Mucous membrane type nucleic acid vaccine adjuvant and preparation method of vaccine preparation thereof

Abstract

The invention discloses a mucous membrane type nucleic acid vaccine adjuvant and a preparation method of a vaccine preparation thereof, the adjuvant is a nucleic acid component and has the effects of enhancing mucous membrane immune response and promoting immune cell activation and chemotactic factor release in the mucous membrane, so that the immune effect of a nucleic acid vaccine at the mucous membrane part is improved. By means of the LNP system, efficient and lasting mucosal immune response is achieved, a brand new solution is provided for development of mucosal nucleic acid vaccines, and the LNP system is expected to be applied to treatment of various infectious diseases and cancers.

Description

Technical Field

[0001] The present invention relates to the field of vaccines, in particular to a mucosal nucleic acid vaccine adjuvant, and also to a preparation method and application of a vaccine preparation. Background Art

[0002] Nucleic acid vaccines, with their high efficiency, safety, and rapid development, have shown great potential in responding to emerging infectious diseases and treating certain cancers. In recent years, the successful application of the new coronavirus vaccine has vividly demonstrated the key role of nucleic acid vaccines in global public health and has become a milestone in the development of modern vaccine technology. However, the research and application of nucleic acid vaccines in the field of mucosal immunity still face a series of challenges, especially in the core issue of how to effectively stimulate mucosal immune responses, and no substantial breakthroughs have been made.

[0003] The mucosa is the main portal for many pathogens to invade the host. Therefore, it is particularly important to develop nucleic acid vaccines that can effectively stimulate immune responses in the mucosal area. Compared with traditional intramuscular vaccines, mucosal vaccines have unique advantages: they can not only initiate immune responses in the first line of defense against pathogen invasion, but also induce systemic and local dual immunity. However, existing technologies have obvious shortcomings in the mucosal delivery of nucleic acid vaccines. Although traditional lipid nanoparticles (LNPs) as nucleic acid vaccine delivery systems have been widely used and successful in the intramuscular route, they show low efficiency in mucosal delivery and are difficult to produce mucosal immune responses of sufficient intensity. This limitation has greatly restricted the widespread application of mucosal nucleic acid vaccines.

[0004] In the development of mucosal vaccines, the choice of adjuvant is crucial. The role of adjuvants is not only to enhance the immunogenicity of vaccines, but also a key factor in determining the recruitment, activation and sustained response of immune cells in mucosal sites. Effective adjuvants can promote the activation of antigen-presenting cells in the mucosal environment and improve the effectiveness of vaccines. However, the current selection of adjuvants available for mucosal nucleic acid vaccines is very limited. These adjuvants either have safety issues or are insufficiently active in the mucosal environment, making it difficult to meet the dual needs of safety and efficiency at the same time.

[0005] To meet this challenge, the development of new mucosal nucleic acid vaccine adjuvants has become an urgent task. These new adjuvants can include both synthetic and natural sources. Synthetic adjuvants can optimize their immunostimulatory properties through precise design and achieve controllable regulation of immune responses, thereby achieving a balance between safety and efficacy. Natural adjuvants, on the other hand, have unique advantages in stimulating natural immune responses due to their wide sources and good biocompatibility. For example, some natural ingredients from plants and their derivatives, such as licorice extract. In addition, combining synthetic and natural adjuvants to further improve the efficiency of mucosal immunity through synergistic effects is also a promising research direction.

[0006] In this invention, we proposed a vaccine adjuvant delivered by LNP, and explored the combination of novel adjuvants and delivery systems by integrating advanced material science and immunology theories. This novel composition not only helps to improve the immunogenicity of the vaccine, but also reduces the amount of adjuvant while maintaining high safety and controllable immune response, providing an innovative idea for the design of future vaccines. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a mucosal nucleic acid vaccine adjuvant; the second object of the present invention is to provide a nucleic acid vaccine based on the mucosal nucleic acid vaccine adjuvant; the third object of the present invention is to provide a method for preparing the nucleic acid vaccine.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] 1. A nucleic acid vaccine prepared based on an adjuvant-lipid composition, comprising the following components:

[0010] (1) an active agent, wherein the active agent is a component comprising a nucleic acid;

[0011] (2) cationic lipids;

[0012] (3) non-cationic lipids;

[0013] (4) Vaccine adjuvants.

[0014] 2. A mucosal nucleic acid vaccine adjuvant, characterized in that: the vaccine adjuvant is a component that can significantly improve the immune response ability of the nucleic acid vaccine in the mucosal area, and comprises at least one selected from the following: AS03, MF59, monophosphoryl lipid A, miniaturized monophosphoryl lipid A, ophiopogon saponin D, saponin QS21, CpG oligonucleotide, 2'3'-cyclic GMP-AMP, 3'3'-cyclic GMP-AMP, licorice extract or all-trans retinoic acid, 1-100 μg / dose, preferably 2-20 μg / dose.

[0015] Preferably, the active agent is messenger RNA, self-amplifying RNA, circular RNA, small interfering RNA, short hairpin RNA, micro RNA, primary miRNA, antisense oligonucleotide, plasmid DNA, single-stranded DNA, double-stranded DNA, nucleic acid aptamer, CRISPR-related nucleic acid, single guide RNA and double-stranded RNA.

[0016] Preferably, the cationic lipid of the present invention is an ionizable cationic lipid, a cholesterol-derived cationic lipid or a permanent cationic lipid.

[0017] Preferably, the non-cationic lipid is a neutral lipid or a zwitterionic lipid.

[0018] Preferably, the cationic lipid accounts for 20 mol%-80 mol% of the total lipids; and the non-cationic lipid accounts for 10 mol%-50 mol% of the total lipids.

[0019] Preferably, the nucleic acid vaccine is in the form of solution, dry powder, atomization or spray.

[0020] Preferably, the adjuvant-lipid composition for delivering mRNA is administered via the respiratory tract, or orally, or intramuscularly, or intravenously, or subcutaneously, or intratumorally.

[0021] 3. The preparation method of the nucleic acid vaccine comprises the following steps:

[0022] (1) Preparation of organic phase: An ethanol solution containing SM-102, an ethanol solution containing DSPC, an ethanol solution containing cholesterol, and an ethanol solution containing DMG-PEG2K were mixed at a molar ratio of 49:20.1:30:0.9 and diluted with ethanol to a total lipid concentration of 33.3 mg / mL;

[0023] (2) Preparation of aqueous phase: Dilute the mRNA with citrate buffer, mix well and set aside;

[0024] (3) The aqueous phase and the organic phase are mixed in a volume ratio of 3:1, with a total flow rate of 4-20 mL / min, the mixed solution is added to a dialysis bag and dialyzed in PBS, and after filtering with a filter membrane, lipid nanoparticles (particle size 50-200 nm, encapsulation efficiency >80%) that stably encapsulate mRNA are obtained; the mRNA is Fluc-mRNA or OVA-mRNA.

[0025] (4) Dissolve the adjuvant in PBS solution and filter through a filter membrane; mix the mRNA lipid nanoparticles of step (3) with the adjuvant solution at a volume ratio of 1:1-8:1; let stand, filter through a filter membrane, and store at 2-8°C.

[0026] The beneficial effects of the present invention are as follows: the present invention discloses a mucosal nucleic acid vaccine adjuvant, which is a nucleic acid component and has the function of enhancing mucosal immune response, and promoting the activation of immune cells and the release of chemokines in the mucosa, thereby improving the immune effect of nucleic acid vaccines in the mucosal part. Through this LNP system, an efficient and lasting mucosal immune response is achieved, providing a new solution for the development of mucosal nucleic acid vaccines, and is expected to be applied to the treatment of various infectious diseases and cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0028] Figure 1 is the hydrodynamic particle size of different formulations;

[0029] Figure 2 is the polydispersity coefficient of different formulation solutions;

[0030] Figure 3 is the nucleic acid encapsulation efficiency of different preparations;

[0031] Figure 4 The expression of F-luc reporter gene in mouse lung tissue;

[0032] Figure 5 is the optical density of OVA-specific serum IgG;

[0033] Figure 6 is the optical density of OVA-specific bronchoalveolar lavage fluid (BALF) sIgA;

[0034] Figure 7 is the optical density of OVA-specific nasal lavage fluid (NLF) sIgA;

[0035] Figure 8 is the alanine aminotransferase content in serum;

[0036] Fig. 9 is the aspartate aminotransferase content in serum;

[0037] Fig.10 is the blood urea nitrogen content in serum;

[0038] Fig.11 is the serum creatinine content;

[0039] Fig.12 Immunization and sampling plan for mice;

[0040] Fig.13 It is a line graph of the time-weight change of mice;

[0041] Fig.14 is the mouse survival curve;

[0042] Fig.15 is the lung weight of mice;

[0043] Fig.16 Representative images of lung metastases;

[0044] Fig.17 is the mRNA expression of mouse tyrosine phosphatase type 1 (Tyrp1). DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0046] Example 1. Preparation of a vaccine containing lipid nanoparticles (LNP)

[0047] The composition of the vaccine containing lipid nanoparticles (LNP) includes the following components:

[0048] (1) an active agent or therapeutic agent, which is a component comprising a nucleic acid; the nucleic acid is selected from at least one of the following: messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), primary miRNA, antisense oligonucleotide (ASO), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), nucleic acid aptamer, CRISPR-related nucleic acid, single guide RNA (sgRNA) and double-stranded RNA (dsRNA);

[0049] (2) cationic lipids, the cationic lipids comprising at least one selected from the group consisting of: ionizable cationic lipids, cholesterol-derived cationic lipids, and permanent cationic lipids; the cationic lipids account for 20 mol% to 80 mol% of the total lipids present in the composition;

[0050] (3) non-cationic lipids; the non-cationic lipids are selected from neutral lipids and zwitterionic lipids, preferably the non-cationic lipids comprise cholesterol or phospholipids; the non-cationic lipids account for 10 mol% to 50 mol% of the total lipids in the composition;

[0051] (4) adjuvant; the adjuvant is selected from the following components: AS03, MF59, monophosphoryl lipid A (mPLAs), miniaturized monophosphoryl lipid A (mPLA-sm), ophiopogon saponin D (OPD), saponin QS21 (QS21), CpG oligonucleotide

[0052] (CpG), 2'3'-cyclic GMP-AMP (2'3'-cGAMP), 3'3'-cyclic GMP-AMP (3'3'-cGAMP), licorice extract (GLA), all-trans retinoic acid (ATRA), 1-100 μg / dose, preferably 2-20 μg / dose.

[0053] In the present invention, the active agent or therapeutic agent is preferably mRNA, and its preparation method is as follows: in vitro transcribed mRNA (IVT-mRNA) encoding a specific antigen or therapeutic protein can also be used to deliver any modified or unmodified mRNA, or mRNA with a naturally occurring sequence or codon optimization. As a non-limiting example, the mRNA encoding firefly luciferase (Fluc-mRNA) and the melanoma-related antigen OVA protein mRNA (OVA-mRNA) described herein. It can be synthesized as follows: construct a recombinant DNA plasmid containing a T7 promoter, 5'UTR, ORF, 3'UTR and a 3' poly (A) tail of about 100 nucleotides in length; prepare a linearized DNA template by enzyme digestion or PCR amplification; perform in vitro transcription with a linearized DNA template, and then add a 5' cap structure (Cap 1) to prepare. After purification, the IVT-mRNA band size and purity are determined by gel electrophoresis.

[0054] The method for preparing the vaccine containing lipid nanoparticles (LNP) is as follows:

[0055] (1) Preparation of organic phase: 50 mg / mL ethanol solution of SM-102, 50 mg / mL ethanol solution of DSPC, 50 mg / mL ethanol solution of cholesterol, and 50 mg / mL ethanol solution of DMG-PEG2K were mixed at a molar ratio of 49:20.1:30:0.9, and diluted with ethanol to a total lipid concentration of 33.3 mg / mL;

[0056] (2) Preparation of aqueous phase: Dilute the mRNA to a final concentration of 0.17 mg / mL with 50 mM citrate buffer (pH 4.0) and mix well for later use;

[0057] (3) The aqueous phase and the organic phase were rapidly mixed at a volume ratio of 3:1 using a microfluidic device with a total flow rate of 12 mL / min. The mixed solution was added to a dialysis bag (dialysis bag MWCO: 8000-14000) and dialyzed in 1×PBS (pH 7.4) for 18 h (ambient temperature was room temperature or 4°C). After filtration with a 0.22 μm filter membrane, lipid nanoparticles stably encapsulating mRNA were obtained. The final concentration of Fluc-mRNA or (OVA-mRNA) encapsulated in the prepared LNP preparation was approximately 0.12 mg / mL.

[0058] (4) Preparation of adjuvant vaccine preparation: The adjuvant was dissolved in 1×PBS solution to 2 mg / mL, filtered through a 0.22 μm filter membrane, and set aside. The prepared and purified LNP solution was mixed with the above adjuvant solution at a volume ratio of 4:1; after standing at room temperature for 20 min, filtered through a 0.22 μm filter membrane, and stored at 2-8°C; the final concentration of Fluc-mRNA or (OVA-mRNA) encapsulated in the prepared LNP+adjuvant preparation was about 0.10 mg / mL, and the final concentration of the adjuvant was 0.4 mg / mL.

[0059] Example 2 is a diagram showing the physical characterization results of the nucleic acid adjuvant vaccine preparation (particle size, dispersion coefficient PDI, encapsulation efficiency EE).

[0060] The particle size and PDI of the formulations were measured using dynamic light scattering (DLS) at 25°C using a Zetasizer Nano ZS instrument.

[0061] The encapsulation efficiency of mRNA was evaluated using the Quant-iTTM RiboGreen RNA detection kit (Thermo Fisher Scientific). The samples were dispersed in 1×TE buffer containing 2% Triton X-100, and the content of free mRNA and total mRNA was determined. Five concentrations of mRNA standards were prepared using 1×TE buffer. An equal volume of sample or mRNA standard was mixed with RiboGreen reagent (1:200 dilution) in a transparent bottom black plate and incubated at room temperature for 3 min. The fluorescence intensity was measured using an ELISA reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. EE (%) = [(fluorescence of total mRNA - fluorescence of free mRNA) / fluorescence of total mRNA] × 100%. The RiboGreen assay method is essentially as described in an article published by Heyes et al. Journal of Controlled Released, 2005, 107: 276-287.

[0062] The results showed that compared with the LNP control group (without adjuvant), the particle size of the preparation prepared by the LNP mixed adjuvant group was Figure 1 ), particle size distribution ( Figure 2 ) and encapsulation efficiency ( Figure 3 ) did not change significantly, indicating that the addition of adjuvants had no effect on the formulation characterization.

[0063] Example 3: Transfection efficiency study

[0064] The transfection efficiency of mRNA encoding firefly luciferase (Fluc-mRNA) in mice was studied 6 hours after the nucleic acid adjuvant vaccine preparation and LNP control preparation were administered by intratracheal spray (it).

[0065] Preparation of preparations: The method described in "Example 1" was used to prepare the adjuvant-containing Fluc-mRNA preparation and the LNP control preparation.

[0066] During isoflurane inhalation anesthesia, a high-pressure spray needle was used to spray a nucleic acid adjuvant vaccine preparation or LNP preparation with an mRNA dose of 2.5μg mRNA / 50μL and an adjuvant concentration of 20μg / 50μL into the trachea of ​​a single mouse. Unless otherwise specified, this dose was used for all experiments of the same type. Six hours after administration, the D-luciferase substrate provided by PerkinElmer was injected intraperitoneally (ip), and the fluorescent signal in the mouse lungs was detected using the IVIS spectral imaging system. The results showed that compared with the LNP control preparation group, the adjuvant-containing preparation was able to significantly increase the level of Fluc-mRNA expression of luciferase in the lungs 6 hours after intratracheal spray (it) administration ( Figure 4 ).

[0067] Example 4: Immune response effect of spray route

[0068] Antigen-specific humoral immune response induced in mice after intratracheal spray (it) inoculation with a nucleic acid adjuvant vaccine preparation loaded with mRNA encoding melanoma-associated antigen OVA protein (OVA-mRNA, SEQ ID NO.1).

[0069] Preparation of preparations: The method described in "Example 1" was used to prepare adjuvanted OVA-mRNA preparations and LNP control preparations.

[0070] During isoflurane inhalation anesthesia, a high-pressure spray needle was used to spray a nucleic acid adjuvant vaccine preparation or LNP preparation with an mRNA dose of 2.5 μg mRNA / 50 μL into the trachea of ​​a single mouse. The first vaccination time was counted as "Day 0", and a booster immunization was performed on the 21st day using the same vaccination route, the same preparation, and the same dose. 28 days after the first immunization, the serum (Serum) of the mice, as well as the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) of the mice were collected to detect the ability of the adjuvanted OVA-mRNA preparation to induce humoral immune responses and mucosal immune responses in the mice. The results are shown in Figure 5 The results showed that compared with the LNP control preparation group, the preparations containing adjuvants were able to significantly increase the level of OVA antigen-specific humoral immune response in serum after 28 days of intratracheal spray (it) administration, among which mPLA-sm had the strongest enhancement effect.

[0071] Example 5: Immune response effect via nasal route

[0072] Study on the efficient adaptability of nucleic acid adjuvant vaccine formulations in mediating OVA-mRNA production after nasal (in) administration.

[0073] Preparation of vaccine preparations: The method described in "Example 1" was used to prepare adjuvanted OVA-mRNA preparations and LNP control preparations.

[0074] Serum, bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) were collected from mice, and then the secretion level of OVA antigen-specific sIgA in BALF and NLF was detected. The results are shown in Figure 2. Figure 6 and Figure 7 The results showed that compared with the LNP control group, the adjuvant-containing preparations were able to significantly increase the secretion level of OVA antigen-specific sIgA in BALF and NLF after 28 days of intratracheal spray (it), among which mPLA-sm had the strongest effect.

[0075] Example 6: Safety study of nucleic acid adjuvant vaccine preparations in mice

[0076] Preparation of preparations: The method described in "Example 1" was used to prepare adjuvanted OVA-mRNA preparations and LNP control preparations.

[0077] During isoflurane inhalation anesthesia, a high-pressure spray needle was used to spray the nucleic acid adjuvant vaccine preparation or LNP preparation with a mRNA dose of 2.5μg mRNA / 50μL into the trachea of ​​a single mouse. The mouse serum was collected 6 hours after administration to test the safety of the preparation in mice. The results showed that the alanine aminotransferase (AAT) level in the mouse serum was significantly increased 6 hours after intratracheal spray (it) administration of the LNP control preparation and the adjuvant preparation. Figure 8 ), aspartate aminotransferase ( Fig. 9 )、blood urea nitrogen( Fig.10 ) and creatinine ( Fig.11 ) contents were within the normal range.

[0078] Example 7. Establishment of a melanoma lung metastasis model and tumor evaluation after vaccination of mice with different vaccine formulations

[0079] Preparation of preparations: Adjuvant vaccine preparations containing adjuvants mPLA-sm and OVA-mRNA and LNP control preparations containing OVA-mRNA were prepared using the method described in the above-mentioned "Preparation Example 1".

[0080] 6-8 week old C57BL / 6 mice were selected and randomly divided into three groups: LNP group, mPLA-sm-LNP group, and control group (PBS). On day 0, 50 μL of the immunization dose was given to each mouse by intratracheal spray (it), 50 μL of LNP preparation was given to the LNP group, 50 μL of LTA1-modified LNP preparation was given to the mPLA-sm-LNP group, and 50 μL of PBS was given to the control group (PBS). On day 14, 50 μL of the corresponding immunization preparation was given again by intratracheal spray (it) in the same manner as the primary immunization for secondary immunization. On day 21, 1×10 6 luciferase-expressing melanoma B16-F10-LUC cells were used to establish a lung melanoma metastasis model ( Fig.12 Within 21 days after inoculation of melanoma cells (i.e., Day 21 to Day 42), D-luciferase substrate provided by PerkinElmer was injected intraperitoneally (ip) regularly, and the fluorescent signal in the lungs of mice was detected using the IVIS spectral imaging system to evaluate the tumor growth in the lungs of mice. At the same time, during the process of tumor growth monitoring, the weight changes of mice were recorded weekly. The results are shown in Fig.13 The results showed that the weight change of mice in the adjuvant-containing formulation group was smaller than that in the LNP control formulation group. The survival of mice was monitored as shown in Fig.14 The results showed that the survival rate of mice in the adjuvant-containing preparation group was higher than that in the LNP control preparation group. On the 42nd day of the experiment, all mice were euthanized, and the lungs of the mice were dissected and weighed. The results are shown in Fig.15 The results showed that the lung weight of mice in the adjuvant-containing preparation group increased less than that in the LNP control preparation group. The number of lung melanoma metastases was statistically analyzed, and the results were shown in Fig.16 The results showed that compared with the LNP control group, the adjuvant-containing group had fewer lung melanoma metastases. The mouse lung tissues were collected, total RNA was extracted, and the expression of melanoma-specific gene tyrosine phosphatase type 1 (Tyrp1) was measured using RT-qPCR technology to evaluate the effects of different treatment regimens on lung melanoma metastasis. The results are shown in Fig.17 The results showed that compared with the LNP control group, the mice in the adjuvant-containing group produced less tyrosine phosphatase type 1 (Tyrp1).

[0081] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A nucleic acid vaccine prepared based on an adjuvant-lipid composition, characterized in that: The components include: (1) an active agent, wherein the active agent is a component comprising a nucleic acid; (2) cationic lipids; (3) non-cationic lipids; (4) Vaccine adjuvants.

2. The vaccine adjuvant according to claim 1, characterized in that: The vaccine adjuvant is a component that can significantly improve the immune response ability of the nucleic acid vaccine in the mucosal area, and includes at least one selected from the following: AS03, MF59, monophosphoryl lipid A, miniaturized monophosphoryl lipid A, ophiopogon saponin D, saponin QS21, CpG oligonucleotide, 2'3'-cyclic GMP-AMP, 3'3'-cyclic GMP-AMP, licorice extract or all-trans retinoic acid, and derivatives of the above substances.

3. The nucleic acid vaccine based on the adjuvant-lipid composition according to claim 2, characterized in that: The dosage of the nucleic acid vaccine is 1-100 μg / dose.

4. The nucleic acid vaccine prepared based on the adjuvant-lipid composition according to claims 1-3, characterized in that: The active agents are messenger RNA, self-amplifying RNA, circular RNA, small interfering RNA, short hairpin RNA, micro RNA, primary miRNA, antisense oligonucleotides, plasmid DNA, single-stranded DNA, double-stranded DNA, nucleic acid aptamers, CRISPR-associated nucleic acids, single guide RNA and double-stranded RNA.

5. The nucleic acid vaccine prepared based on the adjuvant-lipid composition according to claims 1-3, characterized in that: The cationic lipid is an ionizable cationic lipid, a cholesterol-derived cationic lipid or a permanent cationic lipid.

6. The nucleic acid vaccine prepared based on the adjuvant-lipid composition according to claims 1-3, characterized in that: The non-cationic lipid is a neutral lipid or a zwitterionic lipid.

7. The nucleic acid vaccine prepared based on the adjuvant-lipid composition according to claims 1-3, characterized in that: The cationic lipid accounts for 20 mol%-80 mol% of the total lipids; the non-cationic lipid accounts for 10 mol%-50 mol% of the total lipids.

8. The nucleic acid vaccine prepared based on the adjuvant-lipid composition according to claims 1-3, characterized in that: The nucleic acid vaccine is in the form of solution, dry powder, atomization or spray.

9. The nucleic acid vaccine prepared based on the adjuvant-lipid composition according to claims 1-3, characterized in that: The adjuvant-lipid composition for delivering mRNA is delivered and administered via the respiratory tract, or orally, or intramuscularly, or intravenously, or subcutaneously, or intratumorally.

10. The method for preparing the nucleic acid vaccine according to any one of claims 1 to 6, characterized in that: The composition preparation method comprises the following steps: 1) adding the adjuvant to a solution containing the nucleic acid and / or a solution containing the lipid, and 2) mixing the solution containing nucleic acid and the solution containing lipid, thereby forming the composition; or The method comprises: 1) mixing the solution containing the nucleic acid and the solution containing the lipid to form lipid nanoparticles encapsulating the nucleic acid, and 2) adding the adjuvant to the lipid nanoparticle solution loaded with the nucleic acid to form the composition; or The method comprises: 1) preforming the lipid and adjuvant into nucleic acid adjuvant-lipid nanoparticles; and 2) mixing the solution containing the nucleic acid with the adjuvant-lipid nanoparticle solution to form the composition; Or the method comprises: 1) in a solution comprising the lipid, allowing the lipid to pre-form a nucleic acid-free lipid nanoparticle; and 2) mixing a solution containing the nucleic acid and the adjuvant with the lipid nanoparticle solution, Thereby forming the composition. According to the water-solubility and lipid-solubility characteristics of the adjuvant, an aqueous solution or lipid solution containing nucleic acid is added, and then the mRNA solution (aqueous phase) and the lipid solution (organic phase) are mixed; or the mRNA solution (aqueous phase) and the lipid solution (organic phase) are first mixed, and after purification, lipid nanoparticles LNP that stably encapsulate mRNA are obtained, and then the LNP is mixed with the adjuvant.

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