MRNA vaccine adjuvant based on pyroptosis induction and application thereof

Abstract

The invention relates to an mRNA (messenger ribonucleic acid) vaccine adjuvant based on pyroptosis induction. The vaccine adjuvant consists of ionizable lipid, cholesterol, phospholipid, a PEG (polyethylene glycol) derivative and mRNA containing a GSDMD-NT (glutathione-co-dimethylformamide) gene sequence for coding 1-277 amino acids, the amino acid sequence of the GSDMD-NT gene sequence is as shown in SEQ ID NO: 1. The invention successfully develops the mRNA vaccine adjuvant based on pyroptosis induction. The adjuvant molecule and an mRNA vaccine have the same physical characterization attribute, the adjuvant molecule is easy to prepare, and a finished product can be obtained only in one day. And the mRNA vaccine adjuvant can cause rapid, inflammatory and split pyroptosis by expressing and accumulating GSDMD-NT effector molecular protein in cells, and is a universal and efficient pyroptosis induction mode.

Description

Technical Field

[0001] The present invention belongs to the field of mRNA vaccines, and particularly relates to mRNA vaccine adjuvants for promoting vaccine efficacy based on cell pyroptosis induction and applications thereof. Background Art

[0002] Cancer has always been a difficult problem to overcome worldwide. It seriously endangers human health and affects the development of the global economy. Every year, a large amount of money is invested in cancer research around the world. Although certain results have been achieved, there are still many problems that need to be further solved. According to recent global statistics, breast cancer has surpassed lung cancer to become the most common cancer, with approximately 2.3 million new cases in 2020. Although most patients with early breast cancer have a good prognosis after treatment, about 20% to 30% of patients still have in situ recurrence or distant metastasis within 2 years after the diagnosis of the primary tumor, with poor prognosis and decreased quality of life. To date, surgery is still the main treatment for solid tumors. Despite the continuous advancement and updating of surgical techniques and equipment, 30% to 40% of patients still have tumor recurrence or metastasis within 5 years, mainly due to residual tumor cells at the surgical margin and temporary immunosuppression during wound healing.

[0003] In addition to surgical resection, existing cancer treatment methods include chemotherapy, radiotherapy, and immunotherapy to remove cancer cells from the body. Radiotherapy and chemotherapy have low targeting and serious side effects, causing great pain to patients; while immunotherapy is the only method in modern science that can completely remove cancer cells. It has great advantages over traditional treatment methods and was named the top of the top ten scientific breakthroughs by Science magazine in 2013, and has broad prospects.

[0004] Tumor vaccines are a type of immunotherapy, the principle of which is to inject tumor antigens into tumor patients in various forms to overcome the immunosuppression caused by the tumor, activate the patient's own immune system, induce the body to produce cellular and humoral immune responses, and thus control or eliminate the tumor. Compared with other immunotherapies, tumor vaccines have incomparable advantages.

[0005] Tumor vaccines can be divided into four categories based on the formulation method, namely tumor cell vaccines, vaccines based on viruses and bacteria, peptide protein vaccines, and nucleic acid vaccines. Nucleic acid vaccines are the third generation of vaccines after inactivated vaccines, live attenuated vaccines, and genetically engineered recombinant protein vaccines, and are divided into two types: DNA vaccines and RNA vaccines. Compared with other types of tumor vaccines, mRNA tumor vaccines can be generated by in vitro transcription using linearized DNA templates, without the need for complicated in vitro culture procedures, allowing mRNA vaccines to be produced more quickly and efficiently, and are an ideal platform for tumor vaccine preparation; and compared with DNA vaccines, mRNA vaccines can be translated and efficiently express antigens once they enter the cytoplasm, without the risk of insertion and integration into the genome.

[0006] At present, the in vitro transcription preparation method of mRNA molecules and the effective delivery and expression system in vivo have been developed to a fairly mature level. However, the anti-tumor specific T cell immune response effect stimulated by mRNA vaccines is not sufficient, and the maintenance time is not long-lasting, which is a key technical challenge in this field. At present, the main strategy is to improve the expression level by optimizing the sequence design, to fuse and express multiple antigen epitopes to provide broad-spectrum immune stimulation, to adopt a sequential immunization method, etc., but these methods are still limited in improving the efficacy of mRNA vaccines.

[0007] In order to promote the better effectiveness of mRNA vaccines, the innovation of new strategies, methods and concepts is particularly important. Summary of the invention

[0008] In order to develop an adjuvant suitable for mRNA vaccines that promotes anti-tumor immune effects, the present invention proposes an mRNA vaccine adjuvant based on cell pyroptosis induction and its application.

[0009] In a first aspect, the present invention provides an mRNA vaccine adjuvant based on cell pyroptosis induction, wherein the vaccine adjuvant is composed of ionizable lipids, cholesterol, phospholipids, PEG derivatives and mRNA containing a GSDMD-NT gene sequence encoding amino acids 1 to 277; the amino acid sequence encoded by the GSDMD-NT gene sequence is shown in SEQ ID NO: 1.

[0010] In one embodiment of the present invention, the mRNA is composed of a T7 transcription promoter, a 5'UTR, a GSDMD-NT gene sequence encoding amino acids 1 to 277, a 3'UTR, and a polyA tail;

[0011] Wherein, the nucleic acid sequence of the 5'UTR is shown in SEQ ID NO:2;

[0012] The nucleic acid sequence of the 3'UTR is shown in SEQ ID NO:3;

[0013] The nucleic acid sequence of the polyA tail is shown in SEQ ID NO:4.

[0014] The 5'UTR is derived from human hemoglobin α2 subunit, and the 3'UTR is derived from a non-coding sequence encoding 12S ribosomal RNA base of mitochondria.

[0015] In a preferred embodiment of the present invention, the mRNA further comprises a polyC tail having a sequence as shown in SEQ ID NO:5.

[0016] The second aspect of the present invention provides a method for preparing the above-mentioned mRNA vaccine adjuvant, the preparation method comprising the following steps:

[0017] (1) Plasmid construction:

[0018] Using pVAX1 as a plasmid vector, the GSDMD-NT gene sequence was loaded to construct a recombinant plasmid;

[0019] (2) Plasmid linearization:

[0020] The recombinant plasmid was digested with restriction endonuclease Bsa I at 37°C for 1 h, and the linearized plasmid was recovered using a DNA purification kit;

[0021] (3) In vitro transcription synthesis of mRNA molecules:

[0022] The linearized plasmid, dNTPs, RNA polymerase, cap analog and buffer in step (2) were mixed, reacted at 37°C for 3 h, and then DNase I was added to digest the DNA template to obtain GSDMD-NT mRNA;

[0023] (4) Combined lipid nanoparticles:

[0024] Ionizable lipids, cholesterol, phospholipids, PEG derivatives, anhydrous ethanol and GSDMD-NT mRNA are prepared to obtain lipid nanoparticles encapsulating GSDMD-NT mRNA and having a particle size of 100 to 200 nm, namely mRNA vaccine adjuvants.

[0025] The third aspect of the present invention provides an application of preparing a tumor vaccine using the above-mentioned mRNA vaccine adjuvant or the mRNA vaccine adjuvant prepared by the above-mentioned preparation method.

[0026] In one embodiment of the present invention, the tumor vaccine is a tumor mRNA vaccine.

[0027] The fourth aspect of the present invention provides an mRNA vaccine based on cell pyroptosis induction, wherein the mRNA vaccine includes the above-mentioned mRNA vaccine adjuvant or the mRNA vaccine adjuvant prepared by the above-mentioned preparation method, and also includes antigen lipid nanoparticles containing mRNA encoding tumor antigens or pathogen antigens.

[0028] In one embodiment of the present invention, the particle size of the antigen lipid nanoparticles is 100 to 200 nm.

[0029] In another embodiment of the present invention, the mRNA encoding the tumor antigen or pathogen antigen is composed of a T7 transcription promoter, a 5'UTR derived from the human hemoglobin α2 subunit, a Norovirus S domain gene sequence, a linker, a gene sequence of the tumor antigen or pathogen antigen, a 3'UTR derived from a non-coding sequence of the mitochondrial 12S ribosomal RNA base, and a polyA tail;

[0030] Wherein, the nucleic acid sequence of the 5'UTR is shown in SEQ ID NO: 2,

[0031] Specific: GAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC (5'→3')

[0032] The nucleic acid sequence of the 3'UTR is shown in SEQ ID NO: 3,

[0033] Specific:

[0034] CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCC

[0035] CCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGAC

[0036] ACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGC

[0037] AGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC(5'→3')

[0038] The nucleic acid sequence of the polyA tail is shown in SEQ ID NO: 4,

[0039] Specific:

[0040] AAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (5'→3')

[0041] The mRNA further comprises a polyC tail, and the nucleic acid sequence of the polyC tail is shown in SEQ ID NO: 5.

[0042] Specific:

[0043] TGCATCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAAAGGCTCTTTCAGAGCCACCAGAATT(5'→3')

[0044] In another embodiment of the present invention, the amino acid sequence encoded by the Norovirus S domain fused with the HPV E7 tumor antigen gene sequence is shown in SEQ ID NO: 6;

[0045] pfizer-Norovirus S-E7:

[0046] MGGRRVRWEVYISRALWLTREPTAYWLIEMVSIVIRLTIGNKLVFFWSPQTQREPATMKMASNDASPSDG

[0047] STANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFVQAPGGEFTVSPANAPGEILWSAPLGP

[0048] DLNPYLSHLARMYNGYAGGFEVQVILAGNAFTAGKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPV

[0049] LIPLPDVRNNFYHYNQSNDSTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVEGSGSG

[0050] SMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (stop codon)

[0051] In another embodiment of the present invention, the pathogen antigen is selected from the E7 antigen of HPV, and the amino acid sequence of the E7 antigen is shown in SEQ ID NO:7.

[0052] pfizer-E7:

[0053] MGGRRVRWEVYISRALWLTREPTAYWLIEMVSIVIRLTIGNKLVFFWSPQTQREPATMHGDTPTLHEYML DLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (stop codon)

[0054] Among them, the amino acid sequence encoded by the connecting arm is GSGSGS.

[0055] In another embodiment of the present invention, the tumor antigen is selected from the antigen of at least one tumor selected from colon cancer, liver cancer, pancreatic cancer, breast cancer, bladder cancer, fibrosarcoma, kidney cancer, leukemia, lung cancer, white lymphoma, lymphoma, melanoma, myeloma, neuroblastoma, plasmacytoma and prostate cancer.

[0056] In a specific embodiment of the present invention, in the mRNA vaccine, the nucleic acid mass ratio of the mRNA vaccine adjuvant to the antigen lipid nanoparticles is 1:9.

[0057] The fifth aspect of the present invention provides a pharmaceutical composition, which includes the above-mentioned mRNA vaccine adjuvant and antigen lipid nanoparticles.

[0058] In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0059] The term "pharmaceutically acceptable" indicates that the pharmaceutical composition can be administered to a subject without causing adverse physiological reactions that would interfere with the administration of the pharmaceutical composition. For example, "pharmaceutically acceptable excipients" refer to excipients useful in preparing generally safe, non-toxic, and desirable pharmaceutical compositions. Preferably, examples of these excipients or diluents include, but are not limited to, water, saline, Ringer's solution, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used.

[0060] The term "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and agents for delayed absorption, etc. that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride are included in the pharmaceutical composition. Of course, pharmaceutically acceptable carriers may also include trace amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, to extend the shelf life or efficacy of the antibody.

[0061] The sixth aspect of the present invention provides a method for using a pharmaceutical composition, comprising administering an mRNA vaccine adjuvant and an antigen lipid nanoparticle separately or simultaneously.

[0062] In one embodiment of the present invention, the administration of mRNA vaccine adjuvant and antigen lipid nanoparticles is injection.

[0063] Advantages and beneficial effects of the present invention:

[0064] The present invention has successfully developed an mRNA vaccine adjuvant based on cell pyroptosis induction; the adjuvant molecule has the same physical properties as the mRNA vaccine, is easy to prepare, and can be obtained in just one day. In addition, the mRNA vaccine adjuvant can cause rapid, inflammatory, and lytic cell pyroptosis by expressing and accumulating GSDMD-NT effector molecule proteins inside cells, which is a universal and efficient way to induce cell pyroptosis.

[0065] The mRNA vaccine adjuvant of the present invention can cooperate with the antigen to promote the effect, and is a very suitable adjuvant form for mRNA vaccine application. By optimizing the ratio and exploring the vaccine preparation method, we can efficiently induce the occurrence of cell pyroptosis while ensuring sufficient antigen expression, and can provide a strong pyroptosis immune stimulation effect. It stimulates strong systemic anti-tumor cell immunity and effectively reshapes the local tumor microenvironment, thereby achieving significant tumor inhibition.

[0066] The mRNA vaccine adjuvant of the present invention has an adjuvant dosage of 1 / 10 of the total nucleic acid, which is sufficient to produce a good pyroptotic adjuvant vaccine effect, thereby achieving the best anti-tumor promoting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0068] Figure 1 Schematic diagram of the pVAX1-GSDMD-NT plasmid elements in Example 1.

[0069] Figure 2 The preparation flow chart of LNP-GSDMD-NT mRNA and the characterization result chart in Example 1; wherein Figure 2 A is the preparation process of LNP-GSDMD-NT mRNA. Figure 2 B is the agarose gel electrophoresis of plasmid linearization (1 is the original plasmid, 1# is the plasmid after linearization) and the agarose gel electrophoresis of RNA bands after in vitro transcription and purification (1 to 4 are all repeated GSDMD-NT-RNA bands); Figure 2 C is the morphology and size of LNP-GSDMD-NT mRNA observed by transmission electron microscopy and the magnified transmission electron microscopy image with a scale of 200 nm; Figure 2 D is the encapsulation efficiency of different mRNAs, among which the encapsulation efficiency of GSDMD-NT mRNADE is 93.5%; Figure 2 E shows the size range and distribution of LNP-GSDMD-NT mRNA observed by transmission electron microscopy.

[0070] Figure 3 The results of LNP-GSDMD-NT mRNA expression identification and cell pyroptosis induction in Example 2 are shown in FIG. Figure 3 A is a typical cell pyroptosis picture after 293T was co-incubated with LNP-GSDMD-NTmRNA, scale bar 10 μm; Figure 3 B is Western blot detection of protein expression of GSDMD-NT molecules at different incubation times; Figure 3 C and 3E are the cell death rates of pyroptosis observed at different times using the apoptosis flow cytometry antibody Annexin-V+7-AAD+ on different cells. Figure 3 D is the release of lactate dehydrogenase (LDH) after incubation with 293T for different time periods; WT wild-type cells and cells incubated with LNP-LUC were used as controls;

[0071] Data are expressed as mean ± standard deviation (SD) and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0072] Figure 4 middle, Figure 4 A is a schematic diagram of the release of immunogenic mediators mediated by LNP-GSDMD-NT mRNA-induced cell pyroptosis in Example 3, showing the biological phenomena and immunological characteristics that may occur when pyroptosis occurs; Figure 4 B is the release of ATP at different times; Figure 4 C is ELISA to detect the release of inflammatory cytokines IL-18, IL-1β, IL-33 and IL-6 during pyroptosis of 293T cells; Figure 4 D and 4E are ELISA and Western blot detection of HMGB1 release in the supernatant of 293T cell pyroptosis, respectively;

[0073] Data are expressed as mean ± standard deviation (SD) and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0074] Figure 5 This is an experimental diagram showing that LNP-GSDMD-NT mRNA induced cell pyroptosis can promote the maturation of BNDCs and enhance the phagocytosis of pyroptotic cell fragments in Example 4; wherein, Figure 5 A is a schematic diagram of the experimental design of pyroptotic and non-pyroptotic cells promoting BMDCs maturation and APC phagocytosis of pyroptotic cell fragments; Figure 5 B shows the experimental grouping for detecting BMDCs maturation, LPS was used as a positive control, and 293TLNP-LUC was used as a non-pyroptotic isotype control; Figure 5 C is a statistical analysis of the expression of maturation markers CD80, CD86, MHC I and MHC II in CD11c+BMDCs; Figure 5 D is the fluorescence image of 293T pyroptotic cells stained with Annexin-V-FITC and PI; Figure 5 E is the fluorescence localization image of Raw264.7 and DC2.4 engulfing the debris of dead cells;

[0075] Data are expressed as mean ± standard deviation (SD) and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0076] Figure 6 This is the safety assessment of the LNP-GSDMD-NT mRNA molecular adjuvant in Example 5 in mice; wherein, Figure 6 A is the immunization flow chart of mice injected with different doses of GSDMD-NT molecular adjuvant; Figure 6 B is the weight changes of mice recorded at different time points (n=3); Figure 6 C is the weight of spleen tissue after sampling at different time points (n=3); Figure 6 D is the photos of lymph nodes of mice taken at different time points; Figure 6 E is HE sections of lymph nodes of mice in different dose groups at 24h (100×); Figure 6 F is HE sections of muscles of mice in different dose groups at 24 h (200×); Figure 6 G is HE sections of spleens of mice in different dose groups at 24 h (200×); Figure 6 HE sections of livers of mice in different dose groups at H24h (200×);

[0077] The areas indicated by white dotted circles represent the areas where inflammatory immune cells infiltrate and accumulate;

[0078] Data are expressed as mean ± standard deviation (SD). (B) Two-way ANOVA, (C) One-way ANOVA, Tukey multiple comparisons were used for statistical analysis. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0079] Figure 7 This is a diagram of the GSDMD-NT mRNA dosage exploration experiment in Example 6; wherein, Figure 7 A: chemiluminescence detection of LUC expression in 293T cells 1 h after mRNA transfection; Figure 7 B is the detection of LDH release 6 h after cell pyroptosis (n=3); Figure 7 C is the chemiluminescence detection of LUC expression after LNP-mRNA nanoparticles were co-incubated with 293T cells for 6 h; Figure 7 D is the detection of LDH release 12 h after cell pyroptosis (n=3); Figure 7 E is a schematic diagram of in vivo imaging monitoring of fluorescence expression intensity 24 hours after mice were injected with LNP-LUC vaccines of different dosage ratios;

[0080] Data are expressed as mean ± standard deviation (SD) and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0081] Figure 8 This is the anti-tumor effect experiment of different formulations of GSDMD-NT mRNA in Example 7; wherein, Figure 8 A is the immunization schedule of LNP-mRNA vaccine in TC-1 tumor model; Figure 8 B is a schematic diagram of the LNP-mRNA vaccine combination preparation and grouping; Figure 8 C is the tumor growth curve (n=5); Figure 8 D is the weight statistics of tumors in each group (n=5); Figure 8 E is the weight statistics of spleen in each group (n=5); Figure 8 F is the image of tumors in each group (n=5);

[0082] Data are expressed as mean ± standard deviation (SD), and statistical analysis was performed using two-way ANOVA for (C) and one-way ANOVA for (D&E) with Tukey's multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0083] Fig. 9 This is an experiment on the anti-tumor effect of different formulations of GSDMD-NT in Example 8; wherein, Fig. 9 A is a diagram of the vaccine immunization program for mice; Fig. 9 B is a diagram showing the specific grouping and vaccine ratio; Fig. 9 C is the tumor growth curve of mice; Fig. 9 D is a tumor picture of mice, and the red circle represents the elimination of the tumor; Fig. 9 E is the proportion of CD3+CD4+ T cells in the spleen analyzed by flow cytometry; Fig. 9 F is the proportion of IFN-γ+ cells in CD3+CD8+ T cells in the spleen analyzed by flow cytometry; Fig. 9 G is the proportion of CD69b+NK1.1+NK cells in the spleen analyzed by flow cytometry; Fig. 9 H and Fig. 9 I was flow cytometry analysis of the proportion of Th1 (CD3+CD4+IFN-γ+) T cells and Th2 (CD3+CD4+IL-4+) T cells in the spleen; Fig. 9 J and Fig. 9K respectively represents the images and statistical quantification of IFN-γ secretion by infiltrating lymphocytes in tumor tissues by ELISPOT analysis; Fig. 9 L is the proportion of MDSC (CD11b+Gr1+) in tumors analyzed by flow cytometry; Fig. 9 M and Fig. 9 N is the proportion of M1 macrophages (CD11b+F4 / 80+CD86+) and M2 macrophages (CD11b+F4 / 80+CD206+) in tumors analyzed by flow cytometry; Fig. 9 N is the proportion of IL-12-releasing macrophages (CD11b+F4 / 80+IL-12+) in tumors analyzed by flow cytometry; Fig. 9 O is the proportion of M1 TAM cells releasing IL-12 analyzed by flow cytometry.

[0084] Data are expressed as mean ± standard deviation (SD), and statistical analysis was performed using two-way ANOVA for (C) and one-way ANOVA for (D&E) with Tukey's multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. DETAILED DESCRIPTION

[0085] In the following examples, unless otherwise specified, all experimental instruments and raw materials involved are commercially available.

[0086] definition

[0087] Pyroptosis, originally known as inflammatory caspase-dependent cell death. It is a "new" type of programmed cell death that has been discovered and confirmed in recent years and is precisely regulated. It is characterized by cell swelling and membrane rupture. Its effector molecules, the Gasdermins family of proteins, oligomerize to perforate the membrane, destroy the ion homeostasis of the cell, and eventually cause the cell to osmotic collapse. In this process, a large number of inflammatory cytokines (mainly IL-1β, IL-18) and danger signal molecules are released to activate the immune system, which plays an important regulatory role in the body's anti-infection and anti-tumor immune responses. The executor of cell pyroptosis is the Gasdermins protein family with a pore-forming effect. Currently, there are 6 homologous genes in humans, namely GSDMA, GSDMB, GSDMC, GSDME (DFNA5) and Pejvakin (DFNB59). Studies have shown that the N-terminal domain with pore-forming activity and the C-terminal inhibitory domain constitute the Gasdermins protein molecule. After being hydrolyzed and cleaved, the intramolecular inhibition is released and the active N-terminal domain is generated, which will be inserted into the cell membrane and oligomerize to form pyroptotic cell pores. In addition to Pejvakin, the N-terminal domains of ectopically expressed GSDMA~E all have membrane perforation activity, which preferentially targets cell membrane acidic phospholipids (phosphatidylinositol and cardiolipin). Since phosphatidylinositol is only present in the cytoplasmic leaflet of the cell membrane, its N-terminal domain can only induce intracellular pyroptosis. Adding activated Gasdermins outside the cell does not cause cell membrane lysis; cell pyroptosis can be divided into the classical pathway mediated by caspase-1 and the non-classical pathway mediated by caspase-4, -5 or -11. As a macromolecular protein complex, inflammasomes play a vital role in the classical and non-classical cell pyroptosis pathways. They provide a platform for the cleavage and maturation of inflammatory caspases. It is noteworthy that in recent years, it has been found that pyroptosis is also mediated by apoptotic caspases, granzymes, and elastases.

[0088] Vaccine adjuvants , refers to auxiliary substances that are injected into the body together with the vaccine or in advance and can non-specifically enhance the body's immune response to antigens or change the type of immune response. Adjuvants can be immunogenic or non-immunogenic.

[0089] Application, refers to a method for delivering a composition to a subject or patient. The method of administration can be selected to target delivery (e.g., specifically deliver to) a specific area or system of the body. For example, administration can be parenteral (e.g., subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection, and any suitable infusion technique), oral, transdermal or intradermal, intradermal, rectal, intravaginal, local (e.g., by powders, ointments, creams, gels, lotions and / or drops), mucosa, nasal cavity, oral cavity, intestinal, vitreous, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation and / or inhalation; as oral spray and / or powder, nasal spray and / or aerosol, and / or by portal vein catheter.

[0090] dNTPs , refers to dATP, dCTP, dGTP, and dUTP.

[0091] Cap analogs , refers to a non-polymerizable dinucleotide with a cap function because it promotes translation or localization, and / or prevents degradation of the RNA molecule when incorporated into the 5' end of the RNA molecule. Non-polymerizable means that the cap analog will only be incorporated at the 5' end because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase. Cap analogs include, but are not limited to: a chemical structure selected from the group consisting of m7GpppG, m7GpppA, and m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G) or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and its tetraphosphate derivatives).

[0092] The cell lines used in the present invention are shown in the table below.

[0093] Table 1

[0094] Cell lines name source 4T1 Mouse breast cancer cells Tumor Cell Bank of Chinese Academy of Medical Sciences TC-1 Mouse cervical cancer cells Chinese Academy of Sciences Cell Bank DC2.4 Mouse dendritic cells Chinese Academy of Sciences Cell Bank Raw264.7 Mouse mononuclear macrophages ATCC 293T Human embryonic kidney cells Chinese Academy of Sciences Cell Bank L929 Mouse fibroblasts Chinese Academy of Sciences Cell Bank 3T3 Mouse embryonic fibroblasts Chinese Academy of Sciences Cell Bank

[0095] Culture method: 4T1, TC-1, DC2.4, and L929 cells were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum, and 293T, Raw264.7, and 3T3 cells were cultured in DMEM complete medium containing 10% fetal bovine serum. 1% penicillin-streptomycin double antibody was added to the complete medium and cultured in a 37°C constant temperature cell culture incubator containing 5% CO2.

[0096] The experimental instruments used in the present invention are shown in the table below.

[0097] Table 2

[0098]

[0099] The raw materials used in the present invention are shown in the following table.

[0100] Table 3

[0101]

[0102]

[0103] The amino acid sequence of the control experimental group designed by the present invention is as follows:

[0104] The amino acid sequence encoded by the firefly luciferase gene sequence is shown in SEQ ID NO: 8;

[0105] Specific:

[0106] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKR

[0107] YGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQ

[0108] KKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHR

[0109] TACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSA

[0110] LLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDK

[0111] PGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYW

[0112] DEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKL (stop codon)

[0113] The amino acid sequence encoded by the HPV E6 and E7 tumor antigen fusion gene sequence is shown in SEQ ID NO:9;

[0114] Specific:

[0115] MGGRRVRWEVYISRALWLTREPTAYWLIEMVSIVIRLTIGNKLVFFWSPQTQREPATMHQKRTAMFQDPQ

[0116] ERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYR

[0117] HYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRR

[0118] ETQLGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (stop codon)

[0119] The design principle of cancer vaccines is to induce tumor-specific cellular immunity by providing effective antigens and powerful immunostimulants, helping the body restore its ability to identify and eliminate tumor cells. In order to improve the problem of weak immunogenicity and short duration of immune response of mRNA vaccines, we believe that the addition of adjuvants is very necessary.

[0120] In addition to having highly immunogenic and specific antigen molecules and an excellent delivery system, a good vaccine design also needs to be paired with an effective vaccine adjuvant to exert stronger immune protection and therapeutic effects. These are also three important areas that have received much attention in vaccine research. The present invention has built a delivery platform for antigen presentation, optimized the antigen form, and also carried out conversion production between vaccine forms, highlighting the flexibility of our vaccine technology platform. On this basis, the study of adjuvants is also very necessary. Although nanoparticle vaccines and mRNA vaccines both have self-adjuvant activity, they are still not comprehensive, and the additional addition of adjuvants is crucial for the antigen to exert its effect. Therefore, the present invention further develops adjuvants that match antigen vaccines.

[0121] Example 1 Preparation and Characterization of LNP-GSDMD-NT mRNA

[0122] Prepare LNP-GSDMD-NT mRNA as follows:

[0123] (1) Plasmid construction: pVAX1 was used as the plasmid vector to directly synthesize Figure 1 The gene sequence shown is specifically composed of a T7 transcription promoter, a 5'UTR derived from human hemoglobin α2 subunit (HBA2), a GSDMD gene sequence of amino acids 1 to 277 (GSDMD-NT), a 3'UTR derived from a non-coding sequence of the mitochondrial 12S ribosomal RNA base, and a poly(A) tail. The linearization restriction site is Bsa I. The amino acid sequence of the N-terminal active domain (GSDMD-NT) of the cell pyroptosis effector molecule protein GSDMD is shown in SEQ ID NO: 1, which can produce a cell pyroptosis effector molecule;

[0124] Specifically, the amino acid sequence of GSDMD-NT (N-terminus → C-terminus) is:

[0125] MPSAFEKVVKNVIKEVSGSRGDLIPVDSLRNSTSFRPYCLLNRKFSSSRFWKPRYSCVNLSIKDILEPSAPE

[0126] PEPECFGSFKVSDVVDGNIQGRVMLSGMGEGKISGGAAVSDSSSASMNVCILRVTQKTWETMQHERHLQ

[0127] QPENKILQQLRSRGDDLFVVTEVLQTKEEVQITEVHSQEGSGQFTLPGALCLKGEGKGHQSRKKMVTIPA

[0128] GSILAFRVAQLLIGSKWDILLVSDEKQRTFEPSSGDRKAVGQRHHGLNVLAALCSIGKQLSLLSD

[0129] (2) Plasmid linearization: Figure 2 As shown, the plasmid was digested with restriction endonuclease Bsa I at 37°C for 1 hour, and 1% agarose gel electrophoresis was used to identify whether the plasmid template was linearized. Lane 1 is the band of the original plasmid, and lane 1# is the plasmid lane linearized after digestion. The results show that the plasmid template is completely linearized after digestion ( Figure 2 B left); then the linearized plasmid template was recovered using a DNA purification recovery kit and the DNA concentration was determined for later use.

[0130] (3) In vitro transcription to synthesize mRNA molecules: Figure 2 As shown, the required substrate, template, enzyme, cap analog and buffer were added according to the instructions of the in vitro transcription kit, and DNase I was added to digest the DNA template after reacting at 37°C for 3 hours; the synthesized mRNA molecules were identified by 1% agarose gel electrophoresis, and lanes 1, 2, 3, and 4 were duplicate wells of the synthesized mRNA molecules, and the results showed a single band ( Figure 2 B right). Then, the mRNA molecules were recovered using an RNA purification and recovery kit and the concentration was determined for later use.

[0131] (4) Lipid nanoparticles (LNP) encapsulate mRNA molecules: Figure 2 As shown, the LNP solution was prepared according to Pfizer's LNP formula and ratio, and the mRNA solution and LNP solution were prepared according to the microfluidic packaging instructions of Maianna to prepare LNP-mRNA; under the electron microscope, uniform round nanoparticles were observed - LNP-GSDMD-NT mRNA ( Figure 2 C); Encapsulation efficiency is above 90% ( Figure 2 D); the particle size is evenly distributed around 100-200 nm ( Figure 2 E).

[0132] Example 2 Expression and Identification of LNP-GSDMD-NT mRNA and Induction of Cell Pyroptosis

[0133] The LNP-GSDMD-NT mRNA obtained in step (4) of Example 1 was co-incubated with a human renal epithelial cell line (293T) at a dose of 2 μg. After 4 h, 8 h, and 12 h, cells were collected for quantitative Western blot detection. The expression of GSDMD-NT protein increased with time ( Figure 3 B).

[0134] In the present invention, we attempted to induce cell pyroptosis in vivo by intramuscular injection of LNP-GSDMD-NT mRNA molecules. We conducted simulation experiments in vitro with various types of cells, including human renal epithelial cell line (293T), mouse TC-1 cervical cancer cell line (TC-1), 4T1 breast cancer cell line (4T1), normal mouse L929 fibroblast cell line (L929), 3T3 embryonic fibroblast cell line (3T3), and mouse immune cell DC2.4 dendritic cell line (DC2.4), Raw264.7 macrophage cell line (Raw264.7), etc. We injected the above cells at 5×10 5 The cells were incubated with 4μg of LNP-GSDMD-NT mRNA vaccine with nucleic acid content. During the period, the state of cell pyroptosis was observed under a microscope. It was recorded that a large number of pyroptotic cells appeared in 293T cells around 10h, followed by DC2.4 and Raw264.7, while other cells showed a large number of cell death after 24 to 48h. This may be because 293T cells are a kind of tool cells suitable for transfection, and the immune cells that perform antigen presentation have a strong ability to phagocytose and take up nanoparticles, so there is such a difference. In order to better show the typical cell morphology of pyroptosis, the picture of 293T cells was partially enlarged. Compared with the control picture (LNP-LUC), the field of view of the experimental group (LNP-GSDMD-NT mRNA) was basically full of dead cells, and showed a typical cell pyroptosis morphology of swelling and rounding ( Figure 3 A).

[0135] Next, WT wild-type cells and cells incubated with LNP-LUC were used as controls and the apoptosis flow cytometry antibody Annexin-V was used to analyze the apoptosis + 7-AAD + The cells that underwent pyroptosis for 12h, 24h, and 48h were stained, and flow cytometry statistical analysis showed that the rate of cell pyroptosis increased with time ( Figure 3 C. Figure 3 E).

[0136] In general, the LNP-GSDMD-NT mRNA molecules we prepared can induce pyroptosis in various cells in vitro, which also lays the foundation for in vivo research in mice.

[0137] Example 3 LNP-GSDMD-NT mRNA induces cell pyroptosis and mediates the release and expression of immunogenic mediators

[0138] In this example, we investigated and analyzed the classic immunogenic mediators of cell pyroptosis mediated by LNP-GSDMD-NT mRNA. We used the mRNA technology platform to encapsulate the pyroptosis effector molecule GSDMD-NT into a drug form of lipid nanoparticles, which were delivered into the body to induce cell pyroptosis and enhance the body's immune activity by releasing and expressing immunogenic mediator molecules ( Figure 4 A), the corresponding supernatant of pyroptotic cells was collected, and the release of LDH was first detected to increase over time ( Figure 3 D).

[0139] ATP is a molecule in the early stage of ICD. The occurrence of pyroptosis promotes the release of ATP ( Figure 4 B). Next, we used ELISA to examine the release of inflammatory cytokines when 293T tool cells underwent pyroptosis. According to literature reports, pyroptosis can trigger the secretion of inflammatory cytokines IL-1β and IL-18, and the same conclusion was confirmed here. In addition, we also examined the secretion of the "alarmin" molecule IL-33 and the classic inflammatory cytokine IL-6, which increased over time ( Figure 4 C). As the most important signaling molecule that plays the role of "eat me", high mobility group protein B1 (HMGB1) was quantitatively detected by ELISA and Western blot. The results showed that the more severe the cell death, the more HMGB1 was released ( Figure 4 D and Figure 4 E).

[0140] Example 4 LNP-GSDMD-NT mRNA induced cell pyroptosis can promote the maturation of BNDCs and enhance the phagocytosis of pyroptotic cell debris

[0141] Pyroptosis is a newly discovered form of immunogenic cell death (ICD) that can render cells highly immunogenic. Pyroptotic cells can release danger signal molecules to recruit and activate dendritic cells (DCs).

[0142] First, LNP-GSDMD-NT and isotype control LNP-LUC were added to 293T cells. The cells that can undergo pyroptosis and produce immune stimulation are not limited to 293T. Then we used FITC-labeled Annexin-V phospholipid membrane staining antibody and nucleic acid molecule PI dye to stain the cells in order to better visualize the phagocytosis of pyroptotic cell fragments by APC ( Figure 5 A) During the experiment, we used LPS to stimulate BMDCs as a positive control, and 293T LNP-LUC Cells are isotype controls that do not undergo pyroptosis, while 293T LNP-GSDMD-NTPyroptotic cell group ( Figure 5 B). After the above-treated cells were co-incubated with BMDCs for 12 h, we washed with PBS to remove the pyroptotic cells, collected BMDCs for flow cytometry staining, and used the expression of CD86 molecules as a representative graph of flow cytometry analysis. The results showed that the addition of exogenous cells could stimulate the expression of BMDCs co-stimulatory molecules CD80 / CD86 and antigen presenting molecules MHC I / MHC II. The maturity of BMDCs in the pyroptotic cell group was the highest and statistically significant ( Figure 5 C) When using fluorescence to visualize cell pyroptosis, cell membrane fragments stained green and nuclear DNA molecules stained red are clearly seen. LNP-LUC Unbroken cells cannot be stained and no fluorescent signal is seen ( Figure 5 D). We then added the stained cells to Raw264.7 and DC2.4 cells, incubated them for 5 hours, washed them with PBS to remove the floating cells, fixed them with DAPI to stain the nuclei, and observed under a fluorescence microscope that the pyroptotic 293T cell fragments were engulfed, while intact 293T cells were not engulfed ( Figure 5 E).

[0143] In summary, through our ingenious experimental design, we have demonstrated the process of pyroptotic cells being phagocytosed and taken up by antigen-presenting cells such as Raw264.7 and DC2.4, and also proved that pyroptotic cells promote the maturation of BMDCs.

[0144] Example 5 Safety evaluation of LNP-GSDMD-NT mRNA in mice

[0145] The GSDMD-NT molecule is a toxic protein that can only form oligomers from the inside of the cell to pierce the cell membrane. The inflammatory environment it creates and the immune stimulation it brings will also cause pyroptosis side effects if they exceed a certain level. Therefore, when we want to use it as a vaccine adjuvant, we must first consider its safety. While ensuring that it can stimulate a good effect and is within a controllable safety range, we tried an exploratory experiment on the adjuvant dose, and selected a dose gradient of 1μg, 4μg, and 10μg for intramuscular injection in mice. Here we define 10μg as an extremely large adjuvant dosage. Monitor the changes in the status of the mice 12h, 24h, 48h, 72h, 96h, and 120h after injection ( Figure 6 A). Weighing the body weight revealed that the mice became slightly lighter 24 hours after the adjuvant injection and then recovered to maintain an upward trend. The change in the 10μg dose group was the most obvious, but it was not statistically significant ( Figure 6 B). During the sampling of spleen tissue at each time point, we also recorded its weight, and the results showed no difference between them ( Figure 6 C).

[0146] Existing technical studies have shown that mRNA and eGFP protein can be observed in macrophages, fibroblasts and adipocytes at the injection site 6h and 24h after intramuscular injection of LNP-mRNA vaccine. In muscle fibers, no eGFP mRNA or eGFP protein was observed at both detection points. The concentration of eGFP mRNA in different immune tissues is distributed from high to low as follows: spleen>draining lymph nodes>injection site>plasma>liver. Therefore, our research also focuses on the changes in these organs. First of all, for the injection site, we did observe symptoms of redness, swelling and inflammation of the muscle tissue during the sampling process, but this was limited to high doses of 4μg and 10μg. LNP-GSDMD-NT will soon reach the draining lymph nodes from the injection site. We removed the draining lymph nodes on the side close to the injection site at each time point and took pictures. We found that the 4μg and 10μg dose groups showed obvious swelling and redness at 24-48h, and then returned to normal ( Figure 6 D). HE sections of draining lymph nodes were also taken 24 hours after injection of different doses, and it was observed that the lymph nodes were significantly damaged as the dose increased ( Figure 6 E). Similarly, we selected the time point of 24h when the changes were most obvious, and performed HE sections on the intramuscular injection site, spleen tissue that mediates systemic immune response, and liver tissue that performs metabolic functions. Compared with the 10μg irrelevant molecule LUC group, the 1μg adjuvant group was not much different from it, but the 4μg and 10μg doses clearly caused muscle damage and inflammatory cell infiltration ( Figure 6 F). The spleen also seems to have an increased infiltration of immune cells ( Figure 6 G). For the liver, no obvious difference was seen in the slices, and it seemed to be the same as the control group ( Figure 6 H).

[0147] In summary, when using GSDMD-NT molecular adjuvant, as long as the dosage is controlled within a certain range, more serious side effects can be avoided. Combined with literature reports, we speculate that GSDMD-NT molecules will not damage muscle tissue, and they mainly have toxic effects on immune cells, which also proves its protective effect on tissues and organs from another perspective. The death of immune cells can be understood as a new concept of dying for a good reason. The value of death it triggers is greater to some extent, helping the body to stimulate a cascade of immune effects.

[0148] Example 6 Exploration of the dosage of LNP-GSDMD-NT mRNA

[0149] Vaccine adjuvants and antigens must be in the same space and time to synergize with each other to enhance the immune effect of the vaccine. In this study, both antigens and adjuvants were prepared by encapsulating LNPs in the form of mRNA, which enables the antigens and adjuvants to exert their effects in the same space and time.

[0150] The only thing we need to pay attention to is that the expression of GSDMD-NT molecules will lead to cell death, which will affect the expression of antigens to a certain extent. Therefore, in this balance between sufficient antigen expression and the ability to induce pyroptosis adjuvant effect, we need to explore a more appropriate ratio between the two.

[0151] First, we explored the ratio of a certain nucleic acid dose at the cellular level. By transfecting mRNA, we used LUC (amino acid sequence such as SEQ ID NO: 8) to replace the antigen for quantitative visualization of antigen expression. Similarly, we used the antigen N-E7 (amino acid sequence such as SEQ ID NO: 6) to replace the GSDMD-NT molecule and matched the same amount of LUC molecules as an isotype control. Specifically, we used four nucleic acid dose ratios (900ngLUC: 100ngGSDMD-NT / N-E7, 800ngLUC: 200ngGSDMD-NT / N-E7, 500ngLUC: 100ngGSDMD-NT / N-E7, 500ngLUC: 500ngGSDMD-NT / N-E7) to perform 293T cell transfection experiments. In our early transfection of GSDMD-NT molecules, it only took 30min to 1h for cell pyroptosis to occur. Although the transfection dose of GSDMD-NT molecules was greatly reduced here, the occurrence of cell pyroptosis was still very rapid and severe. Therefore, we comprehensively considered and tested the expression of LUC 1 hour after transfection. The results showed that when N-E7 antigen molecules were replaced with GSDMD-NT when transfected with LUC, the expression of LUC was affected, and the presence of only 100ng of GSDMD-NT molecules led to a very significant decrease in the expression of LUC ( Figure 7 A), 6 hours after transfection, we also performed LDH detection to characterize the degree of cell pyroptosis ( Figure 7 B). Since we were going to use LNP-GSDMD-NT to conduct experimental research in mice in the later stage, we also used the same combination method, using LNP and cells to co-incubate and conduct the same test, but the detection of LUC was delayed to 6h, and the detection of LDH was delayed to 12h. Here we also obtained the same conclusion as above ( Figure 7C&D). According to the same theoretical system, we selected the three ratios in the above experiment and conducted in vivo studies in mice. We injected the vaccine into the hind leg muscle of mice with a total amount of 5 μg of nucleic acid. After 24 hours, we injected potassium salt of fluorescein as a substrate in the same area to stabilize the reaction for 30 minutes. Then, we performed in vivo imaging of the mice to capture the fluorescence expression intensity ( Figure 7 E). The results showed that only 1 / 10 of GSDMD-NT had a significant effect on the expression of LUC, which is consistent with the conclusions obtained from the cell level experiment. The experimental results showed that the presence of GSDMD-NT affects the expression of antigens, and the higher the dose, the greater the effect on antigen expression ( Figure 7 E).

[0152] In addition, we also conducted a set of experiments, increasing the total amount of nucleic acid to 10μg. When 1 / 10 of the GSDMD-NT molecules were present, the fluorescence expression intensity of LUC was significantly affected. However, under the same conditions, its fluorescence intensity was significantly higher than the previous 5μg total amount of nucleic acid. This also confirms that this effect can be offset by increasing the antigen injection dose.

[0153] In summary, according to the minimum ratio in the experiment, that is, when the GSDMD-NT molecule accounts for only 1 / 10, it has a significant inhibitory effect on the expression of LUC, which indirectly reflects that the intensity of cell pyroptosis is sufficient. In subsequent experiments, we can use such a dosage ratio to conduct a preliminary anti-tumor effect evaluation.

[0154] Example 7 Preliminary Evaluation of the Use of LNP-GSDMD-NT mRNA as a Vaccine Adjuvant

[0155] A mouse TC-1 tumor model was established, where a total vaccine dose of 10 μg was injected each time, 9 μg of E6E7 tumor antigen (amino acid sequence as shown in SEQ ID NO: 9) and 1 μg of GSDMD-NT adjuvant molecule were selected for vaccine combination preparation, and injected once every three days ( Figure 8 A). In the process of vaccine preparation, we explored two combined preparation methods. One is that before encapsulating and preparing LNP, we mixed the nucleic acid molecules of the two together in the above ratio and prepared them into one LNP through microfluidics, which was named LNP (E6E7 + GSDMD-NT); the other is that the two nucleic acid molecules were prepared into two LNPs through microfluidics separately and then mixed according to the concentration to prepare a vaccine named LNP-E6E7 + LNP-GSDMD-NT.

[0156] As isotype controls, in the adjuvant effect group (G2), we used 9 μg of LUC as a substitute for the antigen molecule to supplement the same nucleic acid dose, and in the antigen group (G3), we used 1 μg of LUC as a substitute for the adjuvant molecule to supplement the same nucleic acid dose ( Figure 8 B). During this period, the growth of mouse tumors was monitored, and the results showed that there was no difference between the adjuvant effect group and the PBS group (G1), while tumor growth was significantly inhibited when antigen molecules were present. When antigens were combined with adjuvants, only the LNP-E6E7+LNP-GSDMD-NT group showed a statistical difference from the antigen alone group, indicating that the GSDMD-NT molecule helped the tumor antigen to exert a better tumor inhibition effect; for the two vaccine preparation methods, the method of encapsulating into two LNPs and then mixing them was better than the previous method of mixing nucleic acid molecules ( Figure 8 C), the difference between the tumor weight column statistics and tumor images can also be seen, and the spleen weight also shows a corresponding trend ( Figure 8 D&E&F). For the construction of the vaccine platform, it is very necessary to try to compare the preparation methods of the two. The method of preparing two nucleic acid molecules into one LNP obviously reduces the economic and time costs of LNP purification and concentration in the later stage. However, this more economical vaccine preparation method is obviously not feasible in the tumor treatment effect. We speculate that the probability of two nucleic acid molecules entering a cell in one LNP is the same, and the translation expression of the GSDMD-NT molecule will significantly affect the state of the cell, which leads to a greater impact on the expression of the antigen. Separating into two LNPs will increase the probability of entering two different cells, which reduces the mutual influence between the two to a certain extent.

[0157] Example 8 Evaluation of the anti-tumor immune effect of the optimized ratio of GSDMD-NT and tumor antigens

[0158] The antigen Nov S-E7 was used for the experiment, and three different doses of 1μg, 2μg and 4μg were selected as the dosage of GSDMD-NT molecular adjuvant. In order to achieve the ideal tumor inhibition effect, the Nov S-E7 antigen dose was 9μg. The total amount of nucleic acid was controlled at 13μg when immunizing animals. As for the insufficient part, it was supplemented with irrelevant molecules such as LNP-LUC. Finally, LNPs with three nucleic acid molecules were encapsulated by microfluidics and prepared into mixed vaccines according to the expected ratio. The immunization procedure is consistent with the above. In the TC-1 tumor-bearing mouse model, when the tumor volume grows to about 50-100mm 3 When the mice were injected with different LNP-mRNA vaccines ( Fig. 9A&B). The anti-tumor ability was evaluated by measuring the tumor volume of the mice. The tumor volume was significantly suppressed after the third injection of the LNP-mRNA vaccine, and cure began to appear on the 27th day. The mouse tumor tissues were collected and weighed on the 30th day. The results showed that the N+G3 vaccine group had the best anti-tumor effect, with 5 / 14 tumors cleared; 2 / 14 tumors in the N+G2 vaccine group regressed, while only one mouse in the N+G1 vaccine group regressed. Overall, the tumor growth of mice was significantly inhibited in the presence of Nov S-E7 antigen; among them, the combined treatment group with the addition of GSDMD-NT molecular adjuvant had better effect than the Nov S-E7 antigen group alone, and the three doses were statistically significant compared with it. In addition, the GSDMD-NT molecular adjuvant alone also had a significant tumor inhibitory effect at a higher dose of 4μg, while the 1μg dose had basically no effect ( Fig. 9 C). The corresponding spleen weight was also statistically recorded and was negatively correlated with tumor size ( Fig. 9 D). This also indirectly shows that a series of reactions triggered by the expression of a certain dose of GSDMD-NT molecules can promote anti-tumor immune effects.

[0159] Of course, the evaluation of immunological indicators is very important for defining the effectiveness of the vaccine and the strength of the response. We evaluated conventional anti-tumor immunological indicators. Specifically, flow cytometric analysis of lymphocytes in the spleen showed that the vaccine group with the Nov S-E7 antigen could significantly increase the proportion of CD3+CD4+T cells in the spleen and the proportion of IFN-γ+ cells in CD3+CD8+T cells in the spleen after injection, among which the N+G3 vaccine group had the most obvious increase and the most significant statistical difference ( Fig. 9 E&F). We further used flow cytometry to analyze the proportion of NK cells (CD69b+NK1.1+) in the spleen, the proportion of Th1 T cells (CD3+CD4+IFN-γ+) in the spleen, and the proportion of Th2 T cells (CD3+CD4+IL-4+). The results showed that except for the 1μg low-dose GSDMD-NT molecular adjuvant group, the N+G3 vaccine group had the most obvious increase, and the statistical difference was the most significant ( Fig. 9 G&H&I).

[0160] In addition to the systematic immune effect evaluation, the specific situation inside the tumor can better reflect the strength of the anti-tumor immune response. Although the tumor was very small at the end of our experiment, since there were 14 mice in each group, at least 9 of them had tumors, we combined them into four groups to separate the lymphocytes inside the tumor. 49-57The specific immune response was again confirmed in the IFN-γ-ELISPOT test, in which the N+G3 group had the highest average number of spots and the highest IFN-γ release ( Fig. 9 J). Similarly, flow cytometry analysis showed that in the presence of Nov S-E7 antigen, immunosuppressive MDSC (CD11b + Gr1 + ) decreased, except for the single antigen group, the others showed certain statistical differences ( Fig. 9 L). In addition, the type of macrophages in the tumor also affects the anti-tumor immune effect. Generally, there are more M2 macrophages in the tumor. Many studies have shown that only when the proportion of M1 macrophages increases can the immunosuppressive tumor microenvironment be reversed. Here, we used flow cytometry to examine the ratio of the two types of macrophages in tumor tissues. M1 macrophages (CD11b + F4 / 80 + CD86 + ) showed an upward trend only in the N+G3 vaccine group, probably because the tumors in this group were the smallest and its anti-tumor microenvironment was established and maintained until the detection time point ( Fig. 9 M). Interestingly, M2 macrophages (CD11b + F4 / 80 + CD206 + ) showed a decreasing trend in the presence of GSDMD-NT molecular adjuvant, and the Nov S-E7 antigen group alone was also statistically different from the other three doses of combined treatment groups ( Fig. 9 N). TAMs are the main immune cells in tumor tissues, among which M1 TAMs release a large amount of IL-12, which is a multifunctional pro-inflammatory cytokine that can promote CTLs to secrete more IFN-γ and enhance tumor killing. Here, we also detected tumor-associated macrophages that release IL-12, and the results showed that their proportion increased in mice treated with GSDMD-NT molecular adjuvant combined with antigen ( Fig. 9 O), which is consistent with the previous results of CTL detection. The secretion of pro-inflammatory cytokine IL-12 can induce strong T cell immunity to suppress tumor growth.

[0161] In light of the present disclosure, all methods disclosed and claimed herein may be performed and executed without undue experimentation. Although the compositions and methods of the present invention have been described in terms of preferred aspects, it will be apparent to those skilled in the art that variations may be applied to the methods described herein and the steps of the methods or the sequence of steps of the methods without departing from the concept, spirit and scope of the present invention. More specifically, it is apparent that certain reagents that are both chemically and physiologically related may be substituted for the reagents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

Claims

1. An mRNA vaccine adjuvant based on cell pyroptosis induction, characterized in that The vaccine adjuvant is composed of ionizable lipids, cholesterol, phospholipids, PEG derivatives and mRNA containing a GSDMD-NT gene sequence encoding amino acids 1 to 277; the amino acid sequence of the GSDMD-NT gene sequence is shown in SEQ ID NO:

1.

2. The mRNA vaccine adjuvant according to claim 1, characterized in that The mRNA consists of a T7 transcription promoter, a 5'UTR, a GSDMD-NT gene sequence encoding amino acids 1 to 277, a 3'UTR, and a polyA tail; Wherein, the nucleic acid sequence of the 5'UTR is shown in SEQ ID NO:2; The nucleic acid sequence of the 3'UTR is shown in SEQ ID NO:3; The nucleic acid sequence of the polyA tail is shown in SEQ ID NO:

4.

3. The mRNA vaccine adjuvant according to claim 2, characterized in that The mRNA also includes a polyC tail with a sequence as shown in SEQ ID NO:

5.

4. A method for preparing an mRNA vaccine adjuvant as claimed in any one of claims 1 to 3, characterized in that The preparation method comprises the following steps: (1) Plasmid construction: Using pVAX1 as a plasmid vector, the GSDMD-NT gene sequence was loaded to construct a recombinant plasmid; (2) Plasmid linearization: The recombinant plasmid was digested with restriction endonuclease Bsa I at 37°C for 1 h, and the linearized plasmid was recovered using a DNA purification kit; (3) In vitro transcription synthesis of mRNA molecules: The linearized plasmid, dNTPs, RNA polymerase, cap analog and buffer in step (2) were mixed, reacted at 37°C for 3 h, and then DNase I was added to digest the DNA template to obtain GSDMD-NT mRNA; (4) Combined lipid nanoparticles: Ionizable lipids, cholesterol, phospholipids, PEG derivatives, anhydrous ethanol and GSDMD-NT mRNA are prepared to obtain lipid nanoparticles with a particle size of 100 to 200 nm encapsulating GSDMD-NT mRNA, namely mRNA vaccine adjuvants.

5. An mRNA vaccine based on cell pyroptosis induction, characterized in that The mRNA vaccine includes the mRNA vaccine adjuvant described in any one of claims 1 to 3 or the mRNA vaccine adjuvant prepared by the preparation method described in claim 4, and also includes antigen lipid nanoparticles containing mRNA encoding tumor antigens or pathogen antigens.

6. The mRNA vaccine according to claim 5, characterized in that The particle size of the antigen lipid nanoparticles is 100-200 nm.

7. The mRNA vaccine according to claim 5, characterized in that The mRNA encoding the tumor antigen or pathogen antigen is composed of a T7 transcription promoter, a 5'UTR, a Norovirus S domain gene sequence, a linker, a gene sequence of the tumor antigen or pathogen antigen, a 3'UTR, and a polyA tail; Wherein, the nucleic acid sequence of the 5'UTR is shown in SEQ ID NO:2; The nucleic acid sequence of the 3'UTR is shown in SEQ ID NO:3; The nucleic acid sequence of the polyA tail is shown in SEQ ID NO:

4.

8. The mRNA vaccine according to claim 7, characterized in that The amino acid sequence encoded by the Norovirus S domain fused with the HPV E7 tumor antigen gene sequence is shown in SEQ ID NO: 6; the amino acid sequence encoded by the connecting arm is GSGSGS.

9. The mRNA vaccine according to any one of claims 5 to 8, characterized in that The pathogen antigen is selected from the E7 antigen of HPV, and the amino acid sequence of the E7 antigen is shown in SEQ ID NO:

7.

10. The mRNA vaccine according to claim 9, characterized in that In the mRNA vaccine, the nucleic acid mass ratio of the mRNA vaccine adjuvant to the antigen lipid nanoparticles is 1:9.

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