mRNA vaccine adjuvants based on pyroptosis induction and uses thereof

Abstract

A kind of mRNA vaccine adjuvant based on pyroptosis induction, the vaccine adjuvant is composed of ionizable lipid, cholesterol, phospholipid, PEG derivative and mRNA containing GSDMD-NT gene sequence encoding 1-277 amino acids;The amino acid sequence of the GSDMD-NT gene sequence is shown as SEQ ID NO:1.The present application successfully develops a kind of mRNA vaccine adjuvant based on pyroptosis induction;The adjuvant molecule has the same physical characterization attribute as mRNA vaccine, is easy to prepare, and only one day is needed to obtain finished product.And the mRNA vaccine adjuvant can cause rapid, inflammatory, lysis type pyroptosis by expressing and accumulating GSDMD-NT effector molecule protein in cell, is a general and efficient pyroptosis induction mode.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of mRNA vaccine, and particularly relates to an mRNA vaccine adjuvant based on pyroptosis induction to promote vaccine efficacy and application thereof. BACKGROUND

[0002] Cancer has been a worldwide problem that is difficult to overcome, which seriously endangers human health and affects the development of global economy. A large amount of funds is invested in cancer research every year, although there has been some success, but there are still many problems to be further solved. According to the recent global statistics, breast cancer has surpassed lung cancer to become the most common cancer, with about 2.3 million new cases in 2020. Although most early breast cancer patients have good prognosis after treatment, about 20% to 30% of patients have in situ recurrence or distant metastasis within 2 years after diagnosis of primary tumor, with poor prognosis and decreased quality of life. So far, surgery is still the main treatment for solid tumors. Although surgical techniques and equipment are constantly improving and updating, there are still 30% to 40% of patients who have tumor recurrence or metastasis within 5 years, the main reason is that there are tumor cells remaining in the surgical margin and temporary immunosuppressive state during wound healing.

[0003] In addition to surgical resection, the existing methods for treating cancer also include chemotherapy (referred to as chemotherapy), radiotherapy (referred to as radiotherapy) and immunotherapy to remove cancer cells in the body. Radiotherapy and chemotherapy have low targeting and large side effects, causing great pain to patients; and immunotherapy is the only method in modern technology that can completely remove cancer cells, which has great advantages over traditional treatment methods, and was ranked first in the top ten scientific breakthroughs by Science magazine in 2013, with broad prospects.

[0004] Tumor vaccine is a kind of immunotherapy, whose principle is to inject tumor antigens into tumor patients in various forms, overcome the immunosuppressive state caused by tumors, activate the patient's own immune system, and induce the body to produce cellular and humoral immune responses, so as to control or eliminate tumors. Compared with other immunotherapies, tumor vaccine has incomparable advantages.

[0005] Tumor vaccines can be mainly divided into four categories according to the preparation method, namely tumor cell vaccine, virus and bacteria based vaccine, polypeptide protein vaccine and nucleic acid vaccine. Nucleic acid vaccine is the third generation vaccine after inactivated vaccine, attenuated live vaccine and genetically engineered recombinant protein vaccine, which is divided into DNA vaccine and RNA vaccine. Compared with other types of tumor vaccines, mRNA tumor vaccine can use linear DNA template to generate in vitro transcription, without complicated in vitro culture process, so that mRNA vaccine can be produced more quickly and efficiently, which is an ideal platform for tumor vaccine preparation; and compared with DNA vaccine, mRNA vaccine can be translated and efficiently expressed in cytoplasm without the risk of insertion and integration into the genome.

[0006] At present, the in vitro transcription preparation method of mRNA molecule and the effective delivery expression system in vivo have been quite mature. However, the anti-tumor specific T cell immune response effect stimulated by mRNA vaccine is not enough, and the sustained time is not long enough, which is the key technical challenge in this field. At present, the main strategies are to improve the expression level by optimizing the sequence design, to provide broad-spectrum immune stimulation by fusing the expression of multiple antigen epitopes, and to adopt the inoculation mode of sequential immunization, but these ways are still limited in improving the efficacy of mRNA vaccine.

[0007] In order to promote the better function of mRNA vaccine, a new strategy method and the innovation of concept are particularly important. SUMMARY

[0008] In order to develop an adjuvant suitable for mRNA vaccine to promote anti-tumor immune effect, the present application provides a mRNA vaccine adjuvant based on cell pyroptosis induction and application thereof.

[0009] In a first aspect, the present application provides a mRNA vaccine adjuvant based on cell pyroptosis induction, which is composed of ionizable lipid, cholesterol, phospholipid, PEG derivative and mRNA containing GSDMD-NT gene sequence encoding amino acids 1-277; the amino acid sequence encoded by the GSDMD-NT gene sequence is shown as SEQ ID NO: 1.

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

[0011] The nucleic acid sequence of the 5'UTR is shown as SEQ ID NO: 2;

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

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

[0014] The 5'UTR is derived from the human hemoglobin alpha 2 subunit, and the 3'UTR is derived from the non-coding sequence of the 12S ribosomal RNA base encoded by mitochondria.

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

[0016] In a second aspect of the present application, a preparation method of the above mRNA vaccine adjuvant is provided, and the preparation method comprises the following steps:

[0017] (1) Plasmid construction:

[0018] A recombinant plasmid is constructed by using pVAX1 as a plasmid vector and loading a GSDMD-NT gene sequence;

[0019] (2) Plasmid linearization:

[0020] After the recombinant plasmid is digested by restriction endonuclease Bsa I at 37℃ for 1h, the linearized plasmid is recovered by using a DNA purification recovery kit;

[0021] (3) In vitro transcription to synthesize mRNA molecules:

[0022] After the linearized plasmid of step (2), dNTPs, RNA polymerase, cap analogs and buffer are uniformly mixed and reacted at 37℃ for 3h, DNA template is digested by DNAse I to obtain mRNA of GSDMD-NT;

[0023] (4) Combination of lipid nanoparticles:

[0024] The mRNA of GSDMD-NT is prepared to obtain lipid nanoparticles encapsulating the mRNA of GSDMD-NT and having a particle size of 100-200nm, i.e., the mRNA vaccine adjuvant.

[0025] In a third aspect of the present application, an application of a tumor vaccine prepared by using the above mRNA vaccine adjuvant or the mRNA vaccine adjuvant prepared by the above preparation method is provided.

[0026] In an embodiment of the present application, the tumor vaccine is a tumor mRNA vaccine.

[0027] In a fourth aspect of the present application, an mRNA vaccine based on pyroptosis induction is provided, which comprises the mRNA vaccine adjuvant or the mRNA vaccine adjuvant prepared by the preparation method described above, and further comprises an antigen lipid nanoparticle containing mRNA encoding a tumor antigen or a pathogen antigen.

[0028] In an embodiment of the present application, the antigen lipid nanoparticle has a particle size of 100-200 nm.

[0029] In another embodiment of the present application, the mRNA encoding the tumor antigen or the pathogen antigen comprises a T7 transcription promoter, a 5'UTR derived from human hemoglobin alpha 2 subunit, a Norovirus S domain gene sequence, a linker, a gene sequence of the tumor antigen or the pathogen antigen, a 3'UTR derived from a non-coding sequence of mitochondrial encoded 12S ribosomal RNA, and a polyA tail.

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

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

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

[0033] Specifically:

[0034] CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCC

[0035] CCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGAC

[0036] ACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGC

[0037] AGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC (5'→ 3')

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

[0039] Specifically,

[0040] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (5'→ 3')

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

[0042] Specifically,

[0043] TGCATCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAAAGGCTCTTTTCAGAGCCACCAGAATT (5'→ 3')

[0044] In another embodiment of the present application, the amino acid sequence encoded by the Norovirus S domain fusion HPV E7 tumor antigen gene sequence is shown as 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 application, the pathogen antigen is selected from an E7 antigen of HPV, and the amino acid sequence of the E7 antigen is shown as SEQ ID NO: 7.

[0052] pfizer-E7:

[0053] MGGRRVRWEVYISRALWLTREPTAYWLIEMVSIVIRLTIGNKLVFFWSPQTQREPATMHGDTPTLHEYML DLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (stop codon)

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

[0055] In another embodiment of the present application, the tumor antigen is selected from an antigen of at least one tumor of 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 application, in the mRNA vaccine, the nucleic acid mass ratio of the mRNA vaccine adjuvant to the antigen lipid nanoparticle is 1:9.

[0057] In a fifth aspect of the present application, a pharmaceutical composition is provided, which comprises the mRNA vaccine adjuvant and the antigen lipid nanoparticle described above.

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

[0059] The "pharmaceutically acceptable" indicates that the pharmaceutical composition can be administered to a subject without producing an adverse, physiologically- mediated response in the subject that outweighs the medicinal benefits provided by the pharmaceutical composition. For example, "pharmaceutically acceptable excipients" refer to excipients useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and physiologically tolerable, and preferably, examples of these excipients or diluents include, but are not limited to: water, saline, Ringer's solution, dextrose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, and liposomes and non-aqueous vehicles such as fixed oils can also be used.

[0060] The term "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Specific examples can be one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, and the like, are included in the pharmaceutical composition. Of course, the pharmaceutically acceptable carrier can also include minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or potency of the antibody.

[0061] In a sixth aspect of the present application, a method for using a pharmaceutical composition is provided, comprising administering mRNA vaccine adjuvant and antigen lipid nanoparticles separately or simultaneously.

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

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

[0064] The present application successfully develops an mRNA vaccine adjuvant based on pyroptosis induction; the adjuvant molecule has the same physical characterization as the mRNA vaccine, is easy to prepare, and only one day is needed to obtain the finished product. And the mRNA vaccine adjuvant can cause rapid, inflammatory, and lytic pyroptosis by expressing and accumulating GSDMD-NT effector molecule protein inside the cell, which is a general and efficient pyroptosis induction method.

[0065] The mRNA vaccine adjuvant of the present application can cooperate with antigens to promote effects, which is a very mRNA vaccine application adjuvant form. Through optimization of the ratio and exploration of the vaccine preparation method, we can efficiently induce the occurrence of pyroptosis while ensuring sufficient antigen expression, which can provide strong pyroptosis immune stimulation effect. Strong systemic anti-tumor cell immunity is stimulated, the local tumor microenvironment is effectively reshaped, and thus significant tumor inhibition is achieved.

[0066] The mRNA vaccine adjuvant of the application can produce good pyroptosis adjuvant vaccine effect and achieve the best anti-tumor promotion effect at an adjuvant amount of 1 / 10 of the total nucleic acid. BRIEF DESCRIPTION OF DRAWINGS

[0067] The application will be further described below in conjunction with the drawings and examples.

[0068] Figure 1 Figure 1 is a schematic diagram of the pVAX1-GSDMD-NT plasmid element in Example 1.

[0069] Figure 2 Figure 1 is a schematic diagram of the pVAX1-GSDMD-NT plasmid element in Example 1. Figure 2 A is the preparation process of LNP-GSDMD-NT mRNA, Figure 2 B is a linearized plasmid agarose gel electrophoresis diagram (1 is the original plasmid, 1# is the linearized plasmid) and an agarose gel electrophoresis diagram of RNA bands after in vitro transcription and purification (1-4 are all repeated GSDMD-NT-RNA bands) ; Figure 2 C is a transmission electron microscope observation of the morphology and size of LNP-GSDMD-NT mRNA and a transmission electron microscope magnification diagram with a scale of 200 nm; Figure 2 D is the encapsulation rate of different mRNA, wherein the encapsulation rate of GSDMD-NT mRNA DE is 93.5%; Figure 2 E is a transmission electron microscope observation of the size range and distribution of LNP-GSDMD-NT mRNA.

[0070] Figure 3 Figure 2 is a result diagram of LNP-GSDMD-NT mRNA expression identification and cell pyroptosis induction in Example 2; wherein Figure 3 A is a typical cell pyroptosis picture after 293T and LNP-GSDMD-NT mRNA co-incubation, with a scale of 10 μm; Figure 3 B is a Western blot detection of GSDMD-NT molecular protein expression at different incubation times; Figure 3 C and 3E are observations of cell death rates of different cells using apoptosis flow cytometry antibodies Annexin-V+7-AAD+ at different times, Figure 3 D is the lactate dehydrogenase (LDH) release situation of 293T co-incubation at different times; wherein WT wild type cells and LNP-LUC incubated cells are used as controls;

[0071] Data are expressed as mean ± SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. 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 In summary, Figure 4 A is a schematic diagram of LNP-GSDMD-NT mRNA inducing the release of cell pyroptosis-mediated immunogenic mediators, 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 the release of inflammatory cytokines IL-18, IL-1β, IL-33 and IL-6 in 293T cells by ELISA detection when pyroptosis occurs; Figure 4 D and 4E are the release of HMGB1 in the supernatant of 293T cells by ELISA and Western blot detection when pyroptosis occurs, respectively;

[0073] Data are expressed as mean ± SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. 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 A schematic diagram of experiments in Example 4 that LNP-GSDMD-NT mRNA induced cell pyroptosis can promote the maturation of BNDCs and enhance the phagocytosis of pyroptosis cell fragments; wherein, Figure 5 A is a schematic diagram of the experimental design for promoting BMDCs maturation and APC phagocytosis of pyroptosis cell fragments by pyroptosis and non-pyroptosis cells; Figure 5 B is the experimental grouping for detecting the maturation of BMDCs, LPS as a positive control, 293T LNP-LUC as a non-pyroptosis homologous control; Figure 5 C is a statistical analysis chart of the expression of maturation markers CD80, CD86, MHC I and MHC II in CD11c+BMDCs; Figure 5 D is a fluorescence picture of Annexin-V-FITC and PI staining of 293T pyroptosis cells; Figure 5 E is a fluorescence localization picture of Raw264.7 and DC2.4 phagocytosis of pyroptosis cell fragments;

[0075] Data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test. 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 For safety evaluation of LNP-GSDMD-NT mRNA molecule adjuvant in mice in Example 5; wherein, Figure 6 A is the immunization flow chart of mice injected with different doses of GSDMD-NT molecule adjuvant; Figure 6 B is the weight change of mice 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 photograph of lymph nodes of mice at different time points; Figure 6 E is the HE section of lymph nodes of mice in different dose groups at 24h (100x); Figure 6 F is the HE section of muscle of mice in different dose groups at 24h (200x); Figure 6 G is the HE section of spleen of mice in different dose groups at 24h (200x); Figure 6 H is the HE section of liver of mice in different dose groups at 24h (200x);

[0077] The area where the white dotted line circle indicates represents the area of inflammatory immune cell infiltration aggregation;

[0078] Data are expressed as mean ± SD. (B) Two-way ANOVA was used, (C) one-way ANOVA was used, and Tukey's multiple comparison test was used for statistical analysis. 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 For GSDMD-NT mRNA dose-finding experiment diagram in Example 6; wherein, Figure 7 A is the detection of LUC expression by chemiluminescence method after transfection of 293T cells with mRNA molecules for 1h; Figure 7 B is the detection of LDH release after pyroptosis of cells for 6h (n = 3); Figure 7 C is the detection of LUC expression by chemiluminescence method after co-incubation of LNP-mRNA nanoparticles with 293T cells for 6h; Figure 7 D is the detection of LDH release after pyroptosis of cells for 12h (n = 3); Figure 7 E is a schematic diagram of monitoring the fluorescence expression intensity by in vivo imaging after injection of LNP-LUC vaccine with different dose ratios into mice for 24h;

[0080] Data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test. 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 Anti-tumor effect experiment of different formulations of GSDMD-NT mRNA in Example 7; wherein, Figure 8 A is the immunization program of LNP-mRNA vaccine in TC-1 tumor model; Figure 8 B is a schematic diagram of the combination of LNP-mRNA vaccine 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 spleens in each group (n = 5); Figure 8 F is the image of tumors in each group (n = 5);

[0082] Data are expressed as mean ± SD. (C) Two-way ANOVA was used, (D & E) One-way ANOVA was used, and Tukey's multiple comparison test was used for statistical analysis. Significant differences are as follows: ns, not significant, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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

[0084] Data are expressed as mean ± standard deviation (SD), (C) two-way ANOVA, (D & E) one-way ANOVA, Tukey's multiple comparisons for statistical analysis. 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, the experimental instruments and materials involved, in the absence of a specific description, are commercially available.

[0086] DEFINITIONS

[0087] AdministrationPyroptosis, initially known as inflammatory caspase-dependent cell death, is a recently discovered and confirmed 'new' programmed cell death mode with precise regulation. It is characterized by cell swelling and membrane rupture. The effector molecule Gasdermins family protein oligomerization destroys the ion homeostasis of the membrane and ultimately causes the osmotic collapse of the cell. In this process, a large amount of inflammatory cytokines (mainly IL-1β and IL-18) and danger signal molecules are released to activate the immune system, which plays an important role in regulating the body's anti-infection immune response and anti-tumor immune response. The executor of cell pyroptosis is the Gasdermins protein family with pore-forming effect. There are currently six homologous genes in humans, namely GSDMA, GSDMB, GSDMC, GSDME (DFNA5) and Pejvakin (DFNB59). Studies have shown that the N-terminal domain and C-terminal inhibition domain with pore-forming activity make up the Gasdermins protein molecule. After being hydrolyzed and cut, the N-terminal domain is released and becomes active, which can insert into the cell membrane and oligomerize to form a pyroptotic cell pore. Except for Pejvakin, the N-terminal domain of GSDMA-E expressed in ectopic has membrane perforation activity, and it preferentially targets the cell membrane acidic phospholipid (phosphatidylinositol and cardiolipin). Because phosphatidylinositol only exists in the cytoplasmic leaflet of the cell membrane, its N-terminal domain can only induce intracellular pyroptosis, and the addition of activated Gasdermins outside the cell will not cause 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, inflammasome plays a crucial role in the classical and non-classical cell pyroptosis pathways, providing a platform for the cleavage and maturation of inflammatory caspases. It is worth noting that in recent years, cell pyroptosis has also been found to be mediated by apoptotic caspases, granzymes and elastases.

[0088] dNTPs Adjuvant refers to a helper substance that can non-specifically enhance the body's immune response to antigens or change the type of immune response when injected into the body together with or before the vaccine. Adjuvants can be immunogenic or non-immunogenic.

[0089] Cap analogsis meant to refer to a method of delivering the composition to a subject or patient. The method of administration can be chosen to target (e.g., specifically deliver to) a particular region 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, as well as any suitable infusion techniques), oral, transdermal or intradermal, intracutaneous, rectal, intravaginal, topical (e.g., via powders, ointments, creams, gels, lotions and / or drops), mucosal, nasal, buccal, enteral, vitreous, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation and / or inhalation; as oral sprays and / or powders, nasal sprays and / or aerosols, and / or through a portal vein catheter.

[0090] Cell lines is meant to refer to dATP, dCTP, dGTP, dUTP.

[0091] Name is meant to refer to a non-polymerizable dinucleotide with a capping function, as it facilitates translation or localization, and / or prevents degradation of the RNA molecule when incorporated at the 5' end of the RNA molecule. Non-polymerizable means that the cap analog will only be incorporated at the 5' end, as it does not have a 5' triphosphate and thus 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, m7GpppC; an unmethylated cap analog (e.g., GpppG); a dimethylated cap analog (e.g., m2,7GpppG), a trimethylated cap analog (e.g., m2,2,7GpppG), a dimethylated symmetric cap analog (e.g., m7Gpppm7G), or an anti-reverse cap analog (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG, and tetraphosphate derivatives thereof).

[0092] Cell lines used in the present application, see the following table

[0093] Table 1

[0094] Source Mouse breast cancer cells Tumor Cell Bank of Chinese Academy of Medical Sciences 4T1 TC-1 Mouse cervical cancer cells Cell Bank of Chinese Academy of Sciences DC2.4 Mouse dendritic cells Cell Bank of Chinese Academy of Sciences Raw264.7 Mouse monocyte macrophages ATCC Human embryonic kidney cells Cell Bank of Chinese Academy of Sciences 293T Mouse fibroblasts Cell Bank of Chinese Academy of Sciences L929 Mouse embryonic fibroblasts Cell Bank of Chinese Academy of Sciences 3T3 Figure 1 Figure 2

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

[0096] The experimental instruments used in the application are shown in the following table.

[0097] Table 2

[0098]

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

[0100] Table 3

[0101]

[0102]

[0103] The amino acid sequence of the control experiment group involved in the application is as follows:

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

[0105] Specifically:

[0106] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKR

[0107] YGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQ

[0108] KKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHR

[0109] TACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSA

[0110] LLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDK

[0111] PGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYW

[0112] DEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKL (stop codon)

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

[0114] Specifically:

[0115] MGGRRVRWEVYISRALWLTREPTAYWLIEMVSIVIRLTIGNKLVFFWSPQTQREPATMHQKRTAMFQDPQ

[0116] ERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYR

[0117] HYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRR

[0118] ETQLGSGSGSMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (stop codon)

[0119] The design principle of cancer vaccine is to induce tumor-specific cellular immunity by providing effective antigens and strong immune stimulants, helping the body to restore the ability to recognize and clear tumor cells. In order to improve the problem of weak immunogenicity and short duration of immune response of mRNA vaccine, we think that the addition of adjuvant is very necessary.

[0120] Good vaccine design requires not only highly immunogenic and specific antigen molecules and excellent delivery systems, but also effective vaccine adjuvants to exert stronger immune protection and therapeutic effects. This is also the three important fields of concern in vaccine research. The present application builds an antigen-presenting delivery platform, optimizes the antigen form, and also carries out the conversion production between vaccine forms, highlighting the flexibility of our vaccine technology platform. On this basis, the research of adjuvants is also very necessary. Although the nanoparticle vaccine and the mRNA vaccine both have the activity of self-adjuvant, but it is not comprehensive, and the additional addition of adjuvant is crucial for the antigen to exert effect. Therefore, the present application further develops the adjuvant matching the antigen vaccine.

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

[0122] The LNP-GSDMD-NT mRNA was prepared according to the following steps:

[0123] (1) Plasmid construction: The gene sequence shown in Figure 2 was directly synthesized by using pVAX1 as a plasmid vector, specifically consisting of a T7 transcription promoter, a 5'UTR derived from human hemoglobin a2 subunit (HBA2), a GSDMD gene sequence (GSDMD-NT) of amino acids 1-277, a 3'UTR derived from a non-coding sequence of mitochondrial encoded 12S ribosomal RNA, and a poly(A) tail. The linearization enzyme cutting 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 be a cell pyroptosis effector molecule;

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

[0125] MPSAFEKVVKNVIKEVSGSRGDLIPVDSLRNSTSFRPYCLLNRKFSSSRFWKPRYSCVNLSIKDILEPSAPE

[0126] PEPECFGSFKVSDVVDGNIQGRVMLSGMGEGKISGGAAVSDSSSASMNVCILRVTQKTWETMQHERHLQ

[0127] QPENKILQQLRSRGDDLFVVTEVLQTKEEVQITEVHSQEGSGQFTLPGALCLKGEGKGHQSRKKMVTIPA

[0128] GSILAFRVAQLLIGSKWDILLVSDEKQRTFEPSSGDRKAVGQRHHGLNVLAALCSIGKQLSLLSD

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

[0130] (3) In vitro transcription to synthesize mRNA molecules: As shown in Figure 2 , the required substrates, templates, enzymes, cap analogs and buffers were added according to the instructions of the in vitro transcription kit, and DNase I was added to digest the DNA template after 3h of reaction at 37℃. 1% agarose gel electrophoresis was used to identify the synthesized mRNA molecules. Lanes 1, 2, 3 and 4 were repeated wells of the synthesized mRNA molecules, and the results showed a single band Figure 2 B right). Then the mRNA molecules were recovered with an RNA purification recovery kit and the concentration was determined for standby use.

[0131] (4) Lipid nanoparticle (LNP) encapsulated mRNA molecules: As shown in Figure 2 , LNP solution was prepared according to the LNP formulation and ratio of Pfizer, and mRNA solution and LNP solution were prepared according to the microfluidic encapsulation instructions of Maynana to prepare LNP-mRNA. Under electron microscopy, it was observed that the LNP-GSDMD-NT mRNA was uniform round nanoparticles Figure 2 C); the encapsulation efficiency was more than 90% Figure 3 D); and the particle size was uniformly distributed at about 100-200nm Figure 3 E).

[0132] Example 2 Expression identification and cell pyroptosis induction of LNP-GSDMD-NT mRNA

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

[0134] In the present application, we try to induce cell pyroptosis in vivo by injecting LNP-GSDMD-NT mRNA molecules into the muscle. Through simulation experiments in vitro with various types of cells, including human kidney epithelial cell line (293T), mouse TC-1 cervical cancer cell line (TC-1), 4T1 breast cancer cell line (4T1), mouse normal L929 fibroblast cell line (L929), 3T3 embryonic fibroblast cell line (3T3), and mouse immune cells DC2.4 dendritic cell line (DC2.4), Raw264.7 macrophage cell line (Raw264.7), etc., we incubated the above cells with 5x10 5 pyroptosis, and recorded that 293T cells appeared a large number of pyroptosis cells at about 10h, followed by DC2.4 and Raw264.7, while other cells appeared a large number of cell death at 24 to 48h. It may be because 293T cells are a suitable tool for transfection, and immune cells that perform antigen presentation have strong phagocytosis of nano-form particles, so there is such a difference. In order to better show the typical cell morphology of pyroptosis, the picture of 293T cells was enlarged, compared with the control picture (LNP-LUC), the experimental group (LNP-GSDMD-NT mRNA) basically filled the field of view with dead cells, and showed the typical cell pyroptosis morphology of swelling and rounding Figure 3 A).

[0135] Next, using WT wild-type cells and cells incubated with LNP-LUC as controls, we used the apoptosis flow antibody Annexin-V + 7-AAD + to stain various cells that occurred pyroptosis at 12h, 24h, 48h, and the flow cytometry analysis showed that the ratio of cell pyroptosis increased with time Figure 4 C, Figure 3 E).

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

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

[0138] In this example, we performed a classical immunogenic mediator analysis of LNP-GSDMD-NT mRNA-mediated pyroptosis. We used the mRNA platform to encapsulate the pyroptosis effector molecule GSDMD-NT into a drug form of a lipid nanoparticle, which was delivered to induce cell pyroptosis in vivo to enhance the body's immune activity by releasing and expressing immunogenic mediator molecules. Figure 4 A), and collected the corresponding pyroptotic cell supernatant. First, we detected the increasing release of LDH over time Figure 4 D).

[0139] ATP is a molecule in the early stage of ICD, and the occurrence of pyroptosis promotes the release of ATP Figure 4 B). Next, we used ELISA to investigate the release of inflammatory cytokines from 293T tool cells undergoing pyroptosis. According to literature reports, the occurrence of pyroptosis can trigger the secretion of inflammatory cytokines IL-1β and IL-18, and here the same conclusion was again confirmed. In addition, we also investigated the increasing secretion of "alarmin" molecule IL-33 and classical inflammatory cytokine IL-6 over time Figure 4 C). As the most important signal molecule playing the role of "eat me", high mobility group protein B1 (HMGB1), we used ELISA and Western blot to quantitatively detect the release and accumulation of HMGB1 in the supernatant, and the results showed that the more severe the cell death, the more HMGB1 was released Figure 5 D and Figure 5 E).

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

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

[0142] First, we added LNP-GSDMD-NT and the same control LNP-LUC to 293T cells, and here the cells that can undergo pyroptosis to produce immune stimulation are not limited to 293T. Next, we used FITC-labeled Annexin-V phospholipid membrane staining antibodies and nucleic acid dye PI to stain the cells, in order to better visualize the phagocytosis of APC by pyroptotic cell fragments Figure 5 A). During the experiment, we used LPS-stimulated BMDCs as a positive control, 293T LNP-LUC cells were the same control without pyroptosis, and 293T LNP-GSDMD-NTPyroptotic cell group Figure 5 B) After co-incubation of the treated cells with BMDCs for 12h, we removed the pyroptotic cells with PBS wash and collected the BMDCs for flow cytometry staining. The expression of CD86 molecule was used as a representative figure for flow cytometry analysis. The results showed that the addition of exogenous cells could stimulate the expression of costimulatory molecules CD80 / CD86 and antigen-presenting molecules MHC I / MHC II in BMDCs, among which the maturity of the pyroptotic cell group was the highest and statistically significant Figure 5 C) When visualizing cell pyroptosis with fluorescence, it can be clearly seen that the cell membrane fragments are dyed green, and there are also red-stained nuclear DNA molecules. For 293T LNP-LUC Cells that are not broken cannot be stained and no fluorescence signal can be seen Figure 6 D) Then we added these dyed cells to Raw264.7 and DC2.4 cells, incubated for 5 hours, and then removed the floating cells with PBS wash, fixed DAPI for nuclear staining, and observed under a fluorescence microscope that the pyroptotic 293T cell fragments were phagocytosed, while the intact 293T cells were not phagocytosed Figure 6 E).

[0143] In summary, through our ingenious experimental design, we have shown the process of pyroptotic cell phagocytosis by Raw264.7 and DC2.4 antigen-presenting cells, and also proved that pyroptotic cells promote the maturation of BMDCs.

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

[0145] GSDMD-NT molecule is a toxic protein that can only form oligomers inside the cell to punch the cell membrane, and the inflammatory environment it creates and the immune stimulation it brings will exceed a certain extent and also trigger pyroptosis side effects. Therefore, when we want to use it as a vaccine adjuvant, we must first consider its safety. In order to ensure that it can stimulate good effects and within a controllable safety range, we tried to explore the dose of the adjuvant, and selected 1 μg, 4 μg, 10 μg dose gradient for intramuscular injection in mice. Here we define 10 μg as an extremely large dose of adjuvant. We monitored the changes in the state of mice at 12h, 24h, 48h, 72h, 96h, 120h after injection Figure 6 A) By weighing the body weight, we found that the mice became slightly lighter 24h after injection of the adjuvant, and then recovered and maintained an upward trend, among which the change in the 10 μg dose group was the most obvious, but not statistically significant Figure 6 B) During the process of taking the spleen tissue at each time point, we also recorded its weight, and the results showed that there was no difference between them Figure 6 C)

[0146] The prior art studies show that: 6h and 24h after LNP-mRNA vaccine intramuscular injection, mRNA and eGFP protein can be observed in macrophages, fibroblasts and adipocytes at the injection site. In muscle fibers, eGFP mRNA or eGFP protein was not observed at two detection points. The eGFP mRNA concentration in different immune tissues is distributed from high to low as follows: spleen > draining lymph node > injection site > plasma > liver. Therefore, our study also focuses on the changes in these organs. First, for the injection site, we did observe the symptoms of redness and inflammation of the muscle tissue during the sampling process, but this was limited to high doses of 4μg and 10μg. LNP-GSDMD-NT quickly reached the draining lymph nodes from the injection site, and we took pictures of the draining lymph nodes on the side close to the injection site at each time point. 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). At the same time, we performed HE sectioning of the draining lymph nodes 24h after injection of different doses, and observed that the lymph nodes were significantly damaged as the dose increased( Figure 6 E). Similarly, we selected the most obvious change time point of 24h, and performed HE sectioning of the muscle injection site, the spleen tissue mediating systemic immune response, and the liver tissue performing metabolic function. Compared with the 10μg irrelevant molecule LUC group, the 1μg adjuvant group showed no significant difference, but the 4μg and 10μg doses significantly caused inflammatory cell infiltration in muscle damage( Figure 6 F). For the spleen, it also seemed to increase the aggregation and infiltration of immune cells( Figure 7 G). For the liver, no obvious difference was observed from the section, which seemed to be the same as the control group( Figure 7 H).

[0147] In summary, when using the GSDMD-NT molecular adjuvant, as long as the dose is controlled within a certain range, serious side effects can be avoided. Combined with literature reports, we speculate that the GSDMD-NT molecule does not damage the muscle tissue, and its main toxic effect is on immune cells. From another perspective, it also proves its protective effect on tissues and organs, and the death of immune cells can be understood as a new concept of dying in place. The value of the death it triggers is greater to some extent, which helps the body to trigger a cascading immune effect.

[0148] Example 6 LNP-GSDMD-NT mRNA dose exploration

[0149] Vaccine adjuvants and antigens can synergistically enhance vaccine immune effects in the same space-time. In this study, both antigens and adjuvants are in the form of mRNA for LNP packaging preparation, which can realize the effect of antigens and adjuvants in the same space-time.

[0150] The only thing to note is that the expression of GSDMD-NT molecules will cause cell death, which will affect the expression of antigens to some extent, so in this balance of both sufficient amount of antigen expression and the ability to induce pyroptosis adjuvant effect, we need to explore the more appropriate proportion between the two.

[0151] First of all, we carried out a certain nucleic acid dose ratio exploration at the cellular level. By transfecting mRNA, we used LUC (amino acid sequence as SEQ ID NO: 8) to replace the antigen to quantitatively visualize the expression of the antigen, and the same amount of LUC molecule was used as a homotypic control to replace the GSDMD-NT molecule to match the antigen N-E7 (amino acid sequence as SEQ ID NO: 6). Specifically, we used four nucleic acid dose ratios (900ng LUC:100ng GSDMD-NT / N-E7, 800ng LUC:200ng GSDMD-NT / N-E7, 500ng LUC:100ng GSDMD-NT / N-E7, 500ng LUC:500ng GSDMD-NT / N-E7) to transfect 293T cells. Our previous transfection of GSDMD-NT molecules resulted in the occurrence of cell pyroptosis only in 30min to 1h, and 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 considered the expression of LUC at 1h after transfection, and the results showed that when N-E7 antigen molecules were replaced by GSDMD-NT, the expression of LUC was affected, and only 100ng of GSDMD-NT molecules resulted in a very significant reduction in LUC expression amount Figure 7 A), at 6h after transfection we also detected LDH to characterize the degree of cell pyroptosis Figure 7 B). Based on the fact that we later used LNP-GSDMD-NT to conduct experimental research in mice, we also used the same matching method to use LNP to co-incubate with cells to conduct the same detection, except that the detection of LUC was delayed to 6h, and the detection of LDH was delayed to 12h. Here we also got the same conclusion as above Figure 7C&D). Following the same theoretical system, we selected three combinations from the above experiments to conduct in vivo studies in mice, with a total of 5 μg of nucleic acid injected into the hind leg muscles of mice, and 24 hours later, the same site was injected with potassium luciferin as a substrate to stabilize the reaction for 30 minutes before taking live mouse images to capture the fluorescence intensity Figure 8 E). The results showed that only 1 / 10 of GSDMD-NT had a significant impact on the expression of LUC, consistent with the conclusion obtained from the cell level experiment. The experimental results showed that the presence of GSDMD-NT would affect the expression of antigens, and the higher the dose, the greater the impact on antigen expression Figure 8 E).

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

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

[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, and here a total of 10 μg of vaccine dose was used for each injection, 9 μg of E6E7 tumor antigen (amino acid sequence as shown in SEQ ID NO: 9) and 1 μg of GSDMD-NT adjuvant molecules were used to prepare the vaccine combination, and the injection was performed every three days Figure 8 A). In the process of vaccine preparation, we explored two ways of combination preparation, one is that before being wrapped into LNP, we mix the two nucleic acid molecules together according to the above ratio and prepare them into an LNP through microfluidic control, named LNP(E6E7+GSDMD-NT); the second is to prepare two LNP through microfluidic control respectively, and then mix them according to the concentration to prepare a vaccine, named LNP-E6E7+LNP-GSDMD-NT.

[0156] As a homotypic control, we used 9 pg of LUC as a replacement of the antigenic molecule to complement the same nucleic acid dose in the adjuvant effect group (G2) and 1 pg of LUC as a replacement of the adjuvant molecule to complement the same nucleic acid dose in the antigen group (G3) Figure 8 B). During this period, the growth of the tumors in the mice was monitored, and the results showed that there was no difference between the adjuvant effect group and the PBS group (G1), while the tumor growth was significantly inhibited when the antigenic molecule was present. When the antigen and the adjuvant were combined, 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 achieve better tumor inhibition effect; for the two vaccine preparation methods, the method of mixing two LNP was better than the previous method of mixing nucleic acid molecules Figure 9 C), the tumor weight columnar statistics results and the tumor pictures also showed the differences between them, and the spleen weight also showed the corresponding trend Figure 9 D&E&F). It is necessary to compare the two preparation methods for the construction of the vaccine platform, and the method of preparing two nucleic acid molecules into one LNP obviously reduces the economic and time cost of LNP purification and concentration in the later stage. However, this more economical vaccine preparation method is obviously not suitable for tumor treatment effect. We guess that the probability of the two nucleic acid molecules entering one cell in one LNP is the same, while the translation and expression of the GSDMD-NT molecule will significantly affect the state of the cell, which leads to the expression of the antigen being more affected. Separating into two LNP increases the probability of entering two different cells, to some extent, reducing the mutual influence between them.

[0157] Example 8 Evaluation of the Anti-tumor Immune Effect of the Optimized Ratio of GSDMD-NT and Tumor Antigen

[0158] The experiment was carried out with the antigen Nov S-E7, and 1 pg, 2 pg and 4 pg were selected as the dosage of the GSDMD-NT molecule adjuvant, and the Nov S-E7 antigen dosage was 9 pg to achieve the ideal tumor inhibition effect, and the total amount of nucleic acid in the immune animals was controlled at 13 pg, and the insufficient part was supplemented with LNP-LUC irrelevant molecule, and finally the three nucleic acid molecules were wrapped into LNP by microfluidic to prepare mixed vaccines according to the expected ratio. The immune program was consistent with the foregoing, and in the TC-1 tumor-bearing mouse model, when the tumor volume grew to about 50-100 mm 3 , the mice were injected with different LNP-mRNA vaccines in the hind leg muscles Figure 9A&B). Anti-tumor ability was evaluated by measuring the tumor volume of mice, which was significantly inhibited after the third injection of LNP-mRNA vaccine, and the cure began to appear on the 27th day. The tumor tissues of mice were collected and weighed on the 30th day, and the results showed that the anti-tumor effect of the N+G3 vaccine group was the best, and 5 / 14 tumors were cleared; 2 / 14 tumors in the N+G2 vaccine group regressed, and only one mouse tumor regressed in the N+G1 vaccine group. Overall, the tumor growth of mice with Nov S-E7 antigen was significantly inhibited; among them, the effect of the combined treatment group with GSDMD-NT molecular adjuvant was better than that of the Nov S-E7 antigen alone group, and the three doses were statistically significant. In addition, the GSDMD-NT molecular adjuvant alone also had a significant tumor inhibition effect at a higher dose of 4 μg, while the 1 μg dose had almost no effect Figure 9 C). The corresponding spleen weight was also statistically recorded, which was negatively correlated with tumor size Figure 9 D). This also indirectly shows that a certain dose of GSDMD-NT molecular expression can trigger a series of reactions that can promote anti-tumor immune effects.

[0159] Of course, the evaluation of immunological indicators is very important to define the effectiveness of the vaccine and the strength of the response, and we evaluated the conventional anti-tumor immunological indicators. Specifically, flow cytometry analysis of lymphocytes in the spleen showed that the vaccine group with Nov S-E7 antigen could significantly up-regulate 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 rose most obviously, with the most statistically significant difference Figure 9 E&F). Further, we analyzed the proportion of NK cells (CD69b+NK1.1+) in the spleen and the proportion of Th1 type T cells (CD3+CD4+IFN-γ+) and Th2 type T cells (CD3+CD4+IL-4+) in the spleen by flow cytometry. The results showed that except for the 1 μg low-dose GSDMD-NT molecular adjuvant group, the N+G3 vaccine group rose most obviously, with the most statistically significant difference Figure 9 G&H&I).

[0160] In addition to the evaluation of systemic immune effects, the specific situation inside the tumor can better reflect the strength of the anti-tumor immune response. Although our tumor was very small at the end of the experiment, at least 9 of our 14 mice had tumors, and we combined them into four groups to isolate lymphocytes inside the tumor. Obviously, this E7 49-57The specific immune response was again verified in the IFN-γ-ELISPOT test, in which the average number of spots and the release of IFN-γ were the most in the N+G3 group Figure 9 J) Similarly, the results of flow cytometry showed that the proportion of cells of immunosuppressive MDSC (CD11b + Gr1 + ) decreased in the presence of Nov S-E7 antigen, and there was a certain statistical difference in the rest of the antigen groups except for the single antigen group Figure 9 L) In addition, the macrophage type in the tumor also affected the exertion of anti-tumor immune effect. Generally, there were more M2 type macrophages in the tumor, and many studies have shown that only when the proportion of M1 type macrophages increases can the immunosuppressive tumor microenvironment be reversed. Here, we examined the proportion of two types of macrophages in the tumor tissue by flow cytometry. The proportion of M1 type macrophages (CD11b + F4 / 80 + CD86 + ) showed an upward trend only in the vaccine group of N+G3, which may be because the tumor of this group was the smallest, and its anti-tumor microenvironment was established and maintained until the detection time point ​ M) Interestingly, the proportion of M2 type macrophages (CD11b + F4 / 80 + CD206 + ) showed a downward trend in the presence of GSDMD-NT molecular adjuvant, and there was a statistical difference between the single Nov S-E7 antigen group and the other three dose combination therapy groups ​ N) TAM is the main immune cell in tumor tissue, and M1 type TAM releases a large amount of IL-12, which is a multifunctional pro-inflammatory cytokine that can promote CTL to secrete more IFN-γ and enhance the killing ability of tumor. Here, we also detected the IL-12 releasing tumor-associated macrophages, and the results showed that their proportion increased in mice treated with GSDMD-NT molecular adjuvant combined with antigen ​ O) This also supports the previous results of CTL detection, and the secretion of pro-inflammatory cytokine IL-12 can trigger strong T cell immune inhibition of tumor growth.

[0161] In accordance with the present disclosure, all of the methods disclosed and claimed herein can be performed and executed without undue experimentation in light of the present disclosure. While this application has been described in terms of preferred aspects, it will be apparent to those of ordinary skill in the art that variations can be applied to the methods described herein without departing from the concept, spirit and scope of the application. More specifically, it will be apparent to one of ordinary skill in the art that certain agents chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications according to the skills of those in the art, viewed in terms of the present disclosure, are to be embraced within the spirit and scope of the application defined by the following claims.

Claims

1. A pyroptosis-induced mRNA vaccine, characterized in that... The mRNA vaccine includes an mRNA vaccine adjuvant and antigen lipid nanoparticles containing a first mRNA encoding an E7 tumor antigen fused to the norovirus S domain for HPV. The mRNA vaccine adjuvant is composed of ionizable lipids, cholesterol, phospholipids, PEG derivatives, and a second mRNA containing a gene sequence encoding GSDMD-NT; the amino acid sequence of GSDMD-NT is shown in SEQ ID NO:

1. The amino acid sequence of the norovirus S domain fused with the HPV E7 tumor antigen is shown in SEQ ID NO:

6.

2. The mRNA vaccine according to claim 1, characterized in that... The second mRNA consists of a T7 transcription promoter, a 5'UTR, a gene sequence encoding GSDMD-NT, a 3'UTR, and a polyA tail; 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 according to claim 2, characterized in that... The second mRNA also includes a polyC tail as shown in SEQ ID NO:

5.

4. The mRNA vaccine according to any one of claims 1 to 3, characterized in that... The preparation method of the vaccine adjuvant includes the following steps: (1) Plasmid construction: Using pVAX1 as a plasmid vector, the gene sequence encoding GSDMD-NT 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 hour, and the linearized plasmid was recovered using a DNA purification and recovery kit. (3) In vitro transcription to synthesize mRNA molecules: The linearized plasmid, dNTPs, RNA polymerase, cap analog and buffer from step (2) were mixed and reacted at 37°C for 3 h. Then, DNA enzyme I was added to digest the DNA template to obtain the second mRNA. (4) Combination lipid nanoparticles: lipid nanoparticles with a particle size of 100-200 nm encapsulated with second mRNA are prepared by mixing ionizable lipids, cholesterol, phospholipids, PEG derivatives, anhydrous ethanol and second mRNA, i.e. mRNA vaccine adjuvants.

5. The mRNA vaccine according to claim 1, characterized in that... The antigen lipid nanoparticles have a particle size of 100–200 nm.

6. The mRNA vaccine according to claim 1, characterized in that... The first mRNA consists of a T7 transcription promoter, a 5'UTR, a norovirus S domain gene sequence, a linker arm, a gene sequence encoding the E7 tumor antigen of HPV, a 3'UTR, and a polyA tail; 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.

7. The mRNA vaccine according to claim 6, characterized in that... The amino acid sequence encoded by the linker arm is GSGSGS.

8. The mRNA vaccine according to any one of claims 5 to 7, characterized in that... The amino acid sequence of the HPV E7 tumor antigen is shown in SEQ ID NO:

7.

9. The mRNA vaccine according to claim 8, characterized in that... In the mRNA vaccine, the mass ratio of mRNA vaccine adjuvant to nucleic acid in antigen lipid nanoparticles is 1:9.

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