Self-assembled ferritin nanovaccine targeting tuberculosis subunit antigens, preparation method and application thereof

Abstract

The application provides a self-assembled ferritin nanovaccine targeting tuberculosis subunit antigens and a preparation method and application thereof, and belongs to the technical field of biological medicines. The application provides a subunit nanoparticle, selects ESAT-6 (E6), CFP-10 (C10) and ESAT-6-CFP-10 (EC) proteins as antigen target points, takes ferritin as a potential presentation carrier of TB epitope target points, fuses the E6, C10 and EC antigens into the ferritin, successfully self-assembles into a uniform epitope nanovaccine, has better immunogenicity, and can trigger a high-efficiency cellular immune response and immune memory. The application also uses an expression system method to complete expression and production of the subunit nanoparticle, and has economy and is suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a self-assembled ferritin nano-vaccine targeting subunit antigens of tuberculosis and a preparation method and application thereof. BACKGROUND

[0002] Tuberculosis (TB) is a chronic and highly infectious disease caused by Mycobacterium tuberculosis (Mtb) infection, which can be transmitted through the respiratory tract and seriously endanger global human health. In recent years, with the emergence of drug-resistant / multidrug-resistant tuberculosis, human immunodeficiency virus (HIV) infection / acquired immunodeficiency syndrome (AIDS) combined with tuberculosis, and the application of immunosuppressive agents, the goal of "ending the global tuberculosis epidemic" proposed by WHO in the "Ending Tuberculosis Strategy" by 2035 still has a long way to go. As the only vaccine approved for the prevention of tuberculosis, Bacillus Calmette-Guerin (BCG) can effectively prevent tuberculous meningitis and miliary tuberculosis in children. BCG has slowed down the trend of the spread of tuberculosis to some extent, but its preventive effect in adults is not certain and varies greatly (0-80%).

[0003] At present, self-assembled nanoparticles play an important role in vaccine development. Compared with monomeric antigens, the affinity between the surface of nanoparticles and cell membranes helps immune cells to anchor antigens more efficiently, and these nanoparticles can serve as both a vaccine delivery platform and an adjuvant. However, there has been no report on the fusion expression and immunogenicity of TB antigens. SUMMARY

[0004] The present application provides a self-assembled ferritin nano-vaccine targeting subunit antigens of tuberculosis and a preparation method and application thereof, which uses ferritin as a universal platform for TB candidate vaccines to improve the immunogenicity of BCG and existing subunit vaccines and induce strong cellular immunity.

[0005] The present application provides a subunit nanoparticle, which comprises a subunit antigen isolated from tuberculosis and a self-assembled carrier, and the self-assembled carrier comprises ferritin.

[0006] In a preferred mode of the present application, the subunit antigen isolated from tuberculosis comprises any one of the following: ESAT-6, CFP-10, and a fusion protein, wherein the fusion protein is a fusion protein of ESAT-6 and CFP-10.

[0007] The application also provides a construction method of the subunit nanoparticle, comprising the following steps: connecting a subunit antigen separated from tuberculosis and a ferritin through a connecting peptide to construct the subunit nanoparticle.

[0008] In a preferred mode of the application, the ferritin comprises a ferritin derived from Helicobacter pylori.

[0009] The application also provides a recombinant vector for expressing the subunit nanoparticle.

[0010] The application also provides a recombinant engineering cell comprising the recombinant vector and expressing the subunit nanoparticle.

[0011] The application also provides a production method of the subunit nanoparticle, comprising the following steps: inserting a gene encoding the subunit nanoparticle into a basic skeleton vector to obtain a recombinant vector;

[0012] transforming a host cell with the recombinant vector to construct a recombinant engineering cell;

[0013] inducing the recombinant engineering cell to express the subunit nanoparticle.

[0014] In a preferred mode of the application, when the subunit nanoparticle is produced by a prokaryotic expression method, the basic skeleton vector comprises pET-28a(+) and the host cell comprises Escherichia coli.

[0015] The application also provides an application of the subunit nanoparticle or the subunit nanoparticle produced by the production method in preparing a tuberculosis nanoparticle vaccine.

[0016] The application also provides a vaccine for preventing and / or treating tuberculosis, wherein the effective component comprises the subunit nanoparticle or the subunit nanoparticle produced by the production method.

[0017] Beneficial effects: the application provides a subunit nanoparticle, selects ESAT-6 (E6), CFP-10 (C10) and ESAT-6-CFP-10 (EC) proteins derived from a religion of difference (RD) of Mtb as antigen target points, utilizes the high self-assembly capacity of ferritin, and takes ferritin as a potential presentation carrier of TB epitope target points, fuses the E6, C10 and EC antigens into the ferritin, and successfully self-assembles into a uniform epitope nanoparticle vaccine.

[0018] The application also uses the method of expression system to complete the expression and production of the subunit nanoparticles, such as the high-efficiency expression of the subunit nanoparticles by the prokaryotic system in the examples, which is economical and suitable for large-scale production. The subunit nanoparticles of the application have better immunogenicity than the conventional subunit vaccine, and can trigger high-efficiency cellular immune response and immune memory. It is proved that the ferritin of the application can be used as a universal platform of TB candidate vaccine to further improve the immunogenicity of BCG and existing subunit vaccines.

[0019] The application adopts the BCG heterologous enhancement strategy, and subcutaneously injects subunit proteins and nanoparticles. The HE staining results show that the nano-vaccine has good safety. Compared with BCG and traditional subunit vaccines, the spleen cells of the mice immunized with EFCF and ECF show stronger cell proliferation level after being stimulated by antigens. ELISpot and cytokine level show that the nano-vaccine promotes IFN-γ recall reaction and strongly stimulates the secretion of various TB immune-related cytokines including Th1 type cytokines. The results of multicolor flow cytometry detection show that the nano-particle vaccine represented by ECF induces immune memory mainly with IFN-γ + T CM / T EM , CD4 + IL-2 + T EM and CD8 + IL-2 + T CM mainly. In summary, in the context of heterologous enhancement immunization, the mice immunized with EF, CF and ECF show acceptable safety and stronger cellular immune response, induce immune memory mainly with IFN-γ and significantly stimulate the secretion of various TB immune-related cytokines. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a figure of recombinant protein expression and nanoparticle characterization analysis, in which A: design of target protein expression plasmid; B: SDS-PAGE electrophoresis analysis of purified recombinant protein, lane M: protein standard molecular weight; lane 1: ESAT-6; lane 2: CFP-10; lane 3: EC fusion protein; lane 4: Fer; lane 5: ESAT6-Fer; lane 6: CFP10-Fer; lane 7: EC-Fer; C: transmission electron microscope (TEM) observation of nanoparticles; D: dynamic light scattering (DLS) analysis of nanoparticles; E: particle size and molecular weight; F: SDS-PAGE analysis of ferritin and antigen-coupled nanoparticles after 1, 3 and 5 freeze-thaw cycles; G: SDS-PAGE electrophoresis analysis of ferritin and antigen-coupled nanoparticles stored at the specified temperature for 4 weeks;

[0021] Figure 2 Figure 1 is a graph of immunization schedule, immunization dose and lymphocyte proliferation test results, wherein A: immunization schedule; B: immunization dose; C: lymphocyte proliferation test;

[0022] Figure 3 Figure 2 is a graph of the frequency of functional antigen-specific central memory T cells (Tcm) in immunized mice, wherein A: CD4 IFN-γ Tcm; B: CD4 IL-2 Tcm; C: CD8 IFN-γ Tcm; D: CD8 IL-2 Tcm; CM + + CM + + CM + + CM + + CM ;

[0023] Figure 4 Figure 3 is a graph of the frequency of functional antigen-specific effector memory T cells (Tern) in immunized mice, wherein A: CD4 IFN-γ Tern; B: CD4 IL-2 Tern; C: CD8 IFN-γ Tern; D: CD8 IL-2 Tern; EM + + EM + + EM + + EM + + eM ;

[0024] Figure 5 Figure 4 is a graph of the number of spot forming cells (SFC) producing IFN-γ per million cells determined by ELISpot, wherein A: ELISpot detection of IFN-γ secreting spots; B: ELISpot detection statistical analysis of IFN-γ secretion;

[0025] Figure 6 Figure 5 is a graph of the results of tuberculosis disease related cytokine secretion levels, wherein A: IFN-γ secretion expression level; B: TNF-α secretion expression level; C: IL-2 secretion expression level; D: IL-6 secretion expression level; E: IL-10 secretion expression level; F: IL-12 secretion expression level; G: IL-17 secretion expression level; H: IL-23 secretion expression level;

[0026] Figure 7 ​​​​​​​​​​​​​​​​​​​​​​​​Fig. 1 is a diagram of results of transcription levels of tuberculosis-related cytokines, in which A: transcription expression level of IFN-γ; B: transcription expression level of TNF-α; C: transcription expression level of IL-2; D: transcription expression level of IL-17;

[0027] Figure 8 Fig. 2 is a diagram of results of H&E staining. DETAILED DESCRIPTION

[0028] The present application provides a subunit nanoparticle, comprising a subunit antigen isolated from tuberculosis and a self-assembled carrier, wherein the self-assembled carrier comprises a ferritin.

[0029] In one preferred embodiment of the present application, the subunit antigen isolated from tuberculosis comprises any one of ESAT-6 (E6), CFP-10 (C10) and a fusion protein (EC) of ESAT-6 and CFP-10. The ESAT-6 of the present application is a major T cell immunodominant antigen in the RD region purified from the filtrate of short-term culture of Mtb, which is closely related to the increase of virulence of Mtb and mutation, and inhibits the degradation and killing of immune cells to Mtb through various pathways, and promotes the long-term chronic migratory infection of Mtb in vivo. The GenBank of E6 of the present application is YP_178023, and the GenBank of C10 is NP_218391. The fusion protein of E6 and C10 of the present application is connected through a flexible linker, and the connection sequence is N-terminal-E6-linker-C10-C-terminal. In one embodiment, the linker is (G4S)3.

[0030] The ferritin of the present application is a protein nanoparticle platform for antigen presentation and immune stimulation, which is composed of 24 subunits arranged in an octahedral symmetry, can induce high-efficiency immune response, and has significant thermal stability, chemical stability and low heterogeneity. In one preferred embodiment of the present application, the ferritin comprises a ferritin derived from Helicobacter pylori, and the GenBank is NP_223316.

[0031] The present application also provides a construction method of the above-mentioned subunit nanoparticle, comprising the following steps: connecting the subunit antigen isolated from tuberculosis and the ferritin through a connecting peptide to construct the subunit nanoparticle.

[0032] In one embodiment of the present application, ESAT-6, CFP-10 and EC are respectively fused with the ferritin derived from Helicobacter pylori through a flexible linker, wherein the flexible linker can be (G4S)3. In one embodiment of the present application, the subunit antigen is fused to the N-terminal of the ferritin, and then the flexible linker is added to the C-terminal of the ferritin.

[0033] The present application also provides a recombinant vector for expressing the subunit nanoparticle.

[0034] The basic backbone vector of the recombinant vector can be a prokaryotic expression vector, such as pET-28a(+) used in the examples, and can also be a commonly used vector of other expression systems. In one embodiment of the present application, the gene encoding the subunit nanoparticle is connected to the Nco I and Xho I restriction sites of the pET-28a(+).

[0035] The present application also provides a recombinant engineering cell containing the above-mentioned recombinant vector and expressing the above-mentioned subunit nanoparticle.

[0036] In a preferred mode of the present application, prokaryotic bacteria are used as host cells, such as E. coli used as host bacteria in one embodiment, and the above-mentioned recombinant vector is transformed into the host bacteria to obtain recombinant E. coli that can express the subunit nanoparticle.

[0037] The present application also provides a production method of the above-mentioned subunit nanoparticle, which comprises inserting the gene encoding the subunit nanoparticle into a basic backbone vector to obtain a recombinant vector;

[0038] The host cell is transformed with the recombinant vector to construct a recombinant engineering cell;

[0039] The recombinant engineering cell is induced to express the subunit nanoparticle.

[0040] In one preferred mode of the present application, when the subunit nanoparticle is produced by a prokaryotic expression method, the basic backbone vector comprises pET-28a(+) and the host cell comprises E. coli. In the examples of the present application, the prokaryotic expression vector pET-28a(+) containing His tag at the C-terminal is double-digested with restriction enzymes Nco I and Xho I, and the E6, C10 and EC sequences are fused with H. pylori ferritin (GenBank: NP_223316) through GSG linker, respectively. The target sequences and pET-28a(+) are connected with DNA ligase, and are named as ESAT6-Fer (EF), CFP10-Fer (CF) and ESAT6-CFP10-Fer (ECF), respectively.

[0041] The present application also provides the use of the above-mentioned subunit nanoparticle or the subunit nanoparticle produced by the above-mentioned production method in the preparation of a tuberculosis nanoparticle vaccine.

[0042] The application fuses E6, C10 and EC antigens into ferritin, successfully constructs a subunit nanoparticle capable of preparing epitope nanovaccine; in the context of heterologous booster immunization, mice immunized with EF, CF and ECF show acceptable safety and stronger cellular immune response, induce immune memory dominated by IFNTM and significantly stimulate the secretion of various tuberculosis immune-related cytokines.

[0043] The application also provides a vaccine for preventing and / or treating tuberculosis, and the effective component comprises the above-mentioned subunit nanoparticle or the subunit nanoparticle produced by the above-mentioned production method.

[0044] In order to further illustrate the application, the self-assembled ferritin nanovaccine targeting tuberculosis subunit antigens, the preparation method and the application provided by the application are described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the application.

[0045] The materials and reagents used in the examples of the application are all conventional commercially available reagents in the art, such as experimental animals: SPF female C57BL / 6 mice (6-8 weeks old, weighing 18-20 g) are purchased from Jiangsu Jucu Yakang Co., Ltd., and the mice are randomly divided into cages. The mice are placed in separate ventilated cages, and the environmental temperature is 22-25℃, the relative humidity is 45-65%, and the light / dark cycle is 12h / 12h. The animals can freely eat and drink water throughout the experiment. The animal experiments involved in this paper are approved by the Animal Ethics Committee of Bengbu Medical University (Approval No.: 2022127).

[0046] Experimental strains: E. coli Top10 clone strain is used for recombinant plasmid cloning, and BL21(DE3) expression strain is used for protein expression. E. coli is inoculated in LB agar medium containing kanamycin (50 μg / mL, Kan+) and cultured at 37℃ in a 5% CO2 environment.

[0047] Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using Tukey's multiple comparison test in SPSS Version 29.0. Data were visualized in GraphPad Prism software (version 9.0). Each group was compared pairwise. P<0.05 was considered statistically significant (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05).

[0048] Example 1

[0049] 1.1 Plasmid construction

[0050] The ESAT-6, CFP-10, and EC gene sequences of the Mtb H37Rv standard strain (ATCC 27294) were synthesized by Nanjing Zhongding Biotechnology Co., Ltd. The prokaryotic expression vector pET-28a(+) containing a His tag at the C-terminus was double-digested using restriction endonucleases Nco I and Xho I. The ESAT-6, CFP-10, and EC sequences were fused with Helicobacter pylori ferritin (GenBank: NP_223316) via the GSG linker, and the target sequences were ligated to pET-28a(+) using DNA ligase, named ESAT6-Fer(EF), CFP10-Fer(CF), and ESAT6-CFP10-Fer(ECF), respectively.

[0051] This invention fuses E6, C10, or EC fusion proteins to the N-terminus of ferritin using an antigen-ferritin linker (G4S)3, and then adds GSG and a 6×His-tag to the C-terminus of ferritin. A schematic diagram of the expression plasmid construction is shown below. Figure 1 As shown in Figure A. The constructed expression vector was converted into competent cells, induced with IPTG, and then purified using a Ni-NTA column to obtain purified ESAT-6, CFP-10, EC, Fer, ESAT-6-Fer, CFP-10-Fer, and EC-Fer proteins. SDS-PAGE electrophoresis results are shown below. Figure 1 As shown in Figure B, the purified protein band is single and consistent with the predicted target protein size. Figure 1 (E).

[0052] 1.2 Expression and purification of recombinant proteins and nanoparticles

[0053] The successfully constructed plasmid was transformed into competent cells of *E. coli* expression strain BL21(DE3), and evenly spread on Kan+LB plates. The plates were incubated at 37°C for 16 hours. Single-clone positive strains were then selected and cultured in 600 mL of LB liquid medium containing 50 μg / mL kanamycin in a constant-temperature shaker at 37°C until the bacterial culture reached OD500. 600 When the pH reaches 0.6–0.8, 0.2 mM IPTG is added to the culture medium, and expression is induced overnight at 16°C. The induced bacterial culture is collected and sonicated. The supernatant is loaded into a Ni-IDA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-IDA Binding-Buffer. The column is washed with Ni-IDA Binding-Buffer and Ni-IDA Ashing-Buffer (20 mM Tris-HCl, 30 mM imidazole, 0.15 M NaCl, pH 8.0) until the effluent OD... 280Values reached baseline, the target protein was eluted slowly with Ni-IDA Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0), and the effluent was collected. The collected protein solution was added to a dialysis bag and dialyzed against PBS overnight. The protein was collected and the protein concentration was determined by BCA method, and stored at -80 °C for later use.

[0054] 1.3 Identification and characterization of the protein

[0055] Recombinant proteins and nanoparticles were detected by 12% SDS-PAGE and Western-blot to verify the purity and specificity of the proteins. The morphology and size distribution of the self-assembled nanoparticles were observed and detected by TEM and DLS. To determine the temperature / thawing stability of the nanoparticles, each assembled nanoparticle was placed in an environment of -80 °C until the solution was completely frozen, and then taken out to incubate at room temperature until completely thawed. The process was defined as 1 freeze-thaw cycle. After 1, 3 and 5 freeze-thaw cycles, each sample was subjected to SDS-PAGE analysis. To study the storage stability, all self-assembled nanoparticle solutions were stored at -80 °C, -20 °C, 4 °C and 24 °C for 4 weeks, respectively, and whether there was protein degradation was preliminarily evaluated by SDS-PAGE.

[0056] TEM, DLS and SDS-PAGE were used to characterize and verify the nanoparticles (NPs). Transmission electron microscopy observed uniformly dispersed NPs, confirming that the structures of all NPs were similar to the purified ferritin particles, indicating that they could all be self-assembled into ferritin nanoparticles with uniform size. Figure 1 DLS measured the size distribution of the NPs Figure 1 , and the results showed that the size of the antigen-loaded nanoparticles was larger than that of the ferritin monomers Figure 1 .

[0057] The present application performed SDS-PAGE analysis on the proteins under the conditions of multiple freeze-thaw cycles and different temperature storage. The gel results showed that after 1, 3 and 5 freeze-thaw cycles and storage at different temperatures for 4 weeks, the corresponding bands of the nanoparticles remained clear without obvious dispersion, indicating that the proteins were probably not degraded Figure 1 , F and G).

[0058] 1.4 Mouse immunization scheme

[0059] The mice were acclimated for one week before the experiment, and all immunogens were diluted in sterile PBS, keeping the total volume at 200 μL. The mice were divided into eight groups (5 mice per group), and immunized once every two weeks for a total of three times Figure 2 , A). At the beginning of the experiment, the first group of mice (i.e., week 0) was subcutaneously injected with 2 x 106 BCG or self-assembled nanoparticles (SA-NPs) (A) and (B) respectively. Figure 2 BCG. Other mice using the "prime-boost" strategy were subcutaneously inoculated with protein antigens or corresponding self-assembled nanoparticles at weeks 2 and 4 respectively after the prime immunization with BCG.

[0060] 1.5 Lymphocyte proliferation assay

[0061] Forty-two days after the first inoculation, mice were euthanized and spleens were aseptically collected. Spleen lymphocytes were isolated using a mouse spleen lymphocyte isolation kit. Isolated spleen lymphocytes were diluted to 3x10 6 cells / mL with RPMI-1640 medium containing 10% fetal bovine serum (FBS) and seeded into 96-well plates at a concentration of 2x10 5 cells / well. Cells were stimulated with 20 μg / mL of recombinant ESAT-6, CFP-10 and EC fusion proteins respectively, 2 mg / mL ConA and untreated cells (with RPMI-1640 medium containing FBS only) were used as positive and negative controls. Cells were incubated at 37°C in a 5% CO2 incubator for 48 h and then incubated for another 4 h. OD 490 values of the stimulated wells - average OD 490 values of the blank control wells) / (average OD 490 values of the negative control wells - average OD 490 values of the blank control wells). 490

[0062] Two weeks after the last immunization, cells were stimulated with ESAT-6, CFP-10 and EC recombinant proteins respectively and SI was introduced to evaluate the level of lymphocyte proliferation. The results are shown in Figure 6. Figure 2 ​As shown in Figure C: Regardless of the type of stimulant used, the lowest proliferative capacity was observed only in the Fer group. Following recombinant protein stimulation, lymphocyte proliferation levels in both the monomeric antigen and NPs groups were higher than in the BCG group. In summary, cell proliferation in the NPs-inoculated group was superior to that in the monomeric antigen group. However, when CFP-10 was used as a stimulus, the levels in the BEF group were similar to, but not higher than, those in the BC10 group and the EC fusion protein group. This does not mean that NPs cannot effectively promote cell proliferation. Classical immunology suggests that, under normal immune function and without considering immune exemption, cells are rapidly activated or proliferate when the body encounters the same antigen stimulus again. This result may be because CFP-10 induces this effect more strongly, hence the similarity between the BEF and BC10 groups. Importantly, the proliferation level in the BCF group was significantly higher than that in the BC10 group, indicating that ferritin-loaded immunogens further promote cell proliferation after immunogen identification. When mice were stimulated with ESAT-6, EC, and ConA, the SI in the BEF and BCF groups was significantly higher than that in mice inoculated with EC fusion protein, which still indicates that the proliferation induced by single antigen-loaded Fer is stronger than that of ordinary fusion antigen subunit vaccines.

[0063] To verify whether nanoparticle vaccines are capable of providing early or sustained protective immunity, this invention compared the number of specific memory cells in the spleen of each group based on secreted effector cytokines and memory T cell subsets. The results are as follows: Figure 3 As shown: in CD4 + IFN-γ + T CM ( Figure 3 (A) and CD4 + IFN-γ + T EM ( Figure 4 In aspect A), regardless of the protein used as the stimulant, the number of antigen-specific memory T cells induced by the nanovaccine group was significantly higher than that in the corresponding ordinary antigen group. When CFP-10 stimulated cells, the highest number of CD8 cells was observed in the BEF group. + IFN-γ + T CM ( Figure 3 C) and CD4 + IL2 + T EM ( Figure 4 (B). Similarly, this invention also detected that when ESAT-6 was stimulated, CD4 in the BCF group... + IL2 + T EM The highest level ( Figure 4 (B) It is speculated that this may be due to cross-immunity caused by the co-transcription of ESAT-6 and CFP-10 at the gene level.

[0064] As the most classic tuberculosis vaccine, the advantages of BCG can still be seen in flow cytometry results. For example, when cells are stimulated with ESAT-6 and EC, the CD8+ level in the BCG group is significantly higher. + IFN-γ + T CM ( Figure 3 C) and CD8 + IL-2 + T EM ( Figure 4 (D) is dominant. Besides CD8 + IL-2 + T EM ( Figure 4 In addition to the above, this invention still yielded the highest level of antigen-specific memory T cells in the Fer nanovaccine group, a phenomenon particularly evident in the BECF group. Although CD4+ cells were stimulated by ESAT-6 or CFP-10, + IL-2 + T EM The frequency was not the highest in the BECF group ( Figure 4 (B), but it is at least one of the BEF or BCF groups, which also illustrates the advantage of Fer as a TB antigen carrier.

[0065] In summary, under the immunization program of this invention, regardless of the recombinant protein used for stimulation, the ferritin nanovaccine can significantly stimulate CD4. + IFN-γ + T M (including T) CM and T EM CD8 + IFN-γ + T EM The proliferation of CD4 and the increase of CD4 when CFP-10 or EC are used as stimulation. + IL-2 + T CM and CD8 + IL-2 + T CM frequency ( Figure 3 (B and D in the middle).

[0066] 1.6 Enzyme-linked immunospot assay

[0067] The number of antigen-specific immune cells secreting IFN-γ was detected using a commercially available mouse IFN-γ pre-coated ELISPOT kit. First, PVDF membranes were activated with universal serum-free medium, and then 3 × 10⁻⁶ cells were added to each well. 5cells were stimulated with 15 μg / mL of recombinant ESAT-6, CFP-10 and EC fusion protein, respectively. Untreated cell suspension and mixture containing 500 ng / mL PMA and 10 ng / mL ionomycin were used as negative and positive controls. ELISpot plates were incubated at 37 °C in 5% CO2for 16 h, then the procedure was followed as described and the spot forming cells (SFC) of antigen-specific IFN-γ secretion were observed, finally the number of SFC was counted using IRIS™ ELISpot Reader.

[0068] ELISpot results are shown in Figure Figure 5 , the empty Fer group had almost no response and the BCG group had weak response after stimulation with relevant recombinant antigens. The BECF group reached the highest level regardless of the degree of protein stimulation. The BEF group induced a higher number of IFN-γ SFC than other protein groups by ESAT-6, and the secretion level was significantly higher than that of the BCG group Figure 5 A). Similarly, similar results were observed for CFP-10 and EC stimulation. Overall, the number of IFN-γ secreting cells in the spleen of mice in the EF-Fer, C10-Fer or EC-Fer booster group was generally higher than that in the subunit vaccine immunization group, and this trend was particularly evident in the BECF group Figure 5 B). The above results show that the heterologous booster vaccination strategy using nanotuberculosis antigens can significantly enhance the ability of BCG to stimulate cells to secrete IFN-γ, and the nanotuberculosis vaccine shows a higher degree of specificity and potency in response to antigen restimulation, and it is possible to secrete more effector molecules compared with traditional subunit vaccines.

[0069] 1.7 Enzyme-linked immunosorbent assay

[0070] To comprehensively verify the ability of spleen lymphocytes of each group of mice to secrete TB immune-related cytokines under the stimulation of TB antigens ESAT-6, CFP-10 and EC, the secretion levels of various TB-related cytokines in the culture supernatant of mouse spleen lymphocytes were detected by using commercial ELISA detection kits. 3 x 10 6 cells / well and 15 μg / mL of recombinant protein were incubated at 37 °C, 5% CO2for 24 h to detect IL-2, and incubated for 48 h to detect the secretion levels of IFN-γ, TNF-α, IL-6, IL-10, IL-12, IL-17 and IL-23. After the culture was completed, the cell culture supernatant was collected and detected using the corresponding commercial pre-coated ELISA kit. Within the first 15 min after the color reaction was completed, the OD 450The secretion levels of cytokines IFN-γ, TNF-α, IL-6, IL-2, IL-10, IL-12, IL-17 and IL-23 were analyzed.

[0071] Results of multiple cytokine secretion levels in cell culture supernatant as follows Figure 6 As shown, mouse lymphocytes inoculated with TB nanoantigen elicited IFN-γ and IL-2 responses more significantly after stimulation than BCG and monomeric proteins. Figure 6 (A and C). A stronger secretion trend similar to IL-2 was also detected in TNF-α ( Figure 6 (B). Therefore, ferritin nanocarriers can help TB antigens stimulate a stronger Th1 immune response, thereby significantly improving the immunogenicity of BCG.

[0072] Furthermore, when ESAT-6 stimulation was used, this invention observed that only the BECF group had the highest levels of IL-6 and IL-12, while the IL-6 levels in all booster immunization groups were higher than those in the BCG group, the latter being similar to IL-10 secretion under EC stimulation conditions. Figure 6 (DF). Regarding CFP-10-stimulated IL-6 and ESAT-6-stimulated IL-10, the BEF, BCF, and BECF levels in the three nanoantigen groups were significantly higher than those in the BCG group and the booster immunization group with the corresponding common antigen. Figure 6 (D and E).

[0073] The IL-17 levels obtained after booster immunization induced by conventional subunit vaccines were almost identical to those obtained after BCG vaccination alone. Furthermore, after antigen fusion with Fer, regardless of ESAT6, CFP-10, or EC stimulation, the IL-17 levels in the BEF, BCF, and BECF groups were more ideal or significantly higher than those observed in other groups. Figure 6 (G). However, each group secreted only trace amounts of IL-23, with no difference between the groups. Figure 6 (H). This indicates that when mouse spleen cells immunized with ferritin nanotube tuberculosis vaccine encounter the relevant Mtb antigen, they not only produce appropriate amounts of IL-6 and IL-10, but more importantly, they can exhibit a Th1-predominant immune response, and cytokines related to the Th17 response may be secreted through an IL-23-independent pathway.

[0074] 1.8 Flow Cytometry Analysis

[0075] Splenic lymphocytes were divided into 3×10 6Cells were harvested by centrifugation, washed with pre-chilled PBS, and blocked with 1 μL of TruStain FcX™ Plus Mouse Anti-CD16 / 32 Monoclonal Antibody to block Fc receptors. Cells were then stained with 2 μL of FITC-labeled anti-mouse CD3ε, 1.5 μL of PE-labeled anti-mouse CD4, 1.5 μL of APC / Fire™ 750-labeled anti-mouse CD8a, 2.5 μL of APC-labeled anti-mouse CD44, and 1.5 μL of PE / Cyanine 7-labeled anti-mouse CD62L monoclonal antibodies. Cells were then fixed with FluoroFix Buffer, permeabilized with PermWash Buffer, and then intracellularly stained with 5 μL of APC-labeled anti-mouse IFN-γ and 1 μL of Brilliant Violet 421-labeled anti-mouse IL-2 monoclonal antibodies. Cells were then washed with PBS and resuspended in FACS Buffer before being analyzed using a DxPAthena™ Flow Cytometer. TM TM TM + + + + CM + high EM + low

[0076] Figure 3 Figure 4 + CM EM + + EM + + CM

[0077] 1.9 Real-time quantitative PCR

[0078] Total RNA was extracted from 10 7 cells using the TRIzol method. The OD​​​​​​​​​​​​​​​​​​​​​​​​​260 / 280 and OD 260 / 230 The concentration and purity of RNA were detected. Then cDNA synthesis was performed by reverse transcription PCR using One-Step gDNA Removal and cDNA Synthesis SuperMix. qPCR was performed using Green qPCR SuperMix to evaluate the transcription levels of cytokines IFN-γ, TNF-α, IL-2 and IL-17.2 -ΔΔCT Method for calculating mRNA expression.

[0079] The mRNA transcription of IFN-γ, TNF-α, IL-2 and IL-17 is shown in A-D of Figure 7 Figure 2, the transcription and secretion levels of cytokines (IFN-γ, TNF-α, IL-2 and IL-17) of BE6, BC10 and BEC groups are similar, indicating that ESAT-6 and CFP-10 failed to enhance the BCG-induced cytokine transcription under the immunization scheme before TB protein stimulation. The results obtained by BEF, BCF, especially BECF group are significantly higher than those of BCG or mice receiving conventional subunit protein vaccine, indicating that ferritin nanoparticles help antigens to enhance the transcription level of Th1 cytokine mRNA without TB protein stimulation. Therefore, the cytokine transcription level verified by qPCR is roughly similar to the secretion trend after protein stimulation.

[0080] 1.10 H&E Staining

[0081] The heart, liver, spleen, lung and kidney of mice in each group were taken, fixed with 4% paraformaldehyde, dehydrated with different concentrations of ethanol gradient, embedded with paraffin, uniformly cut into 6 μm thin sections, and stained with H&E to evaluate the safety of ferritin vaccine.

[0082] In order to detect the safety of the vaccine and whether it will cause damage to the important organs of mice, the present application not only records the behavior of mice before and after vaccination, but also removes the relevant organs such as heart, liver, spleen, lung and kidney of euthanized mice, and observes the pathological state of each organ section under a microscope by HE staining. No inflammatory reaction is found in the injection site and surrounding area after two rounds of intensive immunization, the respiration is normal, the hair condition is good, the eating and drinking conditions are normal, and no mice die during the whole process. This shows that the vaccine does not cause adverse reactions in the whole body of mice. Then, the general condition and pathological sections of each organ are continuously and closely observed. The morphology of each organ is generally normal under naked eye observation. Under the microscope, there is no obvious inflammatory cell infiltration in each organ Figure 8). Specifically, the myocardial fibers of each group of mice were normal, and no eosinophils and plaque formation were observed. No basement membrane thickening, inflammatory exudation and cell proliferation were observed in glomeruli and renal tubules. The number of central veins in the hepatic lobules and portal areas was single and no deflection was observed; the morphology of liver cells was normal, and no ballooning degeneration, acidophilic degeneration and abnormal substance deposition were observed. The lung parenchyma and interstitium were normal; no effusion was observed in the alveolar cavity, the alveolar septum was normal, the tissue structure of each level of bronchus was clear, and no granulomatous inflammatory lesions were observed. The boundary between the red pulp area and the white pulp area in the spleen was clear; a large number of red blood cells and megakaryocytes were observed in the red pulp area, accompanied by a small amount of lymphoid follicle; the white pulp area contained lymphoid follicles, and the germinal center was observed in the center; the cell proliferation in the perivascular lymph sheath was obvious. These results show that the vaccine does not cause systemic symptoms in mice, and no toxic effects on important organs are observed, confirming that the nano-tuberculosis vaccine constructed in the application has tolerability and safety in mice.

[0083] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A subunit nanoparticle characterized in that, The subunit nanoparticle comprises a subunit antigen isolated from tuberculosis and a self-assembling carrier comprising ferritin; The subunit antigen isolated from tuberculosis comprises any one of ESAT-6, CFP-10 and a fusion protein of ESAT-6 and CFP-10; The ferritin is derived from ferritin of Helicobacter pylori.

2. A recombinant vector expressing the subunit nanoparticle of claim 1.

3. A recombinant engineering cell comprising the recombinant vector of claim 2.

4. The method of producing subunit nanoparticles of claim 1, wherein, The recombinant vector is obtained by inserting a gene encoding the subunit nanoparticle into a basic backbone vector; The recombinant engineering cell is constructed by transforming a host cell with the recombinant vector; The subunit nanoparticle is expressed by inducing the recombinant engineering cell.

5. The production method according to claim 4, characterized by, When the subunit nanoparticle is produced by a prokaryotic expression method, the basic backbone vector comprises pET-28a(+) and the host cell comprises Escherichia coli.

6. Use of the subunit nanoparticle of claim 1 or the subunit nanoparticle produced by the production method of claim 4 or 5 in the preparation of a tuberculosis nanoparticle vaccine.

7. A vaccine for tuberculosis, characterized by comprising the recombinant mycobacterium of claim 1. The active ingredient comprises the subunit nanoparticle of claim 1 or the subunit nanoparticle produced by the production method of claim 4 or 5.

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