, the mice were transferred to IVC cages in an animal biosafety level 3 laboratory; after anesthesia with Shutai 50, the 9 groups of mice were infected with Mycobacterium bovis (200 CFU) by intranasal drops; on the 28th day after infection, the mice were killed, and the lungs and spleens were collected for organ bacterial load detection. The gross lesions of the lungs and spleens of the 9 groups of mice after infection are shown in the attached figure. Figure 6 The results of organ bacterial load detection showed that the bacterial load in the lungs and spleens of mice immunized with CW-MnJ(α) and CW-MnJ(β) was significantly lower than that of the control group (PBS group), among which the bacterial load in the lungs was not significantly different from that of the BCG immunization group. It is worth noting that the bacterial load in the spleens of mice immunized with CW-MnJ(α) was significantly reduced compared with that of the BCG immunization group (see attached Figure 7 This indicates that both CW-MnJ (α) and CW-MnJ (β) subunit vaccines have very good immune protection effects, especially the immune protection effect of CW-MnJ (α) in the spleen is even better than that of BCG.

[0137] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a novel tuberculosis subunit vaccine, characterized in that: The following steps are involved: S1, culture the attenuated strain of Mycobacterium bovis to obtain the bacteria; S2, extraction of cell wall components of attenuated strains of Mycobacterium bovis; S3, identifying whether the extracted cell wall contains polysaccharides and determining their concentration; S4, cell wall components were formulated into subunit vaccines using manganese adjuvants; S5, mice were immunized with subunit vaccine by intranasal administration; S6, challenge and protection experiments were performed on immunized mice to evaluate the immune effect of the vaccine.

2. The method according to claim 1, characterized in that The S1 step specifically includes: Take 100 μl of the attenuated strain of Mycobacterium bovis preserved in the laboratory and spread it on 7H10 solid medium; The coated culture medium was placed in a 37°C water-tight constant temperature incubator and cultured for 14-30 days to obtain an appropriate amount of bacteria.

3. The method according to claim 1, characterized in that The S4 step includes the preparation of the following two subunit vaccines: CW-MnJ(α): Mix 10 μg of polysaccharide-containing cell wall components with 10 μg (100 μL) of MnJ(α) adjuvant and emulsify for 5 minutes for mouse immunization. The antigen and adjuvant must be prepared and used immediately. CW-MnJ(β): Mix 10 μg of cell wall components with polysaccharide content and 50 μg of MnJ(β) adjuvant with a concentration of 5 mg / mL at a ratio of 1:

5. Mix well on a vortexer and let stand for 3 minutes. If no stratification is performed, it can be used for immunization.

4. The method according to claim 1, characterized in that The S6 step specifically includes: Mice immunized three times were infected intranasally with a virulent strain of Mycobacterium bovis; The mice were autopsied 4 weeks after infection, and lung and spleen samples were collected to detect bacterial loads to evaluate the protective effect of the vaccine.

5. A method for evaluating the immune effect of a novel tuberculosis subunit vaccine on mice, characterized in that: The following steps are involved: The prepared subunit vaccine was used to immunize mice by intranasal drops; After immunization, a challenge experiment was performed to infect mice to evaluate the protective effect of the vaccine; Lung and spleen tissue samples were collected from mice at set times after infection; The bacterial load in the lungs and spleen was measured and the immune effect was analyzed.

6. The method according to claim 1, characterized in that The mouse immunization procedure includes three nasal drop immunizations, with one week between each immunization to enhance the mouse's specific immune response and ensure the effectiveness of the protective effect.

7. The method according to claim 1, characterized in that The challenge experiment specifically involved infecting mice that had been immunized three times with a virulent strain of Mycobacterium bovis through nasal drops, and dissecting the mice 4 weeks after infection to collect lung and spleen samples for bacterial load testing.

8. The method according to claim 1, characterized in that: The bacterial load in the lungs and spleen of mice is quantitatively analyzed through the data acquisition and analysis system to generate an immune effect evaluation report to facilitate scientific verification and comparative analysis of the protective effect of the vaccine.

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