Vaccine adjuvant encoded by nucleic acid and application of vaccine adjuvant in novel crown mRNA (messenger Ribonucleic Acid) vaccine

Abstract

The invention discloses a vaccine adjuvant coded by nucleic acid and application of the vaccine adjuvant in a novel crown mRNA vaccine, nucleic acid coded mRNA is used as an adjuvant to prepare a nano preparation containing the nucleic acid coded adjuvant, the mRNA can be effectively protected from being degraded, stable delivery of nucleic acid is ensured, and compared with a traditional mRNA vaccine preparation, the vaccine adjuvant has higher transfection efficiency. Meanwhile, the adjuvant vaccine coded by nucleic acid can effectively express specific immunomodulatory protein in animal bodies, humoral immune response, mucosal immune response and cellular immune response are enhanced at the same time, and the immune protection effect of resisting virus invasion of the bodies is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of vaccines, specifically to vaccine adjuvants encoded by nucleic acids, and also to the use of vaccine adjuvants in new crown mRNA vaccines. Background Art

[0002] In 2019, the global pandemic of the novel coronavirus (SARS-CoV-2) caused millions of infections and deaths, posing huge challenges to global public health, economy and social life. As a result, the development of an effective COVID-19 vaccine has become an urgent task for scientists and pharmaceutical companies around the world. Nucleic acid therapy, especially mRNA technology, has shown great potential and advantages in this vaccine race. mRNA vaccines are a new type of vaccine that induces an immune response by delivering messenger RNA encoding pathogen antigens in the body. During the COVID-19 pandemic, mRNA vaccines stood out for their rapid development, flexible manufacturing and strong immune response, successfully curbing the spread of the virus. The mRNA COVID-19 vaccines developed by Pfizer / BioNTech and Moderna have been widely used and recognized around the world. The success of mRNA vaccines has made people pay more attention to the potential application of nucleic acid therapy in infectious diseases, cancer and other diseases.

[0003] Although the application of mRNA vaccines has made important breakthroughs, further improving its immune effect still faces challenges. As an important component for enhancing the immunogenicity and immune response of vaccines, immune adjuvants are crucial to improving the protective efficacy and durability of vaccines. Adjuvants can enhance immune responses through various mechanisms, including prolonging the existence of antigens, promoting the uptake and presentation of antigens, and activating immune cells. Although traditional adjuvants such as aluminum salts and water-in-oil emulsions are widely used in protein and peptide vaccines, how to design effective adjuvants to enhance their immune effects in mRNA vaccines is still a hot topic and difficulty in research. Traditional adjuvants often have problems such as large dosage and significant side effects, which limit their application in nucleic acid vaccines. Nucleic acid-encoded adjuvants deliver mRNA encoding specific immunomodulatory proteins together with target antigen mRNA into cells, allowing the cells to express adjuvant proteins themselves, thereby enhancing immune responses locally or systemically in the body. This strategy can not only accurately regulate the expression level of adjuvants and reduce the amount of adjuvants used, but also improve the immunogenicity and safety of vaccines.

[0004] In this invention, we propose a nucleic acid-encoded adjuvant delivered by LNP to further optimize the effect of the new crown mRNA vaccine. This new composition contains mRNA encoding a specific immunomodulatory protein and target antigen mRNA co-encapsulated in LNP. Through appropriate ratios and dosages, it can significantly enhance the immune response of the vaccine while maintaining high safety and controllable immune effects, providing an innovative and effective strategy for the design and application of future nucleic acid vaccines. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a vaccine adjuvant encoded by nucleic acid; the second object of the present invention is to provide a vaccine preparation prepared based on the vaccine adjuvant encoded by nucleic acid; the third object of the present invention is to provide a method for preparing the vaccine preparation.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. A nucleic acid-encoded immune adjuvant composition, comprising:

[0008] (A) Nucleic acids encoding specific immunomodulatory proteins (vaccine adjuvants), i.e. nucleic acid adjuvants;

[0009] (B) an active agent or therapeutic agent, preferably the active agent or therapeutic agent is a nucleic acid;

[0010] (C) cationic lipids;

[0011] (D) Non-cationic lipids.

[0012] Preferably, the nucleic acid adjuvant of the present invention comprises one or more nucleic acid molecules, which encode one or more immunomodulatory proteins selected from the following: heat-sensitive enterotoxin A subunit, heat-sensitive enterotoxin B subunit, cholera toxin A subunit, cholera toxin B subunit, interleukin-12, interleukin-2, interleukin-7, interleukin-15, granulocyte-macrophage colony stimulating factor, thymic stromal lymphopoietin, and cyclic GMP-AMP synthase.

[0013] Preferably, the amino acid sequence of the immunomodulatory protein encoded by the nucleic acid adjuvant is as shown in SEQ ID NO: 1-11, or a variant having ≥80% homology with any amino acid sequence encoded in SEQ ID NO: 1-11 and maintaining immune activity, and the sequence immunomodulatory protein can enhance, promote or regulate the immune response in vivo or in vitro, and is thus used as an immune adjuvant.

[0014] Preferably, the nucleic acid adjuvant coding region sequence of the present invention is selected from SEQ ID NO: 14-24, or a variant having ≥ 80% homology with any nucleic acid sequence in SEQ ID NO: 14-24 and maintaining immunological activity.

[0015] The present invention preferably comprises: the nucleic acid adjuvant encodes therapeutic agents such as therapeutic proteins, vaccine antigens, antibodies, regulators, gene repair factors, etc. The nucleic acid comprises at least one selected from the following: messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer (aptamer), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acids, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activated crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).

[0016] Preferably, the mass ratio of the nucleic acid adjuvant to the therapeutic agent encoding the target antigen is 1:1-1:5, preferably 1:3. The dosage per dose is between 1 μg and 50 μg, preferably 1 μg to 10 μg.

[0017] Preferably, the composition is administered by any one or more of the following routes: intratracheal spray, nasal instillation, intramuscular injection, subcutaneous injection, intravenous injection, oral administration, skin administration, ocular administration or other suitable administration routes. Preferably, the cationic lipid is selected from ionized lipids, permanently positively charged lipids or a combination thereof; the non-cationic lipid is selected from cholesterol, neutral phospholipids, polyethylene glycol-lipid (PEG-lipid), block copolymers or a combination thereof.

[0018] 2. A method for preparing the vaccine preparation, wherein the vaccine preparation is obtained by mixing a vaccine adjuvant and a nucleic acid therapeutic agent encoding a target antigen and then encapsulating the mixture in lipid nanoparticles. The method comprises the following steps:

[0019] a) dissolving a therapeutic agent encoding a target antigen and a nucleic acid adjuvant as described above in an aqueous buffer;

[0020] b) rapidly mixing the aqueous phase with an organic phase containing lipid components by nanomixing or microfluidic control technology to form a lipid nanoparticle LNP complex;

[0021] c) adjusting the particle size of the LNP complex to 20-150 nm, and controlling the encapsulation efficiency to be no less than 80%;

[0022] d) collecting and purifying the LNP complex to obtain a vaccine preparation loaded with a nucleic acid adjuvant.

[0023] 3. Use of the nucleic acid-encoded immune adjuvant composition as described above in the preparation of a drug for enhancing the immune response of a nucleic acid vaccine, wherein the nucleic acid vaccine is selected from nucleic acid vaccines against viruses, bacteria, tumors or other pathogens.

[0024] The beneficial effects of the present invention are as follows: the present invention provides a vaccine adjuvant encoded by nucleic acid and its application in the new crown mRNA vaccine, and uses nucleic acid-encoded mRNA as an adjuvant to prepare lipid nanoparticles containing nucleic acid-encoded adjuvants, which can effectively protect mRNA from degradation and ensure stable delivery of nucleic acids, and have higher transfection efficiency than traditional LNP systems. At the same time, nucleic acid-encoded adjuvant vaccines can effectively express specific immunomodulatory proteins in mice, while enhancing humoral and cellular immune responses, significantly improving the immune protection effect against viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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:

[0026] Figure 1 The results of physical characterization of nucleic acid adjuvant vaccine preparations (a: particle size comparison between nucleic acid adjuvant vaccine preparations and LNP preparations; b: PDI comparison between nucleic acid adjuvant vaccine preparations and LNP preparations; c: encapsulation efficiency EE% comparison between nucleic acid adjuvant vaccine preparations and LNP preparations);

[0027] Figure 2 The results of nucleic acid adjuvant ratio and dosage screening are shown in Figure 1 (a: nucleic acid adjuvant ratio screening comparison; b: nucleic acid adjuvant vaccine preparation dosage screening comparison);

[0028] Figure 3To evaluate the immune response of mice (a: comparison of humoral immunity level of mice after vaccination with nucleic acid adjuvant vaccine and LNP preparation; b: comparison of IgA level of alveolar lavage fluid of mice after vaccination with nucleic acid adjuvant vaccine and LNP preparation; c: comparison of IgA level of nasal lavage fluid of mice after vaccination with nucleic acid adjuvant vaccine and LNP preparation; d: comparison of IFN-γ secretion level in lung lymphocytes of mice after vaccination with nucleic acid adjuvant vaccine and LNP preparation; e: comparison of IL-17 secretion level in lung lymphocytes of mice after vaccination with nucleic acid adjuvant vaccine and LNP preparation; f: comparison of CD4 +TCM cell ratio comparison; g: comparison of CD4+TEM cell ratio in mouse lung lymphocytes after nucleic acid adjuvant vaccine and LNP preparation inoculation; h: comparison of CD4+TRM cell ratio in mouse lung lymphocytes after nucleic acid adjuvant vaccine and LNP preparation inoculation; i: comparison of CD8+TCM cell ratio in mouse lung lymphocytes after nucleic acid adjuvant vaccine and LNP preparation inoculation; j: comparison of CD8+TEM cell ratio in mouse lung lymphocytes after nucleic acid adjuvant vaccine and LNP preparation inoculation; k: comparison of CD8+TRM cell ratio in mouse lung lymphocytes after nucleic acid adjuvant vaccine and LNP preparation inoculation);

[0029] Figure 4 To evaluate the protective effect (a: the trend of changes in mouse body weight after intranasal vaccination with nucleic acid adjuvant vaccine and LNP control preparation following attack by SARS-CoV-2 variants; b: comparison of viral RNA loads in the lungs and nasal nasal concha of mice on the 3rd and 12th days after attack with nucleic acid adjuvant vaccine and LNP; c: the trend of body weight changes in each experimental group after contact with donor hamsters infected with Omicron BA.5 variant; d: determination of viral RNA load in nasal washes of recipient hamsters vaccinated with different vaccine groups after cohabitation). DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1. Preparation of in vitro transcribed mRNA

[0032] The formulations described in this embodiment can be used to deliver any mRNA, in particular in vitro transcribed mRNA (IVT-mRNA) encoding a specific antigen or therapeutic protein. The formulations of the present invention can also be used to deliver any modified or unmodified mRNA, or mRNA with a naturally occurring sequence or codon optimization. As non-limiting examples, the amino acid sequences encoding heat-sensitive enterotoxin mRNA (LT-mRNA), cholera toxin (CT-mRNA), heat-sensitive enterotoxin A subunit (LTA-mRNA), heat-sensitive enterotoxin B subunit (LTB-mRNA), cholera toxin A subunit (CTA-mRNA), cholera toxin B subunit (CTB-mRNA), interleukin 12 (IL-12-mRNA), interleukin 2 (IL-2-mRNA), interleukin 7 (IL-7-mRNA), interleukin 15 (IL-15-mRNA), granulocyte-macrophage colony-stimulating factor (GM-CSF-mRNA), thymic stromal lymphopoietin (TSLP-mRNA), and cyclic GMP-AMP synthase (cGAS-mRNA) described herein are as shown in SEQ ID NO.1 to SEQ ID NO.11, and the nucleotide sequences are as shown in SEQ ID NO.14 to SEQ ID As shown in NO.24, the mRNA encoding firefly luciferase (Fluc-mRNA) and the RBD antigen (RBD-mRNA) encoding the spike protein receptor binding domain of the new coronavirus SARS-CoV-2 are shown in SEQ ID NO.12 to SEQ ID NO.13. 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 size and purity of the IVT-mRNA band are determined by gel electrophoresis.

[0033] It can also be a variant that has ≥80% homology with the amino acid sequence encoded by any one of SEQ ID NOs: 1-11 and maintains immune activity. The sequence immunomodulatory protein can enhance, promote or regulate the immune response in vivo or in vitro, and can be used as an immune adjuvant; its nucleotide sequence has ≥80% homology with the nucleic acid sequence encoded by any one of SEQ ID NOs: 14-24 and maintains immune activity.

[0034] Example 2. Preparation of nucleic acid adjuvant vaccine preparation

[0035] Preparation of organic phase: lipids, including cationic lipids and non-cationic lipids, were weighed using a 1 / 100,000 balance. Specifically, lipids SM102, DSPC, Cholesterol and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 49:20.1:30:0.9 and mixed, and the total lipid concentration was diluted with ethanol to 33.3 mg / mL. The mixture was incubated in a 37°C metal bath for more than 20 min.

[0036] Preparation of aqueous phase: Dilute OVA-mRNA and adjuvant mRNA with 50 mM citrate buffer (pH 4.0) to a final concentration of 0.17 mg / mL and mix at a mass ratio of 1:1-1:5 for later use.

[0037] The aqueous phase and the organic phase were quickly 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 hours (ambient temperature was room temperature or 4°C) to obtain lipid nanoparticles with a particle size of 20-150 nm and stable encapsulation (encapsulation rate not less than 80%) of adjuvant mRNA.

[0038] The experimental animal species is Balb / C, and the administration routes include but are not limited to endotracheal intubation (it), nasal instillation (in), and intramuscular injection (im).

[0039] The mass ratio of the vaccine adjuvant and the mRNA encoding the target antigen in the present invention can be 1:1-1:5.

[0040] The composition was administered by intratracheal spray (it): During isoflurane inhalation anesthesia, a high-pressure spray needle was used to spray the 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. Unless otherwise specified, this dose was used in all experiments of the same type.

[0041] The composition was administered via the nasal route (in): the nucleic acid adjuvant vaccine formulation or LNP formulation was administered to Balb / c mice at an mRNA dose of 2.5 μg mRNA / 40 μL. During isoflurane inhalation anesthesia, the solution was applied in the form of a drop to the nostril of a single animal, and the formulation was naturally inhaled. Unless otherwise specified, all experiments of the same type used this dose.

[0042] The composition was administered by intramuscular injection (im): the skin surface of the outer thigh muscle of Balb / c mice was disinfected with 75% alcohol cotton balls, and the muscle was quickly pierced with a syringe to inject the nucleic acid adjuvant vaccine preparation or LNP preparation with an mRNA dose of 5μg mRNA / 100uL. Unless otherwise specified, all experiments of the same type used this dose.

[0043] The dosage used in the present invention can be between 1 μg and 50 μg.

[0044] Example 3: Physical characterization of nucleic acid adjuvant vaccine preparation (particle size, dispersion coefficient PDI, encapsulation efficiency EE)

[0045] The particle size of the formulations was measured using dynamic light scattering (DLS) at 25 °C using a Zetasizer Nano ZS instrument. Figure 1 , a) and PDI( Figure 1 , b). Determination of encapsulation efficiency: The mRNA encapsulation efficiency was evaluated using the Quant-iTTM RiboGreen RNA Assay Kit (ThermoFisher Scientific). PHL samples were dispersed in 1×TE buffer containing 2% Triton X-100, and the contents of free mRNA and total mRNA were determined ( Figure 1 , c). Five concentrations of mRNA standards were prepared using 1×TE buffer. An equal volume of PVA 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 a microplate 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%.

[0046] The results showed that compared with the traditional preparation LNP, the particle size of the adjuvant vaccine preparation was between 80-100nm, the PDI was between 0.10-0.18, the mRNA encapsulation rate was between 80-98%, and the preparation was superior to the LNP preparation in terms of physical characterization and drug loading.

[0047] Example 4: Comparative screening of nucleic acid adjuvant ratios

[0048] Preparation of preparations: Adjuvant vaccine preparations containing Fluc-mRNA and LNP control preparations containing Fluc-mRNA were prepared using the method described in "Example #1" above. (A) Control group: LNP (Fluc-mRNA without adjuvant); (B) LTA-mRNA experimental group (Fluc-mRNA:LTA-mRNA=1:1-5:1).

[0049] Balb / C mice (6-8 weeks old) were selected and randomly divided into groups of 3. During isoflurane inhalation anesthesia, a high-pressure spray needle was used to spray mRNA at a dose of 2.5 μg mRNA / 50uL of nucleic acid adjuvant vaccine preparation containing Fluc-mRNA or LNP preparation containing Fluc-mRNA into the trachea of ​​a single mouse. Unless otherwise specified, this dose was used for all experiments of the same type. The results showed that 6 hours after intratracheal spray (it) administration, a relatively low level of firefly luciferase activity was detected in the lung tissue of mice in the control group (LNP preparation containing only Fluc-mRNA); in each experimental group containing mRNA adjuvant, the luciferase activity produced by Fluc-mRNA was significantly higher than that of the control group ( Figure 2 a). Further comparison of the results of different ratios showed that when the mass ratio of Fluc-mRNA to mRNA adjuvant was 3:1, the luciferase level in the lungs was the highest, and the "3:1" ratio had the best synergistic effect.

[0050] Example 5. Comparative screening of dosages of nucleic acid adjuvant vaccine preparations

[0051] Preparation of preparations: The method described in the above "Example #1" was used to prepare LTA adjuvant vaccine preparations containing Fluc-mRNA (SEQ ID NO.12) and LNP control preparations containing Fluc-mRNA.

[0052] Control group: LNP preparation containing only Fluc-mRNA (without adjuvant), fixed dose of 2.5 μg;

[0053] Experimental group: Fluc-mRNA:LTA mRNA adjuvant = 3:1, total mRNA 2.5-5 μg;

[0054] The results showed that 6 hours after intratracheal spray (it) administration, relatively low levels of firefly luciferase activity were detected in the lung tissue of mice in the control group (LNP preparation containing only Fluc-mRNA); while in each experimental group containing mRNA adjuvant, the luciferase activity produced by Fluc-mRNA was significantly higher than that of the control group ( Figure 2 b). Further comparison of different dosages showed that when the total dosage of Fluc-mRNA and mRNA adjuvant was 3 μg, the luciferase level in the lung was the highest, with the best synergistic effect.

[0055] Example 6. Evaluation of mouse immune response

[0056] (1) Evaluation of the immune response of mice after inoculation of nucleic acid adjuvant vaccine preparations and LNP control preparations via the nasal route (in). Preparation of preparations: The adjuvant vaccine preparation containing RBD-mRNA (SEQ ID NO. 13) and the LNP control preparation containing RBD-mRNA were prepared using the method described in "Example 1" above.

[0057] The immunization dose was 3.0ug RBD-mRNA / mouse. The first vaccination time was counted as "day 0", and the same vaccination route, same preparation and same dose were used for booster immunization on day 21. Serum, bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) of mice were collected 28 days after the first immunization, and the humoral immune response, mucosal immune response and cellular immune response of the body were tested respectively. The results are shown in Figure 3 shown.

[0058] The ELISA method was used to measure the serum of mice vaccinated with adjuvant vaccine preparations. At 28 days, the IgG antibody levels in the serum and the sIgA antibody levels in the BALF and NLF of mice vaccinated with adjuvant vaccine preparations were significantly higher than those in the LNP group. We further used the enzyme-linked immunospot technique (EliSpot) to analyze the ability of mouse lung lymphocytes to produce cytokines interferon-γ (IFN-γ) and interleukin-17 (IL-17). 28 days after intranasal inoculation (in) of the adjuvant vaccine preparation, the ability of mouse lung lymphocytes to secrete IFN-γ and IL-17 was far greater than that of the control group of LNP preparations vaccinated intranasally. Flow cytometry was used to analyze the central memory T cells (Tcm), effector memory T cells (Tem), and lung tissue resident memory T cells (T R m) Subgroups. After intranasal inoculation (in) with adjuvanted vaccine preparations, CD8 + Tcm and CD4 + Tcm cells, CD8 + Tem and CD4 + TEM cells, CD8 + T R m and CD4 + T R The proportion of m cells was much higher than that of LNP preparations inoculated intranasally (in). The results showed that adjuvant vaccine preparations could induce the proliferation and differentiation of central memory T cells, resident memory T cells and effector memory T cells in the lung tissue of mice.

[0059] (2) The protective effects of nucleic acid adjuvant vaccine preparations and LNP control preparations against SARS-CoV-2 variants in mice after intranasal (in) vaccination and against close contact transmission in hamsters.

[0060] Preparation: The LTA1 adjuvant vaccine preparation containing RBD-mRNA and the LNP control preparation containing RBD-mRNA were prepared by the method described in "Example 1" above. The nucleic acid adjuvant vaccine and the LNP control preparation were inoculated intranasally, and then the weight changes of mice were detected. The change trend after the SARS-CoV-2 variant was as follows Figure 4 As shown in a. The results showed that mice vaccinated with PVA vaccine did not experience significant weight loss compared with LNP, and showed 100% complete protection.

[0061] Comparison of viral RNA loads in the lungs and nasal turbinates of mice with nucleic acid adjuvant vaccine and LNP on days 3 and 12 after challenge Figure 4 As shown in middle b. The results showed that the levels of viral RNA in the lungs of mice vaccinated with the LTA1 adjuvant vaccine formulation on the 3rd day after challenge and in the nasal turbinates on the 12th day after challenge were significantly lower than those in the LNP group.

[0062] The weight change trends of each experimental group after contact with donor hamsters infected with Omicron BA.5 variant are as follows Figure 4 As shown in c. Its potential benefit against omicron transmission was further evaluated using hamsters in a close contact transmission protection model. The results showed that no weight loss was observed in the exposed hamsters after exposure to the virus donor. The viral load in the nasal wash samples collected from the PBS, LNP and LTA1 groups on the third day showed that the LTA1 group significantly prevented the colonization of omicron virus compared with PBS and LNP, showing the lowest viral load in the nasal turbinates of mice ( Figure 4 , d).

[0063] 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-encoded immune adjuvant composition, characterized in that: The composition comprises: (A) nucleic acid encoding a specific immunomodulatory protein, i.e., a nucleic acid adjuvant; (B) an active agent or therapeutic agent, preferably the active agent or therapeutic agent is a nucleic acid; (C) cationic lipids; (D) Non-cationic lipids.

2. The nucleic acid-encoded vaccine adjuvant composition according to claim 1, wherein the nucleic acid adjuvant comprises one or more nucleic acid molecules, and the nucleic acid molecules encode one or more immunomodulatory proteins selected from the following: heat-sensitive enterotoxin A subunit, heat-sensitive enterotoxin B subunit, cholera toxin A subunit, cholera toxin B subunit, interleukin-12, interleukin-2, interleukin-7, interleukin-15, granulocyte-macrophage colony-stimulating factor, thymic stromal lymphopoietin, and cyclic GMP-AMP synthase.

3. The nucleic acid-encoded vaccine adjuvant composition according to claim 2, characterized in that: The amino acid sequence of the immunomodulatory protein encoded by the nucleic acid adjuvant is as shown in SEQ ID NO: 1-11, or a variant having ≥80% homology with any amino acid sequence encoded in SEQ ID NO: 1-11 and maintaining immune activity. The sequence immunomodulatory protein can enhance, promote or regulate the immune response in vivo or in vitro, and is thus used as an immune adjuvant.

4. The nucleic acid-encoded vaccine adjuvant composition according to claim 3, wherein the nucleic acid adjuvant coding region sequence is selected from SEQ ID NO: 14-24, or a variant having ≥ 80% homology with any nucleic acid sequence in SEQ ID NO: 14-24 and maintaining immunological activity.

5. The nucleic acid-encoded vaccine adjuvant composition according to claims 1-4, characterized in that: The nucleic acid adjuvant encodes therapeutic agents such as therapeutic proteins, vaccine antigens, antibodies, regulators, gene repair factors, etc. The nucleic acid comprises at least one selected from the following: messenger RNA, self-amplifying RNA, circular RNA, plasmid DNA, single-stranded DNA, double-stranded DNA, deoxyribozymes, ribozymes, nucleic acid aptamers, clustered regularly interspaced short palindromic repeats-related nucleic acids, single guide RNA, CRISPR-RNA, trans-activated crRNA, guide RNA, single-stranded RNA and double-stranded RNA.

6. The nucleic acid-encoded immunoadjuvant composition according to claims 1 and 2, characterized in that: The mass ratio of the nucleic acid adjuvant to the therapeutic agent encoding the target antigen is 1:1-1:5, and the dosage per dose is between 1 μg and 50 μg.

7. The nucleic acid-encoded immunoadjuvant composition according to claims 1 and 2, characterized in that: The composition is administered by any one or more of the following routes: intratracheal spray, nasal drip, intramuscular injection, subcutaneous injection, intravenous injection, oral administration, skin administration, ocular administration or other appropriate administration routes.

8. The nucleic acid-encoded immune adjuvant composition according to claim 1, characterized in that: The cationic lipid is selected from ionized lipids, permanently positively charged lipids or a combination thereof; the non-cationic lipid is selected from cholesterol, neutral phospholipids, polyethylene glycol-lipids, block copolymers or a combination thereof.

9. The method for preparing the vaccine preparation according to any one of claims 4 to 8, characterized in that: The vaccine preparation is obtained by mixing a vaccine adjuvant and a nucleic acid therapeutic agent encoding a target antigen and encapsulating them in lipid nanoparticles. It includes the following steps: a) dissolving a therapeutic agent encoding a target antigen and a nucleic acid adjuvant as described in claims 2 and 3 in an aqueous buffer; b) rapidly mixing the aqueous phase with an organic phase containing lipid components by nanomixing or microfluidic control technology to form a lipid nanoparticle LNP complex; c) adjusting the particle size of the LNP complex to 20-150 nm, and controlling the encapsulation efficiency to be no less than 80%; d) collecting and purifying the LNP complex to obtain a vaccine preparation loaded with a nucleic acid adjuvant.

10. Use of the nucleic acid-encoded immune adjuvant composition according to claim 1 in the preparation of a medicament for enhancing the immune response of a nucleic acid vaccine, wherein the nucleic acid vaccine is selected from nucleic acid vaccines against viruses, bacteria, tumors or other pathogens.

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