Lipid nanoparticles comprising nucleic acid immunostimulatory molecules, methods of making and uses thereof

Abstract

The present application provides a lipid nanoparticle containing a nucleic acid immunostimulatory molecule, which comprises the following components: a nucleic acid immunostimulatory molecule, an ionizable lipid compound, a phospholipid, a structural lipid, and a PEGylated lipid. The present application also provides a preparation method of the lipid nanoparticle, a use in preparing a vaccine adjuvant, and a vaccine composition comprising the lipid nanoparticle. The lipid nanoparticle containing a nucleic acid immunostimulatory molecule of the present application has particularly excellent immune enhancement effect on recombinant protein vaccines, and is suitable for use as a recombinant protein vaccine adjuvant.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and relates to vaccine adjuvant preparation technology, specifically to lipid nanoparticles containing nucleic acid-based immunostimulatory molecules, their preparation methods, and applications. Background Technology

[0002] With the development of vaccine technology, especially biotechnology, vaccine antigens are becoming increasingly pure and safer, but their immunogenicity is also weakening, making adjuvants even more important. Adjuvants can non-specifically enhance the immune response to antigens and alter the type of immune response. The development of vaccines for malaria, HIV, pneumonia, and tumors places higher demands on adjuvants, requiring not only the induction of humoral immune responses but also strong cellular immune responses, while the development of new adjuvants is becoming increasingly difficult. Since 2010, only AS01 has been approved for widespread use. B However, it also faces many challenges, such as difficulty in obtaining raw materials and safety concerns. Therefore, the need for developing novel, safe, and effective adjuvants remains urgent in this field.

[0003] The successful launch of mRNA COVID-19 vaccines has made lipid nanoparticles (LNPs) the undisputed preferred carrier for the safe and effective delivery of mRNA into target cells. LNPs can efficiently encapsulate and protect nucleic acid molecules such as mRNA, preventing their degradation by nucleases in vivo, while effectively mediating the entry of nucleic acid molecules into target cells, achieving transient and efficient expression of the target protein. The successful application of LNPs in COVID-19 mRNA vaccines, with multiple mRNA-LNP vaccines already in clinical trials, fully demonstrates the reliable technological basis for LNP delivery.

[0004] Nucleic acid immunostimulant molecules mainly include three types: 1. CpG ODN (CpG oligodeoxynucleotides), which are TLR9 agonists, activating TLR9 and inducing Th1 immune responses; 2. dsDNA (double-stranded DNA), viral, bacterial, or artificially synthesized dsDNA, which activate the cGAS-STING pathway, induce type I interference, and enhance immune responses; 3. dsRNA (double-stranded RNA), such as Poly(I:C) (polyinosinic-polycytidylic acid), which are TLR3 agonists and induce humoral and cellular immune responses. Nucleic acid immunostimulant molecules have weak immune effects when used alone due to their easy degradation in vivo and difficulty in delivery into cells. Nucleic acid immunostimulant molecules can only activate innate immunity but cannot produce antibodies against specific proteins. Therefore, they cannot be used as therapeutic or preventative agents on their own and must be used in combination with specific antigens to enhance the immune response to those antigens.

[0005] Currently, it has been noted that nucleic acid-based immunostimulating molecules can be loaded onto LNPs to prepare vaccine adjuvants, but problems remain, such as low delivery efficiency and poor simultaneous enhancement of humoral and cellular immune responses. Therefore, there is still an urgent need to develop LNP-based vaccine adjuvants that can efficiently encapsulate and deliver nucleic acid-based immunostimulating molecules to achieve significantly enhanced humoral and cellular immune responses. Summary of the Invention

[0006] To address the aforementioned problems in existing technologies, the inventors conducted in-depth research on the compatibility between the components of lipid nanoparticles and nucleic acid-based immunostimulatory molecules, seeking a lipid nanoparticle delivery system that can maximize the immunostimulatory effect of nucleic acid-based immunostimulatory molecules. Based on this, the present invention designs lipid nanoparticles containing specific components to encapsulate nucleic acid-based immunostimulatory molecules (e.g., CpG ODN, Poly(I:C)) within the lipid nanoparticles. This not only effectively prevents the degradation of nucleic acid-based immunostimulatory molecules by nucleases in vivo and promotes their entry into cells, but also maximizes the immunostimulatory effect, thereby achieving excellent humoral and cellular immune responses. The lipid nanoparticles containing nucleic acid-based immunostimulatory molecules of the present invention exhibit particularly excellent immunostimulatory effects for recombinant protein vaccines and are suitable for use as adjuvants in recombinant protein vaccines.

[0007] Specifically, the present invention provides: (1) A lipid nanoparticle containing nucleic acid-based immunostimulatory molecules, comprising the following components: Nucleic acid-based immunostimulatory molecules; The structure of the ionizable lipid compound is shown below:

[0008] Phospholipids; Structural lipids; and Polyethylene glycol-modified lipids.

[0009] (2) The lipid nanoparticles according to (1), wherein the nucleic acid immunostimulatory molecules are selected from CpG oligodeoxynucleotides and double-stranded RNA.

[0010] (3) The lipid nanoparticles according to (2), wherein the sequence of the CpG oligodeoxynucleotide is 5'-TGACTGTGAACGTTCGAGATGA-3'.

[0011] (4) The lipid nanoparticles according to (2), wherein the double-stranded RNA is polyinosinic acid.

[0012] (5) The lipid nanoparticles according to (1), wherein the phospholipid is selected from 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-distearyl-sn-glycerol-3-phosphate-rac-glycerol (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1-stearoyl-2-myristoyl-sn-glycerol-3-phosphate choline (SMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and mixtures thereof; preferably, the phospholipid is 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC).

[0013] (6) The lipid nanoparticles according to (1), wherein the structural lipid is selected from cholesterol, sitosterol, campesterol, stigmasterol, brassosterol and mixtures thereof; preferably, the structural lipid is cholesterol.

[0014] (7) The lipid nanoparticles according to (1), wherein the molar ratio of the ionizable lipid compound to the phospholipid, the structural lipid and the polyethylene glycol-modified lipid is (40-65):(5-17.5):(35-55):(1.0-2.0), preferably 48.5:10:40:1.5.

[0015] (8) The lipid nanoparticles according to (1), wherein the nitrogen / phosphorus ratio of the ionizable lipid compound and the nucleic acid immunostimulatory molecule is 2:1 to 12:1, preferably 5:1.

[0016] (9) The lipid nanoparticles according to (1), wherein the size of the lipid nanoparticles is 70 nm to 100 nm and the polydispersity index of the lipid nanoparticles is less than 0.15.

[0017] (10) A method for preparing lipid nanoparticles containing nucleic acid-based immunostimulatory molecules according to any one of (1)-(9), comprising the following steps: (a) Preparation of an aqueous solution containing nucleic acid-based immunostimulatory molecules: (b) Prepare an organic phase solution containing ionizable lipid compounds, phospholipids, structured lipids, and polyethylene glycol-modified lipids; and (c) Using a microfluidic device to mix the aqueous solution and the organic solution to obtain a solution containing lipid nanoparticles containing nucleic acid-like immunostimulatory molecules.

[0018] (11) According to the method of (10), wherein in step (c), the microfluidic device is configured to make the flow rate ratio of the organic phase solution to the aqueous phase solution 1:3 to 1:5 for the mixing.

[0019] (12) The method according to (10) further includes, after step (c), an ultrafiltration step of the solution containing the lipid nanoparticles containing nucleic acid immunostimulatory molecules.

[0020] (13) According to the method described in (10), the encapsulation rate of the nucleic acid immunostimulatory molecule is above 95%.

[0021] (14) Use of lipid nanoparticles containing nucleic acid immunostimulatory molecules according to any one of (1)-(9) in the preparation of vaccine adjuvants.

[0022] (15) According to the use described in (14), wherein the vaccine adjuvant enhances humoral and cellular immune responses to the recombinant protein vaccine antigen.

[0023] (16) According to the use described in (15), the recombinant protein vaccine antigen is selected from recombinant herpes zoster virus protein and hepatitis B surface antigen.

[0024] (17) A vaccine composition comprising: Lipid nanoparticles containing nucleic acid-based immunostimulatory molecules according to any one of (1)-(9); and Recombinant protein vaccine antigen.

[0025] (18) The vaccine composition according to (17), wherein the recombinant protein vaccine antigen is selected from recombinant herpes zoster virus protein and hepatitis B surface antigen.

[0026] Compared with the prior art, the present invention has the following advantages and positive effects: Through in-depth research on lipid nanoparticle vaccine adjuvants, the inventors discovered a lipid nanoparticle with a specific composition containing nucleic acid immunostimulatory molecules. When used as an adjuvant for recombinant protein vaccines, it can significantly enhance the humoral and cellular immune responses of recombinant protein vaccine antigens. This lipid nanoparticle containing nucleic acid immunostimulatory molecules contains an ionizable lipid compound BMO28 with a specific structure, and contains phospholipids (e.g., 1,2-distearate-sn-glycerol-3-phosphocholine), structural lipids (e.g., cholesterol), and polyethylene glycol-modified lipids, wherein the molar ratio of BMO28 to phospholipids, structural lipids, and polyethylene glycol-modified lipids is (40-65):(5-17.5):(35-55):(1.0-2.0).

[0027] More specifically, the lipid nanoparticles of the present invention containing nucleic acid-based immunostimulatory molecules have the following advantages: 1. The ionizable lipid compound BMO28 carries a positive charge under acidic conditions, which can effectively electrostatically bind negatively charged nucleic acid immunostimulant molecules. Under the combined action of specific phospholipids, structural lipids, and polyethylene glycol-modified lipids, it forms stable and uniformly sized lipid nanoparticles containing nucleic acid immunostimulant molecules. The particle size is 70 nm to 100 nm, and the polydispersity index is below 0.15. This encapsulates the nucleic acid immunostimulant molecules within the lipid nanoparticles, preventing premature degradation of the nucleic acid immunostimulant molecules and making them difficult to deliver into cells. It can also significantly reduce the amount of nucleic acid immunostimulant molecules required.

[0028] 2. In particular, when used as an adjuvant for recombinant protein vaccines, it was unexpectedly discovered that the lipid nanoparticles of the present invention, containing nucleic acid-like immunostimulatory molecules with the specific composition described above, can significantly enhance the immunostimulatory effect of nucleic acid-like immunostimulatory molecules, inducing a balanced humoral and cellular immune response. They are particularly suitable as adjuvants for recombinant protein vaccine antigens (e.g., recombinant herpes zoster virus, hepatitis B surface antigen). Compared with the control adjuvant aluminum adjuvant, the vaccine adjuvant of the present invention significantly enhances both humoral and cellular immune responses to recombinant protein vaccines. When used in combination with recombinant herpes zoster virus protein gE, the antibody titer of the vaccine adjuvant of the present invention can be up to 11.31 times that of using aluminum adjuvant, and the proportion of gE-specific CD4+ T cells is 4.47 times that of using aluminum adjuvant. When used in combination with hepatitis B surface antigen (HBsAg), the antibody titer of the vaccine adjuvant of the present invention can be up to 97.71 times that of using aluminum adjuvant, and the proportion of HBsAg-specific CD4+ T cells is 4.47 times that of using aluminum adjuvant. The percentage of T cells was 3.15 times that of those using aluminum adjuvant, and the percentage of HBsAg-specific CD8+ T cells was 5.79 times that of those using aluminum adjuvant.

[0029] 3. The lipid nanoparticles containing nucleic acid-based immunostimulating molecules of the present invention are simple to prepare, easy to operate, and suitable for large-scale application in vaccine production. Attached Figure Description

[0030] Figure 1 The dynamic light scattering particle size of lipid nanoparticles CpG-LNP, Poly(I:C)-LNP, and control empty LNP containing nucleic acid immunostimulatory molecules according to the present invention is shown.

[0031] Figure 2 The dynamic light scattering polydispersity index (PDI) of lipid nanoparticles CpG-LNP, Poly(I:C)-LNP, and control empty LNP containing nucleic acid immunostimulatory molecules according to the present invention is shown.

[0032] Figure 3 The encapsulation efficiency of lipid nanoparticles CpG-LNP, Poly(I:C)-LNP, and control empty LNP containing nucleic acid immunostimulatory molecules according to the present invention is shown.

[0033] Figure 4 The results of detecting the anti-VZV antibody levels in the serum of mice in each group were shown in the evaluation test of the immunomodulatory effect of recombinant herpes zoster virus gE in Example 3.

[0034] Figure 5 The following is an example of the evaluation test of the immune enhancement effect of recombinant herpes zoster virus gE in Example 3, showing the proportion of gE-specific CD4+ T cells (including single-positive, double-positive, and triple-positive cells of three factors: IFN-γ, IL-2, and TNF-α) in the spleen lymphocytes of mice in each group of mice to the total CD4+ T cells.

[0035] Figure 6 The results of the detection of hepatitis B surface antibody (anti-HBs) levels in the serum of mice in each group were shown in the evaluation test of the hepatitis B surface antigen (HBsAg) immune enhancement effect in Example 3.

[0036] Figure 7 The following is an example of the evaluation test of the immune enhancement effect of hepatitis B surface antigen (HBsAg) in Example 3, showing the proportion of HBsAg-specific CD4+ T cells (including single-positive, double-positive, and triple-positive cells of three factors: IFN-γ, IL-2, and TNF-α) in the spleen lymphocytes of mice in each group of mice to the total CD4+ T cells.

[0037] Figure 8 The following is an example of the evaluation test of the immune enhancement effect of hepatitis B surface antigen (HBsAg) in Example 3, showing the proportion of HBsAg-specific CD8+ T cells (including single-positive, double-positive, and triple-positive cells of three factors: IFN-γ, IL-2, and TNF-α) in the spleen lymphocytes of mice in each group of mice to the total CD8+ T cells. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings through specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0039] The lipid nanoparticles of the present invention, with their specific composition, are particularly suitable for delivering nucleic acid-based immunostimulatory molecules and significantly enhance humoral and cellular immune responses to various types of vaccine antigens.

[0040] In one aspect, the present invention provides lipid nanoparticles comprising nucleic acid-based immunostimulatory molecules, comprising the following components: Nucleic acid-based immunostimulatory molecules; The structure of the ionizable lipid compound BMO28 is shown below:

[0041] Phospholipids; Structural lipids; and Polyethylene glycol-modified lipids.

[0042] The lipid nanoparticles of the present invention, comprising nucleic acid-based immunostimulatory molecules, have a nanoscale size (e.g., 1 nm to 1000 nm) in at least one dimension. In a preferred embodiment, the particle size of the lipid nanoparticles comprising nucleic acid-based immunostimulatory molecules is 70 nm to 100 nm, for example, 70 nm to 90 nm or 75 nm to 85 nm. The polydispersity index (PDI) of the lipid nanoparticles comprising nucleic acid-based immunostimulatory molecules is 0.15 or less, more preferably 0.10 or less.

[0043] In some embodiments, the nucleic acid-based immunostimulatory molecule is selected from CpG oligodeoxynucleotides and double-stranded RNA. In a preferred embodiment, the sequence of the CpG oligodeoxynucleotide may be 5'-TGACTGTGAACGTTCGAGATGA-3', which is a synthetically produced CpG oligodeoxynucleotide, also known as ODN 1018; the double-stranded RNA may be polyinosinic acid (Poly(I:C)), which is a synthetically produced interferon inducer that stimulates human cells to produce endogenous interferon.

[0044] The specific components of the lipid nanoparticles containing nucleic acid immunostimulatory molecules of the present invention are matched with each other, unexpectedly exhibiting excellent effects particularly suitable for enhancing the immune response to recombinant protein vaccine antigens. Specifically, the molecular structure of the ionizable lipid compound BMO28 is particularly suitable for effectively binding nucleic acid immunostimulatory molecules (e.g., CpG oligodeoxynucleotides, double-stranded RNA) and efficiently delivering these nucleic acid immunostimulatory molecules to enhance the immune response. In some embodiments, the ionizable lipid compound BMO28 accounts for 40% to 65% of the total molar percentage of the lipid nanoparticles, preferably 40% to 63%, more preferably 46% to 50%, for example 48.5%, based solely on the total molar percentage of the components of the lipid nanoparticles that do not contain nucleic acid immunostimulatory molecules.

[0045] In some embodiments, the nitrogen / phosphorus ratio of the ionizable lipid compound to the nucleic acid immunostimulatory molecule is 2:1 to 12:1, preferably 5:1.

[0046] The lipid nanoparticles of the present invention, comprising nucleic acid-based immunostimulatory molecules, further comprise phospholipids. In some embodiments, the phospholipids are selected from 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-distearyl-sn-glycerol-3-phosphate-rac-glycerol (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1-stearoyl-2-myristoyl-sn-glycerol-3-phosphate choline (SMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and mixtures thereof. In a preferred embodiment, the phospholipid is DSPC.

[0047] In some implementations, the phospholipids account for 5% to 17.5% of the total molar number of the lipid nanoparticles that do not contain nucleic acid-based immunostimulatory molecules, preferably 10%.

[0048] The lipid nanoparticles of the present invention, comprising nucleic acid-based immunostimulatory molecules, further comprise structural lipids. In some embodiments, the structural lipids are selected from cholesterol, sitosterol, campesterol, stigmasterol, brassosterol, and mixtures thereof. In a preferred embodiment, the structural lipid is cholesterol.

[0049] In some embodiments, the structural lipids account for 35% to 55% of the total molar percentage of the lipid nanoparticles, preferably 35% to 48.5%, more preferably 38.5% to 45%, based solely on the total molar percentage of the components of the lipid nanoparticles that do not contain nucleic acid-like immunostimulatory molecules; for example, 40%.

[0050] The lipid nanoparticles containing nucleic acid-based immunostimulatory molecules of the present invention further contain polyethylene glycol-modified lipids, wherein the polyethylene glycol-modified lipids are selected from one or more of the following: methoxy polyethylene glycol bis(tetradecyl acetamide) (ALC-0159), dimyristoylglycerol-3-methoxy polyethylene glycol 1000 (PEG1000-DMG), dimyristoylglycerol-3-methoxy polyethylene glycol 5000 (PEG5000-DMG), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (PEG2000-DMG), distearate phosphatidylethanolamine polyethylene glycol 2000 (PEG2000-DSPE), and dimyristoyl phosphate ethanolamine polyethylene glycol 2000 (PEG2000-DMPE); preferably, the polyethylene glycol-modified lipid is PEG2000-DMG.

[0051] In some embodiments, the percentage of polyethylene glycol-modified lipids in the lipid nanoparticles is 1.0% to 2.0%, preferably 1.5%, based solely on the total molar number of all components of the lipid nanoparticles that do not contain nucleic acid-based immunostimulatory molecules.

[0052] In a preferred embodiment, the lipid nanoparticles of the present invention comprising nucleic acid immunostimulatory molecules are composed of the following components: (i) nucleic acid immunostimulatory molecules; (ii) ionizable lipid compound BMO28; (iii) phospholipids; (iv) structural lipids; and (v) polyethylene glycol-modified lipids.

[0053] In some embodiments, the molar ratio of the ionizable lipid compound BMO28 to phospholipids, structured lipids and polyethylene glycol-modified lipids may be (40-65):(5-17.5):(35-55):(1.0-2.0), preferably 48.5:10:40:1.5.

[0054] In another aspect, the present invention provides a method for preparing the above-mentioned lipid nanoparticles containing nucleic acid-based immunostimulatory molecules, comprising the following steps: (a) Preparation of an aqueous solution containing nucleic acid-based immunostimulatory molecules: (b) Prepare an organic phase solution containing ionizable lipid compounds, phospholipids, structured lipids, and polyethylene glycol-modified lipids; and (c) Using a microfluidic device to mix the aqueous solution and the organic solution to obtain a solution containing lipid nanoparticles containing nucleic acid-like immunostimulatory molecules.

[0055] In some embodiments, in step (a), an aqueous buffer solution containing nucleic acid immunostimulant molecules can be prepared, for example, a citrate-sodium citrate buffer solution (pH=4.2). The resulting aqueous solution has a concentration of 10-1 nucleic acid immunostimulant molecules. g / mL to 300 g / mL, preferably 14 g / mL g / mL up to 261 g / mL, for example, 33 g / mL.

[0056] In some embodiments, in step (b), an organic phase solution containing an ionizable lipid compound, phospholipid, structural lipid, or polyethylene glycol-modified lipid can be prepared separately using an organic solvent, for example, methanol, ethanol, and / or isopropanol; then, the organic phase solutions are mixed according to the molar ratio of the ionizable lipid compound to the phospholipid, structural lipid, and polyethylene glycol-modified lipid to obtain an organic phase solution containing the ionizable lipid compound, phospholipid, structural lipid, and polyethylene glycol-modified lipid, wherein the total concentration of each solute can be from 0.5 mg / mL to 3.0 mg / mL, for example, 1.0 mg / mL.

[0057] In some embodiments, in step (c), the microfluidic device is configured to mix the organic phase solution with the aqueous phase solution at a flow rate ratio of 1:3 to 1:5, preferably 1:3. The microfluidic device (e.g., a microfluidic instrument) enables the mixing of the organic phase solution and the aqueous phase solution to form lipid nanoparticles containing nucleic acid-like immunostimulatory molecules with a narrow particle size distribution and stable structure, having a particle size of 70 nm to 100 nm, for example 70 nm to 90 nm or 75 nm to 85 nm, and a polydispersity index of 0.15 or less, more preferably 0.10 or less.

[0058] In some embodiments, the preparation method according to the present invention further includes, after step (c), a step of diluting and / or ultrafiltration of the solution containing the lipid nanoparticles encapsulating nucleic acid immunostimulatory molecules. The dilution step may be performed using a buffer solution, such as a phosphate buffer solution. The ultrafiltration step may be performed using an ultrafiltration device known in the art (e.g., an ultrafiltration tube, an ultrafiltration membrane, etc.) to ultrafilter the solution of lipid nanoparticles encapsulating nucleic acid immunostimulatory molecules to 1 / 4 to 1 / 10 of the original solution volume.

[0059] In this document, the term "encapsulation efficiency" refers to the proportion of nucleic acid immunostimulant molecules encapsulated within lipid nanoparticles out of all nucleic acid immunostimulant molecules. In some embodiments, according to the preparation method of the present invention, the encapsulation efficiency of the nucleic acid immunostimulant molecules is 95% or higher, for example, 96% or higher, 97% or higher, or 98% or higher.

[0060] In another aspect, the present invention provides the use of lipid nanoparticles containing nucleic acid-based immunostimulating molecules in the preparation of vaccine adjuvants. Using lipid nanoparticles containing nucleic acid-based immunostimulating molecules according to the present invention as vaccine adjuvants can significantly enhance humoral and cellular immune responses to recombinant protein vaccine antigens.

[0061] In some implementations, the recombinant protein vaccine antigen may be selected from recombinant herpes zoster virus protein (e.g., varicella-zoster virus glycoprotein E) and hepatitis B surface antigen.

[0062] In another aspect, the present invention provides a vaccine composition comprising lipid nanoparticles containing nucleic acid-based immunostimulatory molecules according to the invention; and a recombinant protein vaccine antigen. In some embodiments, the recombinant protein vaccine antigen is selected from recombinant herpes zoster virus proteins (e.g., varicella-zoster virus glycoprotein E) and hepatitis B surface antigen.

[0063] In some embodiments, the vaccine composition of the present invention further comprises excipients. Excipients refer to components other than vaccine antigens and adjuvants, which do not have an immune-activating effect but instead play an auxiliary role such as stabilizing pH, protecting proteins, inhibiting bacteria, and regulating osmotic pressure. Excipients include (but are not limited to) buffers, osmotic pressure regulators, protein protectants, stabilizers, surfactants, antibacterial preservatives, solvents, etc.

[0064] Depending on the properties of the specific recombinant protein vaccine antigen, a mixture of the antigen solution and the solution containing lipid nanoparticles with nucleic acid-like immunostimulatory molecules can be prepared immediately before administering the vaccine composition and administered to the subject, for example, prepared immediately 1 minute to 2 hours before administration, such as within 1 hour, 30 minutes, 10 minutes, or 2 minutes before administration; alternatively, the vaccine composition can be formulated into a vaccine preparation that can be stored for a certain period of time and administered to the subject, with storage time ranging from 1 day to 1 year, for example, it can be stored for 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 1 month, 3 months, 6 months, or 1 year.

[0065] The vaccine composition of the present invention can be formulated for oral, intravenous, intramucosal, intraperitoneal, intramuscular, transdermal, intradermal, subcutaneous, or intranasal administration, or for inhalation. Preferably, the vaccine composition is formulated for injection, for example, intramuscular injection.

[0066] The following examples further explain or illustrate the content of the present invention, but these examples should not be construed as limiting the scope of protection of the present invention.

[0067] Example

[0068] Unless otherwise specified, the experimental methods used in the following examples are all performed using standard experimental procedures, operations, materials and conditions in the field.

[0069] Unless otherwise stated, the raw materials used in the embodiments of this application are commercially available.

[0070] The sources of the materials and instruments used in the examples are as follows: The sequence of the CpG ODN is 5'-TGACTGTGAACGTTCGAGATGA-3', CAS Registry No.: 937402-51-2, synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0071] Poly(I:C) was prepared by Sinopharm Biotechnology Research Institute Co., Ltd.: Polyinosinic acid and polycytidylic acid powders were dissolved separately in phosphate-buffered saline (PBS) to a fixed concentration. Equal volumes of the dissolved polyinosinic acid and polycytidylic acid solutions were measured, shaken in a constant temperature water bath at 42°C for 30 minutes, and then filtered sterilely to obtain the final product. Polyinosinic acid and polycytidylic acid were purchased from Kaiping Qianniu Biochemical Pharmaceutical Co., Ltd.

[0072] The ionizable lipid compound BM028 was synthesized with reference to the invention patent with patent number ZL202411204254.5, the entire contents of which are incorporated herein by reference.

[0073] Cholesterol was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number: 06040010300.

[0074] 1,2-Disteaaroylphosphatidylcholine (DSPC) was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number: 06030001100.

[0075] 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG) was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number: 06020112402.

[0076] Recombinant herpes zoster virus protein gE (varicella-zoster virus glycoprotein E), abbreviated as gE: prepared by Sinopharm Biotechnology Research Institute Co., Ltd.

[0077] Hepatitis B surface antigen (HBsAg): provided by Beijing Institute of Biological Products Co., Ltd.

[0078] HEK-Blue hTLR3 cells were purchased from Invivogen, product number: hkb-htlr3.

[0079] The detection medium was purchased from Invivogen, product number: hb-det2.

[0080] Dynamic light scattering instrument: NANO-8S90, purchased from Malvern.

[0081] ELISA reader: MULTISKAN MK3, purchased from Thermo.

[0082] Flow cytometer: CytoFLEX LX, purchased from Beckman Coulter.

[0083] Example 1: Preparation of lipid nanoparticles containing nucleic acid-based immunostimulatory molecules according to the present invention

[0084] (1) Preparation of aqueous solution

[0085] CpG ODN was dissolved in 20 mM citrate-sodium citrate buffer (pH=4.2) to prepare an aqueous solution with a concentration of 33 µg / mL. Poly(I:C) used an ultrafiltration tube with a molecular weight cutoff of 30 kDa (purchased from Millipore) to replace the buffer with 0.02 M phosphate-buffered saline (PBS) at pH=7.2, adjusting the concentration to 33 µg / mL aqueous solution.

[0086] (2) Preparation of organic phase solution

[0087] Ionizable lipid compounds BMO28, cholesterol, DSPC, and PEG2000-DMG were dissolved in anhydrous ethanol (purchased from Sinopharm Chemical Reagent Co., Ltd., product number: 10009218) to prepare solutions with a concentration of 1.0 mg / mL. Then, the corresponding volumes of the four solutions were taken according to the molar ratio of BMO28:DSPC:cholesterol:PEG2000-DMG = 48.5:10:40:1.5 and mixed to prepare an organic phase solution with a total concentration of 1.0 mg / mL.

[0088] (3) Microfluidic synthesis

[0089] The organic phase solution containing the lipid mixture and the aqueous phase solution containing nucleic acid immunostimulant molecules (CpG ODN or Poly(I:C)) were mixed at a flow rate of 1:3 using a microfluidic instrument (NEXSTAR). The total flow rate was 8-12 mL / min. Finally, the prepared lipid nanoparticles encapsulated with nucleic acid immunostimulant molecules were diluted with 20 times their volume of phosphate buffer solution (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2.0 mM NaH2PO4, pH 7.3 ± 0.1), and the solution was ultrafiltered to 1 / 4 to 1 / 10 of the original aqueous phase volume using an ultrafiltration tube with a molecular weight cutoff of 30 kDa (Millipore).

[0090] Lipid nanoparticles containing CpG ODN (CpG-LNP) and lipid nanoparticles containing Poly(I:C) (Poly(I:C)-LNP) were prepared using the above method. In addition, lipid nanoparticles without nucleic acid-based immunostimulatory molecules (empty LNP, i.e., using citrate-sodium citrate buffer solution (pH=4.2) as the aqueous phase solution) were also prepared as a control using the above method.

[0091] The particle size and polydispersity index (PDI) of the prepared CpG-LNP, Poly(I:C)-LNP, and LNP were measured using a dynamic light scattering instrument. The encapsulation efficiency of the nucleic acid immunostimulatory molecules was characterized using RiboGreen reagent (Thermo Fisher Scientific, Invitrogen™, R11491).

[0092] Figure 1 The dynamic light scattering particle sizes of CpG-LNP, Poly(I:C)-LNP, and empty LNP (control) are shown, with CpG-LNP and Poly(I:C)-LNP having a particle size of approximately 80 nm, and empty LNP having a particle size slightly less than 80 nm.

[0093] Figure 2 The dynamic light scattering polydispersity index (PDI) of CpG-LNP, Poly(I:C)-LNP, and empty LNP (control) is shown. The PDI of all three LNPs is less than 0.10.

[0094] Figure 3 The encapsulation efficiency of CpG-LNP, Poly(I:C)-LNP, and empty LNP (control) is shown. The encapsulation efficiency of CpG-LNP and Poly(I:C)-LNP is above 95%, while the encapsulation efficiency of empty LNP is 0.

[0095] The CpG-LNP and Poly(I:C)-LNP samples were placed at 2-8℃ for 3 months. The particle size of each sample did not change significantly, the PDI remained below 0.10, and the encapsulation efficiency remained above 95%. This indicates that the lipid nanoparticles containing nucleic acid immunostimulatory molecules of the present invention are not only easy to prepare, but also maintain good stability, making them suitable for large-scale application in vaccine production.

[0096] Example 2: Preparation of the vaccine composition according to the present invention

[0097] Using CpG-LNP or Poly(I:C)-LNP prepared in Example 1 as vaccine adjuvants, vaccine compositions were prepared by combining different recombinant protein vaccine antigens, as follows: Take the recombinant protein vaccine antigen (HBsAg or gE), mix it with the solution containing CpG-LNP or Poly(I:C)-LNP prepared in Example 1, add 0.02 M PBS pH=7.2 until the concentration of LNP in the solution is 7 μg / mL to 180 μg / mL, mix well and it is ready for use. The vaccine composition according to the present invention can be stored at 2-8°C for no more than 48 hours.

[0098] Example 3: Evaluation of the immune-enhancing effect of lipid nanoparticles containing nucleic acid-based immunostimulatory molecules according to the present invention

[0099] (1) Evaluation of the immune-enhancing effect of recombinant herpes zoster virus gE

[0100] The experimental mice were female BALB / c mice, 6-8 weeks old, weighing 16-18 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Immunization groups were set up as follows: gE, gE+Al, gE+CpG, gE+Poly(I:C), gE+CpG-LNP, and gE+Poly(I:C)-LNP (see Table 1), with 8 mice randomly assigned to each group. Primary immunization was performed on day 0, and booster immunization was performed on day 21, with an immunization dose of 5 μg / mouse. On day 35, blood was collected from the eyeballs, and the mice were sacrificed, and their spleens were harvested. Serum varicella-zoster virus antibody (anti-VZV antibody) levels were detected using enzyme-linked immunosorbent assay (ELISA), and cellular immunity levels were detected using flow cytometry.

[0101] The anti-gE antibody titer was determined by indirect ELISA using a 20 PFU / well VZV-coated sample. Intracellular cytokines were detected by flow cytometry. Mouse spleen lymphocytes were isolated and the cell concentration was adjusted to 1... 10 7Cells / mL, 100 μL / well added to a 96-well plate, followed by the addition of varicella-zoster virus peptide (VZV peptide, synthesized by Sangon Biotech (Shanghai) Co., Ltd.) to a final concentration of 1.25 μg / mL. The plate was incubated at 37°C for 2 hours. Then, 0.2 μL / well of Golgi apparatus blocking agent (purchased from BD, product number: 51-2301KZ) was added, and the plate was incubated at 37°C for 4 hours. The cells were then stained with PerCP Rat anti-mouse CD4 antibody (purchased from BioLegend, product number: 100432), and washed twice with PBS containing 2% fetal bovine serum. After fixation and permeabilization, intracellular cytokines were simultaneously labeled with PE Rat anti-mouse IFN-γ (BioLegend, product number: 505808), APC Rat anti-mouse IL-2 (BioLegend, product number: 503810), and PE-Cy7 Rat anti-mouse TNF-α antibody (BioLegend, product number: 506324). Cells were washed twice with fixation and permeabilization buffer (BD, product number: 554714), and then resuspended in 200 μL of flow cytometry loading buffer (PBS containing 2% fetal bovine serum). Flow cytometry was used for analysis, and the results were analyzed using FlowJO software: the cumulative percentages of single-positive, double-positive, and triple-positive cells for IFN-γ, IL-2, and TNF-α in CD4+ T cells.

[0102] Table 1. Grouping for evaluating the immunomodulatory effect of recombinant herpes zoster virus protein gE.

[0103] Note: "-" indicates none or not present. CpG represents CpG ODN.

[0104] Figure 4The results of detecting anti-VZV antibody levels in the serum of mice in each group are shown. For the serum antibody levels of mice 14 days after two injections, there was no statistically significant difference between the gE+CpG-LNP group and the gE+Poly(I:C)-LNP group (P=0.0527). The antibody level in the gE+CpG-LNP group was significantly higher than that in the gE+CpG group (P<0.0001), and the antibody level in the gE+Poly(I:C)-LNP group was significantly higher than that in the gE+CpG group. The antibody levels in the gE+Poly(I:C) group (P<0.0001), gE+CpG-LNP group, and gE+Poly(I:C)-LNP group were significantly higher than those in the gE+Al group (P=0.0001 and P<0.0001, respectively). The antibody titer in the gE+CpG-LNP group was 5.19 times that of the gE+Al group, and the antibody titer in the gE+Poly(I:C)-LNP group was 11.31 times that of the gE+Al group.

[0105] Figure 5 The proportion of gE-specific CD4+ T cells (including single-positive, double-positive, and triple-positive cells of IFN-γ, IL-2, and TNF-α) in splenic lymphocytes of mice in each group was shown. There was no statistically significant difference in gE-specific CD4+ T cell levels between the gE+CpG-LNP group and the gE+Poly(I:C)-LNP group (P=0.6405). The gE-specific CD4+ T cell levels in the gE+CpG-LNP group were significantly higher than those in the gE+CpG group (P<0.0001), and the gE-specific CD4+ T cell levels in the gE+Poly(I:C)-LNP group were significantly higher than those in the gE+Poly(I:C) group (P=0.0092). The gE-specific CD4+ T cell levels in the gE+CpG-LNP group and the gE+Poly(I:C)-LNP group were significantly higher than those in the gE+Poly(I:C) group. The levels of T cells were significantly higher in the gE+Al group than in the gE+Al group (P<0.0001, P=0.0054, respectively). Specifically, the proportion of gE-specific CD4+ T cells in the gE+CpG-LNP group was 3.95 times that in the gE+Al group, and the proportion of gE-specific CD4+ T cells in the gE+Poly(I:C)-LNP group was 4.47 times that in the gE+Al group.

[0106] In the case of using gE antigen, the results of humoral immunity (reflected by the level of anti-VZV antibodies in serum) and cellular immunity (reflected by the level of gE-specific CD4+ T cells) confirmed that the LNP of the present invention containing CpG oligodeoxynucleotides or polyinosinic acid (Poly(I:C)) can significantly enhance the immune-enhancing effect, and the humoral immune response and cellular immune response generated are significantly higher than those of the control aluminum adjuvant.

[0107] (2) Evaluation of the immune-enhancing effect of hepatitis B surface antigen (HBsAg)

[0108] The experimental mice were female BALB / c mice, 6-8 weeks old, weighing 16-18 g. Immunization groups were established: HBsAg, HBsAg+A1, HBsAg+CpG-LNP, and HBsAg+Poly(I:C)-LNP (see Table 2), with 8 mice randomly assigned to each group. Primary immunization was performed on day 0, and booster immunization was performed on day 21, with an immunization dose of 1 μg / mouse. On day 35, blood was collected from the eyeballs, and the mice were sacrificed, and their spleens were harvested. Serum hepatitis B surface antibody (anti-HBs) levels were detected using ELISA, and cellular immunity levels were detected using flow cytometry.

[0109] Serum anti-HBs levels were quantitatively detected by serially diluting serum samples with sample diluent (5% BSA, i.e., PBS solution of 5% bovine serum albumin). The anti-HBs levels of each serum sample were then measured using a hepatitis B virus surface antibody quantitative assay kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd., product number: 808). A double logarithmic curve was plotted using the anti-HBs antibody content and corresponding optical density (OD) values ​​of standards (10 mIU / mL-160 mIU / mL) to derive a linear regression equation. The logarithmic value of the OD value of the test sample was substituted into the regression equation to obtain the corresponding anti-HBs antibody content. Positive result determination: a sample with an anti-HBs level higher than 10 mIU / mL was considered positive, and a sample with a level lower than 10 mIU / mL was considered negative. Intracellular cytokines were detected by flow cytometry. Mouse spleen lymphocytes were isolated and the cell concentration was adjusted to 1... 10 7 Cells / mL, 100 μL / well added to a 96-well plate, with a final concentration of 1.25 μg / mL of HBsAg peptide added. Incubate at 37°C for 2 hours, then remove and add 0.2 μL / well of Golgi inhibitor, incubate at 37°C for 4 hours. Remove from the plate. Stain the surface of lymphocytes with PerCP Rat anti-mouse CD4 and CD8 antibodies, washing twice with buffer. After fixation and permeabilization, simultaneously label intracellular cytokines with PERat anti-mouse IFN-γ, APC Rat anti-mouse IL-2, and PE-Cy7 Rat anti-mouse TNF-α antibodies. Wash twice with fixation and permeabilization buffer, resuspend cells in 200 μL of flow cytometry loading buffer, and analyze using flow cytometry. Analyze the results using FlowJO software: the cumulative percentage of single-positive, double-positive, and triple-positive cells for IFN-γ, IL-2, and TNF-α in CD4+ T cells or CD8+ T cells.

[0110] Table 2. Grouping for Evaluation of HBsAg Immunostimulation Effect

[0111] Note: "-" represents none or not.

[0112] Figure 6 The results of the detection of specific anti-HBs antibody levels in the serum of mice in each group are shown. For the serum antibodies of mice in each group 14 days after two injections, the antibody level of the HBsAg+Poly(I:C)-LNP group was slightly higher than that of the HBsAg+CpG-LNP group (P=0.0107). The antibody levels of the HBsAg+CpG-LNP group and the HBsAg+Poly(I:C)-LNP group were significantly higher than those of the HBsAg+A1 group (P=0.0008 and P=0.0001, respectively). The anti-HBs antibody titer of the HBsAg+CpG-LNP group was 21.03 times that of the HBsAg+A1 group, and the anti-HBs antibody titer of the HBsAg+Poly(I:C)-LNP group was 97.71 times that of the HBsAg+A1 group.

[0113] Figure 7 The proportion of HBsAg-specific CD4+ T cells (including single-positive, double-positive, and triple-positive cells of IFN-γ, IL-2, and TNF-α) in splenic lymphocytes of mice in each group was shown. There was no statistically significant difference in HBsAg-specific CD4+ T cell levels between the HBsAg+CpG-LNP group and the HBsAg+Poly(I:C)-LNP group (P=0.8603). However, the HBsAg-specific CD4+ T cell levels in the HBsAg+CpG-LNP group and the HBsAg+Poly(I:C)-LNP group were significantly higher than those in the HBsAg+A1 group (P=0.0089 and P=0.0029, respectively). Specifically, the proportion of HBsAg-specific CD4+ T cells in the HBsAg+CpG-LNP group was 3.02 times that in the HBsAg+A1 group, and the proportion in the HBsAg+Poly(I:C)-LNP group was significantly higher. The proportion of T cells was 3.15 times that of the HBsAg+Al group.

[0114] Figure 8The proportion of HBsAg-specific CD8+ T cells (including single-positive, double-positive, and triple-positive cells of IFN-γ, IL-2, and TNF-α) in splenic lymphocytes of mice in each group was shown. The HBsAg-specific CD8+ T cell level in the HBsAg+CpG-LNP group was slightly higher than that in the HBsAg+Poly(I:C)-LNP group (P=0.0338). The HBsAg-specific CD8+ T cell levels in the HBsAg+CpG-LNP and HBsAg+Poly(I:C)-LNP groups were significantly higher than those in the HBsAg+A1 group (P<0.0001, P=0.0007, respectively). Specifically, the proportion of HBsAg-specific CD8+ T cells in the HBsAg+CpG-LNP group was 9.68 times that in the HBsAg+A1 group, and the proportion in the HBsAg+Poly(I:C)-LNP group was significantly higher than that in the HBsAg+A1 group. The proportion of T cells was 5.79 times that of the HBsAg+Al group.

[0115] In the use of HBsAg antigen, humoral immunity (reflected by anti-HBs levels in serum) and cellular immunity (reflected by HBsAg-specific CD4+ T and CD8+ T cell levels) assays confirmed that LNPs containing CpG oligodeoxynucleotides or polyinosinic acid (Poly(I:C)) could significantly enhance immune enhancement, producing humoral and cellular immune responses that were significantly higher than those of the control aluminum adjuvant.

[0116] Comparative Example 1: Lipid nanoparticles containing nucleic acid-based immunostimulatory molecules prepared using other ionizable lipid compounds and their characterization results.

[0117] Using the same method as in Example 1, lipid nanoparticles (Poly(I:C)-LNP) containing Poly(I:C) were prepared using different ionizable lipid compounds (the molecular structures corresponding to the compound numbers and the preparation methods are described in the invention patent with patent number ZL202411204254.5), DSPC, and cholesterol. The composition and characterization results are shown in Table 3.

[0118] Table 3. Composition and characterization results of lipid nanoparticles containing Poly(I:C)

[0119] As shown in Table 3, the particle size, PDI and encapsulation efficiency of the lipid nanoparticles containing Poly(I:C) prepared using ionizable lipid compounds BMO20, BMO23, BMO31 and BMO37 are comparable to those of CpG-LNP and Poly(I:C)-LNP containing BMO28 in Example 1.

[0120] The co-transformation of human Toll-like receptor 3 (TLR3) gene and secreted embryonic alkaline phosphatase (SEAP) reporter gene in cell lines is currently the most specific method for analyzing the in vitro biological activity of Poly(I:C). This cell line can be specifically stimulated by Poly(I:C), activating SEAP reporter gene expression through an effector pathway, catalyzing substrate color development, and analyzing activity by detecting absorbance. The test samples were diluted to a Poly(I:C) concentration of 100 ng / mL. Water for bacterial endotoxin testing was used as a negative control. The lipid nanoparticle samples containing Poly(I:C) listed in Table 3 were added to 96-well plates, 20 μL per well, with at least four replicates. HEK-Blue hTLR3 cells were diluted to 2.8 × 10⁻⁶ in Detection medium. 5 Cells / mL, 180 μL / well (5 × 10⁻⁶ cells / mL, 180 μL / well) 4 (1 cell / well) was added to a 96-well cell culture plate containing the sample to be tested, and incubated at 37°C with 5% CO2 for 20 hours. The absorbance of the sample well at 630 nm was detected using an ELISA reader.

[0121] Table 4 shows the results of the five Poly(I:C)-LNPs listed in Table 3, as well as Poly(I:C) alone, activating the in vitro TLR3 cell activity. The Poly(I:C)-LNP prepared with BM028 showed the highest absorbance at 630 nm, exhibiting the highest optical density (OD), confirming that the Poly(I:C)-LNP prepared with compound BM028 has the highest in vitro TLR3 cell activation activity. The OD 630 nm absorbance of Poly(I:C)-LNPs prepared using compounds BM020, BM023, BM031, and BM037 was lower than that of Poly(I:C)-LNP containing BM028, but higher than that of Poly(I:C) alone.

[0122] Table 4. Results of TLR3 cell activation in vitro based on the five Poly(I:C)-LNPs in Table 3, and Poly(I:C) alone.

Claims

1. A lipid nanoparticle comprising nucleic acid-based immunostimulatory molecules, comprising the following components: Nucleic acid-based immunostimulatory molecules; The structure of the ionizable lipid compound is shown below: Phospholipids; Structural lipids; and Polyethylene glycol-modified lipids.

2. The lipid nanoparticles according to claim 1, wherein the nucleic acid immunostimulatory molecule is selected from CpG oligodeoxynucleotides and double-stranded RNA.

3. The lipid nanoparticles according to claim 2, wherein the sequence of the CpG oligodeoxynucleotide is 5'-TGACTGTGAACGTTCGAGATGA-3'.

4. The lipid nanoparticles according to claim 2, wherein the double-stranded RNA is polyinosinic acid.

5. The lipid nanoparticles according to claim 1, wherein the phospholipid is selected from 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-distearyl-sn-glycerol-3-phosphate-rac-glycerol (DSPG), 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA), 1-stearoyl-2-myristoyl-sn-glycerol-3-phosphate choline (SMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and mixtures thereof; preferably, the phospholipid is 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC).

6. The lipid nanoparticles according to claim 1, wherein the structural lipid is selected from cholesterol, sitosterol, campesterol, stigmasterol, brassosterol, and mixtures thereof; preferably, the structural lipid is cholesterol.

7. The lipid nanoparticles according to claim 1, wherein the molar ratio of the ionizable lipid compound to the phospholipid, the structural lipid and the polyethylene glycol-modified lipid is (40-65):(5-17.5):(35-55):(1.0-2.0), preferably 48.5:10:40:1.

5.

8. The lipid nanoparticles according to claim 1, wherein the nitrogen / phosphorus ratio of the ionizable lipid compound to the nucleic acid immunostimulatory molecule is 2:1 to 12:1, preferably 5:

1.

9. The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles have a particle size of 70 nm to 100 nm and the polydispersity index of the lipid nanoparticles is less than 0.

15.

10. A method for preparing lipid nanoparticles containing nucleic acid-based immunostimulatory molecules according to any one of claims 1-9, comprising the following steps: (a) Preparation of an aqueous solution containing nucleic acid-based immunostimulatory molecules: (b) Prepare an organic phase solution containing ionizable lipid compounds, phospholipids, structured lipids, and polyethylene glycol-modified lipids; and (c) Using a microfluidic device to mix the aqueous solution and the organic solution to obtain a solution containing lipid nanoparticles containing nucleic acid-like immunostimulatory molecules.

11. The method according to claim 10, wherein in step (c), the microfluidic device is configured to perform the mixing by setting the flow rate ratio of the organic phase solution to the aqueous phase solution to 1:3 to 1:

5.

12. The method of claim 10, further comprising, after step (c), an ultrafiltration step of the solution containing the lipid nanoparticles comprising nucleic acid-like immunostimulatory molecules.

13. The method according to claim 10, wherein the encapsulation rate of the nucleic acid immunostimulatory molecule is 95% or higher.

14. Use of lipid nanoparticles containing nucleic acid-based immunostimulatory molecules according to any one of claims 1-9 in the preparation of vaccine adjuvants.

15. The use according to claim 14, wherein the vaccine adjuvant enhances humoral and cellular immune responses to the recombinant protein vaccine antigen.

16. The use according to claim 15, wherein the recombinant protein vaccine antigen is selected from recombinant herpes zoster virus protein and hepatitis B surface antigen.

17. A vaccine composition comprising: Lipid nanoparticles comprising nucleic acid-based immunostimulatory molecules according to any one of claims 1-9; and Recombinant protein vaccine antigen.

18. The vaccine composition of claim 17, wherein the recombinant protein vaccine antigen is selected from recombinant herpes zoster virus protein and hepatitis B surface antigen.

Images