A vaccine adjuvant containing llo me and vaccine compositions and uses thereof

Abstract

The application belongs to the technical field of biotechnology, and discloses a vaccine adjuvant containing LLOMe, a vaccine composition and application thereof. The application uses LLOMe as a vaccine adjuvant, which can enhance the activity of dendritic cells (DCs) when used in combination with a vaccine, promote the promotion of vaccine antigens, and improve the prevention and treatment effect of the vaccine. The application first discloses the technical content of LLOMe as a vaccine adjuvant, and the vaccine obtained by combining LLOMe with antigens can significantly enhance the anti-tumor cell immunity, thereby obtaining effective immune activity and treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a vaccine adjuvant containing LLOMe, its vaccine composition, and its application. Background Technology

[0002] For existing tumor tissue, the human immune system mainly responds through cytotoxic T lymphocytes (also known as CD8). + T cells directly induce tumor cell death or indirectly kill tumor cells by secreting cytotoxic granules. Vaccines are biological products that induce a specific immune response in the human body, thereby inhibiting or eliminating pathogens and protecting the body. After active inoculation with a tumor vaccine, the vaccine activates or enhances the immune system's specific surveillance and clearance functions against tumors through its antigens, thus exerting a tumor-suppressive effect without causing other adverse effects on the body. The use of adjuvants can enhance the CD8+ induced by the vaccine antigen. + An effective means of enhancing the strength and persistence of T-cell responses.

[0003] As early as the beginning of the last century, insoluble aluminum salts of alum were used clinically as vaccine adjuvants. Alum is still used as an adjuvant in inactivated COVID-19 vaccines. After nearly a century of development, five adjuvants—MF59, CpG1018, and AS04, AS03, and AS01 developed by GlaxoSmithKline—have also been used to help enhance the immune activity of vaccines. These substances exert their adjuvant effects through antigen sustained-release, promoting the generation of dendritic cells (DCs), and activating pattern recognition receptors.

[0004] Antigen presentation is a central step in activating anti-tumor T-cell responses in vaccines. It means that an antigen-presenting cell, after taking up and processing an antigen, presents the antigen information (mainly polypeptide fragments) to T cells, thereby activating T cells to specifically recognize and kill tumor cells carrying that antigen. Dendritic cells (DCs) are the most crucial antigen-presenting cells in the body. They possess exceptional capabilities in acquiring and processing antigens and expressing high levels of immune co-stimulatory molecules. They are also the only antigen-presenting cells capable of activating naive T cells. These factors determine the importance of DCs in CD8... + T cells play a crucial role in executing specific immune responses. Experiments have demonstrated that excessive antigen degradation and insufficient dendritic cell (DC) activation occur during vaccine antigen presentation. The absence of DCs severely limits the adjuvant activity of ligands for alum, MF59, and pattern recognition receptors in mice, leading to impaired adaptive immunity. Current therapeutic tumor vaccine development focuses on enhancing DC activation through various methods and strategies to improve antigen presentation efficiency and thus enhance anti-tumor T cell responses.

[0005] Cervical cancer is the fourth leading cause of cancer death among women worldwide. Early-stage cervical cancer is primarily treated with surgical removal, while mid-to-late-stage cancer is mainly treated with radiotherapy supplemented with chemotherapy. However, the 5-year survival rate for cervical cancer recurrence after treatment is only about 17%, highlighting the urgent need for more effective treatment methods. Vaccination against high-risk HPV infection can significantly reduce the incidence of cervical cancer. Currently, widely used HPV vaccines are formulated with virus-like particles formed from the L1 capsid protein of the HPV virus and aluminum salt adjuvants or ASO4 adjuvants. In current clinical practice, while HPV vaccines are effective in preventing cervical cancer, they are not significantly effective in treating cervical cancer caused by HPV infection.

[0006] Existing adjuvants have so far failed to help cervical cancer vaccines achieve satisfactory clinical therapeutic effects. Therefore, there is an urgent need to develop adjuvants with novel functional mechanisms to enhance the efficacy of therapeutic cervical cancer vaccines.

[0007] L-Leucyl-L-Leucine methyl ester (LLOMe) hydrobromide is a dipeptide condensation product of L-leucine methyl ester produced by human monocytes or polymorphonuclear leukocytes. Currently, there are no studies on its use as an adjuvant to enhance the immunizing effect of vaccines. Summary of the Invention

[0008] To overcome the problem of insufficient immunogenicity in existing therapeutic vaccines, the primary objective of this invention is to provide a vaccine adjuvant containing LLOMe. This invention uses LLOMe as a novel active ingredient in vaccine adjuvants. By combining it with a vaccine, its adjuvant effect can effectively enhance the vaccine's immunogenicity, resulting in a significantly enhanced therapeutic effect.

[0009] Another object of the present invention is to provide a vaccine composition containing the above-mentioned vaccine adjuvant.

[0010] Another object of the present invention is to provide the application of LLOMe as a vaccine adjuvant.

[0011] Another object of the present invention is to provide the application of LLOMe in the preparation of vaccines that inhibit the proliferation of tumor cells.

[0012] Another objective of this invention is to provide the application of LLOMe in the preparation of therapeutic cervical cancer vaccines. Through experiments, this invention has found that LLOMe enhances the activity of vaccines in inhibiting cervical cancer proliferation, thus effectively enhancing the therapeutic efficacy of therapeutic cervical cancer vaccines.

[0013] The objective of this invention is achieved through the following solution:

[0014] A vaccine adjuvant containing LLOMe.

[0015] The vaccine adjuvant of the present invention contains LLOMe, which has the characteristics of good biocompatibility, low cost and convenient use. When combined with antigen, it can achieve a significant and excellent effect of enhanced immune activity.

[0016] The present invention also provides a vaccine composition comprising the vaccine adjuvant described herein and an immunogenic amount of antigen.

[0017] Vaccine compositions containing the vaccine adjuvant of the present invention can achieve effectively enhanced immune activity and therapeutic effects.

[0018] The antigens mentioned are natural products purified from pathogens or proteins or peptides artificially produced through methods such as gene recombination. Specifically, they include virions, viral structural proteins, viral non-structural proteins, whole bacterial cells of pathogens, and proteins or glycoproteins derived from pathogens, including infectious antigens and inactivated antigens.

[0019] Furthermore, the antigens may be measles virus, rubella virus, poliovirus, human papillomavirus, hepatitis A virus, influenza A virus antigen, and tumor-associated antigens, etc.

[0020] This invention also provides the application of LLOMe in the preparation of vaccine adjuvants.

[0021] LLOMe can be used as a vaccine adjuvant in combination with vaccines to effectively enhance vaccine activity.

[0022] The present invention also provides the use of LLOMe, or the above-mentioned vaccine adjuvant, or vaccine composition in the preparation of vaccines.

[0023] The present invention also provides the use of LLOMe, or the above-mentioned vaccine adjuvant, or vaccine composition in the preparation of vaccines that inhibit tumor proliferation.

[0024] According to the present invention, applying LLOMe to the preparation of vaccines that inhibit tumor cell proliferation can increase the uptake of vaccine antigens and promote the escape of vaccine antigens from DCs lysosomes to reduce antigen degradation, thereby achieving a significant increase in the activity of inhibiting tumor cell proliferation.

[0025] Furthermore, inhibiting tumor cell proliferation includes pre-vaccination to inhibit tumor development and therapeutic vaccination to inhibit tumor proliferation.

[0026] This invention also provides the use of LLOMe, or the above-mentioned vaccine adjuvant, or vaccine composition in the preparation of therapeutic cervical cancer vaccines. Through experiments, this invention has found that LLOMe enhances the activity of vaccines in inhibiting cervical cancer proliferation, effectively enhancing the therapeutic effect of therapeutic cervical cancer vaccines.

[0027] According to the present invention, combining LLOMe with an antigen can increase the effectiveness of a vaccine.

[0028] Furthermore, the increased efficacy of the vaccine is manifested by enhanced activity of DCs, including increased secretion of inflammatory factors such as IL-1β, IFN-γ, and TNF-α.

[0029] Furthermore, the enhanced vaccine efficacy manifests as increased activation of antigen presentation in dendritic cells (DCs) by the vaccine, which in turn increases the expression of genes involved in the formation of immune synapses in DCs. Specifically, it upregulates the expression of the immune co-stimulatory molecules CD80 and CD86.

[0030] Furthermore, the enhanced vaccine efficacy manifests as a stronger anti-tumor cellular immune response, and further as an increased anti-tumor effect of CD8+ in secondary lymphoid organs and tumor tissues. + The levels of T cells and immune memory T cells.

[0031] This invention discloses for the first time the technical content of LLOMe as a vaccine adjuvant, which can enhance the antigen presentation function of DCs by increasing the expression of immune co-stimulatory molecules, thereby enhancing anti-tumor cell immunity, inhibiting tumor proliferation and development, and thus achieving effectively improved immune activity and therapeutic effect. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The physical parameters of the vaccine after loading with LLOMe are characterized. A is a transmission electron microscopy image of the liposomal vaccine; B is the particle size and polydispersity index of the liposomal vaccine in neutral PBS solution as measured by dynamic light scattering; C is the surface potential of the liposomal vaccine in neutral PBS solution as measured by dynamic light scattering. *P < 0.05 vs. Vehicle, n = 3.

[0034] Figure 2 LLOMe enhances the uptake and utilization of vaccine antigens in DCs.

[0035] Figure 3 LLOMe promotes vaccine activation of dendritic cells (DCs). Image A shows the effect of LLOMe on promoting BMDC migration (scale bar 100 μm); image B shows the statistical analysis of cellular IL-1β, IFN-γ, and TNF-α secretion. *P < 0.05 vs. PBS. #P < 0.05 vs. E7@V, n = 4.

[0036] Figure 4 LLOMe upregulates the immune synaptic function of dendritic cells (DCs). In the graph, A shows the statistical expression of the cellular co-stimulatory molecules CD80 and CD86 (n=4); B shows a representative graph of the expression of the cellular co-stimulatory molecules CD80 and CD86 (n=4). *P<0.05 vs. PBS. # P < 0.05 vs. E7@V.

[0037] Figure 5 To investigate the effect of LLOMe-containing vaccines on the mTORC1 signaling pathway in DC cells.

[0038] Figure 6 To enhance the cellular immune response activated by the LLOMe vaccine in mice. In the figure, A shows the change in subcutaneous tumor volume over time in each group of mice; B shows the statistical graph of tumor tissue mass size in each group of mice. *P < 0.05 vs. PBS. # P < 0.05 vs. E7@V, n = 6.

[0039] Figure 7 To enhance the inhibitory effect of LLOMe vaccine on the proliferation of cervical cancer cells. A shows the flow cytometry analysis of changes in the content of myeloid-derived suppressor cells and cytotoxic T cells in the spleen tissue of mice in each group; B shows the CD4+ content in the spleen tissue of mice in each group. + and CD8 + Flow cytometry analysis of central memory T cell content; C is a flow cytometry analysis of changes in cytotoxic T cell and regulatory T cell content in tumor tissues of mice in each group. *P<0.05 vs PBS; # P<0.05 vs Vehicle; $ P<0.05 vs E7@V; n=4-6. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. Unless otherwise specified, the methods described in the following embodiments are conventional methods, and the materials involved are commercially available unless otherwise specified.

[0042] The molecular formula of LLOMe (L-Leucyl-L-Leucine methyl ester) involved in this invention is C 12 H 27 N2O3, with a molecular weight of 247.35, has the following structural formula:

[0043]

[0044] Example 1

[0045] A liposome-based anti-cervical cancer vaccine loaded with LLOMe was prepared using a thin-film dispersion method, in which the HPV viral protein peptide fragment HPV16 E7 was utilized. 44-62 (Based on the literature ACS Applied Materials & Interfaces 2020 12(49), 54399-54414, synthesized by Jier Biochemical Co., Ltd., with a purity of 98%) as antigen (abbreviated as E7), a liposomal vaccine E7 / LLOMe@Vehicle (E7 / L@V) co-loaded with LLOMe and antigen E7 was prepared.

[0046] Taking the preparation of 1 mL as an example: In a 25 mL round-bottom flask, add 0.33 mg of DOTAP, 0.11 mg of DOPE, 1.4 mg of DSPE-PEG2000, and 0.16 mg of cholesterol dissolved in methanol, and then add 1 mL of methanol to thoroughly mix each lipid solution. Use a rotary evaporator to evaporate the methanol solvent, allowing the dissolved lipids to adhere to the inside of the flask and form a lipid film layer. Then place the flask in a vacuum drying oven and dry at room temperature for 1 hour. After removing the flask, add 25 μL of 1 mg / mL E7 solution, 225 μL of 4 mg / mL LLOMe solution, and 750 μL of PBS solution. Sonicate the flask at 40 kHz and 400 W for 3 min, then vortex for 3 min, repeating the sonication and vortexing process 3 times. Extrude the liposomes obtained in the above steps 11 times in a polycarbonate membrane liposome extruder equipped with a 100 nm pore size. The liposomes, after being extruded into whole particles, were added to the inner tube of an ultrafiltration tube with a 3 kDa cutoff and centrifuged at 14000 × g for 30 min at 4 °C. After centrifugation, a separate outer tube was taken, and the inner tube was carefully inverted into the collection tube. The tube was centrifuged at 100 × g for 3 min to collect the concentrated liposome solution. The collected concentrate was then reconstituted with PBS to restore it to its original volume before centrifugation, transferred to an EP tube, and stored at 4 °C or used for further experiments.

[0047] To observe the morphology of the vaccine, the prepared liposome solution was diluted 20-fold with PBS buffer. The diluted liposome vaccine solution was dropped onto a copper grid with a carbon-coated support membrane and immersed for 30 seconds. This was repeated three times. Excess liquid on the copper grid was then blotted dry with filter paper. A 1% tungsten phosphate solution was then dropped onto one side of the copper grid, covering and immersing it for 30 seconds. Afterward, the liquid was blotted dry with filter paper. The copper grid was allowed to air dry for 2 hours before being used for observation under a transmission electron microscope. The microscope accelerating voltage was adjusted to 100 kV during observation.

[0048] The particle size and surface potential of liposomes were measured using dynamic light scattering (DLS). Specifically, 1 mL of the prepared 2 mg / mL liposome solution was transferred to a micro-volume quartz cuvette and loaded into a laser particle size analyzer. Each sample was measured three times at 25°C, and the Z-Average and Polydispersity Index (PDI) values ​​were recorded. The liposome solution was then transferred to a potential sample cell and loaded into the particle size analyzer. The Zeta detection program was run, and the Zeta Potential value was recorded.

[0049] To determine the drug loading of E7 and LLOMe within the vaccine, standard solutions of E7 and LLOMe at quantitative concentrations were first prepared and diluted to obtain solutions of various concentration gradients. For E7, the absorbance of each concentration of standard solution at 562 nm was measured using the BCA (bicinchoninic acid assay), and a scatter plot of concentration versus absorbance was plotted. The functional relationship between the two was obtained using the least squares method. For LLOMe, high-performance liquid chromatography (HPLC) was used. Specifically, a methanol-water mobile phase gradient of 40 min was used on a C18 column, with separation at 40%-90% methanol, and detection using a 254 nm detector. Standard solutions of each concentration were measured individually, and a scatter plot of characteristic peak height versus sample concentration was plotted. The functional relationship between the two was obtained using the least squares method. 500 μL of the nano-vaccine was placed in an ultrafiltration tube, concentrated to approximately 100 μL, and 2 μL of Triton 100 was added. After mixing, the mixture was allowed to stand for 1 h to rupture the liposomes. After membrane disruption, the liposome sample solutions were quantified using the BCA method and high-performance liquid chromatography (HPLC), respectively, and the drug loading and encapsulation efficiency of both drugs in the original liposome solution before ultrafiltration were calculated accordingly. The obtained data are as follows: Figure 1 As shown in Table 1.

[0050] Table 1

[0051]

[0052] Depend on Figure 1 As shown in Table 1, the liposomal vaccine E7 / LLOMe@Vehicle (E7 / L@V), which co-loads LLOMe and antigen E7, was successfully prepared with a high encapsulation rate.

[0053] Example 2

[0054] (1) Grouping: ① Blank control group: PBS; ② Antigen group: E7; ③ Carrier-loaded antigen group: E7@V; ④ Carrier-loaded adjuvant and antigen group: E7 / L@V.

[0055] (2) A liposome vaccine was prepared using the 5-FAM-labeled antigenic peptide E7, as described in Example 1. DC2.4 cells (mouse bone marrow-derived dendritic cells, purchased from Wuhan Pronosei Biotechnology Co., Ltd., CL-0545) were seeded in confocal dishes. After the cells reached a confluence of 70-80%, they were divided into groups and diluted with PBS and 5-FAM fluorescently labeled E7, E7@V, and E7 / L@V in culture medium (E7 group contained 50 μL of 21.3 μg / mL E7 solution per 1 mL of culture medium; E7@V and E7 / L@V groups contained 50 μL of 2 mg / mL liposome preparation per 1 mL of culture medium). To investigate the survival of the antigen in the cells, the cells were incubated with the fluorescently labeled vaccine for 36 h, then removed, washed twice with PBS, digested with trypsin, washed once with PBS, and stored on ice in the dark for flow cytometry analysis. To investigate lysosomal escape of antigens, Lyso-Tracker reagent was diluted in PBS to prepare a 100 μM stock solution; Hoechst 33342 reagent was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL stock solution, and stored at 4°C in the dark for later use. Cells were cultured for 24 h and then washed twice with PBS. 1 mL of serum-free OPTI-MEM medium was added, and Lyso-Tracker and Hoechst 33342 solutions were added under dark conditions to final concentrations of 100 nM and 10 μg / mL, respectively. After washing, OPTI-MEM medium containing Lyso-Tracker and Hoechst 33342 was added to the dish. Cells were transferred to a 37°C incubator and incubated for 30 min. After incubation, cells were removed, washed twice with PBS, and 1 mL of normal culture medium was added. Fluorescence was observed using a laser confocal microscope. The results are as follows: Figure 2 As shown.

[0056] As shown in the figure, for antigen uptake analysis, the distribution curve of the free E7 group shifted to the right compared to the PBS group, indicating higher fluorescence intensity. The average intracellular fluorescence intensity of the E7@V group was higher than that of the E7 group, and the fluorescence intensity of the E7 / L@V group was even higher than that of the E7@V group, indicating that the E7 / L@V group took up the most E7 antigen. For lysosomal escape analysis, in the E7 group and the adjuvant-free E7@V group, the green fluorescence of antigen E7 almost completely overlapped with the red fluorescence of lysosomes, resulting in a yellow color; however, in the E7 / L@V group, there were partial free green fluorescence that did not overlap with the red lysosomes. Using software for co-localization and quantitative analysis of fluorescence, it can be seen that the curves representing the two fluorescence types completely overlapped in the E7 group and the adjuvant-free E7@V group; however, in the E7 / L@V group, there were areas where the curves did not overlap. This suggests that the LLOMe-loaded vaccine promoted the escape of antigen from lysosomes into the cytoplasm.

[0057] Example 3

[0058] (1) Grouping: ① Blank control group: PBS; ② Antigen group: E7; ③ Adjuvant group: LLOMe; ④ Carrier-loaded antigen group: E7@V; ⑤ Carrier-loaded adjuvant and antigen group: E7 / L@V.

[0059] (2) Obtaining bone marrow-derived dendritic cells (BMDCs) from mice for viability testing: Healthy female mice were euthanized, and the hind limbs were removed at the hip joint. Skin and muscle tissue were separated, while the femur and tibia were preserved intact to prevent contamination. After external disinfection by soaking in 75% ethanol for 5-10 seconds, the BMDCs were transferred to a laminar flow hood and placed on ice. Using sterile instruments, the ends of the tibia and femur were cut off to expose the bone cavity. A syringe was used to draw pre-cooled RPMI-1640 complete culture medium and inserted into the bone to flush the bone marrow into a centrifuge tube on ice. After collection, the bone marrow clumps were separated into single cells by pipetting several times. The centrifuge tubes were removed and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, and the cell clumps were resuspended. The viable cell density was counted using trypan blue staining, and 2 × 10⁶ cells were collected in each 9 cm diameter sterile bacterial culture dish. 6 Live cell seeding. Each dish was filled with 10 mL of culture medium and 20 ng / mL of granulocyte-macrophage colony-stimulating factor (GM-CFS) was added. Cells were cultured in a 5% CO2 incubator. On day 3, 10 mL of fresh culture medium containing 20 ng / mL GM-CFS was added. On day 6, 10 mL of the culture medium was removed and 10 mL of fresh culture medium containing GM-CFS was added. Cells were harvested on day 8.

[0060] (3) Transwell chambers were used to detect the migration of BMDCs after stimulation by the vaccine antigen, exploring the ability of the vaccine to induce BMDCs to migrate to it. First, Transwell culture plates with a 12 μm pore size were prepared. 500 μL of culture medium containing PBS, E7, LLOMe, E7@V, and E7 / L@V was added to the lower culture chamber of the plate according to the groupings (PBS group: 25 μL PBS solution per 500 μL of medium; E7 group: 25 μL E7 solution per 500 μL of medium with a concentration of 21.3 μg / mL; LLOMe group: 25 μL LLOMe solution per 500 μL of medium with a concentration of 130 μg / mL; E7@V and E7 / L@V groups: 25 μL liposome preparation per 500 μL of medium). The obtained BMDCs were prepared into a density of 1×10⁻⁶. 6100 μL of cell suspension was seeded into the upper chamber of the 6-well plate. After culturing the cells normally in an incubator for 6 hours, the culture medium in the lower wells of the 6-well plate was carefully removed. The cells were fixed with 4% paraformaldehyde for 10 min, followed by staining with 0.1 mg / mL crystal violet for 10 min. The cells were then washed twice with PBS to remove unbound dye, and after drying, the cell count in the chamber was photographed using a microscope.

[0061] (4) The secretion of IFN-γ, tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β) by BMDCs was detected using enzyme-linked immunosorbent assay (ELISA). The kits were purchased from Wuhan E-EL-M0037c, IFN-γ (E-EL-M0048c), and TNF-α (E-EL-M3063). The experiment was divided into PBS, E7, LLOMe, E7@V, and E7@V groups. Cells in each group were treated with the corresponding drug for 24 hours, and the cell culture supernatant was collected (PBS group: 50 μL PBS solution per 1 mL of culture medium; E7 group: 50 μL E7 solution at 21.3 μg / mL per 1 mL of culture medium; LLOMe group: 50 μL LLOMe solution at 130 μg / mL per 1 mL of culture medium; E7@V and E7 / L@V groups: 50 μL liposome preparation at 2 mg / mL per 1 mL of culture medium). For each well of a plate coated with the corresponding cytokine antibody, 100 μL of the collected supernatant was added according to the group. After incubation at 37°C for 1.5 hours, the sample liquid was removed, and 100 μL of biotinylated antibody from the kit was added. Incubation continued at 37°C for 1 hour to allow the cytokines to adsorb into the wells and be biotinylated. The wells were then washed three times with washing buffer to remove any remaining unbound antibody. Prepare a horseradish peroxidase conjugate solution. Add 100 μL to each well and incubate at 37°C for 0.5 h. After incubation, wash five times with washing buffer to remove any remaining unbound horseradish peroxidase conjugate. Add 90 μL of substrate solution to each well and incubate at 37°C for 15 min. Then, remove the plate and quickly add 50 μL of stop solution to each well to terminate the reaction. Use a microplate reader to read the absorbance of the liquid at 450 nm. Calculate the corresponding cytokine concentration in each well based on the measured standard curve.

[0062] The results are as follows Figure 3As shown in the figure, the liposomal vaccine E7 / LLOMe@Vehicle (E7 / L@V), which co-loads LLOMe of the present invention and antigen E7, has a significantly enhanced effect on promoting the migration of BDMCs and can significantly promote the secretion of IL-1β, IFN-γ, and TNF-α in cells, and is significantly better than the antigen group. This proves that the vaccine obtained by combining LLOMe as an adjuvant with the antigen has significantly enhanced activity.

[0063] Example 4

[0064] (1) Grouping: ① Blank control group: PBS; ② Antigen group: E7; ③ Carrier-loaded antigen group: E7@V; ④ Carrier-loaded adjuvant and antigen group: E7 / L@V.

[0065] (2) The analysis steps for the expression of T cells of BMDCs binding to the corresponding ligands / co-stimulatory molecules CD80 and CD86 on the cell surface are as follows: The experiment was divided into PBS, E7, E7@V, and E7 / L@V groups. Each group was treated with the corresponding drug for 24 h (the PBS group contained 50 μL of PBS solution per 1 mL of culture medium; the E7 group contained 50 μL of 21.3 μg / mL E7 solution per 1 mL of culture medium; and the E7@V and E7 / L@V groups contained 50 μL of 2 mg / mL liposome preparation per 1 mL of culture medium). After treatment, the cells were collected, counted, and 1×10⁶ cells were collected. 6 Cells were dispersed in staining buffer. 2 μL each of PerCP / Cyanine 5.5-CD11c, PE-CD80, and APC-CD86 antibodies were added to each cell tube, and the mixture was incubated at 4°C for 0.5 h. Four additional cell tubes were used as positive staining tubes. 2 μL each of PBS, PerCP / Cyanine 5.5-CD11c, PE-CD80, and APC-CD86 were added to each tube, and the mixture was incubated at 4°C for 0.5 h. After incubation, the cells were centrifuged at 1500 rpm for 5 min to remove the supernatant. The cells were resuspended in 500 μL of pre-chilled PBS and stored on ice in the dark for flow cytometry analysis.

[0066] The results are as follows Figure 4 As shown in the figure, the liposomal vaccine E7 / LLOMe@Vehicle (E7 / L@V), which co-loads LLOMe of the present invention and antigen E7, has significantly enhanced activity in promoting the expression of CD80 and CD86, and is significantly superior to the antigen group. This demonstrates that the vaccine obtained by combining LLOMe as an adjuvant with the antigen has significantly enhanced activity.

[0067] Example 5

[0068] (1) Grouping: ① Blank control group: PBS; ② Carrier-loaded antigen group: E7@V; ③ Carrier-loaded adjuvant and antigen group: E7 / L@V.

[0069] (2) The experiment was divided into PBS, E7@V, and E7@V groups. Cells in each group were treated with the corresponding drug for 24 hours (the PBS group contained 50 μL of PBS solution per 1 mL of culture medium; the E7@V and E7 / L@V groups contained 50 μL of liposome preparation at 2 mg / mL per 1 mL of culture medium). After treatment, residual culture medium was washed off with PBS, and lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor RIPA was added. Cells were gently shaken on ice for 30 minutes, then scraped off and collected into centrifuge tubes. The supernatant obtained after centrifugation at 12000 rpm for 10 minutes at 4°C was the protein solution. The protein solution was quantified according to the BCA kit instructions, and the sample concentration was adjusted accordingly. 1 / 4 volume of 5x loading buffer was added, mixed well, and boiled in boiling water for 5 minutes before storage at -80°C for later use. Electrophoretic separation and membrane transfer were performed using a Western Bolt experimental kit. Blocking buffer was applied at room temperature for 1 hour, followed by overnight incubation at 4°C with antibodies against p-mTOR, mTOR, p-P70, p-4EBP, and β-Actin. Finally, the membrane was incubated at room temperature with horseradish enzyme-labeled secondary antibody for 1 hour. Fluorescence was excited using ECL chemiluminescence buffer, and imaging was performed using an imager. Results are shown below. Figure 5 As shown in the figure, the liposomal vaccine E7 / LLOMe@Vehicle (E7 / L@V), which co-loads LLOMe of the present invention and antigen E7, has a significantly enhanced effect on promoting the expression of mTORC1 signaling pathway proteins.

[0070] Example 6

[0071] (1) Drug grouping: ① Blank control group: PBS; ② Carrier group: Vehicle; ③ Carrier-loaded antigen group: E7@V; ④ Carrier-loaded adjuvant and antigen group: E7 / L@V.

[0072] (2) Eight-week-old female C57BL / 6J mice were used in the experiment, with six mice in each group. As per the experimental design, tumors were implanted on the lower right side of the mice's backs before vaccination. Specifically, TC-1 cell lines were cultured in 10mm diameter culture dishes using RIPM-1640 + 10% fetal bovine serum. When the cells reached the plateau phase after exponential growth, they were digested with 0.25% trypsin, and the supernatant was removed by centrifugation. The cell pellet was resuspended in pre-chilled PBS, counted using a cell counter, and the cell concentration was adjusted to 1×10⁻⁶. 7 / mL of live cells were collected, and 0.1mL of cell suspension was injected into the tumor graft sites of mice. The day of tumor grafting was designated as day 0. Mouse body weight and tumor volume were monitored every three days. Tumor growth was measured using calipers to determine the longest diameter and the shortest diameter perpendicular to it. Tumor volume was calculated as V = 0.5 × L × W. 2Calculations are performed, where L is the major axis and W is the minor axis. The tumor is expected to grow to an average volume of approximately 50 mm. 3 Treatment began with subcutaneous vaccination of the vaccine on the lower left side of the mouse's back every 7 days, according to the designated groups. The PBS group served as a control, receiving an equal volume of PBS at the vaccination time point. The Vehicle group contained 0.2 mg of the vehicle vector per 100 μL of injection solution. The E7@V group received a liposome solution containing 2 mg / mL of antigenic peptide E7, with 2 μg of E7 per 100 μL of injection solution. The E7 / L@V group received a liposome solution containing both antigenic peptide E7 and the adjuvant LLOMe at equal concentrations, with 2 μg of E7 and 13 μg of LLOMe per 100 μL of injection solution.

[0073] The results are as follows Figure 6 As shown in the figure, the liposomal vaccine E7 / LLOMe@Vehicle (E7 / L@V), which co-loads the LLOMe of this invention and the antigen E7, can rapidly and efficiently eliminate tumors, and its effect is significantly better than that of the antigen group. This proves that the vaccine obtained by combining LLOMe as an adjuvant with the antigen has significantly enhanced activity, and that LLOMe of this invention plays a role in enhancing antigen activity, thereby enhancing the therapeutic effect.

[0074] Example 7

[0075] Drug grouping: ① Blank control group: PBS; ② Vector group: Vehicle; ③ Vector-loaded antigen group: E7@V; ④ Vector-loaded adjuvant and antigen group: E7 / L@V.

[0076] After the mice were euthanized following the observation of tumor growth inhibition in mice, lymphocytes were extracted and isolated from their spleen and tumor tissue for analysis. The spleen tissue was harvested, weighed, and a portion was ground on a 200-mesh screen to separate cells. Cells were treated with 1 mL of erythrocyte lysis buffer for 10 min to remove erythrocytes. The cells were then centrifuged at 1500 rpm for 5 min at 4°C to remove the supernatant. The cell pellet was resuspended in 5 mL of lymphocyte separation medium. The cell suspension was transferred to a 15 mL centrifuge tube, and 0.5 mL of RIPM-1640 medium was slowly added to the top layer. The tube was centrifuged at 800 g for 30 min at room temperature, and the middle white lymphocyte layer was carefully aspirated and transferred to another centrifuge tube. 10 mL of RIPM-1640 medium was added to the cell suspension, and the tube was washed by inversion. The tube was then centrifuged at 250 g for 10 min at room temperature, and the cell pellet was collected.

[0077] The tumor tissue was weighed and recorded after removal, and 0.3g was cut off and further trimmed into pieces of about 1mm. 3The fragments were placed in an EP tube containing 500 μL of Collagenase A (1 mg / mL) solution and digested at 37°C with shaking at 150 rpm for 30 min. After treatment with Collagenase A, tumor tissue was gently ground to separate cells through a 200-mesh filter, and the cells were filtered into 15 mL centrifuge tubes using lymphocyte separation medium. 0.5 mL of RIPM-1640 complete medium was added to the top layer of the centrifuge tube, and the tube was centrifuged at 800 g for 30 min. After centrifugation, the white lymphocyte layer was carefully removed and aspirated. The cells were washed once with RIPM-1640 complete medium and resuspended in 1 mL of cell staining buffer. All cells obtained from the spleen and tumor tissues were fixed with 4% paraformaldehyde at room temperature for 10 min, followed by washing with PBS to remove residual fixative. After cell counting, a certain amount of cells was taken from each tissue and subjected to combined staining with fluorescently labeled antibodies. The cells were incubated at 4°C for 30 min, centrifuged to remove the staining buffer, and resuspended in 300 μL of PBS. The cell staining protocol was as follows: cytotoxic T cells (CTLs) (PE-CD8, APC-IFN-γ); myeloid-derived suppressor cells (MDSCs) (FITC-CD11b, PerCP / Cyanine5.5-Gr-1); central memory T cells (Tcm) (FITC-CD4 / PE-CD8, APC-CD44, PerCP / Cyanine5.5-CD62L); and regulatory T cells (Tregs) (FITC-CD4, PE-Foxp3). Cell typing was performed using flow cytometry, and the multicolor scheme was analyzed after compensation adjustment.

[0078] The results are as follows Figure 7 As shown in the figure, the liposomal vaccine E7 / LLOMe@Vehicle (E7 / L@V), which co-loads LLOMe of this invention and antigen E7, can significantly enhance the anti-tumor activity of CD8 in secondary lymphoid organs and tumor tissues. + The levels of T cells and immune memory T cells were significantly higher than those in the antigen group.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of LLOMe, or vaccine adjuvants containing LLOMe in the preparation of therapeutic cervical cancer vaccines.

2. The use of a vaccine composition comprising the vaccine adjuvant of claim 1 and an immunologically effective amount of antigen E7 in the preparation of a therapeutic cervical cancer vaccine.

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