Tuberculosis VLP nanoparticle vaccine as well as preparation and application thereof

Abstract

The invention relates to the technical field of tuberculosis vaccines, in particular to a tuberculosis VLP nanoparticle vaccine and preparation and application thereof. According to the tuberculosis VLP nanoparticle vaccine, through up-regulation of Th1 type, Th2 type and TH17 type cellular immune response and down-regulation of Treg cellular immune response, the H37Rv growth inhibition ability of splenic lymphocytes and lung cells in vitro is enhanced, and the cellular immune response induced by EPPE + mi3 / AS01E and the H37Rv growth inhibition ability of the cells in vitro are higher than those of BCG and a corresponding subunit vaccine EPPE / AS01E; in addition, the EPPE + mi3 / AS01E can be used as a BCG booster vaccine, and the cell immune response after BCG immunization and the H37Rv growth inhibition capacity of cells in vitro are improved.

Description

Technical Field

[0001] This invention relates to the field of tuberculosis vaccine technology, and in particular to a tuberculosis VLP nanoparticle vaccine and its preparation and application. Background Technology

[0002] Tuberculosis (TB) is a zoonotic infectious disease caused by Mycobacterium tuberculosis (Mtb) infection, transmitted through the respiratory tract, and is one of the major threats to global public health. Researching treatment and intervention strategies for TB is urgently needed. Vaccination is the most cost-effective method for preventing and managing TB. Bovine Bacillus Calmette-Guérin (BCG) is currently the only licensed TB vaccine. However, its drawbacks include insufficient antigen processing and presentation, inability to optimally activate the production of anti-Mtb T cells and regulatory T cells, and inability to provide effective protection for adults. Therefore, there is an urgent need to develop novel TB vaccines that are more effective than BCG, to compensate for BCG's shortcomings, or to enhance BCG-mediated immune responses. Secretory antigens, dormant antigens, latent-associated antigens, and resuscitation-promoting factors of Mtb at different stages of infection have been proposed as promising vaccine candidates. However, a single antigen may not be sufficient to induce comprehensive immune protection against Mtb infection, necessitating a multi-stage antigen vaccination strategy.

[0003] Currently, 17 candidate vaccines are in clinical trials. These vaccines, at different stages of clinical trials, are mainly used to prevent TB infection and morbidity, and to improve TB treatment prognosis. However, existing candidate vaccines have not shown the expected protective effect. Although recombinant protein subunit vaccines have many advantages, their relatively weak immunogenicity makes it difficult to induce a sufficient immune response. Therefore, adjuvants and delivery display systems for subunit vaccines are particularly important. Among the adjuvants approved for use in vaccines, we found that AS01, composed of QS21, liposomes, and the TLR4 agonist monophospholipid A (MPL), can generate an effective and durable antibody response, inducing a higher antigen-specific Th1 response. It currently plays an important adjuvant role in the immunization of the TB clinical vaccine M72 / AS01E. Virus-like particles (VLPs), as a polymer platform, are currently the only nanoparticle (NP) class approved for use in vaccines in humans. Antigens can be safely delivered to the immune system on a highly immunogenic scaffold, preferentially absorbed by dendritic cells (DCs) and macrophages, concentrating antigen uptake on immune cells to achieve a sustained release of the vaccine and induce a protective immune response. Currently, many VLPs are used in vaccines, such as AP205, ferritin, I53-50, and mi3, which have been used for African swine fever, SARS-CoV-2, and HIV. Among them, the porous dodecahedral 60-mer particles mi3, designed based on KDPG from the hyperthermophilic bacterium *Thermosporum tobira*, exhibit better particle uniformity and 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. Spycatcher and SpyTag can spontaneously and irreversibly form heteropeptide covalent bonds in vitro. In most nanocage vaccine platform studies, the SpyTag / Spycatcher system is used to display antigens, counteracting the significant reduction in expression levels caused by direct fusion of antigens to the N-terminus or C-terminus of VLPs. Using the Spycatcher fusion of mi3, the target antigen is linked to the SpyTag peptide. Through the spontaneous coupling of the Spycatcher and SpyTag heteropeptides, the target antigen is displayed on the surface of the Spycatcher-mi3 nanoparticles, forming a "plug-and-play" vaccine platform. Spycatcher-mi3 NP has been used in vaccine development against a variety of pathogens, including SARS-CoV-2, malaria, and influenza. Spycatcher-mi3 is solublely expressed in E. coli with high yields, precise and robust derivatization, and the lack of sequence homology between Spycatcher-mi3 and the human genome reduces potential concerns about the platform inducing autoimmune responses. In conclusion, Spycatcher-mi3 is a promising VLP nanoparticle display tool that can facilitate its widespread application in vaccine development.

[0004] This invention screens antigen genes from different stages of Mtb, including secretion, dormancy, latency, and recovery phases, starting from the whole Mtb genome. It uses the IEDB database to predict MHC-restricted immunodominant peptides and screens out Mtb's dominant T-cell antigens. ELISA is used to assess the immunogenicity of these antigens in Quantiferon (QFT) + PBMCs of subjects. A self-assembled multivalent nanoparticle candidate vaccine based on the SpyCatcher / SpyTag strategy, displaying candidate antigens on mi3 nanoparticles, is constructed. Immunization of C57BL / 6 mice compares the induced significant cellular immune responses and in vitro inhibitory effects on Mycobacterium tuberculosis by BCG vaccine, multivalent mi3 / AS01E NPs candidate vaccine, and their corresponding subunit vaccines. The cellular immune response and in vitro inhibitory effect on Mycobacterium tuberculosis induced by the mi3 / AS01E NPs vaccine are higher than those induced by its corresponding subunit vaccine and BCG vaccine. Furthermore, the cellular immune response and in vitro inhibitory effect on Mycobacterium tuberculosis induced by the mi3 NPs vaccine combined with BCG immunization are higher than those induced by BCG immunization alone. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a tuberculosis VLP nanoparticle vaccine, its preparation, and its application. The cellular immunogenicity induced after immunization of mice and its ability to inhibit the growth of Mycobacterium tuberculosis in vitro are stronger than those of BCG and its corresponding subunit vaccine EPPE / AS01E. Furthermore, the multivalent VLP nanoparticle vaccine EPPE+mi3 / AS01E exhibits the characteristic of synergistically enhancing the cellular immunogenicity induced by BCG and its ability to inhibit the growth of Mycobacterium tuberculosis in vitro.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing a tuberculosis VLP nanoparticle vaccine, comprising the following steps:

[0008] 1) Spycatcher-mi3 and ESAT-6-SpyTag are self-assembled to obtain ESAT-6-mi3;

[0009] Spycatcher-mi3 and PPE57-SpyTag were self-assembled to obtain PPE57-mi3;

[0010] Spycatcher-mi3 and PPE68-SpyTag were self-assembled to obtain PPE68-mi3;

[0011] Spycatcher-mi3 and EsxV-SpyTag were self-assembled to obtain EsxV-mi3;

[0012] 2) Mix ESAT-6-mi3, PPE57-mi3, PPE68-mi3 and EsxV-mi3 obtained in step 1) to obtain multivalent nanoparticles;

[0013] 3) Mix and emulsify the multivalent nanoparticles and AS01E adjuvant described in step 2) to obtain a tuberculosis VLP nanoparticle vaccine.

[0014] Preferably, the amino acid sequence of Spycatcher-mi3 in step 1) is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 6;

[0015] The amino acid sequence of the ESAT-6-SpyTag is shown in SEQ ID No. 2;

[0016] The amino acid sequence of the PPE57-SpyTag is shown in SEQ ID No. 3;

[0017] The amino acid sequence of the PPE68-SpyTag is shown in SEQ ID No. 4;

[0018] The amino acid sequence of the EsxV-SpyTag is shown in SEQ ID No. 5.

[0019] Preferably, the molar ratio of Spycatcher-mi3 to ESAT-6-SpyTag in step 1) is 1:2;

[0020] The molar ratio of Spycatcher-mi3 to PPE57-SpyTag is 1:2.

[0021] The molar ratio of Spycatcher-mi3 to PPE68-SpyTag is 1:3.

[0022] The molar ratio of Spycatcher-mi3 to EsxV-SpyTag is 1:3.

[0023] Preferably, the self-assembly conditions in step 1) include: a temperature of 25°C and a time of 6 hours.

[0024] Preferably, the mass ratio of ESAT-6-mi3, PPE57-mi3, PPE68-mi3 and EsxV-mi3 in step 2) is 1:1:1:1;

[0025] Preferably, the volume ratio of the multivalent VLP nanoparticles and the AS01E adjuvant in step 3) is 1:1.5.

[0026] The present invention also provides a tuberculosis VLP nanoparticle vaccine prepared by the preparation method described in the above technical solution.

[0027] The present invention also provides the application of the tuberculosis VLP nanoparticle vaccine described in the above technical solution in the preparation of anti-tuberculosis drugs.

[0028] This invention also provides the application of the multivalent mi3 VLP nanoparticle vaccine combined with BCG vaccine described above in the preparation of anti-tuberculosis drugs.

[0029] Preferably, the tuberculosis bacteria include Mycobacterium tuberculosis H37Rv.

[0030] The beneficial effects of this invention are:

[0031] This invention provides a tuberculosis VLP nanoparticle vaccine that enhances the ability of spleen lymphocytes and lung cells to inhibit H37Rv growth in vitro by upregulating Th1, Th2, and Th17 cellular immune responses and downregulating Treg cellular immune responses. The cellular immune response induced by EPPE+mi3 / AS01E and its ability to inhibit H37Rv growth in vitro are higher than those induced by BCG and its corresponding subunit vaccine EPPE / AS01E. In addition, EPPE+mi3 / AS01E can be used as a BCG booster vaccine to increase the cellular immune response after BCG immunization and the ability to inhibit H37Rv growth in vitro. Attached Figure Description

[0032] 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.

[0033] Figure 1 SDS-PAGE images of each Mtb dominant antigen and Spycatcher-mi3 after expression and purification; lanes A 1-9 are Spycatcher-mi3, ESPC-SpyTag, nCpnT-SpyTag, PPE57-SpyTag, ESAT-6-SpyTag, Rv1794-SpyTag, Rv3134c-SpyTag, EsxV-SpyTag, HSPX-SpyTag respectively; lanes B 1-3 are PPE29-SpyTag, PPE33-SpyTag, PPE68-SpyTag respectively.

[0034] Figure 2 For the construction, purification and characterization of Spycatcher-mi3;

[0035] Figure 3 Figure showing the self-assembly conditions of Mtb candidate antigen-SpyTag and Spycatcher-mi3;

[0036] Figure 4 Preparation of the multivalent VLP rice particle vaccine EPPE+mi3 / AS01E;

[0037] Figure 5 The levels of cytokines secreted by splenic lymphocytes in mice immunized with the multivalent VLP rice grain vaccine EPPE+mi3 / AS01E;

[0038] Figure 6 The frequency of various T cells and the level of spleen lymphocyte proliferation in mice immunized with multivalent VLP rice grain vaccine EPPE+mi3 / AS01E.

[0039] Figure 7 This image shows the colony count of spleen / lung cells from mice immunized with the multivalent VLP rice grain vaccine EPPE+mi3 / AS01E in vitro using H37Rv. Detailed Implementation

[0040] This invention provides a method for preparing a tuberculosis VLP nanoparticle vaccine, comprising the following steps:

[0041] 1) Spycatcher-mi3 and ESAT-6-SpyTag are self-assembled to obtain ESAT-6-mi3;

[0042] Spycatcher-mi3 and PPE57-SpyTag were self-assembled to obtain PPE57-mi3;

[0043] Spycatcher-mi3 and PPE68-SpyTag were self-assembled to obtain PPE68-mi3;

[0044] Spycatcher-mi3 and EsxV-SpyTag were self-assembled to obtain EsxV-mi3;

[0045] 2) Mix ESAT-6-mi3, PPE57-mi3, PPE68-mi3 and EsxV-mi3 obtained in step 1) to obtain multivalent nanoparticles;

[0046] 3) Mix and emulsify the multivalent nanoparticles and AS01E adjuvant described in step 2) to obtain a tuberculosis VLP nanoparticle vaccine.

[0047] This invention involves self-assembling Spycatcher-mi3 with ESAT-6-SpyTag to obtain ESAT-6-mi3; self-assembling Spycatcher-mi3 with PPE57-SpyTag to obtain PPE57-mi3; self-assembling Spycatcher-mi3 with PPE68-SpyTag to obtain PPE68-mi3; and self-assembling Spycatcher-mi3 with EsxV-SpyTag to obtain EsxV-mi3.

[0048] In this invention, the amino acid sequence of Spycatcher-mi3 is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 6; the amino acid sequence of ESAT-6-SpyTag is shown in SEQ ID No. 2; the amino acid sequence of PPE57-SpyTag is shown in SEQ ID No. 3; the amino acid sequence of PPE68-SpyTag is shown in SEQ ID No. 4; and the amino acid sequence of EsxV-SpyTag is shown in SEQ ID No. 5.

[0049] SEQ ID No. 1:

[0050] MGMSYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGGSGGSGGSGGSMKMEELFKKHKIVAVLRANSVEEAKK KALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE;

[0051] SEQ ID No. 6:

[0052]

[0053] SEQ ID No.2:

[0054] MNHKVHHHHHHMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAGGGGSGGGAHIVMVDAYKPTK;

[0055] SEQ ID No.3:

[0056] MNHKVHHHHHHMHPMIPAEYISNIIYEGPGADSLFFASGQLRELAYSVETTAESLEDELDELDENWKGSSSDLLADAVERYLQWLSKHSSQLKHAAWVINGLANAYNDTRRKVVPPEEIAANREERRRLIASNVAGVNTPAIADLDAQYDQYRARNVAVMNAYVSWTRSALSDLPRWREPPQIYRGGGGGGSGGGAHIVMVDAYKPTK;

[0057] SEQ ID No.4:

[0058] MNHKVHHHHHHMLWHAMPPELNTARLMAGAGPAPMLAAAAGWQTLSAALDAQAVELTARLNSLGEAWTGGGSDKALAAATPMVVWLQTASTQAKTRAMQATAQAAAYTQAMATTPSLPEIAANHITQAVLTATNFFGINTIPIALTEMDYFIRMWNQAALAMEVYQAETAVNTLFEKLEPMASILDPGASQSTTNPIFGMPSPGSSTPVGQLPPAATQTLGQLGEMSGPMQQLTQPLQQVTSLFSQVGGTGGGNPADEEAAQMGLLGTSPLSNHPLAGGSGPSAGAGLLRAESLPGAGGSLTRTPLMSQLIEKPVAPSVMPAAAAGSSATGGAAPVGAGAMGQGAQSGGSTRPGLVAPAPLAQEREEDDEDDWDEEDDWGGGGSGGGAHIVMVDAYKPTK;

[0059] SEQ ID No.5:

[0060] MNHKVHHHHHMTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVLTASDFWGGAGSAACQGFITQLGRNFQVIYEQANAHGQKVQAAGNNMAQTDSAVGSSWAGGGGSGGGAHIVMVDAYKPTK.

[0061] In this invention, the preferred molar ratio of Spycatcher-mi3 to ESAT-6-SpyTag is 1:2. The preferred molar ratio of Spycatcher-mi3 to PPE57-SpyTag is 1:2. The preferred molar ratio of Spycatcher-mi3 to PPE68-SpyTag is 1:3. The preferred molar ratio of Spycatcher-mi3 to EsxV-SpyTag is 1:3. The preferred self-assembly conditions in this invention include a temperature of 25°C and a time of 6 hours.

[0062] This invention mixes ESAT-6-mi3, PPE57-mi3, PPE68-mi3, and EsxV-mi3 to obtain multivalent nanoparticles. In this invention, the preferred mass ratio of ESAT-6-mi3, PPE57-mi3, PPE68-mi3, and EsxV-mi3 is 1:1:1:1. This invention does not impose any particular limitation on the mixing process; as long as the mixture is thoroughly homogenized, it is acceptable.

[0063] This invention involves mixing and emulsifying the multivalent nanoparticles and ASO1E adjuvant to obtain a tuberculosis VLP nanoparticle vaccine. In this invention, the preferred volume ratio of the multivalent nanoparticles to the ASO1E adjuvant is 1:1.5. This invention does not specifically limit the source of the ASO1E adjuvant; commercially available options are acceptable. Similarly, this invention does not specifically limit the emulsification conditions; conventional methods can be used by those skilled in the art.

[0064] The present invention also provides a tuberculosis VLP nanoparticle vaccine prepared by the preparation method described in the above technical solution.

[0065] This invention also provides the application of the tuberculosis VLP nanoparticle vaccine described above in the preparation of anti-tuberculosis drugs. In this invention, the tuberculosis bacillus preferably includes Mycobacterium tuberculosis H37Rv.

[0066] This invention also provides the application of the multivalent mi3 VLP nanoparticle vaccine combined with BCG vaccine described above in the preparation of anti-tuberculosis drugs. In this invention, the tuberculosis bacillus preferably includes Mycobacterium tuberculosis H37Rv.

[0067] 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.

[0068] Example 1

[0069] Screening of Mtb-specific candidate antigens

[0070] (1) In this embodiment, NCBI (https: / / www.ncbi.nlm.nih.gov / protein) and IEDB (http: / / tools.iedb.org / ) were used to screen for the latent, secretory, and active phases of tuberculosis T-cell antigens, as well as immune-related T-cell antigens. Furthermore, authoritative online prediction databases and tools such as GLIPH2 and VaxiJen 2.0 (http: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.html) were used to predict and analyze the epitopes and immunogenicity of the screened T-cell antigens. Ten immunodominant antigens were identified: ESAT-6, EsxV, PPE33, PPE29, PPE57, PPE68, Rv1794, Rv3134c, ESPC, and HSPX. These antigens were successfully expressed in prokaryotes. Figure 1 ).

[0071] (2) In this embodiment, peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of QFT+ (n=80) and QFT- (n=42) subjects using human peripheral blood lymphocyte separation medium (TBDTM-0200, TBD, Tianjin, China). The PBMCs were then separated at a concentration of 3 × 10⁻⁶ cells / mL. 5 Cells / wells were placed in 96-well plates and co-incubated for 24 h with PBS, PHA (phytohemagglutinin) (5 μg / mL), or antigen protein (5 μg / mL), respectively. Cell culture supernatants were collected, and ELISA experiments were performed using the Human Interferonγ ELISA Kit (EK0373, BOSTER, Wuhan, China). Results showed that compared to the control group (PBS), the expression of IFN-γ in QFT+ and QFT- volunteer PBMCs was significantly increased by all 10 antigens. Antigens ESAT-6, EsxV, PPE33, PPE29, PPE57, and PPE68 induced significantly higher IFN-γ expression in QFT+ PBMCs than in QFT-PBMCs; and EsxV, PPE57, and PPE68 induced IFN-γ expression levels higher than or similar to those induced by ESAT-6 in QFT+ PBMCs. Therefore, ESAT-6, EsxV, PPE57, and PPE68, with stronger immunogenicity, were selected as candidate antigens for the vaccine.

[0072] Example 2

[0073] Preparation and characterization of multivalent VLP nanoparticle vaccine EPPE+mi3 / AS01E

[0074] (1) In this embodiment, the amino acid sequences of Spycatcher and mi3 were obtained through NCBI, linked to the GGS adapter in a computer, and the 3D structure of Spycatcher-mi3 was predicted using the SWISS-MODEL database (https: / / swissmodel.expasy.org / interactive). After codon optimization, the nucleotide sequence of Spycatcher-mi3 was inserted into the pET28a(+) plasmid (NcoI and XhoI sites) by Nanjing Zhongding Biotechnology Co., Ltd., and transformed into E. coli cells (BL21(DE3)) in vitro. Spycatcher-mi3 protein was expressed and purified by this invention. The size and purity of the protein were analyzed by SDS-PAGE and Western Blot using a protein molecular weight standard (#26616, Thermo Fisher) as a molecular weight marker. Spycatcher-mi3 was diluted in PBS (pH 7.4). The size distribution of Spycatcher-mi3 was determined by dynamic light scattering (DLS) analysis using a Zetasizer Nano Z instrument, and a distribution map was plotted using Origin 2021. Spycatcher-mi3 was diluted to a concentration of 0.2 mg / mL, placed on a 200-mesh copper grid, stained with 2% uranyl acetate, air-dried, and then observed for morphology using transmission electron microscopy (FE-TEM). Results showed that after codon optimization and purification, the Spycatcher-mi3 protein was obtained with a molecular weight of 40.9 KD. Negative staining TEM images revealed a well-defined and uniform spherical nanocage structure, consistent with the dodecahedral structure predicted by SWISS-MODEL. DLS analysis showed that the average diameter of the Spycatcher-mi3 NPs was 43.2 ± 3.75 nm. Figure 2 ).

[0075] (2) Spycatcher-mi3 and antigen-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 antigen-SpyTag achieved the highest self-assembly efficiency at 25℃ for 6 h in neutral solution. Figure 3The study investigated the different mass ratios of four candidate antigens in the self-assembly of Spycatcher-mi3. It was determined that when the mass ratios of ESAT-6-SpyTag, PPE57-SpyTag, PPE68-SpyTag, and EsxV-SpyTag to SpyCatcher-mi3 protein were 1:2, 1:2, 1:3, and 1:3, the proportions of the self-assembly products ESAT-6-mi3, PPE57-mi3, PPE68-mi3, and EsxV-mi3 were the highest, reaching over 90%, 90%, 80%, and 92%, respectively. Figure 4 ). ESAT-6-mi3, PPE57-mi3, PPE68-mi3 and EsxV-mi3 under optimal self-assembly conditions were mixed in a mass ratio of 1:1:1:1. Figure 4 Multivalent nanoparticles (denoted as EPPE+mi3) were prepared and fully emulsified with nanoliposome adjuvant AS01E (CanSino SPH, Shanghai, China) at a volume ratio of 1:1.5 to form a multivalent VLP nanoparticle vaccine EPPE+mi3 / AS01E. The corresponding subunit vaccine was prepared by mixing candidate antigens ESAT-6, PPE57, PPE68 and EsxV at a mass ratio of 1:1:1:1 and then fully emulsifying them with AS01E.

[0076] Example 3

[0077] Immunogenicity study of multivalent VLP nanoparticle vaccine EPPE+mi3 / AS01E

[0078] (1) Multivalent nanoparticle vaccine EPPE+mi3 / AS01E immunization C57BL / 6 regimen

[0079] The study consisted of six groups (PBS, AS01E, BCG, EPPE / AS01E, EPPE+mi3 / AS01E, and EPPE+mi3 / AS01E+BCG), with six C57BL / 6 mice in each group. In a single vaccine dose, 1×10⁻⁶ mice were administered. 6 C57BL / 6 mice were administered a single dose of CFU BCG via subcutaneous injection in the neck and back of each mouse. The control group received 200 μL of PBS in the same manner. In the vaccine group, each mouse received 10 μg of mixed antigen (EPPE) and 10 μg of NPs (EPPE+mi3) at a volume ratio of 1:1.5, which were then thoroughly emulsified with nanoliposome adjuvant AS01E (120 μL solution containing 75 μg phospholipids + 18.75 μg cholesterol + 3.75 μg MPL + 3.75 μg QS-21). 200 μL of this solution was injected subcutaneously into the groin of each mouse. Immunization was performed three times, two weeks apart, with the last dose being half-dose. In the combined BCG immunization regimen, 1×10⁻⁶ BCG was administered concurrently with the single-dose BCG immunization. 6CFU BCG / each C57BL / 6 mouse was subcutaneously injected into the neck and back. One week later, the mice were given the first immunization with adjuvanted vaccine (EPPE / AS01E, EPPE+mi3 / AS01E) (consistent with the single vaccine immunization dose). The second and third immunizations (the third immunization was at half the dose) were administered subcutaneously in the groin every two weeks. All mice were sacrificed by cervical dislocation four weeks after the last adjuvant vaccine immunization.

[0080] (2) Collection of materials

[0081] Four weeks after the final immunization, blood was collected from the eyeballs, and the animals were euthanized by cervical dislocation. The spleen, lungs, and liver were aseptically collected. The upper right lobe of the lung and the liver lobule were fixed in 4% paraformaldehyde general-purpose tissue fixative (BL539A, Biosharp, Anhui, China). The remaining lung tissue was used for lung cell suspension preparation. The spleen was soaked in neutral PBS for spleen lymphocyte suspension preparation. The spleen was placed in a 35mm culture dish covered with a 70μm cell sieve, and mouse lymphocyte separation medium (7211011, DAKEWE, Shenzhen, China) was added. The spleen was then ground using a 2mL syringe plug, transferred to centrifuge tubes, 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, and resuspended in RPMI 1640 medium containing 10% FBS for later use. Mouse lung tissue was extracted and immersed in a tissue digestion solution containing 775 μL RPMI 1640 medium, 10 mg / mL Collagenase IV (C5138-100 mg, Sigma, USA), and 2000 U / mL DNase I (10104159001, Sigma, USA). The tissue was cut into small pieces and placed in centrifuge tubes containing 3.5 mL of digestion solution. The cells were then digested at 37°C with shaking for 1 hour. After digestion, the cell suspension was filtered through a 200-mesh screen and centrifuged (2000 rpm, 5 min). The supernatant was discarded, and the cells were lysed with erythrocyte lysis buffer, washed with PBS, and centrifuged again. The precipitated cells were resuspended in RPMI 1640 medium containing 10% FBS for later use.

[0082] (3) Luminex detection of kinase expression in spleen lymphocytes of antigen-induced immunized mice

[0083] Mouse lymphocyte suspension (2.5×10) 6 100 μL of a mixture of EPPE antigens (ESAT-6, PPE57, PPE68, and EsxV mixed in a 1:1:1:1 ratio) at a final concentration of 5 μg / mL was added to a 96-well plate. The plate was incubated at 37°C for 48 h in a CO2 incubator, and the cell supernatant was collected. Cells were then cultured according to the Luminex cytokine assay and RayPlex assay. TMThe Mouse Inflammation Array 1 kit (FAM-INF-1-96, RayBio, Guangzhou, China) was used for the experiment. In short, 25 μL RayPlex Multiplex Bead Cocktail was added to each of the following 96-well V-bottom microplates containing 25 μL of CNTRL, 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 and AS01E, the levels of Th1 cytokines (IFN-γ, TNF-α), Th2 cytokines (IL-4 and IL-10), and Th17 cytokines (IL-17 and IL-23) were significantly increased in the subunit vaccines EPPE / AS01E, EPPE+mi3 / AS01E, and EPPE+mi3 / AS01E combined with BCG immunization groups. The expression of Th1, Th2, and Th17 cytokines in the EPPE+mi3 / AS01E group was also significantly higher than that in EPPE / AS01E and BCG immunization alone. The expression of Th1 and Th2 cytokines induced by EPPE+mi3 / AS01E and its combination with BCG immunization was significantly higher than that of BCG immunization alone.

[0084] (4) ELISPOT detection of IFN-γ in antigen-induced immunized mice + / IL-4 + Number of T lymphocytes

[0085] Following the instructions of the FluoroSpot Plus: Mouse IFN-γ / IL-4 (FSP-4146-2, Mabtech, Sweden) kit, FluoroSpot plates were washed, blocked, and adjusted. An stimulant (Anti-CD28 mAb1:1000) was added, followed by the addition of mouse lymphocyte suspension (2.5 × 10⁻⁶). 5100 μL of a mixture of EPPE mixed 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 in a CO2 incubator for 24 h, 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 The results showed that EPPE / AS01E, EPPE+mi3 / AS01E, and EPPE+mi3 / AS01E combined with BCG immunization group showed IFN-γ. + T lymphocytes and IL-4 + The number of T lymphocytes was higher in the EPPE+mi3 / AS01E group than in the PBS and AS01E groups, and the IFN-γ of the EPPE+mi3 / AS01E group was also higher. + T lymphocytes and IL-4 + The number of T lymphocytes was significantly higher than that of EPPE / AS01E and BCG immunization; at the same time, IFN-γ was significantly higher after EPPE+mi3 / AS01E and BCG combined immunization. + T lymphocytes and IL-4 + The number of T lymphocytes was significantly higher than that of BCG alone.

[0086] (5) Flow cytometry was used to detect the T cell populations of antigen-induced immunized mice and MMT was used to detect the proliferation level of spleen lymphocytes.

[0087] Take 2×10 6Splenic lymphocytes were seeded in 24-well plates and stimulated for 6 hours with EPPE mixed antigen at a final concentration of 5 μg / mL. Then, 2 μL of Leukocyte Activation Cocktail with BD GolgiPlug (550583) was added to block stimulation for 6 hours before staining. In short, after centrifugation, cells were collected, resuspended in 1 mL PBS, and 1 μL of L-FixableViabilityStain 780 (565388) was added. The cells were incubated at 4°C in the dark for 20 min. Staining was stopped by adding 1×stainbuffer. After centrifugation, the cells were resuspended in 1×stainbuffer. Separately, 1×Perm / Wash buffer (51-9008100) was mixed with 1 μg / test of FITC Hamster Anti-Mouse CD3e (553061), Ms CD4 PerCP-Cy5.5 (550954), and APCRat Anti-Mouse CD25 (557192) of the total surface antibody. The mixture was incubated at room temperature in the dark for 15 min, and washed with 1×stainbuffer. Cells were resuspended in 1×Fixation / Permeabilization solution (562574), incubated at 4°C in the dark for 40 min, washed with Perm / Washbuffer, centrifuged, and 100 μL of the mixture was collected. Resuspend cells in 1×Perm / Washbuffer. Add 1 μg / test of PE Rat Anti-Mouse IFN-γ (554412) and BV421 Rat Anti-Mouse Foxp3 (562996) intracellular staining antibodies to 1×Perm / Washbuffer, mix well, and incubate at 4°C in the dark for 40 min. Wash and centrifuge with 1×Perm / Washbuffer, resuspend cells in 300 μL of Stain Buffer, and detect by flow cytometry. FlowJo 10 software was used for analysis. All reagents were purchased from BD Biosciences, USA. Figure 6 Compared with PBS and AS01E, the vaccines EPPE / AS01E, EPPE+mi3 / AS01E, and their combination with BCG immunization of mouse lymphocytes and CD4+ were shown to have higher levels of these antibodies. + IFN-γ + Th1 cells were significantly elevated, and EPPE+mi3 / AS01E induced CD4. + IFN-γ + Th1 cells were significantly higher than those EPPE / AS01E and were largely consistent with BCG immunity. EPPE+mi3 / AS01E combined with BCG immunity induced CD4. + IFN-γ +Th1 cell levels were significantly higher than with BCG alone; while compared with PBS and AS01E, the EPPE / AS01E, EPPE+mi3 / AS01E and their combination with BCG immunization groups induced CD4+. + CD25 + FoxP3 + Treg cells were significantly reduced, and also significantly lower than in the BCG group. EPPE+mi3 / AS01E and EPPE / AS01E immunogen-induced CD4+ cells were also significantly reduced. + CD25 + FoxP3 + There was no difference in Treg cell levels.

[0088] MTT assay for splenic lymphocyte proliferation, using 3 × 10⁻⁶ cells. 5 Splenic lymphocytes were seeded into 96-well plates, and mixed 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 MTT (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 The results showed that the proliferation level of spleen lymphocytes immunized with EPPE / AS01E, EPPE+mi3 / AS01E and their combination with BCG was significantly higher than that of PBS and AS01E. Moreover, the proliferation level of spleen lymphocytes in the EPPE+mi3 / AS01E combined with BCG immunization group was significantly higher than that of BCG alone immunization.

[0089] Example 4

[0090] Investigating the ability of the multivalent VLP nanoparticle vaccine EPPE+mi3 / AS01E to inhibit the growth of Mycobacterium tuberculosis in spleen and lung cells in vitro.

[0091] As in Example 3, after immunization with C57BL / 6, spleen lymphocytes and lung cells were isolated. The spleen lymphocytes and lung cells were diluted in antibiotic-free RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum + 10 mM HEPES + 2 mM LL-glutamine. 300 μL of each medium was used to count 2 × 10⁻⁶ cells. 6Cells were added to 24-well plates, with 300 μL / well (50 CFU total) of H37Rv added. The plates were incubated at 37°C for 4 days using a CO2 incubator. The culture was then transferred to 400 μL of ice-cold sterile water in an EP tube and centrifuged at 12,000 rpm for 10 min to lyse the cells. The pellet was collected and resuspended in 500 μL of sterile water. 50 μL of the resuspended pellet was plated onto 7H10 medium (262710, BD, USA) containing 10% OADC enrichment agent (212351, BD, USA). 50 μL of the stock solution was diluted in 450 μL of sterile water and mixed thoroughly. 50 μL of this solution was then plated. The plates were incubated at 37°C for 2-3 weeks for colony counting. Figure 7 The results showed that, compared with PBS and AS01E, EPPE / AS01E and EPPE+mi3 / AS01E, as well as their combination with BCG, significantly inhibited the growth of H37Rv in splenic lymphocytes and lung cells of mice immunized in vitro. The ability of EPPE+mi3 / AS01E-immunized mice to inhibit H37Rv growth in vitro was significantly higher than that of EPPE / AS01E and BCG. At the same time, EPPE+mi3 / AS01E combined with BCG significantly increased the ability of BCG-immunized mice to inhibit H37Rv growth in vitro. There was no significant difference in lung cells.

[0092] This invention uses bioinformatics analysis to obtain 10 dominant Mtb antigens of different stages / types: ESAT-6, EsxV, PPE33, PPE29, PPE57, PPE68, Rv1794, Rv3134c, ESPC, and HSPX. ELISA is used to identify the IFN-γ expression level in PBMCs of subjects with dominant antigen-induced QFT+. Four different types of Mtb antigens, ESAT-6, PPE57, PPE68, and EsxV, are identified. The SpyCatcher / SpyTag system is used to display the four antigens on the surface of mi3 nanoparticles. Combined with the nanoliposome adjuvant AS01E, a multivalent VLP nanoparticle vaccine (EPPE+mi3 / AS01E) is prepared. The multivalent VLP nanoparticle vaccine EPPE+mi3 / AS01E enhances the ability of spleen lymphocytes and lung cells to inhibit H37Rv growth in vitro by upregulating Th1, Th2, and Th17 T lymphocyte immune responses and downregulating Treg cell immune responses. At the same time, the multivalent VLP nanoparticle vaccine EPPE+mi3 / AS01E can be used as a BCG booster vaccine to increase the ability of spleen lymphocytes to inhibit H37Rv growth in vitro after BCG immunization.

[0093] 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 tuberculosis VLP nanoparticle vaccine, characterized in that, Includes the following steps: 1) Spycatcher-mi3 and ESAT-6-SpyTag are self-assembled to obtain ESAT-6-mi3; Spycatcher-mi3 and PPE57-SpyTag were self-assembled to obtain PPE57-mi3; Spycatcher-mi3 and PPE68-SpyTag were self-assembled to obtain PPE68-mi3; Spycatcher-mi3 and EsxV-SpyTag were self-assembled to obtain EsxV-mi3; 2) Mix ESAT-6-mi3, PPE57-mi3, PPE68-mi3 and EsxV-mi3 obtained in step 1) to obtain multivalent nanoparticles; 3) Mix and emulsify the multivalent nanoparticles and AS01E adjuvant described in step 2) to obtain a tuberculosis VLP nanoparticle vaccine.

2. The preparation method according to claim 1, characterized in that, The amino acid sequence of Spycatcher-mi3 described in step 1) is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 6; The amino acid sequence of the ESAT-6-SpyTag is shown in SEQ ID No. 2; The amino acid sequence of the PPE57-SpyTag is shown in SEQ ID No. 3; The amino acid sequence of the PPE68-SpyTag is shown in SEQ ID No. 4; The amino acid sequence of the EsxV-SpyTag is shown in SEQ ID No.

5.

3. The preparation method according to claim 1, characterized in that, Step 1) The molar ratio of Spycatcher-mi3 to ESAT-6-SpyTag is 1:2; The molar ratio of Spycatcher-mi3 to PPE57-SpyTag is 1:

2. The molar ratio of Spycatcher-mi3 to PPE68-SpyTag is 1:

3. The molar ratio of Spycatcher-mi3 to EsxV-SpyTag is 1:

3.

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

5. The preparation method according to claim 1, characterized in that, In step 2), the mass ratio of ESAT-6-mi3, PPE57-mi3, PPE68-mi3 and EsxV-mi3 is 1:1:1:

1.

6. The preparation method according to claim 1, characterized in that, In step 3), the volume ratio of the multivalent nanoparticles to the AS01E adjuvant is 1:1.

5.

7. A tuberculosis VLP nanoparticle vaccine prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the tuberculosis VLP nanoparticle vaccine according to claim 7 in the preparation of anti-tuberculosis drugs.

9. The use of the multivalent mi3 VLP nanoparticle vaccine of claim 7 in combination with BCG vaccine in the preparation of anti-tuberculosis drugs.

10. The application according to claim 8 or 9, characterized in that, The tuberculosis bacteria include Mycobacterium tuberculosis H37Rv.

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