Preparation of novel tuberculosis nanoparticle vaccine Rv1794+mi3 as well as adjuvant screening and application of novel tuberculosis nanoparticle vaccine Rv1794+mi3

Abstract

The invention relates to the technical field of preparation of tuberculosis vaccines, in particular to preparation of a novel tuberculosis nanoparticle vaccine Rv1794 + mi3 as well as adjuvant screening and application of the novel tuberculosis nanoparticle vaccine Rv1794 + mi3. After a mouse is immunized by the Rv1794 + mi3, the immune response reaction of H37Rv infected cells and the anti-tubercle bacillus H37Rv infection capability are stronger than those of a single antigen Rv1794 immunization; the vaccine Rv1794 + mi3 + AS01E is used for immunizing mice to induce immune response to H37Rv infected cells, and the anti-tubercle bacillus H37Rv infection capability is higher than that of vaccines Rv1794 + mi3, Rv1794 + mi3 + AS04D and Rv1794 + mi3 + MF59; the adjuvant nanoparticle vaccine Rv1794 + mi3 + AS01E is used as a BCG booster vaccine, and the short-term cellular immune response reaction induced by BCG immunity and the anti-tubercle bacillus H37Rv infection capability are synergistically increased.

Description

Technical Field

[0001] This invention relates to the field of tuberculosis vaccine preparation technology, and in particular to the preparation of a novel tuberculosis nanoparticle vaccine Rv1794+mi3 and its adjuvant screening and application. Background Technology

[0002] Although significant progress has been made in tuberculosis (TB) vaccine development over the past decade, TB has returned to being the leading cause of death from a single infectious disease globally. BCG is currently the only licensed TB vaccine, making outstanding contributions to controlling TB in children, but it does not provide effective protection for adults, highlighting the urgent need for new and effective vaccines to control TB. Screening for effective immunodominant antigens is crucial for TB vaccine research. We discovered that antigen Rv1794 is an ESX-5 type VII secretion system (T7SS) protein, a predominantly expressed antigen during the Mtb resuscitation phase, and is not currently used in TB vaccine development. Therefore, through analysis of its MHC-restricted epitopes using authoritative tools such as IEDB, and prediction of epitope immunogenicity using VaxiJen v2.0, Rv1794 was identified as a promising TB candidate antigen.

[0003] While recombinant protein subunit vaccines offer numerous advantages, their relatively weak immunogenicity makes it difficult to induce a sufficient immune response. Therefore, the selection of adjuvants and delivery display systems is crucial for vaccine research. Among adjuvants authorized for human use, we found AS01 to be unique among novel adjuvants. Composed of QS21, liposomes, and the TLR4 agonist monophospholipid A (MPL), AS01's advantage lies in its ability to induce a novel immune pathway associated with IFN-γ. Several hours after immunization with the AS01 adjuvanted vaccine, the synergistic effect of MPL and QS-21 mediated by macrophages triggers rapid IFN-γ production by cells in lymph nodes. The AS01 adjuvanted vaccine is crucial for inducing Th1-type functional immunity. AS04 is the first approved human vaccine to combine a TLR agonist with an aluminum adjuvant. AS04 is a complex adjuvant consisting of MPL adsorbed onto an aluminum adjuvant. When a vaccine containing AS04 is injected into the human body, AS04 transiently induces local NF-κB activity and cytokine production. Through TLR4 signaling, it stimulates dendritic cells and monocytes. Activated dendritic cells infiltrate draining lymph nodes, producing cytokines to activate antigen-specific T cells, thereby inducing a T cell immune response. AS04 also increases the frequency of memory B cell production and releases high levels of antibodies. MF59 adjuvant is a widely used oil-in-water adjuvant, primarily used in various subunit vaccines, such as hepatitis B and HIV vaccines. MF59 adjuvant can enhance the immunogenicity and protective effect of vaccines, increasing the immune response rate and titer.

[0004] Nanoparticle-based formulations have proven to be a promising approach for enhancing antigen immunogenicity, with virus-like particles (VLPs) currently being the only approved type of NP for human vaccines. Current research on VLPs used in vaccines shows that mi3, a porous dodecahedral 60-mer particle, exhibits excellent structural stability. The multimeric nature of mi3 VLPs provides dual antigen and adjuvant-like properties, inducing humoral and cellular immune responses, which are crucial for antiviral protection. To overcome the problem of decreased expression levels caused by direct fusion of antigens to the N / C-terminus of VLPs, the SpyTag / SpyCatcher system is often used for antigen display. This method offers faster protein purification and macromolecular assembly. SpyCatcher and SpyTag can rapidly and irreversibly form heteropeptide covalent bonds in vitro, enabling the controlled display of exogenous proteins on nanocages, achieving efficient and specific presentation of exogenous proteins. By fusing SpyCatcher to mi3 VLPs and co-incubating with SpyTag-containing antigens, efficient and controllable antigen display can be achieved, constructing a "plug-and-play" vaccine platform.

[0005] This invention obtains the immunodominant antigen Rv1794, which is displayed on the surface of nanoparticle mi3 using the SpyCatcher / SpyTag system to prepare Rv1794+mi3. It is then fully emulsified with adjuvants AS01E, AS04D and MF59 to prepare vaccines Rv1794+mi3+AS01E, Rv1794+mi3+AS04D and Rv1794+mi3+MF59. This invention demonstrates that mice immunized with Rv1794+mi3 after H37Rv infection exhibit stronger cellular immune responses and greater anti-tuberculosis H37Rv infection capabilities compared to mice immunized with the single antigen Rv1794. Mice immunized with the adjuvant nanoparticle vaccine Rv1794+mi3+AS01E showed stronger cellular immune responses against H37Rv infection and greater anti-tuberculosis H37Rv infection capabilities compared to Rv1794+mi3+AS04D and Rv1794+mi3+MF59. The nanoparticle vaccine Rv1794+mi3+AS01E, when used as a BCG booster vaccine, synergistically enhances the short-term cellular immune response induced by BCG immunization and its anti-tuberculosis H37Rv infection capabilities. This invention shows that the adjuvant nanoparticle vaccine Rv1794+mi3+AS01E has the potential to become a novel candidate TB vaccine and warrants further development and research. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides the preparation, adjuvant screening, and application of a novel tuberculosis nanoparticle vaccine, Rv1794+mi3. Rv1794+mi3 immunization of mice showed a stronger cellular immune response to H37Rv infection and greater anti-tuberculosis H37Rv infection capability compared to immunization with the single antigen Rv1794. Furthermore, the Rv1794+mi3+AS01E vaccine immunization of mice showed a stronger cellular immune response to H37Rv infection and greater anti-tuberculosis H37Rv infection capability compared to vaccines BCG, Rv1794+mi3, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59. The adjuvant nanoparticle vaccine Rv1794+mi3+AS01E was used as a BCG booster vaccine, synergistically increasing the short-term cellular immune response induced by BCG immunization and enhancing the anti-tuberculosis H37Rv infection capability.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing a novel tuberculosis nanoparticle vaccine, Rv1794+mi3, comprising the following steps:

[0009] SpyCatcher-mi3 and Rv1794-SpyTag were self-assembled to obtain a novel tuberculosis nanoparticle vaccine, Rv1794+mi3.

[0010] Preferably, the amino acid sequence of SpyCatcher-mi3 is shown in SEQ ID No. 1;

[0011] The amino acid sequence of the Rv1794-SpyTag is shown in SEQ ID No. 2.

[0012] Preferably, the molar ratio of SpyCatcher-mi3 to Rv1794-SpyTag is 1:2.5.

[0013] Preferably, the self-assembly conditions include a temperature of 25°C and a time of 6 hours.

[0014] This invention provides a novel tuberculosis nanoparticle vaccine, Rv1794+mi3, prepared by the preparation method described above.

[0015] This invention also provides a method for preparing an adjuvant nanoparticle vaccine, comprising the following steps:

[0016] The novel tuberculosis nanoparticle vaccine Rv1794+mi3 described in the above technical solution is fully emulsified with adjuvant to obtain an adjuvant nanoparticle vaccine.

[0017] Preferably, the adjuvant includes AS01E, AS04D, or MF59.

[0018] Preferably, the volume ratio of the tuberculosis nanoparticle vaccine to the adjuvant is 1:1.5.

[0019] The present invention also provides an adjuvant nanoparticle vaccine prepared by the preparation method described in the above technical solution.

[0020] The present invention also provides the application of the novel tuberculosis nanoparticle vaccine Rv1794+mi3 or the adjuvant nanoparticle vaccine described in the above technical solutions in the preparation of anti-tuberculosis drugs.

[0021] The beneficial effects of this invention are:

[0022] The present invention provides a tuberculosis nanoparticle vaccine Rv1794+mi3 and an adjuvant nanoparticle vaccine Rv1794+mi3+AS01E. Results showed that immunization of mice with Rv1794+mi3 induced a stronger cellular immune response to H37Rv infection and enhanced anti-tuberculosis H37Rv infection capabilities compared to immunization with the single antigen Rv1794. Immunization of mice with the Rv1794+mi3+AS01E vaccine induced a stronger cellular immune response to H37Rv infection and enhanced anti-tuberculosis H37Rv infection capabilities compared to vaccines Rv1794+mi3, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59. The adjuvant nanoparticle vaccine Rv1794+mi3+AS01E, when used as a BCG booster vaccine, synergistically increased the short-term cellular immune response induced by BCG immunization and enhanced anti-tuberculosis H37Rv infection capabilities. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0024] Figure 1 Image showing the expression and purification of candidate antigen Rv1794-SpyTag;

[0025] Figure 2 Expression, purification, and TEM images of SpyCatcher-mi3 VLP nanoparticles;

[0026] Figure 3 Figure showing the optimal conditions for the self-assembly of antigen Rv1794-SpyTag and SpyCatcher-mi3.

[0027] Figure 4 The expression levels of various cytokines induced by vaccine immunization of C57BL / 6 mice;

[0028] Figure 5The proportion of INF-γ+ and IL-4+ secreting T cells in lymphocytes after immunization of C57BL / 6 mice with the vaccine;

[0029] Figure 6 To enhance the ability of C57BL / 6 mice immunized with the vaccine to induce lymphocyte proliferation;

[0030] Figure 7 Changes in body weight and survival rate in different groups after C57BL / 6 mice were immunized with the vaccine;

[0031] Figure 8 This is to induce a cellular immune response to H37Rv infection through vaccine immunization;

[0032] Figure 9 The amount of H37Rv bacteria in the lungs of mice immunized with various vaccines was measured. Detailed Implementation

[0033] This invention provides a method for preparing a novel tuberculosis nanoparticle vaccine Rv1794+mi3, comprising the following steps: self-assembling SpyCatcher-mi3 with Rv1794-SpyTag to obtain the novel tuberculosis nanoparticle vaccine Rv1794+mi3.

[0034] In this invention, the amino acid sequence of SpyCatcher-mi3 is shown in SEQ ID No. 1; the amino acid sequence of Rv1794-SpyTag is shown in SEQ ID No. 2.

[0035] SEQ ID No. 1:

[0036] MGMSYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGGSGGSGGSGGSMKMEELFKKHKIVAVLRANSVEEAKK KALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE;

[0037] SEQ ID No. 2:

[0038] MDHHHHHHQQSTRTDITVNVDGFWMLQALLDIRHVAPELRCRPYVSTDSNDWLNEHPGMAVMREQGIVVNDAVNEQVAARMKVLAAPDLEVVALLSRGKLLYGVIDDENQPPGSRDIPDNEFRVVLARRGQHWVSAVRVGNDITVDDVTVSDSASIAALVMDGL ESIHHADPAAINAVNVPMEEMLEATKSWQESGFNVFSGGDLRRMGISAATVAALGQALSDPAAEVAVYARQYRDDAKGPSASVLSLKDGSGGRIALYQQARTAGSGEAWLAICPATPQLVQVGVKTVLDTLPYGEWKTHSRVGGGGSGGGAHIVMVDAYKPTK.

[0039] In this invention, the preferred molar ratio of SpyCatcher-mi3 to Rv1794-SpyTag is 1:2.5. In this invention, the preferred conditions for self-assembly include a temperature of 25°C and a time of 6 hours. In this invention, the self-assembly is preferably carried out in a neutral solution.

[0040] The present invention also provides a novel tuberculosis nanoparticle vaccine Rv1794+mi3 prepared by the preparation method described in the above technical solution.

[0041] This invention also provides the application of the novel tuberculosis nanoparticle vaccine Rv1794+mi3 described above in the preparation of anti-tuberculosis drugs.

[0042] This invention also provides a method for preparing an adjuvanted nanoparticle vaccine, comprising the following steps: fully emulsifying the novel tuberculosis nanoparticle vaccine Rv1794+mi3 described in the above technical solution with an adjuvant to obtain the adjuvanted nanoparticle vaccine. In this invention, the adjuvant preferably includes AS01E, AS04D, or MF59 (all purchased from Shanghai CanSino SPH Co., Ltd.). In this invention, the volume ratio of the novel tuberculosis nanoparticle vaccine Rv1794+mi3 to the adjuvant is preferably 1:1.5. This invention does not have a special limitation on the source of the adjuvant; conventional commercially available adjuvants are acceptable. This invention does not have a special limitation on the emulsification conditions; conventional methods are acceptable to those skilled in the art.

[0043] The present invention also provides an adjuvant nanoparticle vaccine prepared by the preparation method described in the above technical solution.

[0044] The present invention also provides the application of the adjuvant nanoparticle vaccine described in the above technical solution in the preparation of anti-tuberculosis drugs.

[0045] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] Screening and purification of Mtb candidate antigen Rv1794

[0048] (1) In this embodiment, the Rv1794 information was obtained using the online tool mycobrowser. It was found that Rv1794 is an important Mtb ESX-5 type VII secretion system (T7SS) antigen protein and also the dominant antigen during the Mtb resuscitation period. Due to the key role of T7SS / ESX in host-pathogen interaction and its involvement in the basic biological processes of Mycobacterium tuberculosis, T7SS / ESX is expected to become a target for novel anti-tuberculosis drugs. This invention also found that Rv1794 has not yet been used in vaccine research, so Rv1794 was identified as a candidate antigen for research.

[0049] (2) The amino acid sequence of Rv1794 and SpyTag was linked by the flexible linker GGGG SGGG. The sequence was inserted into the plasmid pCZN1(NdeI-XbaI) for codon optimization and then transformed into expression bacteria BL21(DE3) for expression. After IPTG-induced protein expression, 10% SDS-PAGE analysis revealed that the target protein was present in the lysate. After lysing the lysate, the sample was collected and eluted at 60mM, 300mM, and 500mM. The samples were then combined and concentrated through a molecular sieve pg75 to obtain the target protein Rv1794-SpyTag. According to the SDS-PAGE and Western Blot analysis, the molecular weight of the protein was 35.4KD, consistent with the theoretical molecular weight. Through purification and expression, the protein that met the requirements was obtained. Figure 1 ).

[0050] Example 2

[0051] Expression, purification, and TEM characterization of SpyCatcher-mi3 nanoparticles

[0052] In this embodiment, the amino acid sequence of SpyCatcher-mi3 was obtained through NCBI, and after codon optimization, the sequence was inserted into the pET28a(+) plasmid (NcoI and XhoI sites). The sequence was then transformed into *E. coli* cells (BL21(DE3)) in vitro. SpyCatcher-mi3 viral particle protein with a size of 38.4 kDa was obtained after in vitro IPTG-induced expression and purification. The SpyCatcher-mi3 concentration was diluted to 0.2 mg / mL, placed on a 200-mesh copper grid, stained with 2% uranyl acetate, and air-dried before morphological observation by transmission electron microscopy (FE-TEM). TEM results showed that SpyCatcher-mi3 has a pentagonal structure, consistent with the expected dodecahedral structure, indicating successful synthesis of SpyCatcher-mi3 viral particles. Figure 2 ).

[0053] Example 3

[0054] Construction of nanoparticle vaccines Rv1794+mi3 and adjuvant vaccines

[0055] (1) SpyCatcher-mi3 and Rv1794-SpyTag proteins were mixed in neutral PBS solution and self-assembled at different temperatures (4℃, 25℃, and 37℃), times (5 min-72 h), and molar ratios. The mixtures were then subjected to 10% SDS-PAGE gel electrophoresis, stained with Coomassie Brilliant Blue (P0017F, Beyotime, Shanghai, China), washed, and photographed. Protein purity and coupling efficiency were analyzed using Image Lab software. The study determined that SpyCatcher-mi3 and Rv1794-SpyTag achieved the highest self-assembly efficiency at 25℃ for 6 h in neutral solution. Figure 3 The results showed that the mass ratio of Rv1794-SpyTag to SpyCatcher-mi3 self-assemblies was 1:2.5, and the assembly efficiency of the product Rv1794+mi3 reached over 75%. Figure 3 ), then use The Ultra50K ultrafiltration tubes screen out unassembled Rv1794-SpyTag and SpyCatcher-mi3.

[0056] (2) Rv1794+mi3 was fully emulsified with AS01E, AS04D and MF59 (all purchased from CanSino SPH, Shanghai, China) at a volume ratio of 1:1.5 to produce adjuvant nanoparticle vaccines Rv1794+mi3+AS01E, Rv1794+mi3+AS04D and Rv1794+mi3+MF59.

[0057] Example 4

[0058] Investigation of cellular immunogenicity of various vaccines after immunization of C57BL / 6 mice

[0059] (1) C57BL / 6 vaccine immunization regimen

[0060] The study consisted of six groups (PBS, BCG, Rv1794, Rv1794+mi3, Rv1794+mi3+AS01E, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59), with six C57BL / 6 mice in each group. Rv1794 and Rv1794+mi3 were administered subcutaneously to the groin of each mouse at 10 μg / mouse. For the adjuvant vaccines Rv1794+mi3+AS01E, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59, each C57BL / 6 mouse received 10 μg of Rv1794+mi3 mixed with adjuvant AS01E (containing 75 μg phospholipids + 18.75 μg cholesterol + 3.75 μg MPL + 3.75 g...). QS-21), AS04D (120 μL solution containing 7.5 μg MPL + 75 μg Aminium hydroxide), and MF59 (containing 3 mg SQ + 0.3 mg Tween + 0.3 mg Span85) were emulsified at a volume ratio of 1:1.5, and 200 μL was administered subcutaneously to the groin. All vaccines were administered three times, two weeks apart, with the last dose being half a dose. BCG vaccine was administered 1×10 6 CFU was administered subcutaneously to the neck and back of each C57BL / 6 mouse; the control group was administered 200 μL of PBS in the same manner; adjuvanted vaccine was combined with BCG immunization, with one week of BCG immunization followed by one week of adjuvanted nanoparticle vaccine immunization. Five weeks after the last immunization, mice were sacrificed by cervical dislocation.

[0061] (2)Acquisition of materials

[0062] Five weeks after the final immunization, blood was collected from the eyeballs, and the animals were euthanized by cervical dislocation. The spleen was aseptically collected and immersed in neutral PBS for the preparation of splenic lymphocyte suspension. The spleen was placed in a 35 mm culture dish covered with a 70 μm cell sieve, and mouse lymphocyte separation medium (7211011, DAKEWE, Shenzhen, China) was added. The spleen was then ground using a 2 mL syringe plug, transferred to a centrifuge tube, covered with RPMI 1640 medium (Sigma, USA), centrifuged, and the lymphocyte layer was aspirated. The cells were washed once with RPMI 1640, centrifuged again to collect the cells, lysed with erythrocyte lysis buffer, washed with PBS, centrifuged again, and the precipitated cells were resuspended in RPMI 1640 medium containing 10% FBS for later use.

[0063] (3) Luminex detection of kinase expression in spleen lymphocytes of antigen-induced immunized mice

[0064] Mouse lymphocyte suspension (2.5×10) 6 100 μL of a mixture of cells / well and antigen protein Rv1794 at a final concentration of 5 μg / mL was added to a 96-well plate. The plates were incubated at 37°C for 48 h in a CO2 incubator, and the cell supernatant was collected. Cells were then analyzed using Luminex cytokine assays and RayPlex assays. TM The Mouse Inflammation Array 1 kit (FAM-INF-1-96, RayBio, Guangzhou, China) was used for the experiment. In short, 25 μL of RayPlex Multiplex Bead Cocktail was added to each of the following 96-well V-bottom microplates containing 25 μL of LNTRL, Standard, or cell supernatant. The plates were incubated at room temperature for 2 h. The microspheres were washed with 1× Wash Buffer. 25 μL of 1× Biotinylated Detection Antibody Cocktail was added to each well to resuspend the microspheres. The plates were incubated at room temperature for 1 h, washed twice, and then 50 μL of 1× Streptavidin-PE was added to each well. The plates were incubated at room temperature for 30 min, washed once, and then resuspended in 150 μL of 1× Wash Buffer for flow cytometry analysis. Figure 4 Compared with PBS, immunization with Rv1794, Rv1794+mi3, Rv1794+mi3+AS01E, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59 induced significantly high levels of expression of Th1 cytokines (IFN-γ and TNF-α), Th2 cytokines (IL-4), and Th17 cytokines (IL-17). Immunization with the nanoparticle vaccine Rv1794+mi3 induced significant expression of Th1 and Th2 cytokines. The expression of Th1, Th2, and Th17 cytokines induced by the adjuvanted vaccine Rv1794+mi3+AS01E was significantly higher than that induced by BCG, Rv1794+mi3, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59. Simultaneously, the expression of Th1, Th2, and Th17 cytokines induced by the adjuvanted nanoparticle vaccine Rv1794+mi3+AS01E combined with BCG was significantly higher than that induced by BCG alone. Figure 4 Therefore, the Rv1794+mi3 nanoparticle vaccine immunized mice produced strong Th1, Th2 and Th17 cellular immune responses. When combined with the adjuvant AS01E, it induced even better cellular immunogenicity. The adjuvant nanoparticle vaccine Rv1794+mi3+AS01E can be used as a booster vaccine for BCG to synergistically enhance the BCG-induced cellular immune response.

[0065] (4) ELISPOT detection of IFN-γ in antigen-induced immunized mice + / IL-4 + T lymphocyte ratio

[0066] FluoroSpot plates were cleaned, blocked, and regulated according to the ELISpot Plus: Mouse IFN-γ (ALP) (3321-4AST-2, Mabtech, Sweden) and ELISpot Plus: Mouse IL-4 (ALP) (3311-4APW-2, Mabtech, Sweden) kit instructions. Then, mouse lymphocyte suspension (2.5 × 10⁻⁶) was added. 5 A mixture of 100 μL of Rv1794 antigen (cell / well) and a final concentration of 5 μg / mL was added to the well plate. PMA (5 μg / mL) and RPMI 1640 complete medium were used as positive and negative control stimuli, respectively. After incubation at 37°C for 24 h in a CO2 incubator, the experiment was conducted according to the manufacturer's instructions. After color development, the number of analytical spots was recorded using the ImmunoSpot software of the CTL-ImmunoSpot enzyme-linked immunofluorescence spot analyzer. Figure 5 Compared with PBS, immunization of mice with Rv1794, Rv1794+mi3, Rv1794+mi3+AS01E, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59 all induced significant IFN-γ. + T lymphocytes and IL-4 + T lymphocytes; nanoparticle vaccine Rv1794+mi3 immunization against IFN-γ + T lymphocytes and IL-4 + T lymphocytes were significantly higher than those immunized with Rv1794, and the adjuvant vaccine Rv1794+mi3+AS01E induced IFN-γ. + T lymphocytes and IL-4 + T lymphocytes were significantly higher than those immunized by BCG, Rv1794+mi3, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59; simultaneously, the adjuvant nanoparticle vaccine Rv1794+mi3+AS01E combined with BCG-induced IFN-γ + T lymphocytes and IL-4 + T lymphocytes were significantly higher than those immunized with BCG alone. Figure 5 Therefore, this further proves the conclusion of (3) above.

[0067] Example 5

[0068] Investigation on lymphocyte proliferation capacity, body weight and survival rate in mice immunized with various vaccines

[0069] (1) Changes in antigen-induced lymphocyte proliferation capacity after mice were immunized with various vaccines

[0070] Splenic lymphocytes were isolated from C57BL / 6 mice immunized with the same vaccine as in Example 4, and their proliferation was detected by the MTT assay: 3 × 10⁻⁶ cells were collected. 5 Splenic lymphocytes were seeded into 96-well plates, and Rv1794 antigen was added to a final concentration of 5 μg / mL. 100 μL of RPMI 1640 complete medium was added to the negative control wells, and blank control wells were set up (medium only). The plates were incubated at 37°C for 42 h in a 5% CO2 incubator. 20 μL of 5 mg / mL LMTT (M1025, Solarbio, Beijing, China) was added to each well, and the plates were incubated for another 4 h. The liquid in the wells was removed, and 100 μL of DMSO was added to each well. The plates were shaken and mixed for 10 min. The OD490 value was measured, and the spleen cell stimulation index (SI) was calculated as: SI = OD490(OD490 of the stimulation group - OD490 of the blank control) / (OD490 of the negative control - OD490 of the blank control). Figure 6 Compared with PBS, immunization of mice with Rv1794, Rv1794+mi3, Rv1794+mi3+AS01E, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59 all showed significant lymphocyte proliferation capacity. The lymphocyte proliferation capacity of the nanoparticle vaccine Rv1794+mi3 immunization was significantly higher than that of Rv1794 immunization, and the lymphocyte proliferation capacity of the adjuvant vaccine Rv1794+mi3+AS01E immunization was significantly higher than that of BCG, Rv1794+mi3, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59 immunization. Furthermore, the lymphocyte proliferation capacity of the adjuvant nanoparticle vaccine Rv1794+mi3+AS01E combined with BCG immunization was significantly higher than that of BCG alone immunization. Figure 6 ).

[0071] (2) Mouse weight and survival rate after immunization with each vaccine

[0072] C57BL / 6 mice were immunized with the same vaccine as in Example 4, and mouse weight and survival rate were recorded weekly. Figure 7The results showed that, except for the adjuvant nanoparticle vaccine Rv1794+mi3+AS04D, the body weight of C57BL / 6 mice immunized with other vaccines, including Rv1794, Rv1794+mi3, and Rv1794+mi3+AS01E, and their combination with BCG vaccine, increased steadily and normally, with no mouse deaths. One mouse died during the immunization process with the adjuvant nanoparticle vaccine Rv1794+mi3+AS04D, and its body weight fluctuated significantly. The body weight of mice immunized with the adjuvant nanoparticle vaccine Rv1794+mi3+MF59 was lower than that of the other groups, with a lower increase in body weight. Figure 7 Therefore, adjuvant AS01E is a suitable adjuvant for immunization with the nanoparticle vaccine Rv1794+mi3.

[0073] Example 6

[0074] Investigation on the protective response of mice immunized with various vaccines against H37Rv infection

[0075] C57BL / 6 mice were immunized with the same vaccine as in Example 4. Four weeks after the last dose of vaccine, 300 CFU H37Rv / mouse was used to infect the mice. Four weeks after infection, the mice were sacrificed and spleen lymphocytes were isolated from the same samples as in Example 3.

[0076] (1) Luminex detection of splenic lymphocyte kinase expression in Rv1794 antigen-induced immunized mice

[0077] The splenic lymphocytes of mice challenged with H37Rv were analyzed using the same Luminex assay as in Example 4-(3). The results of cytokine expression detection in immunized mice after H37Rv challenge showed... Figure 4 Compared with PBS, immunization of mice with Rv1794, Rv1794+mi3, Rv1794+mi3+AS01E, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59 induced significantly high levels of expression of Th1 cytokines (IFN-γ), Th2 cytokines (IL-4), and Th17 cytokines (IL-17). Immunization with the nanoparticle vaccine Rv1794+mi3 induced significantly higher levels of Th1 and Th2 cytokines than with Rv1... Immunization with the adjuvanted nanoparticle vaccine Rv1794+mi3+AS01E significantly increased the expression of Th1, Th2, and Th17 cytokines compared to BCG, Rv1794+mi3, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59 immunizations. Simultaneously, the combined immunization with the adjuvanted nanoparticle vaccine Rv1794+mi3+AS01E and BCG significantly increased the expression of Th1, Th2, and Th17 cytokines compared to BCG alone. Figure 8Therefore, the Rv1794+mi3 nanoparticle vaccine immunized mice against H37Rv infection elicited strong Th1, Th2, and Th17 cellular immune responses. Combined with the adjuvant AS01E, it induced even better protective cellular immune responses. The Rv1794+mi3+AS01E adjuvant nanoparticle vaccine, as a BCG booster vaccine, synergistically enhanced the BCG-induced protective cellular immune response.

[0078] Example 7

[0079] Investigation on the ability of mice immunized with various vaccines to resist H37Rv infection

[0080] Following immunization with the vaccine as in Example 6, mice were infected with H37Rv. Four weeks later, the mice were sacrificed, and lung homogenates were collected. 5 mL of PBS was added to the lung tissue, and homogenization was performed at 200 rpm for 7 cycles. 100 μL of the homogenate suspension was used for CFU plating: the homogenate was diluted 1:10 with the original solution and 1:100 with PBS. Then, 100 μL of the diluted solution was taken from each diluted sample and spread onto 7H10 medium containing 10% OADC. The samples were incubated upside down in a bacterial incubator for 2-3 weeks. After incubation, colony counting was performed. Figure 9 The results showed that the H37Rv bacterial load in the lungs of mice immunized with antigen Rv1794 was lower than that of mice immunized with PBS. At the same time, the H37Rv bacterial load in the lungs of mice immunized with nanoparticle vaccine Rv1794+mi3 was significantly lower than that of mice immunized with Rv1794. The H37Rv bacterial load in the lungs of mice immunized with Rv1794+mi3+AS01E was significantly lower than that of mice immunized with Rv1794+mi3, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59. In addition, mice co-immunized with adjuvant AS01E and nanoparticle vaccine Rv1794+mi3 had the same anti-tuberculosis infection ability as those immunized with BCG. Furthermore, Rv1794+mi3+AS01E can also be used as a BCG booster vaccine, significantly increasing the protective immune effect of BCG.

[0081] In this embodiment, the dominant antigen Rv1794 was obtained using online tools such as mycobrowser and IEDB. The antigen Rv1794 was displayed on the surface of nanoparticle mi3 using the SpyCatcher / SpyTag system to prepare nanoparticle vaccines Rv1794+mi3. These vaccines were then fully emulsified with adjuvants AS01E, AS04D, and MF59, respectively, to form adjuvant nanoparticle vaccines Rv1794+mi3+AS01E, Rv1794+mi3+AS04D, and Rv1794+mi3+MF59. After immunization of mice, the nanoparticle vaccine Rv1794+mi3 enhanced the anti-tuberculosis H37Rv infection ability by upregulating Th1, Th2, and Th17 cellular immune responses. Furthermore, co-immunization with adjuvant nanoparticle vaccine Rv1794+mi3 and adjuvant AS01E induced Th1, Th2, and Th17 cellular immune responses and enhanced the anti-tuberculosis H37Rv infection ability more strongly than adjuvants AS04D and MF59 after H37Rv infection. Adjuvant AS01E was determined to be a suitable adjuvant for immunization with nanoparticle vaccine Rv1794+mi3. Moreover, the adjuvant nanoparticle vaccine Rv1794+mi3+AS01E, as a BCG booster vaccine, synergistically increased the short-term protective effect of BCG.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a novel tuberculosis nanoparticle vaccine Rv1794+mi3, characterized in that, Includes the following steps: SpyCatcher-mi3 and Rv1794-SpyTag were self-assembled to obtain a novel tuberculosis nanoparticle vaccine, Rv1794+mi3.

2. The preparation method according to claim 1, characterized in that, The amino acid sequence of SpyCatcher-mi3 is shown in SEQ ID No. 1; The amino acid sequence of the Rv1794-SpyTag is shown in SEQ ID No.

2.

3. The preparation method according to claim 1, characterized in that, The molar ratio of SpyCatcher-mi3 to Rv1794-SpyTag is 1:2.

5.

4. The preparation method according to claim 1, characterized in that, The conditions for self-assembly include: a temperature of 25°C and a time of 6 hours.

5. A novel tuberculosis nanoparticle vaccine Rv1794+mi3 prepared by the preparation method according to any one of claims 1 to 4.

6. A method for preparing an adjuvant nanoparticle vaccine, characterized in that, Includes the following steps: The novel tuberculosis nanoparticle vaccine Rv1794+mi3 described in claim 5 is fully emulsified with an adjuvant to obtain an adjuvant nanoparticle vaccine.

7. The preparation method according to claim 6, characterized in that, The adjuvants include AS01E, AS04D, or MF59.

8. The preparation method according to claim 6, characterized in that, The volume ratio of the novel tuberculosis nanoparticle vaccine Rv1794+mi3 to the adjuvant is 1:1.

5.

9. An adjuvant nanoparticle vaccine prepared by the preparation method according to any one of claims 6 to 8.

10. The use of the novel tuberculosis nanoparticle vaccine Rv1794+mi3 according to claim 5 or the adjuvant nanoparticle vaccine according to claim 9 in the preparation of anti-tuberculosis drugs.

Images