Vaccine or pharmaceutical composition based on mycobacterium lipid extract, preparation and application thereof

Abstract

The invention provides a vaccine or a pharmaceutical composition based on a mycobacterium lipid extract as well as preparation and application thereof, and relates to the technical field of biological medicine, the mycobacterium lipid extract is bacterial lipid extracted from mycobacterium thalli (including mycobacterium tuberculosis and nontuberculous mycobacterium) by a common lipid extraction method, and the mycobacterium lipid extract is prepared from the mycobacterium thalli (including mycobacterium tuberculosis and nontuberculous mycobacterium). The mycobacterium lipid extract can be used as a vaccine adjuvant, an immunomodulator, a training immunity inducer, a drug delivery carrier auxiliary component or a targeting ligand, and is suitable for preventing and treating various diseases.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedical technology, and in particular to vaccine or pharmaceutical compositions based on mycobacterial lipid extracts, their preparation and application. Background Technology

[0002] Bacteria and their derivatives have broad application potential in the regulation of immune responses, particularly in vaccine development, anti-tumor immunotherapy, and trained immunity. Exogenous bacterial molecules, such as lipopolysaccharide (LPS), peptidoglycan, and DNA, can enhance antigen presentation, immune cell activation, and the establishment of immune memory by activating pattern recognition receptors (such as TLRs) in the innate immune system, thereby improving vaccine efficacy. Bacterial derivatives can alter the phenotype and function of immune cells (such as monocytes and macrophages), inducing long-term immune memory effects and enhancing the body's defense against various pathogens. In anti-tumor immunity, bacterial derivatives can enhance tumor-specific immune responses by activating the host immune system, particularly by promoting the activation of dendritic cells and T cells. The recent concept of trained immunity reveals that bacterial infection or its derivatives can reshape the epigenetic state of the host immune system, enabling the immune system to provide stronger non-specific protection against future pathogen infections or tumor invasion.

[0003] Bacillus Calmette-Guérin (BCG) is a bacterial vaccine derived from *Mycobacterium bovis* through long-term attenuation culture, primarily used for the prevention of tuberculosis in children. Mycobacterium, to which Bacillus belongs, is a group of slender, branching aerobic bacilli. Medically, they are often classified based on diagnosis and treatment into the tuberculous mycobacterial complex (including *Mycobacterium tuberculosis*, *Mycobacterium bovis*, etc.), non-tuberculous mycobacteria (*Mycobacterium abscessum*, *Mycobacterium smegmatis*, etc.), and *Mycobacterium leprae*. Mycobacteria can trigger a strong immune response in the host's immune system and have become a key focus of research in immunotherapy and vaccine development.

[0004] Among them, the complete Freund's adjuvant, constructed from inactivated Mycobacterium tuberculosis or BCG whole-cell components and mineral oil, is a potent immune adjuvant commonly used to enhance the immune response to animal vaccines. BCG polysaccharide-nucleic acid (BCG) can regulate the host's immune response by enhancing the function of antigen-presenting cells and is also widely used in immunological research. Furthermore, BCG plays an important role in training immunization research. BCG can activate the host's innate immune system, alter the phenotype and function of immune cells, provide cross-pathogen protection, and enhance immune tolerance and immune memory.

[0005] However, current research indicates that BCG-mediated antitumor effects and immune-training effects are only effective when using live strains, which poses a risk of live bacterial spread and infection in immunocompromised populations. Meanwhile, complete Freund's adjuvants prepared using inactivated mycobacterial derivatives are limited to animal immunization due to excessive inflammatory side effects. Therefore, there is an urgent need to achieve highly effective and low-toxicity application of BCG derivatives through advanced formulation technologies. Summary of the Invention

[0006] The present invention provides vaccine or pharmaceutical compositions based on mycobacterial lipid extracts, their preparation and application, to at least partially solve the problems existing in the prior art.

[0007] The first aspect of this invention provides a vaccine system based on mycobacterial lipid extract, the vaccine system comprising: mycobacterial lipid extract and antigen, wherein the mycobacterial lipid extract is bacterial lipid extracted from mycobacterial cells, and the main components of the mycobacterial lipid extract are glycerophospholipids, fatty amides and bioactive glycolipids.

[0008] Optionally, the antigen includes: polypeptide antigen, protein antigen, polysaccharide antigen, and nucleic acid antigen.

[0009] Optionally, the vaccine is a vaccine formulation prepared by using a formulation process to prepare an adjuvant from the mycobacterial lipid extract, which is then co-loaded or mixed with an antigen.

[0010] A second aspect of the present invention provides a pharmaceutical composition based on a mycobacterial lipid extract, the pharmaceutical composition comprising: a mycobacterial lipid extract and a pharmaceutical molecule, wherein the mycobacterial lipid extract is a bacterial lipid extracted from mycobacterial cells, and the main components of the mycobacterial lipid extract are glycerophospholipids, fatty amides and bioactive glycolipids.

[0011] Optionally, the pharmaceutical composition is a nanoparticle, liposome, nanoemulsion, or microemulsion prepared by combining the mycobacterial lipid extract with drug molecules through a nano-sizing process.

[0012] A third aspect of the present invention provides a method for preparing a vaccine system based on mycobacterial lipid extract as described in the first aspect or a pharmaceutical composition based on mycobacterial lipid extract as described in the first aspect, the method comprising: Bacterial lipids were extracted from mycobacterial cells to obtain mycobacterial lipid extract; The mycobacterial lipid extract is prepared into a vaccine system with an antigen, or the mycobacterial lipid extract is prepared into a pharmaceutical composition with drug molecules.

[0013] The fourth aspect of this invention provides the application of a mycobacterial lipid extract in the preparation of vaccines, wherein the mycobacterial lipid extract serves as a vaccine adjuvant to improve the immune efficacy of the vaccine and enhance its humoral and cellular immune effects.

[0014] The fifth aspect of this invention provides the application of a mycobacterial lipid extract in the preparation of a pharmaceutical composition, wherein the mycobacterial lipid extract acts as an auxiliary component of a drug delivery carrier or a targeting ligand in conjunction with drug molecules.

[0015] The sixth aspect of this invention provides the application of a mycobacterial lipid extract in the preparation of an immunomodulator.

[0016] The seventh aspect of this invention provides the application of a mycobacterial lipid extract in the preparation of a training immune inducer.

[0017] In this embodiment of the invention, the lipid extract is a bacterial lipid extracted from mycobacteria (including Mycobacterium tuberculosis and non-tuberculous mycobacteria) using common lipid extraction methods. The mycobacterial lipid extract can be used as a vaccine adjuvant, immunomodulator, immune training inducer, drug delivery carrier auxiliary component or targeting ligand, and is suitable for the prevention and treatment of a variety of diseases. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results of mycobacterial lipid extraction and major component identification experiments are shown in the figure. Figure 2 A schematic diagram showing the morphology characterization results of PLGA nanoparticles containing BCG lipids is presented. Figure 3 A schematic diagram showing the application results of PLGA nanomedicine complex containing BCG lipids is presented. Figure 4 A schematic diagram showing the characterization results of drug-carrying nanoparticles containing BCG lipids is presented. Figure 5 A schematic diagram showing the results of the cell-killing ability evaluation of BCG-containing drug-loaded liposomes is presented. Figure 6 A schematic diagram of the ICD effect results of BCG-containing drug-loaded liposomes is shown. Figure 7A schematic diagram showing the antitumor research results of drug-carrying nanoparticles containing BCG lipids is presented. Figure 8 A schematic diagram showing the morphology characterization results of BCG-containing lipid nanoemulsions is presented. Figure 9 A schematic diagram showing the in vivo APC recruitment and antigen presentation promotion capabilities of BCG lipid nanoemulsion adjuvant is presented. Figure 10 A schematic diagram of the in vivo immunogenicity of BCG lipid nanoemulsion adjuvant – protein antigen results is shown. Figure 11 A schematic diagram of the in vivo immunomodulatory effect of BCG lipid nanoemulsion adjuvant-peptide antigen results is shown. Figure 12 A schematic diagram showing the tumor prevention experimental results of the BCG lipid nanoemulsion vaccine is presented. Figure 13 A schematic diagram showing the tumor inhibition experimental results of the BCG lipid nanoemulsion vaccine is presented. Figure 14 A schematic diagram showing the results of the development of the BCG lipid nanoemulsion viral vaccine is presented. Figure 15 A schematic diagram showing the results of BCG lipid nanoemulsion bacterial vaccine development is provided. Figure 16 A schematic diagram showing the application results of BCG nanoemulsion as a mucosal adjuvant is shown. Figure 17 A schematic diagram illustrating the application results of BCG lipid nanoemulsion as a training immune inducer is shown. Detailed Implementation

[0020] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents and other instruments whose manufacturers are not specified are all commercially available products.

[0022] In this embodiment of the invention, based on the high adjuvant effect of attenuated BCG strains, a biomimetic nanoemulsion with immunomodulatory capabilities is constructed by extracting amphiphilic non-covalent lipids from the cell wall surface of the strain. This nanoemulsion is then combined with bacterial antigens, tumor antigens, and therapeutic small molecule drugs to regulate the body's immune response, and is intended for antibacterial, antitumor, and immune training applications.

[0023] This invention provides a vaccine or pharmaceutical composition based on mycobacterial lipid extract, its preparation, and its application.

[0024] In this embodiment of the invention, the vaccine system includes: a mycobacterial lipid extract and an antigen. The mycobacterial lipid extract is a bacterial lipid extracted from mycobacterial cells. The main components of the mycobacterial lipid extract are glycerophospholipids, fatty amides, and bioactive glycolipids.

[0025] In this embodiment of the invention, mycobacteria include: Mycobacterium tuberculosis (Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis) and non-tuberculous mycobacteria (Mycobacterium abscessus, Mycobacterium smegmatis, Mycobacterium marinum, etc.).

[0026] In this embodiment of the invention, the lipid extraction methods include: organic solvent extraction methods, supercritical carbon dioxide extraction methods, etc.

[0027] In this embodiment of the invention, the antigen includes: polypeptide antigen, protein antigen, polysaccharide antigen, and nucleic acid antigen.

[0028] In this embodiment of the invention, the vaccine includes antiviral vaccines, antibacterial vaccines, and antitumor vaccines, etc.

[0029] In this embodiment of the invention, the vaccine is a vaccine formulation prepared by using a formulation process to prepare an adjuvant from the mycobacterial lipid extract, which is then co-loaded or mixed with an antigen.

[0030] In this embodiment of the invention, the vaccine is a squalene-based mycobacterial lipid nanoemulsion.

[0031] In this embodiment of the invention, the pharmaceutical composition comprises: a mycobacterial lipid extract and a pharmaceutical molecule. The mycobacterial lipid extract is a bacterial lipid extracted from mycobacterial cells. The main components of the mycobacterial lipid extract are glycerophospholipids, fatty amides, and bioactive glycolipids.

[0032] In this embodiment of the invention, the drug molecule includes any clinically applicable drug molecule and imaging molecule.

[0033] In this embodiment of the invention, the pharmaceutical composition is: nanoparticles, liposomes, nanoemulsions, or microemulsions prepared by combining the mycobacterial lipid extract with drug molecules through a nano-sizing process.

[0034] This invention also provides a method for preparing the vaccine system or pharmaceutical composition based on mycobacterial lipid extract described in the above embodiments, characterized in that the method includes: S1, lipids were extracted from mycobacterial cells to obtain mycobacterial lipid extract.

[0035] Taking BCG as an example, step S1 specifically includes: amplifying and culturing the BCG strain using Middlebrook 7H9 (M7H9) medium, harvesting the bacterial cells by centrifugation, and washing three times with sterile water for injection. Weigh 500 mg of BCG cells and place them in a high-pressure resistant glass tube, add 15 mL of a methanol:chloroform (2:1) mixed organic solvent, shake overnight at 37°C, and collect the supernatant by centrifugation. Add another 15 mL of a methanol:chloroform (1:2) mixed organic solvent for a second extraction, collect the supernatant, combine them, filter through a 0.22 µm organic filter membrane, evaporate to dryness using a rotary evaporator, and collect the lipids to obtain the mycobacterial lipid extract.

[0036] S2, the mycobacterial lipid extract is prepared into a vaccine system with an antigen, or the mycobacterial lipid extract is prepared into a pharmaceutical composition with a drug molecule.

[0037] To verify and illustrate the vaccine system or pharmaceutical composition based on mycobacterial lipid extract provided in the embodiments of the present invention, specific embodiments are provided below for illustration: Example 1: Lipid extraction and identification of major components from mycobacteria In this embodiment of the invention, the lipid extract of mycobacteria is derived from BCG strain as an example.

[0038] Experimental Methods: BCG strains were amplified and cultured using Middlebrook 7H9 (M7H9) medium. Cells were harvested by centrifugation and washed three times with sterile water for injection. 500 mg of BCG cells were weighed and placed in a high-pressure glass tube. 15 mL of a methanol:chloroform (2:1) mixture was added, and the mixture was shaken overnight at 37°C. The supernatant was collected by centrifugation. A second extraction was performed by adding 15 mL of a methanol:chloroform (1:2) mixture. The supernatants were collected and combined, filtered through a 0.22 µm organic filter membrane, and evaporated to dryness using a rotary evaporator. The lipids were collected and weighed. The lipids were dissolved in a chloroform:methanol:water (16:8:1) mixture to prepare a 4 mg / mL BCG lipid stock solution, which was stored at -20°C in a sealed container. The presence of residual protein in the lipid stock solution was detected using SDS-PAGE protein electrophoresis and BCA protein assay. Lipids were analyzed using LC-MS in both positive and negative ion modes, and lipids were identified by comparison with Liqidmaps and mycobacterial-specific lipid libraries.

[0039] Figure 1 The results of mycobacterial lipid extraction and major component identification experiments are shown in the figure. Figure 1As can be seen, the extracted BCG lipids dissolved in a solvent:chloroform:methanol:water = 16:8:1 form a clear, pale yellow solution. After rotary evaporation and elution with water, no protein components were detected by SDS-PAGE electrophoresis of the eluent. However, the protein concentration decreased after elution with water, as determined by the BCA method, indicating that the extracted BCG lipids may contain certain water-soluble lipopeptides. Lipomics analysis revealed that the main components of the extracted BCG lipids were glycerophospholipids, fatty amides, and bioactive glycolipids. Glycerophospholipids and fatty amides can serve as auxiliary stabilizing components in nanoparticle formulations, while glycolipids can exert immunomodulatory effects.

[0040] Example 2: Validation of Mycobacterium lipid extract as an auxiliary component of nanomedicine delivery carriers Mycobacteria are intracellular parasitic bacteria that primarily enter cells through surface lipids and protein receptors. Constructing nanomedicine delivery carriers using mycobacterial lipid extracts can inherit the lipid-mediated cell entry mechanism of bacteria, promoting cellular uptake of nanomedicines. Common nanomedicine delivery carriers include PLGA nanoparticles, liposomes, and nanoemulsions.

[0041] (1) PLGA nanoparticles with mycobacterial lipids as an auxiliary component 1) Preparation method and morphology characterization of PLGA nanoparticles Preparation of B-PLGA nanoparticles: 1 mL of lipid extract was evaporated to dryness using a rotary evaporator, and 1 mL of dichloromethane containing 15 mg PLGA was added for redissolution. 300 μL of 0.05% PVA was added, and the mixture was vortexed thoroughly and sonicated at 100W for 2 min to form a water-in-oil pre-emulsion. The pre-emulsion was added to 3 mL of 0.5% PVA, vortexed thoroughly, and sonicated at 180W for 5 min to form a WOW emulsion. Organic reagents were removed by rotary evaporation, and sterile water for injection was added to bring the volume to 4 mL to form PLGA nanoparticles containing BCG lipids, named BP-Nano.

[0042] Figure 2 A schematic diagram showing the morphology characterization results of PLGA nanoparticles is presented. Figure 2 It can be seen that the PLGA nanoparticles containing BCG lipids prepared in this embodiment are smaller and more uniform in size and have a higher surface negative charge than pure PLGA nanoparticles, indicating that BCG lipids can be used as an auxiliary stabilizing component for nanomedicines.

[0043] 2) Application Cases of PLGA Nanomedicine Complexes Containing BCG Lipids Experimental Methods: DID dye (for tracing nanoparticles) or RRX-001 (a hypoxia-selective epigenetic factor used as a radiosensitive or chemotherapeutic agent, inducing apoptosis, overcoming drug resistance in myeloma, and possessing anticancer activity; RRx-001 is also an immune checkpoint inhibitor that downregulates the CD47-SIRP-α signaling pathway) was added to the lipid extraction solution. BR-PLGA and BR-PLGA@DID nanoparticles containing DID dye were prepared using this lipid mixture. Various nanoparticles were incubated with mouse macrophage Raw264.7, mouse colon cancer cells MC38, and mouse lung cancer cell lines TC-1, respectively. Cells in the 2-h incubation group were harvested by trypsin digestion, and nanoparticle uptake was assessed by flow cytometry in the presence of DID dye. Cell viability in the 24-h incubation group was assessed using CCK-8 reagent.

[0044] Figure 3 This diagram illustrates the application results of PLGA nanomedicine complexes containing BCG lipids. Figure 3 It can be seen that, in the three cell lines, the use of DID-labeled blank PLGA nanoparticles (PLGA@DID), PLGA nanoparticles containing E80 (E-PLGA@DID), and BR-PLGA@DID containing BCG lipids showed that BCG lipids promoted the cellular uptake efficiency of nanoparticles, especially with significant differences in the RAW and TC-1 cell lines. Since mycobacteria mainly infect macrophages and epithelial cells, the nanoparticles constructed based on BCG lipids showed higher uptake on macrophages and epithelial cells, which may be related to the higher levels of pathogen pattern molecular receptors on the surface of epithelial cells and macrophages. Meanwhile, in the three cell lines, BR-PLGA nanoparticles containing BCG lipids reduced the toxicity of RRx-001 to macrophages and showed some proliferative effect at low concentrations, but enhanced the cytotoxic effect of RRx-001 on tumor cells MC38 and TC-1, indicating that BCG lipids can improve the cancer cell killing efficiency of anticancer drugs.

[0045] (2) Liposome nanomedicine complex with mycobacterial lipids as an auxiliary component 1) Preparation and characterization of drug-carrying nanoparticles containing BCG lipids This invention employs a thin-film hydration method to prepare liposomes. The specific steps are as follows: DOTAP, DOPC, cholesterol, BCG lipid stock solution, and lipophilic octadecyl-modified oxaliplatinic acid (OOC) were dissolved in chloroform at a mass ratio of 0.5:1.5:1:1:0.5 and mixed thoroughly in a rotary evaporator flask. The solvent was then completely removed using a rotary evaporator under reduced pressure (37°C, 100 rpm), resulting in a uniform thin film forming at the bottom of the flask. After 2 hours of complete evaporation of the organic solvent, 2 mL of sterile water for injection was added to the film for hydration. The hydrated solution was transferred to a 5 mL sterile EP tube and further processed using a probe sonication method (250 W, 10 min, ice bath). This yielded cationic liposomes co-loaded with BCG lipids and OOC, named DBO. The formulation of drug-loaded liposomes without cationic and BCG lipids has a mass ratio of DOPC:cholesterol:OOC = 3:1:0.5, and is named LO. The formulation of drug-loaded liposomes without BCG lipids has a mass ratio of DOTAP:DOPC:cholesterol:OOC = 0.5:2.5:1:0.5, and is named DO. The formulation of drug-loaded liposomes without cationic lipids has a mass ratio of DOPC:cholesterol:BCG lipids:OOC = 2:1:1:0.5, and is named BO. The formulation of liposomes without prodrug OOC has a mass ratio of DOTAP:DOPC:cholesterol:BCG lipids = 0.5:1.5:1:1, and is named DB.

[0046] Figure 4 A schematic diagram showing the characterization results of BCG-containing drug-carrying liposomes is presented. Figure 4 As can be seen, the particle size of the DBO formulation is approximately 140 nm, the potential is approximately +28 mV, and the PDI is around 0.23. Furthermore, the DBO formulation exhibits good stability at both 37°C and 4°C. Under electron microscopy, all three formulations—DO, DB, and DBO—appear as smooth, near-spherical particles.

[0047] 2) Investigation of the cytotoxic ability of BCG-containing drug-carrying nanoparticles Experimental method: Actively growing MB49 cells were seeded into 96-well plates, with 1 × 10⁶ cells seeded per well. 4Cells were cultured in 96-well plates, and 200 µL of complete culture medium was added. After overnight culture, the original culture medium was aspirated, and 100 µL of DMEM complete culture medium containing different concentrations of the formulation and free drug was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 h. The cells were washed once with PBS buffer, and then 100 µL of bubble-free CCK-8 working solution was precisely added to each well. The plates were then incubated in the dark for 1–3 h until a significant color change occurred. The absorbance of each well at 450 nm was measured using a microplate reader, and the cell viability was calculated according to the cell viability formula.

[0048] Figure 5 This diagram illustrates the results of the cell-killing ability assessment of BCG-containing drug-loaded liposomes. Figure 5 It can be seen that the DB carrier containing DOTAP lipids and BCG lipids did not have a significant cytotoxic effect on MB49 cells, while the DBO preparation loaded with prodrugs had the best cell-killing effect compared with other groups.

[0049] 3) ICD effect of BCG-containing lipid nanoparticles Experimental method: MB49 cells in the vigorous proliferation phase were cultured at a density of 5 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 24-well plates. After 12 h of culture, the original culture medium was removed, and culture media containing OXA, LO, DO, and DBO were added, with OXA at ​​a concentration of 10 µM. The 24-well plates were placed in a cell culture incubator (37°C, 5% CO2) and incubated for 24 h. The cell culture supernatant was then collected and centrifuged at 2000 rpm for 3 minutes to obtain the supernatant sample. The concentrations of HMGB1 and ATP in the supernatant were then quantitatively analyzed using an HMGB1-specific ELISA kit and an ATP assay kit, strictly following the manufacturer's instructions.

[0050] Figure 6 A schematic diagram of the ICD effect results of BCG-containing drug-loaded liposomes is shown. Figure 6 It can be seen that the DBO preparation containing BCG lipids can induce more ATP and HMGB1 production in tumor cells MB49 more significantly than the DO preparation without BCG lipids, indicating that the presence of BCG lipids can promote the ICD effect induced by chemical drugs.

[0051] 4) Antitumor research on drug-carrying liposomes containing BCG lipids In this embodiment of the invention, bladder cancer is used as an example for illustration.

[0052] Experimental methods: Healthy female C57BL / 6 mice aged 6-8 weeks were selected as experimental subjects. After hair removal on the right lower abdomen of the mice, 1×10⁻⁶ cells were injected into the hair removal area. 6 MB49-luc cells were collected. Mice were randomly assigned to five experimental groups based on tumor growth status, excluding those without tumor formation or with abnormal tumor volume: a PBS control group (no drug administration), an OXA group (free drug), a plain liposome (LO) group (unmodified), a DOTAP-modified (DO) group (DOTAP and BCG-modified), and a DBO group (DOTAP and BCG-modified). Each group contained six tumor-bearing mice. Intratumoral injections were administered every three days at a volume of 25 µL per mouse, for a total of six administrations. The first administration was recorded as day 1. The dosage per mouse was OXA = 2 mg / kg and BCG = 8 mg / kg.

[0053] Figure 7 This diagram illustrates the antitumor research results of BCG-containing drug-carrying nanoparticles. Figure 7 It can be seen that, compared with the untreated PBS group, the free OXA, LO, and DO groups were able to delay tumor development to some extent in the early stages, but tumor growth became rapid after drug administration was stopped. The DBO group loaded with DOTAP and BCG lipids significantly inhibited tumor growth, and in the later monitoring period, the tumors of three mice even completely disappeared. As shown in Figure C, the DBO group significantly prolonged the median survival of mice (65 days), which was 1.8 times that of the PBS group. This indicates that the combined use of BCG lipids and oxaliplatin prodrug can significantly enhance the anti-tumor effect of chemical drugs, demonstrating the feasibility of the strategy of delivering oxaliplatin prodrug using cationic liposomes loaded with BCG lipids.

[0054] Example 3: Nanoemulsion adjuvant with mycobacterial lipids as the main active ingredient (1) Preparation method and morphology characterization of BCG-containing lipid nanoemulsions Oil phase: 50 mg squalene, 10 mg E80, and 1 / 4 / 8 mg BCG lipids (prepared by rotary evaporation of organic solvent using BCG lipid stock solution) were thoroughly dissolved in 1 mL dichloromethane. 3 mL of an external aqueous phase (prepared with sterile water for injection, containing 0.5% poloxamer F68 and 10 mg / mL sodium deoxycholate) was added. After thorough vortex mixing, the mixture was sonicated at 200 W for 3 seconds, 3 seconds, and 10 minutes. The organic solvent was removed by rotary evaporation, and the volume was adjusted to 4 mL to obtain the BCG lipid-containing nano-adjuvant BNE. A blank nanoemulsion prepared using the same procedure but without the added BCG lipids was named NE. Morphology was characterized using transmission electron microscopy and a particle size analyzer.

[0055] Figure 8A schematic diagram showing the morphology characterization results of BCG-containing lipid nanoemulsions is presented. Figure 8 It can be seen that NE nanoemulsions exhibit a uniform spherical structure of about 150 nm, while BNE nanoemulsions have a particle size of about 160 nm. Due to the amphiphilic BCG lipids on the surface, TEM results show that they have a thin membrane structure.

[0056] (2) Application Cases of Nanoemulsion Adjuvants Containing BCG Lipids 1) Investigation on the in vivo APC recruitment and antigen presentation promotion capabilities of BCG lipid nanoemulsion adjuvant Experimental Methods: Using chicken ovalbumin (OVA) as the model antigen, mice were subcutaneously immunized on day 0 with PBS, OVA, NE+OVA, BNE adjuvant groups with different lipid concentrations: B(20)NE+OVA, B(40)NE+OVA, B(80)NE-OVA, B(80)NE+OVA, and the positive control CFA (complete Freund's adjuvant). Each mouse was administered 50 μL of the OVA antigen and 10 μg of the BCG lipid content. The dosage per mouse after conversion is shown in parentheses. On day 3, mice were euthanized by cervical dislocation, and lymph nodes on the same side as the injection site were isolated, ground, and sieved. The obtained lymphocytes were stained with anti-mouse CD11c-Superbright 600, F4 / 80-PE-Cy5, CD86-APC, and H2Kb-PE. Flow cytometry was used to detect the maturation and cross-presentation efficiency of antigen-presenting cells.

[0057] Figure 9 This diagram illustrates the results of an in vivo APC recruitment and antigen presentation enhancement study using BCG lipid nanoemulsion adjuvant. Figure 9 Three days after subcutaneous injection of each formulation, lymph nodes near the injection site were dissected, ground, and stained for analysis. Flow cytometry analysis of the inguinal lymph nodes revealed that BCG lipids promoted the enrichment of antigen-presenting cells (DCs and macrophages) within the lymph nodes. Although there was no significant difference in DC maturation, the formulations containing BCG lipids showed significantly improved cross-presentation efficiency, even exceeding that of the CFA formulation group. Simultaneously, BCG lipids also promoted the production of M1 macrophages, which is beneficial for the generation of inflammatory cytokines and promotes antigen presentation.

[0058] 2) In vivo immunomodulatory effects of BCG lipid nanoemulsion adjuvant - protein antigen (taking OVA as an example) Experimental Methods: For the protein antigen, chicken ovalbumin (OVA) was used as the model antigen. Mice were subcutaneously immunized with PBS, OVA, NE+OVA, and BNE adjuvant groups with different lipid concentrations: B(20)NE+OVA, B(40)NE+OVA, B(80)NE-OVA, and B(80)NE+OVA on day 0 via paw immunization. Each mouse received 50 μL of OVA antigen and 10 μg of OVA antigen. The dosage per mouse after conversion based on BCG lipid content is shown in parentheses. Boost immunizations were administered on days 14 and 21. On day 30, blood was collected from the orbital sinus to collect mouse serum. The OVA-specific antibodies IgG, IgG1, and IgG2a in the serum isolated from the immunized mice were detected by ELISA. IgG1 and IgG2a reflect Th2 and Th1 immune responses, respectively. On day 42 post-immunization, equal amounts of target cells labeled with high concentration of CFSE dye and incubated with SIINFEKL antigen peptide and internal control cells labeled with low concentration of CFSE dye and incubated with PBS were injected via tail vein. The antigen-specific killing power of CTLs was calculated by flow cytometry to detect the ratio of cells with high concentration of CFSE dye to cells with low concentration of CFSE dye in CFSE-positive cells.

[0059] Figure 10 This diagram illustrates the in vivo immunomodulatory effects of BCG lipid nanoemulsion adjuvant against protein antigens. Figure 10 It can be seen that nanoemulsions containing different BCG lipids have significant adjuvant effects, with the 40 μg BCG lipid group exhibiting the best immune-enhancing effect and showing a clear Th1 bias. CTL experiments showed that the cell-specific killing effect of BCG lipid nanoemulsions gradually increased with increasing lipid concentration, with the 40 μg BCG lipid group showing a similar immune-enhancing effect to the 80 μg group.

[0060] 3) In vivo immunomodulatory effects of BCG lipid nanoemulsion adjuvant - peptide antigen (taking E7 as an example) Experimental Methods: For the peptide antigen, the cervical cancer virus E7 peptide (E744-62: QAEPDRAHYNIVTFCCKCD) was used as the model antigen. CpG adjuvant was added to the nanoemulsion to further enhance its immunoadjuvant effect. The groups were as follows: 1. PBS, 2. E7, 3. NE+E7, 4. NE-CpG+E7 (positive control), 5. BNE+E7, 6. BNE-CpG+E7. Mice were subcutaneously immunized via the paw on day 0, with 50 μL of E7 antigen and 20 μg per mouse. The dosage per mouse after BCG lipid content conversion is shown in parentheses. The experimental group containing CpG received 1 μg per mouse. Booster immunizations were administered on days 14 and 21. Serum was collected from the orbital blood bank on day 20. The E7-specific antibodies IgG, IgG1, and IgG2a were detected in the serum isolated from the immunized mice using ELISA.

[0061] Figure 11 This diagram illustrates the in vivo immunomodulatory effects of BCG lipid nanoemulsion adjuvant on peptide antigens. Figure 11 It can be seen that BCG nanoemulsion combined with CpG has the best immune-enhancing effect. However, for peptides with shorter amino acid sequences, the antibody levels produced are lower due to the lower immunogenicity of the peptides, although the combined group still produced higher antibody levels.

[0062] 4) Tumor prevention experiment using BCG lipid nanoemulsion adjuvant (taking OVA as an example) Experimental Methods: The immunization protocol described in section 2) of this embodiment was used. Seven days after immunization, 5 x 10⁵ EG7-OVA tumor cells were subcutaneously inoculated. Starting from day 7, the tumor volume of mice was measured using calipers, and the survival time of tumor-bearing mice was also assessed.

[0063] Figure 12 This diagram illustrates the experimental results of tumor prevention using BCG lipid nanoemulsion adjuvant. Figure 12 It can be seen that BNE+OVA showed strong tumor-suppressive activity and significantly prolonged the survival time of tumor-bearing mice, while the other groups did not show significant tumor suppression.

[0064] 5) Tumor suppression experiment of BCG lipid nanoemulsion adjuvant (taking OVA as an example) Experimental method: 5 x 10⁵ mice were subcutaneously injected into the right abdomen. 5 EG7-OVA tumor cells, designated as day 0. Mice were subcutaneously immunized with a vaccine containing the OVA antigen in the ipsilateral paw on days 3, 7, and 11. Tumor volume and tumor-bearing survival time were monitored simultaneously starting on day 7.

[0065] Figure 13 This diagram illustrates the tumor inhibition experimental results of BCG lipid nanoemulsion adjuvant. Figure 13 It can be seen that the OVA and NE+OVA groups containing the antigen had a slight delaying effect on tumor growth, while the BNE+OVA group showed the strongest inhibitory ability against tumors. The survival trend of tumor-bearing mice was also consistent with the tumor growth curve, with the median survival of each group being: PBS group: 16 days; OVA group and NE+OVA group: 17.5 days; BNE+OVA group: 22 days.

[0066] 6) Development of viral vaccines using BCG lipid nanoemulsion adjuvants (taking gE antigen as an example) Shingles is an acute infectious skin disease caused by the varicella-zoster virus. Vaccination, which induces virus-specific humoral and cellular immunity, is an effective means and method for preventing shingles. The gE glycoprotein antigen is most abundant on the viral envelope and is the dominant shingles virus antigen.

[0067] Experimental methods: BCG lipid nanoemulsion adjuvant was prepared using the experimental method in Part (1) of this embodiment; B-lipo was prepared as a control by referring to the method of BCG liposomes. The specific method was as follows: 4 mg / mL BCG lipid extraction solution and 8 mg of lecithin dissolved in dichloromethane and 3 mg of cholesterol were thoroughly mixed, dried by rotary evaporator to form a film, sterile water for injection was added and hydrated under ultrasonic assistance, and then BCG liposome adjuvant was formed by ultrasonication at 200 W for 3s / 3s for 10 min, named B-lipo as a control.

[0068] Mice were immunized in two groups: a pre-immunization group receiving BCG lyophilized powder (Chengdu Institute of Biological Products Co., Ltd.) to simulate clinical neonatal BCG vaccination, and a normal treatment group. The pre-immunization group received 1 x 10⁻⁶ BCG via subcutaneous injection at the base of the tail 30 days prior to the immunization. 6 CFU / mouse. All groups were immunized with herpes simplex antigen preparations (gE, NE+ gE, BNE+ gE, B-lipo+ gE) starting on day 30; booster immunizations were given on days 44 and 51.

[0069] Figure 14 This diagram illustrates the results of developing a viral vaccine using BCG lipid nanoemulsion adjuvant. Figure 14 It can be seen that BCG nanoemulsion significantly promotes the production of gE antigen-specific IgG, with a significant difference; increases IgG1 levels; and significantly enhances the production of IgG2a, showing a clear Th1 bias. BCG pre-immunization significantly improves the immune-enhancing effect of BCG lipid-containing nanoadjuvants, including B-lipo, indicating that pre-vaccination with BCG can improve the immune efficacy of BCG lipid-based nanovaccines.

[0070] 7) Development of bacterial vaccines using BCG lipid nanoemulsion adjuvants (taking tuberculosis antigen as an example) Based on the fact that BCG lipids are derived from BCG, this invention designs and expresses the tuberculosis protein antigen ESAT6-CFP10 (EF). C57 mice were immunized according to the immunization method described in section 2) of this embodiment above. Specifically, 50 μL of each preparation was subcutaneously injected into the right paw of the mouse, with immunizations administered on days 0, 14, and 21. Blood samples were collected on day 28 to detect antibodies; on day 29, 1×10⁻⁶ mg / L was administered intravenously. 6 Mice were challenged with CFU H37Ra. On day 44, the mice were sacrificed, and lung tissue from 3 mice in each group was fixed and acid-fast stained to assess the bacterial load in the lungs. The lung tissue from the other three mice in each group was ground and inoculated into M7H10 plates containing kanamycin. After 30 days of culture, colony counts were performed.

[0071] Figure 15 This diagram illustrates the results of developing a bacterial vaccine using BCG lipid nanoemulsion adjuvant. Figure 15 It can be seen that BNE adjuvant significantly increased the levels of EF antigen-specific IgG and IgG2a antibodies. Acid-fast staining of lung tissue showed that the BNE group had the fewest red Mycobacterium tuberculosis spots and nodular foci. Simultaneously, plate colony counting showed that culturable colonies in the spleen and lungs of mice immunized with BNE indicated that the combination of BNE adjuvant and the tuberculosis antigen EF has a protective effect against Mycobacterium tuberculosis infection.

[0072] 8) Application of BCG nanoemulsion as a mucosal adjuvant (taking OVA antigen as an example) Most vaccines, administered via the non-enteric route, elicit a strong systemic humoral immune response, but they induce weak T-cell-mediated immunity and are ineffective in inducing mucosal protection. Given that most pathogens enter the body through the mucosal surface, a vaccine capable of inducing protection at the first point of contact between the host and the pathogen is promising. Mycobacterium tuberculosis causes lung infection by invading through the lung mucosa; this invention utilizes a lipid component of attenuated BCG strains to construct a nano-adjuvant that can elicit a mucosal immune response.

[0073] Experimental Methods: For the protein antigen, chicken ovalbumin (OVA) was used as the model antigen. Mice were immunized intratracheally on day 0 with PBS, MF59+OVA, NE+OVA, MF59-B+OVA loaded with lipids, and B(20)NE+OVA adjuvant. Each mouse received 20 μL of PBS, 10 μg of OVA antigen, and 20 μg of BCG lipids. Boosted immunizations were administered intratracheally on days 14 and 28. On day 34, blood was collected from the orbital rim to collect mouse serum. The OVA-specific antibodies IgG, IgG1, and IgG2a in the serum isolated from the immunized mice were detected using ELISA. On day 35, mice were euthanized by cervical dislocation, and bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF), vaginal lavage fluid, and intestinal extract were collected. The OVA-specific antibody IgA in the mucosal lavage fluid of the immunized mice was detected using ELISA.

[0074] Figure 16 This diagram illustrates the application results of BCG nanoemulsion as a mucosal adjuvant. Figure 16 It can be seen that intratracheal immunization with nanoemulsions can effectively increase serum antibody levels, and the effect is superior to MF59. The addition of BCG lipids can induce increased secretion of IgG2a antibodies in mice, shifting the immune response towards the Th1 type. The nanoemulsion group increased IgA secretion in the pulmonary mucosa. In nasal lavage fluid, vaginal lavage fluid, and intestinal extract, the average level of mucosal IgA antibodies in the NE-B group was higher than that in the commercial MF59 group. These experimental results indicate that BCG nanoemulsions can serve as mucosal adjuvants.

[0075] 9) BCG lipid nanoemulsion as a training immune inducer Training immunity, also known as trained innate immunity, refers to the phenomenon where innate immune cells (macrophages, dendritic cells, granulocytes, etc.) exhibit immune memory characteristics after stimulation, resulting in long-term enhanced immune function and providing non-specific protection. BCG is a commonly used training immune inducer, and the lung metastasis model constructed by intravenous injection of tumor cells is a classic model for evaluating the protective effect of BCG-induced training immunity. This invention extracts lipid components from BCG to construct a nano-adjuvant that can inherit the training immune characteristics of BCG strains while avoiding the in vivo safety issues associated with using live BCG bacteria.

[0076] Experimental methods: Six- to eight-week-old C57 female mice were selected and divided into a group receiving intravenous injection of BCG bacteria (1x10⁻¹²). 6 CFU / animal), subcutaneous BCG bacterial group (1x10) 6 The treatment included three groups: CFU / animal, intravenous NE injection (100 μL / animal, once weekly), and intravenous BNE injection (100 μL / animal, containing 100 μg BCG lipids, once weekly). After 4 weeks, 4 x 10 CFU / animal were injected via tail vein. 5TC-1 cells were collected, and mice were sacrificed after 24 days. Lung tissue was fixed with Bouin's fixative to examine lung metastasis.

[0077] Figure 17 This diagram illustrates the application results of BCG lipid nanoemulsion as a training immune inducer. Figure 17 It can be seen that the intravenously injected BCG group has the best anti-tumor ability, while the subcutaneously injected BCG group has a poor anti-tumor ability. The nanoemulsion constructed using BCG lipids can inherit some of the training immunity of BCG bacteria and also has a certain tumor protection effect, while avoiding the splenomegaly problem in mice caused by intravenously injected BCG.

[0078] Based on the research and exploration of the above embodiments, this invention proposes that mycobacterial lipid extracts can be used as adjuvants in vaccine development, including mucosal adjuvants (anti-tuberculosis vaccine, anti-herpes zoster vaccine, anti-tumor subunit vaccine), as immunomodulators, as immune training inducers, as auxiliary components (surfactants) for drug delivery carriers, and as cell-targeting ligands for nanoparticles (epithelial cell targeting and immune cell targeting). Working together with drug molecules, they are suitable for the prevention and treatment of various diseases.

[0079] Based on the research and exploration of the above embodiments, this invention also proposes the application of mycobacterial lipid extract in vaccine preparation. The mycobacterial lipid extract serves as a vaccine adjuvant to improve the immune effect of the vaccine and enhance its humoral and cellular immune effects.

[0080] Among them, the nanoemulsion adjuvant containing mycobacterial lipid extract can promote the enrichment of antigen-presenting cells (DC cells, macrophages) in lymph nodes. The nanoemulsion adjuvant containing mycobacterial lipid extract has a significantly improved antigen cross-presentation efficiency. At the same time, mycobacterial lipid extract can also promote the production of M1 macrophages, which is beneficial to the generation of inflammatory cytokines and promotes antigen presentation.

[0081] Based on the research and exploration of the above embodiments, this invention also proposes the application of mycobacterial lipid extract in the preparation of pharmaceutical compositions, wherein the mycobacterial lipid extract acts as an auxiliary component of drug delivery carrier or a targeting ligand in conjunction with drug molecules.

[0082] Based on the research and exploration of the above embodiments, this invention also proposes the application of a mycobacterial lipid extract in the preparation of immunomodulators.

[0083] In this embodiment of the invention, the nano-adjuvant containing mycobacterial lipid extract has an immune-enhancing effect, wherein the 40 μg BCG lipid group has the best immune-enhancing effect.

[0084] Based on the research and exploration of the above embodiments, this invention also proposes the application of mycobacterial lipid extract in the preparation of mucosal adjuvants. The mucosal adjuvant is a nanoemulsion containing mycobacterial lipid extract prepared by nanotechnology. The nanoemulsion containing mycobacterial lipid extract is used as a mucosal adjuvant to induce an effective increase in serum antibody levels, induce an increase in IgG2a antibody secretion, make the immune response type biased towards Th1, and increase the secretion of IgA at the mucosa.

[0085] Based on the research and exploration of the above embodiments, this invention also proposes the application of mycobacterial lipid extract in the preparation of training immune inducers. The training immune inducer is a nanoemulsion containing mycobacterial lipid extract. The mycobacterial lipid extract inherits the training immune capacity of mycobacterial cells and has a tumor protective effect.

[0086] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0087] The present invention provides a detailed description of a vaccine or pharmaceutical composition based on mycobacterial lipid extract, its preparation, and its application. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, those skilled in the art will recognize that there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A vaccine system based on mycobacterial lipid extract, characterized in that, The vaccine system comprises: a mycobacterial lipid extract, which is a bacterial lipid extracted from mycobacterial cells, and an antigen, wherein the main components of the mycobacterial lipid extract are glycerophospholipids, fatty amides and bioactive glycolipid components.

2. The mycobacterial lipid extract-based vaccine according to claim 1, characterized in that, The antigen comprises: a polypeptide antigen, a protein antigen, a polysaccharide antigen and a nucleic acid antigen.

3. The mycobacterial lipid extract-based vaccine according to claim 1, characterized in that, The vaccine is a vaccine preparation prepared by formulating the mycobacterial lipid extract as an adjuvant, co-loading or mixing with an antigen through a formulation process.

4. A pharmaceutical composition based on mycobacterial lipid extract, characterized in that, The pharmaceutical composition comprises: a mycobacterial lipid extract, which is a bacterial lipid extracted from mycobacterial cells, and a drug molecule, wherein the main components of the mycobacterial lipid extract are glycerophospholipids, fatty amides and bioactive glycolipid components.

5. The mycobacterial lipid extract pharmaceutical composition according to claim 4, wherein, The pharmaceutical composition is a nanoparticle, a liposome, a nanoemulsion or a microemulsion prepared by nano-technological process of the mycobacterial lipid extract and a drug molecule.

6. A process for the preparation of a mycobacterial lipid extract-based vaccine system as claimed in any one of claims 1 to 3 or a mycobacterial lipid extract-based pharmaceutical composition as claimed in any one of claims 4 to 5, characterized in that, The method comprises: extracting a bacterial lipid from mycobacterial cells to obtain a mycobacterial lipid extract; preparing the mycobacterial lipid extract and an antigen into a vaccine system, or preparing the mycobacterial lipid extract and a drug molecule into a pharmaceutical composition.

7. Use of a mycobacterial lipid extract for the preparation of a vaccine, characterized in that, The mycobacterial lipid extract is used as a vaccine adjuvant to improve the immune effect of a vaccine and enhance the humoral and cellular immune effects of the vaccine.

8. Use of a mycobacterial lipid extract for the preparation of a pharmaceutical composition, characterized in that, The mycobacterial lipid extract is used as a drug delivery carrier auxiliary component or a targeting ligand to co-act with a drug molecule.

9. Use of a mycobacterial lipid extract in the preparation of an immunomodulator.

10. Use of a mycobacterial lipid extract in the preparation of an immune induction agent for training.

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