Nanoparticle adjuvant co-loaded with anion and hydrophobic immune adjuvant, preparation method and application thereof

Abstract

The present invention discloses a nanoparticle adjuvant co-loaded with anionic and hydrophobic immune adjuvants and its application. The nanoparticle adjuvant comprises ionizable lipids, anionic immune adjuvants and / or hydrophobic immune adjuvants and auxiliary lipids, and the auxiliary lipids include neutral auxiliary lipids, cholesterol and pegylated lipids. The present invention uses ionizable lipid materials to encapsulate anionic adjuvants and hydrophobic adjuvants with different functions to prepare a new nanoparticle adjuvant system. The nanoparticle adjuvant system breaks through the limitations of traditional reliance on cationic lipids to load anionic adjuvants and hydrophobic adjuvants; at the same time, the nanoparticle adjuvant immune adjuvant prepared by the present invention has a high encapsulation rate. After being applied to animals, it can produce strong humoral immunity and significantly enhance cellular immunity. The immune effect is better than free antigen / adjuvant mixed injection and existing aluminum adjuvant-containing vaccines and other VZV vaccines currently available on the market.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically, relates to a lipid nanoadjuvant formed by co-loading anionic immune adjuvants and / or hydrophobic immune adjuvants with ionizable lipids, a preparation method thereof, and the application of the lipid nanoadjuvant in a vaccine using varicella-zoster virus (VZV)-related protein gE as an antigen. Background Art

[0002] Immune adjuvants, also known as immune agonists, are nonspecific immune-enhancing substances that, when administered concurrently with or prior to an antigen, can enhance the immunogenicity of the antigen and alter the type of immune response. Currently approved adjuvants for use in human vaccines include aluminum salts and MF59, which can enhance humoral and Th2 immune responses but fail to induce sufficient cytotoxic T lymphocyte responses. Virus-like particles (VLPs) are complex to manufacture and their composition is unclear. The development of therapeutic tumor vaccines urgently requires novel adjuvants with well-defined structures, safety, efficacy, and ease of production.

[0003] In recent years, some new cellular Toll-like receptor agonists can produce strong cellular immune responses, such as TLR4 agonist MPLA, TLR9 agonist CpG-ODN, TLR3 agonist Poly (I: C), TLR7 / 8 agonist IMQ and other molecular adjuvants have been widely used in preventive and therapeutic vaccines. However, due to the small molecular weight of such adjuvants, they are prone to cause systemic inflammatory toxicity and are easily metabolized quickly. By encapsulating molecular adjuvants into nanoadjuvants (usually less than 100 nm) through biomaterials, they can be carried to draining lymph nodes through targeted lymph nodes or through antigen-presenting cells. Nanoadjuvants can avoid adjuvant-induced systemic toxicity and can remain in lymph nodes rich in antigen-presenting cells (APCs) for a long time by targeting lymph nodes and continuously activate immune responses. Moreover, because the biomaterials that encapsulate the adjuvant can greatly increase the density of the adjuvant in the lymph nodes, it can efficiently activate the TLR pathway. In addition, due to the different immune characteristics of different molecular adjuvants, two or more molecular adjuvants are usually used together. Currently, the only clinically approved vaccine is the Shingrix recombinant vaccine developed by GSK, which uses the AS01 adjuvant, which can stimulate strong humoral and cellular immune responses. AS01-loaded MPLA and QS21 are embedded in liposome vesicles through hydrophobic interactions. This method has limitations for loading some anionic adjuvants with good adjuvant effects. Moreover, the preparation of AS01 nanoparticle adjuvants uses the traditional membrane hydration method, which has a complex preparation process and poor controllability. Even though some existing cationic lipids, such as DOTAP and DOTMA, can simultaneously load anionic molecular adjuvants and hydrophobic small molecule adjuvants, their strong positive charge binds the anionic molecular adjuvant too strongly, resulting in low adjuvant release efficiency and reduced adjuvant effect. In addition, due to their strong positive charge, permanent cationic lipid particles will bind to serum proteins in body fluids, resulting in rapid clearance by the body fluid system, shortening the drug half-life. Moreover, liposomes composed of cationic lipids have problems such as thermodynamic instability, which greatly limits their application. At the same time, the high cytotoxicity of such cationic adjuvants makes such cationic lipids unsuitable for the simultaneous delivery of anionic adjuvants and hydrophobic adjuvants. Therefore, there is a need for a nanoparticle adjuvant system that can load anionic adjuvants and hydrophobic adjuvants with different functions and has better loading efficiency and immunological efficiency. Summary of the Invention

[0004] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures described herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0005] As used herein, the term "ionizable lipid" refers to a lipid molecule having both a tertiary amino group or a hydrophilic group and a hydrophobic alkyl chain, and having a pKa between 5.0 and 7.4.

[0006] As used herein, the term "particulate adjuvant" refers to a state of matter characterized by the presence of discrete particles, pellets, beads or granules, regardless of their size, shape or morphology, loaded with the corresponding molecular adjuvant and having a specific geometric shape within a size range.

[0007] As used herein, the term "nanoparticulate adjuvant" refers to particles that are less than 200 nanometers in one dimension (ie, the diameter in the longest dimension of the particle).

[0008] As used in this article, the term "particle size" or "equivalent particle size" means that when a certain physical property or physical behavior of the measured particle is closest to that of a homogeneous sphere (or combination) of a certain diameter, the diameter (or combination) of the sphere is taken as the equivalent particle size (or particle size distribution) of the measured particle.

[0009] As used herein, the term "average particle size" refers to the diameter of an actual particle population composed of particles of varying sizes and shapes, compared to a hypothetical particle population composed of uniform spherical particles. If both have the same overall length, the diameter of the spherical particles is considered the average particle size of the actual particle population. Methods for measuring average particle size are known to those skilled in the art, such as light scattering methods; instruments for measuring average particle size include, but are not limited to, a Malvern particle sizer.

[0010] As used herein, the term "room temperature" refers to 25±5°C.

[0011] As used herein, the term "immune adjuvant" refers to a substance that is administered to the body together with an antigen or in advance and can enhance immunogenicity or change the type of immune response. The immune adjuvant itself can be immunogenic (such as BCG) or non-immunogenic (such as aluminum hydroxide adjuvant). The "anionic immune adjuvant" refers to an immune adjuvant that is negatively charged after ionization in water; the "hydrophobic immune adjuvant" refers to an immune adjuvant that is insoluble in water and can only be dissolved in neutral and non-polar solutions (such as organic solvents).

[0012] As used herein, the term "antigen" or "immunogen" refers to a substance that is capable of inducing a specific immune response in a host. Antigens may include whole organisms (e.g., inactivated, attenuated, or live organisms); subunits or parts of an organism; recombinant vectors containing immunogenic inserts; DNA portions or fragments that are capable of inducing an immune response upon presentation to a host; proteins, glycoproteins, lipoproteins, polypeptides, peptides, antigenic epitopes, haptens, toxins, antitoxins, or any combination thereof.

[0013] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a nanoparticle adjuvant.

[0014] Another object of the present invention is to provide a method for preparing the nanoparticle adjuvant.

[0015] Another object of the present invention is to provide applications of the nanoparticle adjuvant.

[0016] A nanoparticle adjuvant comprises an ionizable lipid, an anionic immune adjuvant and / or a hydrophobic immune adjuvant and a helper lipid, wherein the helper lipid comprises a neutral helper lipid, cholesterol and a polyethylene glycol (PEG)-containing lipid.

[0017] Ionizable lipids are substances with a pKa of 5 to 7, whose structures contain ionizable tertiary or tertiary amino groups, hydrophobic alkyl chains, and functional structural groups. They exhibit different charge properties in different pH environments and have been successfully used in the prior art for the delivery of siRNA and mRNA. The present invention, however, demonstrates that these ionizable lipids can load hydrophobic molecular adjuvants and are negatively charged when the pH is greater than the pKa and positively charged when the pH is less than the pKa. Unlike traditional cationic lipids such as DOTAP and DOTMA, ionizable lipids can form lipid nanoparticle adjuvants by encapsulating negatively charged molecular adjuvants (anionic immunoadjuvants) at low pH. Upon increasing the buffer pH, the surface charge of the lipid nanoparticles is reduced to zero or weakly negative. In body fluids, the lipid nanoparticles are slightly negatively charged, resulting in excellent biocompatibility and the ability to effectively release immunoadjuvants, ensuring efficient loading of weakly charged adjuvants while avoiding burst release or non-release of molecular adjuvants. Finally, simultaneous injection with antigens induces a strong immune response.

[0018] Specifically, ionizable lipids are neutral at physiological pH but positively charged in the acidic environment of endosomes. Their positive charge at pH 4 effectively encapsulates negatively charged adjuvants. After injection, their near-neutral state at physiological pH 7.4 prevents nonspecific interactions with serum proteins and improves circulation time. Furthermore, after endocytosis, particles based on ionizable lipids become protonated in the acidic environment of endosomes and interact with negatively charged endogenous lipids, leading to endosomal membrane destabilization and escape into the cytoplasm. Ionizable lipids significantly improve both efficacy and toxicity profiles.

[0019] The neutral helper lipid supports the formation of the lipid bilayer and stabilizes its structural arrangement; the membrane-fusogenic cholesterol adjusts the integrity and hardness of the lipid membrane, enhancing the stability of the nanoparticles; and the PEGylated lipid, which improves hydrophilicity, is located on the nanoparticle surface, preventing rapid clearance by the immune system, thereby extending circulation time, and preventing aggregation, thereby increasing stability. The lipid components undergo intermolecular interactions and spontaneously organize into a core-shell nanostructured entity, with the PEG lipid forming a shell structure on the outermost layer that encapsulates the core.

[0020] The "ionizable lipid" is not limited to being composed solely of carbon, hydrogen, and nitrogen. Its basic structure has an ionizable tertiary amino or hydroxyl amino hydrophilic head and a hydrophobic alkyl chain tail, with a final pKa of 5 to 7.4. It is also not limited to containing chemical groups such as ester groups, aldehyde groups, carbonyl groups, disulfide bonds, hydrazone bonds, and unsaturated bonds. The hydrophobic alkyl chain is not limited to one or one type.

[0021] Preferably, the ionizable lipid is selected from lipids with an acid dissociation constant (pKa) between 5.0 and 7.4, and its structure has a tertiary amino group or a saturated amino group, a hydrophobic alkyl chain and a functional structural group. The ionizable lipid is selected from FDA-approved highly biocompatible 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA), 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate (SM102), ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC0315); and the ionizable lipid (10Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1- Oxylidenehept-2-yl] octadecan-9-enoyl{R2-1,N-(3-(dimethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)oleamide},(10Z,12Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl] octadecan-9,12-dienamide{R2-2,(9Z,12Z)-N-(3-(dimethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl) 1-oxoheptan-2-yl)octadeca-9,12-dienamide},(10Z,12Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl]octadeca-9,12-dienamide any one or more of {R3-2, (9Z, 12Z)-N-(3-(diethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)octadeca-9,12-dienamide}.

[0022] Further preferably, the ionizable lipid is R3-2.

[0023] Preferably, the anionic immune adjuvant is selected from one or more of natural immune agonists, plant-derived adjuvants or cytokine adjuvants.

[0024] Preferably, the innate immune agonist is a pattern recognition receptor (PRRs) agonist.

[0025] Further preferably, the pattern recognition receptor (PRRs) agonist is selected from one or more of Toll-like receptors (TLRs) agonists, nucleotide-binding oligomerization domain NOD-like receptors (NLRs) agonists, retinoic acid-inducible gene I (RIG-1)-like receptors (RIG-1like receptors, RLRs RLRs) agonists, C-type lectin receptors (CLRs) agonists or intracellular nucleic acid sensor STING agonists.

[0026] Further preferably, the Toll-like receptors (TLRs) agonist is selected from one or more of CPGODNs, ssRNA, 23S rRNA, Pam2csk4, Pam3csk4, FLA, ssPoly (U) or poly (I:C).

[0027] Further preferably, the CpG ODNs include but are not limited to CpG-ODN M362, CpG-ODN 2216, CpG-ODN 1018, CpG-ODN 2006, CpG-ODN 1826, CpG-ODN 2395CpG-ODN 1668, CpG-ODN 2007, CpG-ODN BW006, CpG-ODN SL01, CpG-ODN 1585, CpG-ODN 2336, CpG-ODN SL03, etc.

[0028] Further preferably, the nucleotide binding oligomerization domain NOD-like receptors (NLRs) agonist is selected from one or more of C12-iE-DAP, C14-Tri-LAN-Gly, iE-DAP, Tri-DAP, M-TriDAP, Gram-PGNs, MDP or Murabutide.

[0029] Further preferably, the retinoic acid-inducible gene I (RIG-1)-like receptor (RLRs) agonist is selected from one or more of 3p-hpRNA, 5'ppp-dsRNA, Poly (dA:dT), and poly (I:C) LyoVec.

[0030] Further preferably, the C-type lectin receptor (CLRs) agonist is selected from Beta-glucan peptide or Dectin-I.

[0031] Further preferably, the intracellular nucleic acid sensor STING agonist is selected from one or more of 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP or cAIMP.

[0032] Preferably, the plant-derived adjuvant is selected from one or more of QS-21, quinine or plant lectin.

[0033] Preferably, the cytokine adjuvant is selected from one or more of IFN-α, IL-2, TNF, IFN-γ or GM-CSF.

[0034] Preferably, the hydrophobic immune adjuvant is selected from imiquimod (IMQ), monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), muramyl dipeptide (moradinate), Loxoribine, Gardiquinod, Resiquimod; bacterial adjuvants tetanus toxoid (TT), Escherichia coli heat-labile toxin (LT) and Salmonella flagellin; one or more of squalene, heat shock protein 70 or heat shock protein 90.

[0035] Preferably, the neutral helper lipid is selected from dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), dieucoylphosphatidylcholine (DEPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), One or more of distearoylphosphatidylcholine (DSPC), palmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), egg yolk phosphatidylglycerol (EPG), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG-Na), 1,2-palmitoylphosphatidylglycerol (DPPG-NA), distearoylphosphatidylglycerol (DSPG-Na), dimyristoylphosphatidylglycerol (DMPG-Na), distearoylphosphatidylglycerol (DSPA), and dipalmitoylphosphatidylglycerol (DPPA).

[0036] Preferably, the polyethylene glycol (PEG)-ylated lipid is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 (DPPE-MPEG2000), and dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000 (DPPE-MPEG5000).

[0037] Further preferably, the anionic immune adjuvant is CpG oligodeoxynucleotide and / or plant-derived adjuvant QS21; the hydrophobic immune adjuvant is monophosphoryl lipid A (MPLA) and / or imiquimod IMQ (IMQ).

[0038] Preferably, the molar mass ratio of the ionizable lipid, the anionic immune adjuvant, the hydrophobic immune adjuvant and the helper lipid is 35-65:10-30:10-30:35-65.

[0039] Further preferably, the molar mass ratio of the ionizable lipid, the anionic immune adjuvant, the hydrophobic immune adjuvant and the helper lipid is 3:1:1:3 or 2:1:1:2.

[0040] Preferably, the mass ratio of the ionizable lipid to each adjuvant is 1-2:1-2.

[0041] Further preferably, the mass ratio of the ionizable lipid to each adjuvant is 1:1 or 1.5:1 or 2:1 or 1.5:2.

[0042] Preferably, the molar mass ratio of the ionizable lipid, the neutral helper lipid, the cholesterol and the PEGylated lipid is 44-55:9.4-10:38.5-45:1.5-1.6.

[0043] Further preferably, the molar mass ratio of the ionizable lipid, neutral helper lipid, cholesterol and PEGylated lipid is 55:10:38.5:1.5 or 45.5:10:43:1.5.

[0044] Specifically, the nanoparticle adjuvant is a liposome core-shell structure, with an anionic immune adjuvant in the core and an ionizable lipid, auxiliary lipid and hydrophobic immune adjuvant wrapped on the core in the shell; or the core contains a partial anionic immune adjuvant, the shell is an ionizable lipid, auxiliary lipid and hydrophobic immune adjuvant wrapped on the core, and the particle surface is loaded with another portion of anionic immune adjuvant.

[0045] Preferably, the nanoparticles are approximately spherical.

[0046] Preferably, the particle size of the nanoparticles is 30-200 nm, for example, 30-50 nm, 50-80 nm, 80-100 nm, 100-150 nm or 150-200 nm.

[0047] Preferably, the nanoparticles have a Zeta potential of -10 to +20 mV, such as -10 to -5 mV, -5 to -2 mV, -2 to +2 mV, +2 to +5 mV, +5 to +10 mV, +10 to +15 mV, +15 to +20 mV.

[0048] Preferably, the encapsulation efficiency of the immune adjuvant in the nanoparticles is 70% to 100%, for example, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95% or 95% to 100%.

[0049] The present invention also provides a method for preparing any of the above-mentioned nanoparticle adjuvants, comprising the following steps:

[0050] S1. Providing a solution comprising an ionizable lipid, a helper lipid and a hydrophobic immune adjuvant, and a solution comprising an anionic immune adjuvant;

[0051] S2. A solution containing an ionizable lipid, a helper lipid, and a hydrophobic immune adjuvant is passed through the first channel. Three tubes of solution containing anionic immune adjuvant are passed through the second channel, the third channel, and the fourth channel, respectively. The solutions in the four channels reach the mixing zone and are mixed to obtain a nanoparticle adjuvant solution.

[0052] S3. Dialyze the solvent step by step to obtain an aqueous solution of nanoparticle adjuvant.

[0053] Preferably, the pH of the solution containing the anionic immune adjuvant is 3-5.

[0054] More preferably, the pH of the solution containing the anionic immune adjuvant is 4.

[0055] Preferably, the method is carried out in a device comprising a first channel, a second channel, a third channel, a fourth channel and a mixing zone. In a preferred embodiment, the device is a multi-inlet vortex mixer, such as a four-inlet vortex mixer.

[0056] The multi-inlet vortex mixer of the present invention includes a first component located at the top, a second component located in the middle, and a third component located at the bottom, wherein the first, second, and third components are cylindrical bodies having the same diameter. The first component is provided with multiple channels, the second component is provided with a vortex mixing area and multiple flow guide areas, and the third component is provided with channels. The channels of the first component are in fluid communication with the flow guide areas of the second component. The flow guide areas of the second component are all in fluid communication with the vortex mixing areas. The vortex mixing areas of the second component are in fluid communication with the channels of the third component. A threaded connection device can be used to seal the first, second, and third components.

[0057] In certain embodiments, the first component is provided with a plurality of channels, wherein the upper and lower ends of the channels are respectively located on the upper surface and the lower surface of the first component. In certain embodiments, the cross-sections of the plurality of channels are circular. In certain embodiments, the plurality of channels are respectively connected to external pipes via connecting components.

[0058] In certain embodiments, the upper surface of the second component is recessed to form a plurality of flow-guiding regions and a vortex mixing region. In certain embodiments, the plurality of flow-guiding regions are in fluid communication with the vortex mixing region via grooves provided on the upper surface of the second component. In certain embodiments, the vortex mixing region of the second component is in fluid communication with a channel of the third component via a channel parallel to the axial direction of the second component.

[0059] In certain embodiments, the cross-section of the vortex mixing region is circular and shares a common center with the cross-section of the second component. In certain embodiments, the cross-sections of the plurality of flow guiding regions are circular. In certain embodiments, the number of flow guiding regions in the second component is the same as the number of channels in the first component. In certain embodiments, the plurality of flow guiding regions in the second component are each located directly below the plurality of channels in the first component.

[0060] In certain embodiments, the upper and lower ends of the channel of the third component are respectively located on the upper surface and the lower surface of the third component. In certain embodiments, the cross-section of the channel of the third component is circular. In certain embodiments, the channel of the third component is connected to the external pipeline via a connecting component.

[0061] In certain embodiments, the multi-inlet vortex mixer is made of a rigid material (eg, stainless steel).

[0062] The above device has the characteristics of high throughput and strong controllability. The prepared nanoparticles are evenly distributed and have small particle size, with little difference between batches.

[0063] The above-mentioned technology (FNC) and device are described in the inventor's previous patent application number PCT / US2017 / 014080. It can make the dispersion of the prepared nanoparticles more uniform.

[0064] Preferably, the flow rate of each channel is the same, which is 1 to 40 mL / min, for example, 1 mL / min, 5 mL / min, 8 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 30 mL / min or 40 mL / min.

[0065] More preferably, the flow rate of each channel is 10 mL / min.

[0066] Preferably, the method further comprises step S4: freeze-drying and concentrating the aqueous solution containing the nanoparticles, for example, freeze-drying and concentrating by adding a freeze-protectant.

[0067] Primary infection with the varicella-zoster virus (VZV) manifests as chickenpox (varicella), which remains latent within the host's sensory ganglia. During recurrent VZV infection, the virus travels down sensory nerve axons to the skin cells innervated by these nerves and proliferates. This virus then spreads along the sensory nerve pathways, causing a series of blisters on the skin that resemble a band, hence the name herpes zoster. Herpes zoster (HZ) is most common in adults and the elderly. Currently, there are no specific treatments for varicella and herpes zoster. Vaccination is currently the most effective method for preventing and controlling VZV. Currently, available varicella and herpes zoster vaccines include live attenuated vaccines, subunit vaccines, and DNA vaccines. The safety of traditional inactivated or live attenuated vaccines, as well as the systemic immune storm they induce, are unavoidable in many vaccine systems. Subunit vaccines have attracted considerable attention due to their high safety profile. The gE glycoprotein is one of the most important structural proteins of VZV. It has rich B cell and T cell epitopes, which can stimulate the body to produce immune responses against VZV at the serum immunity level and cellular immunity level. It has been successfully used in subunit herpes zoster vaccines and has shown good results in clinical experiments.

[0068] The nanoparticles of the present invention are capable of eliciting an immune response. The nanoparticle adjuvants obtained above can be combined with VZV antigens to prepare immunogenic compositions for preventing and / or treating diseases associated with varicella-zoster virus infection. In one aspect, the present invention claims the use of the nanoparticle adjuvants in preparing immunogenic compositions for diseases associated with VZV infection.

[0069] Preferably, the disease associated with VZV infection is one or more of varicella and herpes zoster.

[0070] The present invention also provides an immunogenic composition, comprising any one of the above-described nanoparticle adjuvants of the present invention.

[0071] The immunogenic compositions of the invention can be formulated for any suitable route of administration, including, for example, topical, oral, intranasal, intramucosal, intravenous, intradermal, intraperitoneal, subcutaneous, and intramuscular administration.

[0072] Preferably, the immunogenic composition of the invention may be used in a vaccine composition, optionally in combination with an adjuvant and / or (other) suitable carriers.

[0073] Preferably, the immunogenic composition further comprises pharmaceutically acceptable excipients, such as excipients, preservatives, antimicrobial agents and / or additional immune adjuvants.

[0074] Preferably, the immunogenic composition is a vaccine.

[0075] Preferably, the immunogenic composition further comprises a VZV antigen, which is an inactivated / attenuated VZV virus strain, a VZV glycoprotein such as VZV gE glycoprotein, VZV gB glycoprotein, VZV gH glycoprotein, VZV gL glycoprotein, etc., as well as a VZV lipoprotein, polypeptide, peptide, antigenic epitope, hapten, toxin, antitoxin or any combination thereof.

[0076] Preferably, the selected antigens are VZV gE and OKA strains;

[0077] Preferably, the VZV gE glycoprotein is a recombinant protein.

[0078] Among them, the amount of VZV antigen is selected to induce an immune protective response in a typical vaccine without obvious adverse side effects. The amount of antigen can vary with the specific immunogen used. Generally speaking, each dose of vaccine contains 5 to 1000 μg of protein, such as 5 to 200 μg or 20 to 100 μg. For VZV gE recombinant protein, the dosage used in mice is 1 to 25 μg, preferably 2 μg, 5 μg or 20 μg; the dosage used in humans is 10 to 100 μg, preferably 20 μg, 50 μg or 80 μg. For the OKA strain, the dosage used is 100 to 100,000 pfu / 0.5 mL, preferably 10,000 pfu / 0.5 mL, 30,000 pfu / 0.5 mL, 50,000 pfu / 0.5 mL, 70,000 pfu / 0.5 mL, and 100,000 pfu / 0.5 mL.

[0079] Preferably, the immunogenic composition is used to prevent and / or treat diseases associated with VZV infection, such as chickenpox and herpes zoster, in a subject.

[0080] Preferably, the subject is a mammal, such as a bovine, equine, bovine, porcine, canine, feline, rodent, or primate; for example, the subject is a human.

[0081] Preferably, the immunogenic composition further comprises a second immunogenic substance. For example, the immunogenic composition further comprises other VZV proteins besides VZV gE protein. For example, the immunogenic composition further comprises inactivated and attenuated VZV. For example, the immunogenic composition further comprises other pathogenic microorganisms (including live, inactivated, or attenuated) besides VZV. For example, the immunogenic composition further comprises a portion of other pathogenic microorganisms besides VZV.

[0082] Preferably, the VZV antigen and attenuated VZV of the present invention can be used together in a composition to stimulate an immune response to VZV, or used separately—either simultaneously or sequentially—in a prime-boost regimen. The vaccine components can be delivered simultaneously or sequentially in any order. In one embodiment, the VZV antigen or an immunogenic derivative thereof is delivered after delivery of a live attenuated VZN or whole-killed VZV. In another embodiment, the VZV antigen or an immunogenic derivative thereof is delivered after delivery of a live attenuated VZV or whole-killed VZV.

[0083] Preferably, the present invention further relates to a method of preventing and / or reducing the severity of herpes zoster and / or postherpetic neuralgia comprising delivering to an individual at risk for herpes zoster an immunogenic composition comprising live attenuated VZV and a VZV antigen.

[0084] Preferably, in another embodiment, the present invention relates to a method for preventing and / or reducing the severity of herpes zoster and / or postherpetic neuralgia comprising sequentially or simultaneously delivering to an individual at risk of herpes zoster a live attenuated VZV and a VZV antigen.

[0085] In one aspect, the present invention also provides a method for preventing and / or treating a disease associated with VZV infection in a subject, comprising administering to the subject the nanoparticle or immunogenic composition (eg, vaccine) of the present invention.

[0086] Preferably, the disease associated with VZV infection is chickenpox or herpes zoster.

[0087] Preferably, the subject is a mammal, such as a bovine, equine, bovine, porcine, canine, feline, rodent, or primate; for example, the subject is a human.

[0088] In one aspect, the invention provides a method of eliciting or enhancing an immune response to VZV in a subject, comprising administering to the subject a nanoparticle or immunogenic composition (eg, a vaccine) of the invention.

[0089] Preferably, the subject is a mammal, such as bovine, equine, bovine, porcine, canine, feline, rodent, or primate; and the subject is a C57BL / 6 mouse.

[0090] The nanoparticle adjuvant system of the present invention breaks through the limitation of traditional reliance on cationic lipids to load anionic adjuvants and hydrophobic adjuvants, maximizes the adjuvant loading efficiency and immune efficiency, and shows a strong immune effect when used in combination with VZV gE antigen or VZV attenuated / inactivated strains.

[0091] Compared with the prior art, the present invention has the following beneficial effects:

[0092] (1) The present invention prepares a novel nanoparticle adjuvant system by encapsulating anionic adjuvants and hydrophobic adjuvants with different functions using ionizable lipid materials. This nanoparticle adjuvant system breaks through the limitations of traditional methods that rely on cationic lipids to load anionic adjuvants and hydrophobic adjuvants, greatly improving the adjuvant release efficiency and adjuvant effect.

[0093] (2) The nanoparticles prepared by the present invention have the characteristics of high throughput and strong controllability. The prepared nanoparticles have regular morphology, round shape, smooth surface, good dispersibility, and no obvious adhesion, breakage, collapse, etc.; the distribution is uniform and the particle size is small (30-200nm), and the difference between batches is small.

[0094] (3) The immune adjuvant loaded in the nanoparticles of the present invention has a high encapsulation rate (70% to 100%); after the nanoparticle adjuvant is administered to animals, it can produce stronger humoral immunity and significantly enhance cellular immunity, and the immune effect is better than the free form of antigen / adjuvant mixed injection and the existing aluminum adjuvant-containing vaccine and other VZV vaccines available on the market; compared with the existing GSK-developed AS01 and traditional cationic lipid-loaded anionic adjuvants and hydrophobic adjuvants, it can produce higher specific antibodies and can stimulate stronger humoral vaccines and cellular immunity.

[0095] (4) The nanoparticles of the present invention have the function of targeting lymph nodes, which improves the enrichment of the vaccine in the lymph nodes and the uptake of antigen-presenting cells;

[0096] (5) The nanoparticle adjuvant of the present invention can be continuously prepared by a simple method, has stable quality, and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 Schematic diagram of the immunization scheme of the nanoadjuvant of the present invention.

[0098] Figure 2 The percentage of IFN-γ+ / TNF-α+ in peripheral blood CD4+ T cells and CD8+ T cells after immunization with the D01-D11 nanoadjuvant (day 34) indicates that the D01-D11 nanoadjuvant of the present invention increases the expression of IFN-γ and TNF-α by CD4+ and CD8+ lymphocytes, thereby enhancing T cell-mediated cellular immunity.

[0099] Figure 3The percentage of IFN-γ+ / TNF-α+ in peripheral blood CD4+ T cells and CD8+ T cells after immunization with S01-S11 nanoadjuvant (day 34). The experimental results show that the S01-S11 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0100] Figure 4 The percentage of IFN-γ+ / TNF-α+ in peripheral blood CD4+ T cells and CD8+ T cells after immunization with R3201-R3211 nanoadjuvants (day 34). The experimental results show that the R3201-R3211 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0101] Figure 5 The percentage of IFN-γ+ / TNF-α+ in peripheral blood CD4+ T cells and CD8+ T cells after immunization with the A01-A11 nanoadjuvant (day 34). The experimental results show that the A01-A11 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0102] Figure 6 The percentage of IFN-γ+ / TNF-α+ in CD4+ T cells and CD8+ T cells in peripheral blood after immunization with R2101-R2111 nanoadjuvant (day 34). The experimental results show that the R2101-R2111 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0103] Figure 7 The percentage of IFN-γ+ / TNF-α+ in CD4+ T cells and CD8+ T cells in peripheral blood after immunization with R2201-R2211 nanoadjuvants (day 34). The experimental results show that the R2201-R2211 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0104] Figure 8The percentage of IFN-γ+ / TNF-α+ in CD4+ T cells and CD8+ T cells in peripheral blood after immunization with R3101-R3111 nanoadjuvant (day 34). The experimental results show that the R3101-R3111 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0105] Figure 9 The percentage of IFN-γ+ and TNF-α+ in CD4+ T cells and CD8+ T cells in peripheral blood after immunization with F00-F11 (day 34). The experimental results show that free adjuvant can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocyte T cells to a certain extent after immunization of mice, but it is different from the Figure 2-Figure 8 Compared with the nanoadjuvants shown in Figure 3, free adjuvants were less effective than nanoadjuvants in enhancing T cell-mediated cellular immunity.

[0106] Figure 10 ELISPOT assay was used to detect the number of splenocytes secreting specific IFN-γ after immunization with the D01-D11 nanoadjuvant using VZV gE (day 42). The experimental results showed that the D01-D11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0107] Figure 11 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with S01-S11 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the S01-S11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0108] Figure 12 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with R3201-R3211 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the R3201-R3211 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0109] Figure 13 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with A01-A11 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the A01-A11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0110] Figure 14 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with R2101-R2111 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the R2101-R2111 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0111] Figure 15 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with R2201-R2211 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the R2201-R221 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0112] Figure 16 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with R3101-R3111 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the R3101-R3111 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0113] Figure 17 ELISPOT was used to detect the number of cells secreting specific IFN-γ in splenocytes of F00-F11 immunized with VZV gE (day 42). The experimental results showed that free adjuvant could increase the expression of IFN-γ in lymphocytes after immunization of mice, but it was not as effective as that in Figures 10-16 Compared with the nanoadjuvants shown in Figure 3, free adjuvants were less effective than nanoadjuvants in enhancing T cell-mediated cellular immunity.

[0114] Figure 18 Figure 5 is the fluorescence signal intensity in the mouse lymph nodes of each group of nanoadjuvant particles loaded with MPLA and CpG and its corresponding free adjuvant group F02. DETAILED DESCRIPTION

[0115] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0116] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0117] The following Examples 45-77 use four ionizable lipids independently designed by the present invention, and their chemical structures and preparation methods are shown below:

[0118] 1. A method for preparing an ionizable lipid R21: (10Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxohept-2-yl]octadec-9-enoyl {R2-1, N-(3-(dimethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)oleamide};

[0119] At room temperature, 1.0 mmol of 2-ethylhexanal and 1.0 mmol of N,N-dimethyl-1,3-propylenediamine were respectively added to 0.5 mL of methanol solution, and 1.0 mmol of oleic acid was added after the reaction at room temperature for 60 minutes. After the reaction at room temperature for 60 minutes, 0.5 mmol of octadecyl isocyanide was added. The reaction was carried out at 40°C for 24 hours. After the reaction was completed, the product was separated and purified by chromatography column, wherein the mobile phase was a mixture of methanol and dichloromethane.

[0120]

[0121]

[0122] 2. Preparation of ionizable lipid R22: (10Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxohept-2-yl]octadec-9-enoyl {R2-1, N-(3-(dimethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)oleamide};

[0123] At room temperature, 1.0 mmol of 2-ethylhexanal and 1.0 mmol of N,N-dimethyl-1,3-propylenediamine were respectively added to 0.5 mL of methanol solution, and 1.0 mmol of linoleic acid was added after the reaction at room temperature for 60 minutes. After the reaction at room temperature for 60 minutes, 0.5 mmol of octadecyl isocyanide was added. The reaction was carried out at 40°C for 24 hours. After the reaction was completed, the product was separated and purified by chromatography column, wherein the mobile phase was a mixture of methanol and dichloromethane.

[0124]

[0125] 3. Preparation method of ionizable lipid R31: (10Z,12Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl]octadeca-9,12-dienamide {R3-1,N-(3-(diethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)oleamide};

[0126] At room temperature, 1.0 mmol of 2-ethylhexanal and 1.0 mmol of N,N-diethyl-1,3-propylenediamine were respectively added to 0.5 mL of methanol solution, and 1.0 mmol of oleic acid was added after the reaction at room temperature for 60 minutes. After the reaction at room temperature for 60 minutes, 0.5 mmol of octadecyl isocyanide was added, and the reaction was carried out at 40°C for 24 hours. After the reaction was completed, the product was separated and purified by chromatography column, wherein the mobile phase was a mixture of methanol and dichloromethane.

[0127]

[0128] 4. Preparation of ionizable lipid R32: (10Z,12Z)-N-[3-(diethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl]octadeca-9,12-dienamide {R3-2, (9Z,12Z)-N-(3-(diethylamino)propyl)-N-(3-ethyl-1-(octadecylamino)-1-oxoheptan-2-yl)octadeca-9,12-dienamide};

[0129] At room temperature, 1.0 mmol of 2-ethylhexanal and 1.0 mmol of N,N-diethyl-1,3-propylenediamine were respectively added to 0.5 mL of methanol solution, and 1.0 mmol of linoleic acid was added after the reaction at room temperature for 60 minutes. After the reaction at room temperature for 60 minutes, 0.5 mmol of octadecyl isocyanide was added, and the reaction was carried out at 40°C for 24 hours. After the reaction was completed, the product was separated and purified by chromatography column, wherein the mobile phase was a mixture of methanol and dichloromethane.

[0130]

[0131] In addition, in the following examples of the present invention, unless otherwise specified, for the combination of multiple adjuvants, the final mass of each adjuvant is the same, and the mass ratio between any two adjuvants is 1:1.

[0132] The CpG used in the following examples of the present invention is CpG ODN, which is CpG-ODN 1826.

[0133] Example 1 Preparation of adjuvant D01

[0134] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, and DMG-PEG2000 were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, and a 5 mg / mL DMG-PEG2000 ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0135] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0136]

[0137] (3) A mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, and DMG-PEG2000 was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0138] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0139] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0140] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0141] (5) After standing for 2-3 minutes, measure the particle size again.

[0142] Example 2 Preparation of adjuvant D02

[0143] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. The immune adjuvant CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0144] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0145]

[0146] (3) A mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic adjuvant MPLA was placed into syringe 1. The CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0147] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0148] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0149] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0150] (5) After standing for 2-3 minutes, measure the particle size again.

[0151] Example 3 Preparation of adjuvant D03

[0152] (1) The ionizable lipid DLin-MC3-DMA, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic immune adjuvant MPLA, and IMQ were dissolved in ethanol to obtain 50 mg / mL DLin-MC3-DMA ethanol solution, 10 mg / mL DSPC, Chol ethanol solution, 5 mg / mL DMG-PEG2000 ethanol solution, 1 mg / mL MPLA ethanol solution, and 1 mg / mL IMQ ethanol solution.

[0153] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0154]

[0155] (3) A mixed phospholipid solution containing ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, hydrophobic adjuvants MPLA and IMQ was loaded into syringe No. 1, and 50 mM CA buffer solution with a pH of 4 was added to the other three syringes. The four syringes were placed on a high-pressure pump, and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants.

[0156] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0157] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0158] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0159] (5) After standing for 2-3 minutes, measure the particle size again.

[0160] Example 4 Preparation of Adjuvant D04

[0161] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0162] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0163]

[0164] (3) A mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic adjuvant MPLA was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0165] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0166] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0167] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0168] (5) After standing for 2-3 minutes, measure the particle size again.

[0169] Example 5 Preparation of adjuvant D05

[0170] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0171] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0172]

[0173] (3) A mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic adjuvant IMQ was placed in syringe 1. The CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0174] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0175] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0176] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0177] (5) After standing for 2-3 minutes, measure the particle size again.

[0178] Example 6 Preparation of Adjuvant D06

[0179] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0180] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0181]

[0182]

[0183] (3) A mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic adjuvant IMQ was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0184] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0185] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0186] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0187] (5) After standing for 2-3 minutes, measure the particle size again.

[0188] Example 7 Preparation of Adjuvant D07

[0189] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0190] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0191]

[0192] (3) A mixed phospholipid solution containing ionizable lipid DLin-MC3-DMA, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ was loaded into syringe No. 1, and CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump, and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants.

[0193] (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0194] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0195] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0196] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0197] (5) After standing for 2-3 minutes, measure the particle size again.

[0198] Example 8 Preparation of Adjuvant D08

[0199] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0200] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0201]

[0202] (3) A mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic adjuvant MPLA was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0203] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0204] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0205] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0206] (5) After standing for 2-3 minutes, measure the particle size again.

[0207] Example 9 Preparation of Adjuvant D09

[0208] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0209] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0210]

[0211] (3) A mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0212] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0213] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0214] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0215] (5) After standing for 2-3 minutes, measure the particle size again.

[0216] Example 10 Preparation of adjuvant D10

[0217] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0218] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0219]

[0220] (3) A mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0221] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0222] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0223] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0224] (5) After standing for 2-3 minutes, measure the particle size again.

[0225] Example 11 Preparation of adjuvant D11

[0226] (1) The ionizable lipid DLin-MC3-DMA, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL DLin-MC3-DMA ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvants CpG and S21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0227] (2) Prepare the mixture in the ratio of DLin-MC3-DMA:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0228]

[0229] (3) A mixed phospholipid solution containing the ionizable lipid DLin-MC3-DMA, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0230] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0231] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0232] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0233] (5) After standing for 2-3 minutes, measure the particle size again.

[0234] Example 12 Preparation of adjuvant S01

[0235] (1) The ionizable lipid SM102, the auxiliary lipid DSPC, Chol, and DMG-PEG2000 were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, and a 5 mg / mL DMG-PEG2000 ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0236] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0237]

[0238] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, and DMG-PEG2000 was placed in syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0239] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0240] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0241] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0242] (5) After standing for 2-3 minutes, measure the particle size again.

[0243] Example 13 Preparation of Adjuvant S02

[0244] (1) The ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0245] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0246]

[0247]

[0248] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA was placed into syringe 1. CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0249] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0250] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0251] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0252] (5) After standing for 2-3 minutes, measure the particle size again.

[0253] Example 14 Preparation of Adjuvant S03

[0254] (1) The ionizable lipid SM102, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic immune adjuvant MPLA, and IMQ were dissolved in ethanol to obtain 50 mg / mL SM102 ethanol solution, 10 mg / mL DSPC, Chol ethanol solution, 5 mg / mL DMG-PEG2000 ethanol solution, 1 mg / mL MPLA ethanol solution, and 1 mg / mL IMQ ethanol solution.

[0255] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0256]

[0257] (3) A mixed phospholipid solution containing the ionizable lipid SM102, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic adjuvants MPLA and IMQ was loaded into syringe 1. A 50 mM CA buffer solution (pH 4) was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (A 5 mL syringe was used, air bubbles were removed, and the flow rate was set to 10 mL / min.)

[0258] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0259] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0260] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0261] (5) After standing for 2-3 minutes, measure the particle size again.

[0262] Example 15 Preparation of Adjuvant S04

[0263] (1) The ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0264] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0265]

[0266] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0267] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0268] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0269] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0270] (5) After standing for 2-3 minutes, measure the particle size again.

[0271] Example 16 Preparation of Adjuvant S05

[0272] (1) The ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0273] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0274]

[0275] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ was placed in syringe 1. The CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0276] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0277] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0278] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0279] (5) After standing for 2-3 minutes, measure the particle size again.

[0280] Example 17 Preparation of Adjuvant S06

[0281] (1) The ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0282] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0283]

[0284] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ was placed in syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0285] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0286] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0287] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0288] (5) After standing for 2-3 minutes, measure the particle size again.

[0289] Example 18 Preparation of Adjuvant S07

[0290] (1) The ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0291] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0292]

[0293] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ was placed in syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0294] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0295] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0296] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0297] (5) After standing for 2-3 minutes, measure the particle size again.

[0298] Example 19 Preparation of Adjuvant S08

[0299] (1) The ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0300] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0301]

[0302]

[0303] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0304] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0305] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0306] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0307] (5) After standing for 2-3 minutes, measure the particle size again.

[0308] Example 20 Preparation of adjuvant S09

[0309] (1) The ionizable lipid SM102, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0310] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0311]

[0312] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0313] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0314] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0315] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0316] (5) After standing for 2-3 minutes, measure the particle size again.

[0317] Example 21 Preparation of Adjuvant S10

[0318] (1) The ionizable lipid SM102, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0319] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0320]

[0321] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0322] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0323] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0324] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0325] (5) After standing for 2-3 minutes, measure the particle size again.

[0326] Example 22 Preparation of adjuvant SM11

[0327] (1) The ionizable lipid SM102, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL SM102 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0328] (2) Prepare the mixture according to the ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0329]

[0330] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0331] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0332] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0333] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0334] (5) After standing for 2-3 minutes, measure the particle size again.

[0335] Example 23 Preparation of adjuvant R3201

[0336] (1) The ionizable lipid R32, the auxiliary lipid DSPC, Chol, and DMG-PEG2000 were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, and a 5 mg / mL DMG-PEG2000 ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0337] (2) Prepare the mixture in the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0338]

[0339] (3) A mixed phospholipid solution containing the ionizable lipid R32, auxiliary lipids DSPC, Chol, and DMG-PEG2000 was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0340] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0341] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0342] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0343] (5) After standing for 2-3 minutes, measure the particle size again.

[0344] Example 24 Preparation of adjuvant R3202

[0345] (1) The ionizable lipid R32, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0346] (2) Prepare the mixture in the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0347]

[0348] (3) A mixed phospholipid solution containing the ionizable lipid SM102, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA was placed into syringe 1. CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0349] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0350] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0351] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0352] (5) After standing for 2-3 minutes, measure the particle size again.

[0353] Example 25 Preparation of adjuvant R3203

[0354] (1) The ionizable lipid R32, auxiliary lipid DSPC, Chol, DMG-PEG2000, hydrophobic immune adjuvant MPLA, and IMQ were dissolved in ethanol to obtain 50 mg / mL R32 ethanol solution, 10 mg / mL DSPC, Chol ethanol solution, 5 mg / mL DMG-PEG2000 ethanol solution, 1 mg / mL MPLA ethanol solution, and 1 mg / mL IMQ ethanol solution.

[0355] (2) Prepare the mixture in the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0356]

[0357] (3) A mixed phospholipid solution containing the ionizable lipid R32, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was loaded into syringe 1. 50 mM CA buffer solution at pH 4 was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (A 5 mL syringe was used, air bubbles were removed, and the flow rate was set to 10 mL / min.)

[0358] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0359] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0360] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0361] (5) After standing for 2-3 minutes, measure the particle size again.

[0362] Example 26 Preparation of adjuvant R3204

[0363] (1) The ionizable lipid R32, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0364] (2) Prepare the mixture in the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0365]

[0366] (3) A mixed phospholipid solution containing the ionizable lipid R32, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0367] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0368] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0369] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0370] (5) After standing for 2-3 minutes, measure the particle size again.

[0371] Example 27 Preparation of adjuvant R3205

[0372] (1) The ionizable lipid R32, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0373] (2) Prepare the mixture in the ratio of R322:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0374]

[0375] (3) A mixed phospholipid solution containing the ionizable lipid R32, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ was placed in syringe 1. CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0376] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0377] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0378] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0379] (5) After standing for 2-3 minutes, measure the particle size again.

[0380] Example 28 Preparation of adjuvant R3206

[0381] (1) The ionizable lipid R32, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0382] (2) Prepare the mixture in the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0383]

[0384] (3) A mixed phospholipid solution containing the ionizable lipid R32, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ was placed in syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0385] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0386] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0387] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0388] (5) After standing for 2-3 minutes, measure the particle size again.

[0389] Example 29 Preparation of adjuvant R3207

[0390] (1) The ionizable lipid R32, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0391] (2) Prepare the mixture in the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0392]

[0393] (3) A mixed phospholipid solution containing the ionizable lipid R32, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant IMQ was placed in syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0394] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0395] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0396] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0397] (5) After standing for 2-3 minutes, measure the particle size again.

[0398] Example 30 Preparation of adjuvant R3208

[0399] (1) The ionizable lipid R32, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0400] (2) Prepare the mixture in the ratio of R322:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0401]

[0402]

[0403] (3) A mixed phospholipid solution containing the ionizable lipid R32, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant MPLA was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0404] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0405] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0406] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0407] (5) After standing for 2-3 minutes, measure the particle size again.

[0408] Example 31 Preparation of adjuvant R3209

[0409] (1) The ionizable lipid R32, the auxiliary lipid DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0410] (2) Prepare the mixture in the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0411]

[0412]

[0413] (3) A mixed phospholipid solution containing ionizable lipid R32, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0414] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0415] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0416] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0417] (5) After standing for 2-3 minutes, measure the particle size again.

[0418] Example 32 Preparation of adjuvant R3210

[0419] (1) The ionizable lipid R32, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0420] (2) Prepare the mixture in the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0421]

[0422] (3) A mixed phospholipid solution containing ionizable lipid R32, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0423] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0424] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0425] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0426] (5) After standing for 2-3 minutes, measure the particle size again.

[0427] Example 33 Preparation of adjuvant R3211

[0428] (1) The ionizable lipid R32, the auxiliary lipids DSPC, Chol, DMG-PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL R32 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL DMG-PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0429] (2) Prepare the mixture in the ratio of R32:DSPC:Chol:DMG-PEG2000=50:10:38.5:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0430]

[0431] (3) A mixed phospholipid solution containing ionizable lipid R32, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0432] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0433] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0434] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0435] (5) After standing for 2-3 minutes, measure the particle size again.

[0436] Example 34 Preparation of Adjuvant A01

[0437] (1) The ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, and DMG-PEG2000 / ALC0159 were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, and a 5 mg / mL DMG-PEG2000 / ALC0159 ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0438] (2) Add ethanol solution according to different proportions to make a 1 mL mixed phospholipid solution.

[0439]

[0440]

[0441] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, and PEG2000 was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0442] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0443] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0444] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0445] (5) After standing for 2-3 minutes, measure the particle size again.

[0446] Example 35 Preparation of Adjuvant A02

[0447] (1) The ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0448] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0449]

[0450] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic adjuvant MPLA was placed into syringe 1. CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0451] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0452] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0453] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0454] (5) After standing for 2-3 minutes, measure the particle size again.

[0455] Example 36 Preparation of Adjuvant A03

[0456] (1) The ionizable lipid ALC0315, auxiliary lipid DSPC, Chol, PEG2000, hydrophobic immune adjuvant MPLA, and IMQ were dissolved in ethanol to obtain 50 mg / mL ALC0315 ethanol solution, 10 mg / mL DSPC, Chol ethanol solution, 5 mg / mL PEG2000 ethanol solution, 1 mg / mL MPLA ethanol solution, and 1 mg / mL IMQ ethanol solution.

[0457] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0458]

[0459] (3) A mixed phospholipid solution of ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, hydrophobic adjuvant MPLA and IMQ was loaded into syringe No. 1, and 50 mM CA buffer solution with pH = 4 was added to the other three syringes. The four syringes were placed on a high-pressure pump, and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants.

[0460] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0461] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0462] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0463] (5) After standing for 2-3 minutes, measure the particle size again.

[0464] Example 37 Preparation of Adjuvant A04

[0465] (1) The ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0466] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0467]

[0468]

[0469] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic adjuvant MPLA was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0470] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0471] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0472] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0473] (5) After standing for 2-3 minutes, measure the particle size again.

[0474] Example 38 Preparation of Adjuvant A05

[0475] (1) The ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0476] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0477]

[0478] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic adjuvant IMQ was placed in syringe 1. CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0479] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0480] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0481] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0482] (5) After standing for 2-3 minutes, measure the particle size again.

[0483] Example 39 Preparation of Adjuvant A06

[0484] (1) The ionizable lipid ALC0315, the auxiliary lipid DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0485] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0486]

[0487] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic adjuvant IMQ was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0488] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0489] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0490] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0491] (5) After standing for 2-3 minutes, measure the particle size again.

[0492] Example 40 Preparation of Adjuvant A07

[0493] (1) The ionizable lipid ALC0315, the auxiliary lipids DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant IMQ were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC, Chol ethanol solution, a 5 mg / mL PEG2000 ethanol solution, and a 1 mg / mL IMQ ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0494] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0495]

[0496] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic adjuvant IMQ was placed in syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0497] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0498] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0499] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0500] (5) After standing for 2-3 minutes, measure the particle size again.

[0501] Example 41 Preparation of Adjuvant A08

[0502] (1) The ionizable lipid ALC0315, the auxiliary lipids DSPC, Chol, PEG2000, and the hydrophobic immune adjuvant MPLA were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC, Chol ethanol solution, a 5 mg / mL PEG2000 ethanol solution, and a 1 mg / mL MPLA ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0503] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0504]

[0505] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic adjuvant MPLA was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0506] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0507] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0508] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0509] (5) After standing for 2-3 minutes, measure the particle size again.

[0510] Example 42 Preparation of Adjuvant A09

[0511] (1) The ionizable lipid ALC0315, the auxiliary lipids DSPC, Chol, and PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvant CpG was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG solution.

[0512] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0513]

[0514] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. CpG solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0515] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0516] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0517] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0518] (5) After standing for 2-3 minutes, measure the particle size again.

[0519] Example 43 Preparation of Adjuvant A10

[0520] (1) The ionizable lipid ALC0315, the auxiliary lipids DSPC, Chol, and PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvant QS21 was dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL QS21 solution.

[0521] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0522]

[0523]

[0524] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. The other three syringes were filled with QS21 solution. The four syringes were placed on a high-pressure pump and each syringe was passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0525] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0526] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0527] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0528] (5) After standing for 2-3 minutes, measure the particle size again.

[0529] Example 44 Preparation of Adjuvant A11

[0530] (1) The ionizable lipid ALC0315, the auxiliary lipids DSPC, Chol, and PEG2000, and the hydrophobic immune adjuvants MPLA and IMQ were dissolved in ethanol to obtain a 50 mg / mL ALC0315 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, a 5 mg / mL PEG2000 ethanol solution, and a 1 mg / mL MPLA and IMQ ethanol solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0531] (2) Prepare the mixture in the ratio of ALC0315:DSPC:Chol:PEG2000=45.5:10:43:1.5, and add ethanol solution to make a 1 mL mixed phospholipid solution.

[0532]

[0533] (3) A mixed phospholipid solution containing the ionizable lipid ALC0315, auxiliary lipids DSPC, Chol, PEG2000, and hydrophobic adjuvants MPLA and IMQ was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0534] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0535] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0536] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0537] (5) After standing for 2-3 minutes, measure the particle size again.

[0538] Examples 45-55 Preparation of adjuvants R2101-R2111

[0539] (1) The ionizable lipid R21, the auxiliary lipid DSPC, Chol, and DMG-PEG2000 were dissolved in ethanol to obtain a 50 mg / mL R21 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, and a 5 mg / mL DMG-PEG2000 ethanol solution. The hydrophobic immune adjuvant MPLA / IMQ was dissolved in ethanol to obtain a 1 mg / mL MPLA / IMQ solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0540] (2) Add ethanol solution according to different proportions to make a 1 mL mixed phospholipid solution.

[0541] (3) A mixed phospholipid solution containing ionizable lipid R21, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0542] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0543] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0544] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0545] (5) After standing for 2-3 minutes, measure the particle size again.

[0546] (The preparation method, ratio of ionizable lipid to adjuvant, and adjuvant composition of nanoadjuvants R2101-R2111 are similar to those in S01-S11)

[0547] Examples 56-66 Preparation of adjuvants R2201-R2211

[0548] (1) The ionizable lipid R22, the auxiliary lipid DSPC, Chol, and DMG-PEG2000 were dissolved in ethanol to obtain a 50 mg / mL R22 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, and a 5 mg / mL DMG-PEG2000 ethanol solution. The hydrophobic immune adjuvant MPLA / IMQ was dissolved in ethanol to obtain a 1 mg / mL MPLA / IMQ solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0549] (2) Add ethanol solution according to different proportions to make a 1 mL mixed phospholipid solution.

[0550] (3) A mixed phospholipid solution containing ionizable lipid R22, auxiliary lipid DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0551] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0552] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0553] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0554] (5) After standing for 2-3 minutes, measure the particle size again.

[0555] (The preparation method of adjuvant R2201-R2211 nanoadjuvants, the ratio of ionizable lipid to adjuvant, and the adjuvant composition are similar to those in S01-S11)

[0556] Preparation of Examples 67-77 Adjuvants R3101-R3111

[0557] (1) The ionizable lipid R31, the auxiliary lipid DSPC, Chol, and DMG-PEG2000 were dissolved in ethanol to obtain a 50 mg / mL R31 ethanol solution, a 10 mg / mL DSPC and Chol ethanol solution, and a 5 mg / mL DMG-PEG2000 ethanol solution. The hydrophobic immune adjuvant MPLA / IMQ was dissolved in ethanol to obtain a 1 mg / mL MPLA / IMQ solution. The immune adjuvants CpG and QS21 were dissolved in a 50 mM CA solution at pH 4 to obtain a 200 μg / mL CpG / QS21 solution.

[0558] (2) Add ethanol solution according to different proportions to make a 1 mL mixed phospholipid solution.

[0559] (3) A mixed phospholipid solution containing the ionizable lipid R31, auxiliary lipids DSPC, Chol, DMG-PEG2000, and hydrophobic adjuvant was placed into syringe 1. The CpG / QS21 solution was added to the other three syringes. The four syringes were placed on a high-pressure pump and each syringe passed through channels 1 to 4 to obtain nanoparticle adjuvants. (Use a 5 mL syringe, remove bubbles, and set the flow rate to 10 mL / min)

[0560] (4) After standing for 2-3 minutes, perform gradient dialysis:

[0561] 1) The obtained nanoadjuvant was added to a 1000D dialysis bag (record the volume in detail), placed in a 50mM CA buffer solution (pH=6.7±0.1) (1000 volumes), and dialyzed at 400 rpm and 4°C for 4 hours.

[0562] 2) The nanoadjuvant was transferred into a PBS buffer solution (pH = 7.4) (1000 volumes) and dialyzed at 400 rpm and 4°C for 6 hours.

[0563] (5) After standing for 2-3 minutes, measure the particle size again.

[0564] (The preparation method of adjuvant R3101-R3111 nanoadjuvants, the ratio of ionizable lipid to adjuvant, and the composition of the adjuvant are similar to those in S01-S11).

[0565] Test Example 1 Particle Size Test

[0566] The particle size of the nanoadjuvants of Examples 1 to 77 was tested using a Malvern particle size analyzer (with a dynamic light scattering detector), and the results are shown in Tables 1-7 (each Example only shows the adjuvant particle size data for the optimal ratio, and the optimal ratio is determined by the particle size and dispersion coefficient of the nanoparticles, where CLP is an ionizable phospholipid).

[0567] Table 1 Physicochemical properties of nanoadjuvants D01-D11

[0568]

[0569] Results are presented as mean±SD(n=3)

[0570] *Polydispersity index.

[0571] Table 2 Physicochemical properties of nanoadjuvants S01-S11

[0572]

[0573] Table 3 Physicochemical properties of R3201-R3211 nanoadjuvants

[0574]

[0575] Table 4 Physicochemical properties of nanoadjuvants A01-A11

[0576]

[0577] Table 5 Physicochemical properties of R2101-R2111 nanoadjuvants

[0578]

[0579] Table 6 Physicochemical properties of R2201-R2211 nanoadjuvants

[0580]

[0581] Table 7 Physicochemical properties of R3101-R3111 nanoadjuvants

[0582]

[0583] As can be seen from the results in Tables 1-7, the present invention uses ionizable lipid materials to encapsulate anionic adjuvants and / or hydrophobic adjuvants with different functions to prepare a new nanoparticle adjuvant system. The prepared nanoparticles have the characteristics of high throughput, strong controllability, uniform distribution, small particle size (30-200 nm), and small batch-to-batch variability.

[0584] Test Example 2 Calculation of Encapsulation Efficiency of Immune Adjuvant in Nanoparticles

[0585] Take 1 mL of nanoparticle adjuvant solution into a 300 kDa ultrafiltration tube and centrifuge at 4°C and 3000 rpm for 30 min. Take the filtrate and use the Limulus amebocyte lysate kit to detect the content of free MPLA in the filtrate. Use Quant-iT TM OliGreen TMThe CpG content in the filtrate was determined using an ssDNA Assay Kit, and the QS21 and IMQ contents were determined by HPLC. The adjuvant encapsulation efficiency in the nanoparticles was calculated according to the following formula: Adjuvant encapsulation efficiency = w0 - w1 / w0 × 100%, where w0 is the total amount of adjuvant added and w1 is the total amount of free adjuvant in the filtrate.

[0586] The results of the determination of the encapsulation efficiency of the immune adjuvant in the nanoparticles of Example 1-Implementation 77 are shown in Tables 8-14. (The encapsulation efficiency here is the encapsulation efficiency of the nanoadjuvant prepared by screening the optimal ratios in Tables 1-7)

[0587] Table 8 Encapsulation efficiency of adjuvant in D01-D11 nanoparticle adjuvants

[0588]

[0589] Table 9 Encapsulation efficiency of adjuvant in S01-S11 nanoparticle adjuvants

[0590]

[0591] Table 10 Encapsulation efficiency of adjuvant in R3201-R3211 nanoparticle adjuvant

[0592]

[0593] Table 11 Encapsulation efficiency of adjuvant in A01-A11 nanoparticle adjuvants

[0594]

[0595] Table 12 Encapsulation efficiency of adjuvant in R2101-R2111 nanoparticle adjuvant

[0596]

[0597] Table 13 Encapsulation efficiency of adjuvant in R2201-R2211 nanoparticle adjuvant

[0598]

[0599] Table 14 Encapsulation efficiency of adjuvant in R3101-R3111 nanoparticle adjuvant

[0600]

[0601] As shown in Tables 8-14, the present invention utilizes ionizable lipid materials to encapsulate anionic adjuvants and / or hydrophobic adjuvants with different functionalities to create a novel nanoparticle adjuvant system. This nanoparticle adjuvant system overcomes the limitations of conventional methods that rely on cationic lipids to support anionic and hydrophobic adjuvants. The various nanoparticles of the present invention exhibit high encapsulation efficiencies of 70% to 100% for each adjuvant, contributing to the potent immune response induced by the nanoparticles.

[0602] Test Example 3 Evaluation of the immune effect of nanoparticles in mice

[0603] 1. Immunization method

[0604] 5-8 week old female C57BL / 6 mice were randomly divided into 5 groups. First, the adjuvant was mixed with VZV gE antigen and injected subcutaneously or intramuscularly at the base of the tail (dose: VZV gE = 5 μg / mouse, each adjuvant = 5 μg / mouse). Figure 1 Mice were immunized with the immunization protocol in the immunization protocol, and boosted once every 4 weeks for a total of 2 immunizations. Aluminum adjuvant and free adjuvant were used as controls, and the groups were described in Table 15. Similarly, each group of adjuvants was mixed with VZV gE antigen according to Figure 1 Mice were immunized twice according to the immunization schedule in (dosage: VZV gE = 5 μg / mouse, each adjuvant = 5 μg / mouse).

[0605] Table 15 Nomenclature of aluminum adjuvants and other free adjuvants

[0606]

[0607] 2. Evaluation of humoral immunity effect

[0608] (1) Detection of IgG in mouse serum

[0609] Orbital blood was collected on days 28 and 42 after the first immunization, and serum was separated. The titer of IgG in the serum was detected by Elisa.

[0610] Testing process:

[0611] 1) Coat 5 μg / mL of VZV gE recombinant protein antigen in a 96-well plate, 100 μL per well, at 4°C overnight.

[0612] 2) The plate coated overnight was washed three times with 200 μL PBST each time, and blocked with 200 μL 3% BSA at 37°C for 2 h.

[0613] 3) Take 2 μL of immune serum or negative control serum, dilute to 200 μL, then dilute in series, add to the antigen-coated wells, and incubate at room temperature for 2 h.

[0614] 4) Wash 5 times, add IgG-HRP at the working concentration, 100 μL per well, and incubate at room temperature for 2 h.

[0615] 5) Wash five times, add 100 μL TMB substrate to each well, incubate in the dark for 20 min, terminate the reaction with 200 μL 2M H2SO4, and measure the OD value at 450 nm.

[0616] 6) Calculate the titer. If the ratio of the average absorbance value (P) of the specimen well to the average absorbance value (N) of the negative control (Group A) (i.e., P / N) is greater than 2.1, the specimen well is considered positive.

[0617] (2) Detection of IgG1 in mouse serum

[0618] Orbital blood was collected on days 28 and 42 after the first immunization, and the serum was separated. The titer of IgG1 in the serum was detected by Elisa.

[0619] (3) Detection of IgG2c in mouse serum

[0620] Orbital blood was collected on days 28 and 42 after the first immunization, and the serum was separated. The titer of IgG2c in the serum was detected by Elisa.

[0621] The humoral immune effects of the nanoparticle adjuvants described in Examples 1 to 77 are shown in Tables 16-22; the humoral immune effects of Al adjuvant and other free adjuvants are shown in Table 23.

[0622] Table 16 VZV gE-specific IgG antibody titers in the serum of mice in each group on the 28th and 42nd day after the first immunization with nanoparticle adjuvants D01-D11

[0623]

[0624] Table 17 VZV gE-specific IgG antibody titers in the serum of mice in each group on the 28th and 42nd day after the first immunization with S01-S11 nanoparticle adjuvants

[0625]

[0626] Table 18 VZV gE-specific IgG antibody titers in the serum of mice in each group on the 28th and 42nd day after the first immunization with R3201-R3211 nanoparticle adjuvant

[0627]

[0628] Table 19 VZV gE-specific IgG antibody titers in the serum of mice in each group on the 28th and 42nd day after the first immunization with A01-A11 nanoparticle adjuvant

[0629]

[0630] Table 20 VZV gE-specific IgG antibody titers in the serum of each group of mice on the 28th and 42nd day after the first immunization with R2101-R2111 nanoparticle adjuvant

[0631]

[0632] Table 21 VZV gE-specific IgG antibody titers in the serum of mice in each group on the 28th and 42nd day after the first immunization with R2201-R2211 nanoparticle adjuvant

[0633]

[0634] Table 22 VZV gE-specific IgG antibody titers in the serum of mice in each group on the 28th and 42nd day after the first immunization with R3101-R3111 nanoparticle adjuvant

[0635]

[0636] Table 23 VZV gE-specific IgG antibody titers in the serum of mice in each group on the 28th and 42nd day after the first immunization with aluminum adjuvant and free adjuvant (F01-F11)

[0637]

[0638] At the same time, cationic lipid trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP) was used to replace ionizable lipids + cholesterol to prepare cationic nanoadjuvants loaded with different adjuvants and the AS01 adjuvant developed by GSK for comparison. The immune effects were compared with the D01-D04 nanoparticle adjuvants. The results are shown in Table 24:

[0639] Table 24 VZV gE-specific IgG antibody titers in the serum of mice in each group on day 42

[0640]

[0641] As can be seen from the results in Tables 15-23, the various nanoparticle adjuvants of the present invention can produce strong humoral immunity and sufficiently strong VZV gE-specific antibodies after immunization in animals; and the immune effect is better than that of free antigen / adjuvant mixed injection and aluminum adjuvant-containing vaccines.

[0642] At the same time, it can be seen from the results in Table 24 that the nanoparticle adjuvant of the present invention can produce higher specific antibodies than the existing AS01 developed by GSK (the average titer of AS01 produced on day 42 is 9.8×10 5); Compared with traditional cationic lipid-loaded cationic nanoadjuvants with different adjuvants, it can produce higher specific antibodies.

[0643] 3. Evaluation of cellular immune effect

[0644] (1) On the 34th day after the first immunization, blood was collected from the orbital cavity, cultured, and analyzed by flow cytometry to measure cytokines such as INF-gamma and TNF-alpha.

[0645] (2) On the 42nd day after the first immunization, the mice were euthanized, the spleens were removed to separate lymphocytes, and the lymphocytes were stimulated with drugs. The ability of the lymphocytes in the spleen to secrete INF-gamma was detected by elispot.

[0646] The cellular immune effect of the nanoparticle adjuvant is as follows Figure 2-17 As shown:

[0647] Figure 2 The percentage of IFN-γ+ / TNF-α+ in peripheral blood CD4+ T cells and CD8+ T cells after immunization with the D01-D11 nanoadjuvant (day 34) indicates that the D01-D11 nanoadjuvant of the present invention increases the expression of IFN-γ and TNF-α by CD4+ and CD8+ lymphocytes, thereby enhancing T cell-mediated cellular immunity.

[0648] Figure 3 The percentage of IFN-γ+ / TNF-α+ in peripheral blood CD4+ T cells and CD8+ T cells after immunization with S01-S11 nanoadjuvant (day 34). The experimental results show that the S01-S11 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0649] Figure 4 The percentage of IFN-γ+ / TNF-α+ in peripheral blood CD4+ T cells and CD8+ T cells after immunization with R3201-R3211 nanoadjuvants (day 34). The experimental results show that the R3201-R3211 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0650] Figure 5The percentage of IFN-γ+ / TNF-α+ in peripheral blood CD4+ T cells and CD8+ T cells after immunization with the A01-A11 nanoadjuvant (day 34). The experimental results show that the A01-A11 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0651] Figure 6 The percentage of IFN-γ+ / TNF-α+ in CD4+ T cells and CD8+ T cells in peripheral blood after immunization with R2101-R2111 nanoadjuvant (day 34). The experimental results show that the R2101-R2111 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0652] Figure 7 The percentage of IFN-γ+ / TNF-α+ in CD4+ T cells and CD8+ T cells in peripheral blood after immunization with R2201-R2211 nanoadjuvants (day 34). The experimental results show that the R2201-R2211 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0653] Figure 8 The percentage of IFN-γ+ / TNF-α+ in CD4+ T cells and CD8+ T cells in peripheral blood after immunization with R3101-R3111 nanoadjuvant (day 34). The experimental results show that the R3101-R3111 nanoadjuvant of the present invention can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0654] Figure 9 The percentage of IFN-γ+ and TNF-α+ in CD4+ T cells and CD8+ T cells in peripheral blood after immunization with F00-F11 (day 34). The experimental results show that free adjuvant can increase the expression of IFN-γ and TNF-α in CD4+ and CD8+ lymphocyte T cells to a certain extent after immunization of mice, but it is different from the Figure 2-Figure 8 Compared with the nanoadjuvants shown in Figure 3, free adjuvants were less effective than nanoadjuvants in enhancing T cell-mediated cellular immunity.

[0655] Figure 10ELISPOT assay was used to detect the number of splenocytes secreting specific IFN-γ after immunization with the D01-D11 nanoadjuvant using VZV gE (day 42). The experimental results showed that the D01-D11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0656] Figure 11 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with S01-S11 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the S01-S11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0657] Figure 12 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with R3201-R3211 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the R3201-R3211 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0658] Figure 13 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with A01-A11 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the A01-A11 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0659] Figure 14 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with R2101-R2111 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the R2101-R2111 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0660] Figure 15 ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with R2201-R2211 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the R2201-R221 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0661] Figure 16ELISPOT assay was used to detect the number of cells secreting specific IFN-γ in splenocytes immunized with R3101-R3111 nanoadjuvants stimulated with VZV gE (day 42). The experimental results showed that the R3101-R3111 nanoadjuvant of the present invention can increase the expression of IFN-γ in lymphocytes after immunization of mice, thereby enhancing the cellular immunity mediated by T cells.

[0662] Figure 17 ELISPOT was used to detect the number of cells secreting specific IFN-γ in splenocytes of F00-F11 immunized with VZV gE (day 42). The experimental results showed that free adjuvant could increase the expression of IFN-γ in lymphocytes after immunization of mice, but it was not as effective as that in Figures 10-16 Compared with the nanoadjuvants shown in Figure 3, free adjuvants were less effective than nanoadjuvants in enhancing T cell-mediated cellular immunity.

[0663] From above Figure 2-17 Results indicate that immunization with various nanoparticle adjuvants of the present invention significantly enhanced cellular immunity, resulting in increased expression of IFN-γ and TNF-α, surpassing the effects of free antigen / adjuvant mixed injections and vaccines containing aluminum adjuvants. Furthermore, eslispot experiments confirmed that the MPLA- and QS21-loaded nanoadjuvants prepared by the present invention produced higher levels of IFN-γ, demonstrating that the nanoparticle adjuvants prepared by the present invention can stimulate stronger cellular immunity than AS01 adjuvant and traditional cationic nanoadjuvants.

[0664] 4. Lymph Node Imaging Analysis

[0665] To investigate the lymph node accumulation behavior of different adjuvant groups, mice were randomly divided into groups of 3 and injected subcutaneously at the base of the tail with a mixture of adjuvant and antigen (equal doses per mouse: VZV gE = 5 μg / mouse, adjuvant = 5 μg / mouse per group). Each group of animals was euthanized at specific time points, and inguinal and axillary lymph nodes were excised for ex vivo imaging. Fluorescence intensity in the lymph nodes of each animal at different time points was then plotted.

[0666] Figure 18 The fluorescence signal intensity in the lymph nodes of mice injected with nanoparticles loaded with MPLA and CpG and their corresponding free adjuvant group F02 is shown. Figure 18 The results show that the nanoparticles of the present invention have the function of targeting lymph nodes and improve the enrichment of the vaccine in the lymph nodes.

Claims

1. A nanoparticle adjuvant, characterized in that The invention relates to a nanoparticle adjuvant comprising an ionizable lipid, an anionic immune adjuvant, a hydrophobic immune adjuvant and an auxiliary lipid, wherein the auxiliary lipid comprises a neutral auxiliary lipid, cholesterol and a pegylated lipid; the nanoparticle adjuvant is a liposome core-shell structure, wherein the core contains the anionic immune adjuvant and the shell is the ionizable lipid, auxiliary lipid and hydrophobic immune adjuvant wrapped around the core; or the core contains part of the anionic immune adjuvant and the shell is the ionizable lipid, auxiliary lipid and hydrophobic immune adjuvant wrapped around the core, and the particle surface is loaded with another part of the anionic immune adjuvant; the particle size of the nanoparticle adjuvant is 30 to 150 nm; the encapsulation efficiency of the anionic immune adjuvant and / or hydrophobic immune adjuvant is 70% to 100%; and the preparation method of the nanoparticle adjuvant comprises the following steps: S1. Providing a solution comprising an ionizable lipid, a helper lipid and a hydrophobic immune adjuvant, and a solution comprising an anionic immune adjuvant; S2. A solution containing an ionizable lipid, a helper lipid, and a hydrophobic immune adjuvant passes through the first channel, and a solution containing an anionic immune adjuvant passes through the second channel, the third channel, and the fourth channel, respectively. The solutions in the four channels reach the mixing region and are mixed to obtain a nanoparticle adjuvant solution. S3. dialyzing the solvent step by step to obtain an aqueous solution of nanoparticle adjuvant; The ionizable lipid is selected from 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 1-octylnonyl 8-[(2-hydroxyethyl) [6-O-6-(undecanyloxy) hexyl] amino] -octanoate, ((4-hydroxybutyl) azadialkyl) bis (hexane-6,1-diyl) bis (2-hexyldecanoate), (10Z) -N- [3-(dimethylamino) propyl] -N- [3-ethyl-1- (octadecylamino) -1-oxyylidenehept-2-yl] octadec-9-enoyl, (10Z,12Z) -N- [3-(dimethylamino) Any one or more of (10Z,12Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxyylidenehept-2-yl]octadeca-9,12-dienamide, (10Z,12Z)-N-[3-(dimethylamino)propyl]-N-[3-ethyl-1-(octadecylamino)-1-oxyylidenehept-2-yl]octadeca-9,12-dienamide; The anionic immune adjuvant is CpG oligodeoxynucleotide and / or plant-derived adjuvant QS21; The hydrophobic immune adjuvant is monophosphoryl lipid A and / or imiquimod; The neutral helper lipid is distearoylphosphatidylcholine; The PEGylated lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000; The molar mass ratio of the ionizable lipid, the anionic immune adjuvant, the hydrophobic immune adjuvant and the auxiliary lipid is 35-65:10-30:10-30:35-65.

2. The nanoparticle adjuvant according to claim 1, characterized in that The ionizable lipid is a lipid with an acid dissociation constant pKa between 5.0 and 7.

4.

3. The nanoparticle adjuvant according to claim 1, characterized in that The molar mass ratio of the ionizable lipid, the anionic immune adjuvant, the hydrophobic immune adjuvant and the auxiliary lipid is 3:1:1:3 or 2:1:1:

2.

4. The nanoparticle adjuvant according to claim 1, characterized in that The molar mass ratio of the ionizable lipid, the neutral auxiliary lipid, the cholesterol and the PEGylated lipid is 44-55:9.4-10:38.5-45:1.5-1.

6.

5. The nanoparticle adjuvant according to claim 1, characterized in that The pH of the solution containing the anionic immune adjuvant is 3-5.

6. The nanoparticle adjuvant according to claim 1, characterized in that The nanoparticle adjuvant is approximately spherical.

7. The nanoparticle adjuvant according to claim 1, characterized in that The zeta potential of the nanoparticle adjuvant is between -10 and +20 mV.

8. The nanoparticle adjuvant according to claim 1, characterized in that The flow rate of each channel is the same, which is 1 to 40 mL / min.

9. The nanoparticle adjuvant according to claim 8, characterized in that The flow rate of each channel is 10 mL / min.

10. Use of the nanoparticle adjuvant according to any one of claims 1 to 8 in the preparation of an immunogenic composition for diseases associated with varicella-zoster virus infection.

11. An immunogenic composition, characterized in that The immunogenic composition comprises the nanoparticle adjuvant according to any one of claims 1 to 8.

12. The immunogenic composition according to claim 11, characterized in that The immunogenic composition further comprises a pharmaceutically acceptable excipient.

13. The immunogenic composition according to claim 11, characterized in that The immunogenic composition is a vaccine.

14. The immunogenic composition according to claim 11, characterized in that The immunogenic composition also comprises a VZV antigen and attenuated VZV.

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