CN118717965A - Nucleic acid vaccine and nucleic acid immune adjuvant co-carried composite lipid nano preparation and preparation method and application thereof - Google Patents
Nucleic acid vaccine and nucleic acid immune adjuvant co-carried composite lipid nano preparation and preparation method and application thereof Download PDFInfo
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- CN118717965A CN118717965A CN202410734477.6A CN202410734477A CN118717965A CN 118717965 A CN118717965 A CN 118717965A CN 202410734477 A CN202410734477 A CN 202410734477A CN 118717965 A CN118717965 A CN 118717965A
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Abstract
The invention belongs to the field of nucleic acid vaccine preparations, and in particular relates to a nucleic acid vaccine and nucleic acid immune adjuvant co-supported composite lipid nano preparation, wherein the preparation is formed by loading a nucleic acid vaccine on the inside of a lipid carrier with a specific composition and structure and loading a nucleic acid immune adjuvant on the outside of the lipid carrier. After the composite nano preparation is inoculated, the external nucleic acid immune adjuvant is gradually released in a physiological environment, relevant immune cells such as antigen presenting cells and the like are recruited and activated, and the positive nucleic acid vaccine lipid nano particles released by the nucleic acid immune adjuvant can effectively deliver nucleic acid vaccine into the cells and express antigen, and are combined with the immune cells activated by the nucleic acid immune adjuvant to induce stronger specific and non-specific immune responses, so that the composite nano preparation has a better application prospect in the aspects of immunoprophylaxis and treatment of virus infection and the like.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a nucleic acid vaccine and nucleic acid immune adjuvant co-carried composite lipid nano preparation, a preparation method and application thereof.
Background
For the novel coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), vaccination remains the most effective method of preventing and controlling its pandemic. The vaccine types commonly used clinically at present mainly comprise: inactivated viral vaccines, subunit vaccines including proteins and polypeptides, viral vector vaccines, and nucleic acid vaccines including DNA and mRNA.
Inactivated viral vaccines use either dead or inactivated viruses to elicit an immune response in the body and do not regain pathogenicity in the body and cause disease. However, due to stringent culture conditions of the virus, inactivated viral vaccines may require relatively long production times and extensive safety testing. Subunit vaccines have the advantage of carrying Receptor Binding Domains (RBDs) of different variants, realizing the immune protection of multiple variants, and have higher safety and expandability, but have relatively lower immunogenicity and higher production cost. In viral vector vaccines, another relatively safe virus is used as a vector, and the target viral antigen coding gene is delivered into a human body to trigger an immune response without causing diseases, but the immune response of the human body to the viral vector may negatively affect the effectiveness of the vaccine, and the pathogenicity (such as genome insertion) of the viral vector itself may cause a safety problem.
The nucleic acid vaccine is a plasmid DNA or mRNA vector containing a gene sequence for encoding antigen protein, expresses the antigen protein through host cells after immunization and induces a host to generate immune response to the antigen protein, and has unique advantages in the aspects of high-level immune response induction, rapid development and preparation and the like: (1) Inducing a broad immune response without any risk associated with viral replication/infection; (2) Similar to viral infection, cellular and humoral immunity can be stimulated; (3) The production can be developed rapidly when new viruses or virus variants appear, or vectors encoding different antigens can be constructed in a single vaccine; (4) low cost, easy mass production, etc. Due to the advantages of the nucleic acid vaccine, the nucleic acid vaccine becomes a novel vaccine with great development potential after the inactivation/attenuation live vaccine, subunit vaccine and viral vector vaccine, and the effect of the nucleic acid vaccine on infectious disease prevention and control is fully verified.
The nucleic acid vaccine needs to enter cells after immunization to generate antigen proteins, the proteins are released and taken up by Antigen Presenting Cells (APCs) comprising Dendritic Cells (DCs) and the like, and the antigen proteins are presented to CD8+ T cells and CD4+ T cells through MHCI molecules and MHCII molecules, so that initial T cells are activated, proliferated and differentiated into effector T cells, and a specific immunoprotection effect is finally generated. However, since nucleic acid vaccine is a long-chain macromolecule with high-density negative charge, good water solubility and easy degradation, and is difficult to penetrate through cell membrane into cells, the nucleic acid vaccine needs to be combined with corresponding technologies such as virus or non-virus vectors or physical methods including electropore and the like in the immunization process, so that the cell entering capability of the nucleic acid vaccine is improved to generate high-level antigen molecules, and effective immune response is induced. Compared with the virus vector, the non-virus nano-vector has the advantages of lower immunogenicity and pathogenicity, no restriction of gene size and the like. In particular, lipid nanoparticles and the like with good biocompatibility and high delivery efficiency have unique advantages in the immunization of nucleic acid vaccines, and are the most widely used gene delivery vectors at present. In addition to the high level of antigen expression and immune induction achieved by improving the ability of nucleic acid vaccines to enter cells, the use of nucleic acid immune adjuvants with immunomodulating effects, including CpG oligonucleotides and poly (I: C) double-stranded RNA, during immunization can recruit and stimulate proliferation and differentiation of related immune cells (such as DC cells, NK cells, T cells, etc.), enhance antigen presentation and immune induction effects, and achieve more efficient immunoprotection or therapeutic effects.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a nucleic acid vaccine and nucleic acid immunoadjuvant co-supported complex lipid nano-preparation, and a preparation method and application thereof, which are used for solving the problems in the prior art.
To achieve the above and other related objects, the present invention provides a composition comprising a nucleic acid vaccine, which is a vaccine containing plasmid DNA or mRNA encoding an antigen protein, and a nucleic acid immunoadjuvant.
Preferably, the composition has one or more of the following characteristics:
1) The nucleic acid vaccine comprises a nucleic acid fragment shown as SEQ ID No. 1;
2) The nucleic acid immune adjuvant is selected from one or more of CpG oligonucleotide, poly-ICLC or Poly (I: C) double-stranded RNA.
The invention also provides a composite lipid nano preparation co-carried by the nucleic acid vaccine and the nucleic acid immunoadjuvant, wherein the composite lipid nano preparation comprises the composition and a carrier, and the carrier of the composite lipid nano preparation is a lipid membrane formed by self-assembly of lipid molecules.
The invention also provides a preparation method of the composite lipid nano preparation, which comprises any one of the following steps:
1) When the lipid membrane in the composite lipid nano preparation contains a plurality of cationic lipids, cholesterol, phospholipids and pegylated phospholipids, uniformly mixing and incubating cationic lipids, cholesterol, phospholipids and pegylated phospholipid ethanol solution with nucleic acid vaccine aqueous solution, dialyzing to prepare lipid nano particles internally loaded with nucleic acid vaccine, and mixing and incubating the aqueous solution of the lipid nano particles with nucleic acid immune adjuvant aqueous solution to obtain the composite lipid nano preparation orderly loaded with nucleic acid vaccine and nucleic acid immune adjuvant;
2) When the lipid membrane in the composite lipid nano preparation contains a plurality of cationic lipids, ionizable cationic lipids, cholesterol, phospholipids and pegylated phospholipids, uniformly mixing and incubating an ionizable cationic lipid ethanol solution and a nucleic acid vaccine-citric acid aqueous solution, and dialyzing to remove ethanol and citric acid to prepare the lipid nano particles of the internal load nucleic acid vaccine; mixing the lipid nanoparticle aqueous solution with a cationic lipid ethanol solution, and dialyzing to obtain lipid nanoparticles with cationic lipids on the surfaces; finally, mixing and incubating the lipid nano-particles with the cationic lipids on the surfaces and the nucleic acid immune adjuvant aqueous solution to obtain the nucleic acid vaccine and nucleic acid immune adjuvant orderly loaded composite vaccine lipid nano-preparation.
The invention also provides the composite lipid nano preparation prepared by the preparation method.
The invention also provides the application of the composition or the composite lipid nano-preparation in preparing vaccine products
As described above, the nucleic acid vaccine and nucleic acid immunoadjuvant co-carried composite lipid nano preparation, the preparation method and the application thereof have the following beneficial effects:
According to the invention, the nucleic acid vaccine is loaded in the lipid nano-carrier through cationic lipid or ionizable cationic lipid, and then the nucleic acid immunoadjuvant is adsorbed on the surface of the lipid nano-carrier through the original or later-introduced cationic lipid based on electrostatic action, so that the nucleic acid vaccine and nucleic acid immunoadjuvant orderly co-loaded composite lipid nano-preparation is prepared; after immunization, the nucleic acid immune adjuvant on the surface of the composite lipid nano preparation is gradually released and promotes the recruitment, proliferation and maturation of immune related cells under a physiological environment, and the surface positive charges of the nucleic acid vaccine lipid nano particles exposed after the release of the nucleic acid immune adjuvant can improve the cell entering capacity and the expression in the cells of the nucleic acid vaccine, and the nucleic acid immune adjuvant is combined with immune cells which are previously recruited and activated, so that the specific and nonspecific immune response which is remarkably enhanced compared with the single nucleic acid vaccine or the nucleic acid immune adjuvant is realized. Further, taking preparation of a novel coronavirus DNA vaccine/interferon inducer poly (I: C) ordered-loaded composite lipid nano-preparation as an example, application of the preparation in inducing specific and non-specific immune responses such as cytokine and IgA and IgG antibody production aiming at novel coronaviruses is explored. Furthermore, the nucleic acid vaccine and nucleic acid immunoadjuvant orderly loaded composite lipid nano preparation solution can be effectively atomized by a micro-grid vibration atomizer to form 2-6 mu m fogdrops, so that the atomizing immunization of the respiratory tract and the lung can be realized, and better prevention and protection effects on respiratory tract infection viruses such as new coronaviruses and the like can be achieved through mucosal immunization.
Drawings
FIG. 1 shows the structure of a novel coronavirus nucleic acid vaccine of the present invention, the novel coronavirus delta variant S protein expression plasmid (pS), and the structure of the novel coronavirus delta variant S protein expression plasmid are verified by enzyme digestion-agarose gel electrophoresis.
FIG. 2 is a representation of nucleic acid vaccine/cationic lipid nanoparticles and nucleic acid vaccine/ionizable cationic lipid nanoparticles of the present invention.
FIG. 3 is a representation of a cationic lipid-based nucleic acid vaccine and nucleic acid immunoadjuvant co-loaded composite lipid nanocarriers of the present invention. FIG. 4 is a representation of ordered co-supported composite lipid nanocarriers of nucleic acid vaccines and nucleic acid immunoadjuvants based on ionizable cationic lipids (inner core) and cationic lipids (surface) in accordance with the present invention.
FIG. 5 shows the nucleic acid vaccine and nucleic acid immunoadjuvant co-supported composite lipid nanocarrier of the present invention, as verified by nucleic acid immunoadjuvant release-agarose gel electrophoresis.
FIG. 6 shows the detection of the immune activation of macrophages by the nucleic acid vaccine and nucleic acid immunoadjuvant co-supported composite lipid nano-preparation according to the present invention.
FIG. 7 shows antigen expression of transfected cells in vitro of nucleic acid vaccine and nucleic acid immunoadjuvant co-loaded composite lipid nanoformulations of the present invention.
FIG. 8 shows the stimulation induction of cytokines by the pulmonary mucosal immunity of the nucleic acid vaccine and nucleic acid immunoadjuvant co-loaded composite lipid nano-formulation of the present invention.
FIG. 9 shows the generation of IgA specific to S protein of novel coronavirus induced by mucosal immunity in lung of the nucleic acid vaccine and nucleic acid immunoadjuvant co-loaded composite lipid nano-formulation of the present invention.
FIG. 10 shows the generation of specific IgG of S protein of novel coronavirus induced by the mucosa immunization of lung of the nucleic acid vaccine and nucleic acid immunoadjuvant co-loaded composite lipid nano-preparation.
FIG. 11 is a graph showing the vibration atomization effect of the micro-grid of the nucleic acid vaccine and nucleic acid immunoadjuvant co-supported composite lipid nano-carrier.
Detailed Description
The present invention provides a composition comprising a nucleic acid vaccine and a nucleic acid immunoadjuvant.
Further, the nucleic acid vaccine is a vaccine containing plasmid DNA or mRNA encoding an antigenic protein.
Further, the nucleic acid immunoadjuvant is selected from one or more of CpG oligonucleotides, poly-ICLC or Poly (I: C) double stranded RNA.
Preferably, the nucleic acid vaccine is a DNA or RNA vaccine encoding a novel coronavirus S protein.
Further, the nucleic acid vaccine comprises a nucleic acid fragment shown as SEQ ID No. 1.
Preferably, the nucleic acid immunoadjuvant is Poly (I: C).
The invention also provides a composite lipid nano preparation co-carried by the nucleic acid vaccine and the nucleic acid immunoadjuvant, wherein the composite lipid nano preparation contains the composition, and a carrier of the composite lipid nano preparation is a lipid membrane formed by self-assembly of lipid molecules.
Further, the lipid membrane contains one or more of cationic lipids, ionizable cationic lipids, cholesterol, phospholipids and pegylated phospholipids. Preferably, the lipid membrane contains a plurality of cationic lipids, cholesterol, phospholipids and pegylated phospholipids, or contains a plurality of cationic lipids, ionizable cationic lipids, cholesterol, phospholipids and pegylated phospholipids.
Further, the cationic lipid is selected from trimethyl-2, 3-dioleoyloxypropyl ammonium chloride (DOTMA); trimethyl-2, 3-dioleoyloxypropyl ammonium bromide (DOTAP); dimethyl-2, 3-dioleyloxypropyl-2- (2-spermidine carboxamido) ethylammonium trifluoroacetate (DOSPA); trimethyl dodecyl ammonium bromide (DTAB); trimethyl Tetradecyl Ammonium Bromide (TTAB); trimethyl cetyl ammonium bromide (CTAB); dimethyl Dioctadecyl Ammonium Bromide (DDAB); dimethyl-2-hydroxyethyl-2, 3-dioleoyloxypropyl ammonium bromide (DORI); dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE); dimethyl-3-hydroxypropyl-2, 3-dioleyloxypropylammonium bromide (DORIE-HP); dimethyl-4-hydroxybutyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE-HB); dimethyl-5-hydroxypentyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE-HPc); dimethyl-2-hydroxyethyl-2, 3-dicetyl-alkoxypropylammonium bromide (DPRIE); dimethyl-2-hydroxyethyl-2, 3-dioctadecyl-oxypropyl-ammonium bromide (DSRIE); dimethyl-2-hydroxyethyl-2, 3-ditetra-alkoxypropylammonium bromide (dmrii); n- (2-arginyl formyl) -N ', N' -dioctadecyl glycinamide (DOGS); 1, 2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC); 3β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol); lipid poly-L-lysine (LPLL); one or more of Stearylamine (SA).
Preferably, the cationic lipid is trimethyl-2, 3-dioleoyloxypropyl ammonium bromide (DOTAP).
Further, the ionizable cationic lipid is selected from Dlin-DMA, dlin-KC2-DMA, dlin-MC3-DMA or derivatives of the above compounds, C12-200, cKK-E12 or derivatives of the above compounds, ALC-0315, SM-102, TT3 or one or more of the above compounds.
Preferably, the ionizable cationic lipid is DLin-MC3-DMA.
Further, the molecular weight of polyethylene glycol in the polyethylene glycol phospholipid is 50-10000. Specifically, the molecular weight is 50-100, 100-200, 200-400, 400-800, 800-2000, 2000-4000, 4000-6000, 6000-8000 or 8000-10000.
Preferably, the polyethylene glycol in the pegylated phospholipid has a molecular weight of 2000.
Preferably, the pegylated phospholipid is 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000).
Further, the lecithin shown is distearoyl phosphatidylcholine.
Further, the particle size of the composite lipid nano preparation is 80-300 nm. Specifically, the particle size is 80-100, 100-120, 120-150, 150-200, 200-250 or 250-300 nm.
Further, the surface potential of the composite lipid nano preparation is-30 to-5 mV. Specifically, the surface potential is-30 to-25, -25 to-20, -20 to-15, -15 to-10 or-10 to-5.
Further, the nucleic acid vaccine is encapsulated inside the carrier by electrostatic action.
Further, the nucleic acid immunoadjuvant is bound to the surface of the carrier by electrostatic action.
Further, the nitrogen-phosphorus ratio of the nucleic acid vaccine loaded in the composite nano preparation to the cationic lipid or the ionizable cationic lipid is 3-30. Specifically, the nitrogen-phosphorus ratio is 3-5, 5-10, 10-15, 15-20, 20-25 or 25-30.
Preferably, the ratio of nitrogen to phosphorus of the nucleic acid vaccine loaded in the composite nano-formulation to the cationic lipid or the ionizable cationic lipid is 10.
Further, the mass ratio of the nucleic acid immunoadjuvant to the nucleic acid vaccine in the composite lipid nano preparation is 2:1 to 0.02:1. specifically, the mass ratio is 2:1 to 1.5: 1. 1.5:1 to 1: 1. 1:1 to 0.5: 1. 0.5:1 to 0.1: 1. 0.1:1 to 0.05:1 or 0.05:1 to 0.02:1.
In some embodiments, the aforementioned composition or the aforementioned complex lipid nanoformulations further comprise pharmaceutically acceptable excipients. The adjuvants include various excipients and diluents, which are not essential active ingredients and which are not excessively toxic after administration. The adjuvant comprises sterile water or physiological saline, stabilizer, excipient, antioxidant (ascorbic acid, etc.), buffer (phosphoric acid, citric acid, other organic acids, etc.), antiseptic, surfactant (PEG, tween, etc.), chelating agent (EDTA, etc.), or binder. The adjuvant also comprises other low molecular weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharide, monosaccharide, mannitol or sorbitol. The adjuvant is selected from physiological saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose or sugar alcohol isotonic solution when used in injectable aqueous solution. The injectable aqueous solution contains a solubilizing agent. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG) and/or nonionic surfactants (Tween 80 or HCO-50).
The invention also provides a preparation method of the composite lipid nano preparation, when the lipid membrane in the composite lipid nano preparation contains a plurality of cationic lipids, cholesterol, phospholipids and pegylated phospholipids, the preparation method comprises the following steps:
(1) Respectively dissolving nucleic acid vaccine, nucleic acid immune adjuvant and lipid component in solvent to prepare aqueous solution of nucleic acid vaccine, aqueous solution of nucleic acid immune adjuvant and lipid ethanol solution;
(2) Mixing the lipid ethanol solution obtained in the step (1) with a nucleic acid vaccine aqueous solution;
(3) Dialyzing the solution obtained in the step (2) in a dialysis bag overnight to obtain nucleic acid vaccine/cationic lipid composite nano particles;
(4) Mixing and oscillating the nucleic acid immune adjuvant aqueous solution obtained in the step (1) with the nucleic acid vaccine/cationic lipid composite nano particles obtained in the step (3), incubating, and ultrafiltering to remove the free nucleic acid immune adjuvant, thereby obtaining the nucleic acid vaccine based on cationic lipid and the nucleic acid immune adjuvant orderly co-loaded composite lipid carrier.
Further, the mole ratio of the lipid-PEG in the step (1) to the total lipid is 0-1.5%. Specifically, the molar ratio is 0 to 0.1%, 0.1 to-0.2%, 0.2 to 0.4%, 0.4 to 0.8%, 0.8 to 1.2% or 1.2 to 1.5%.
Further, the volume ratio of the ethanol solution to the water solution in the step (2) is 1:3.
Further, the nitrogen-phosphorus ratio of the cationic lipid to the nucleic acid vaccine in the step (2) is 3-30; specifically, the nitrogen-phosphorus ratio is 3-5, 5-10, 10-15, 15-20, 20-25 or 25-30. Preferably, the nitrogen to phosphorus ratio is 10.
Further, the mixing mode of the lipid ethanol solution and the nucleic acid vaccine aqueous solution in the step (2) comprises manual addition mixing or microfluidic mixing, preferably microfluidic mixing.
Further, the dialysis medium in the step (3) is deionized water.
Further, the mass ratio of the nucleic acid immunoadjuvant to the nucleic acid vaccine in the step (4) is 2:1 to 0.02:1. specifically, the mass ratio is 2:1 to 1.5: 1. 1.5:1 to 1:1. 1:1 to 0.5: 1. 0.5:1 to 0.1: 1. 0.1:1 to 0.05:1 or 0.05:1 to 0.02:1.
Further, the incubation temperature in the step (4) is room temperature.
In one embodiment, when the lipid membrane in the complex lipid nanopreparation contains a plurality of cationic lipids, ionizable cationic lipids, cholesterol, phospholipids and pegylated phospholipids, the preparation method comprises the steps of:
(1) Respectively dissolving nucleic acid vaccine, nucleic acid immunoadjuvant and lipid component in citric acid buffer solution, deionized water and absolute ethyl alcohol to prepare corresponding solution;
(2) Mixing the lipid ethanol solution obtained in the step (1) with a nucleic acid vaccine citric acid solution;
(3) Dialyzing the solution obtained in the step (2) in a dialysis bag overnight to remove ethanol components and citric acid, so that the preparation environment becomes neutral, and obtaining PEG modified nucleic acid vaccine/ionizable cationic lipid composite nano particles;
(4) Rapidly mixing the cationic lipid absolute ethyl alcohol solution obtained in the step (1) with the PEG-nucleic acid vaccine/ionizable cationic lipid composite nanoparticle solution prepared in the step (3) and dialyzing overnight to obtain the PEG-nucleic acid vaccine/ionizable cationic lipid composite nanoparticle with cationic lipid inserted on the surface;
(5) Mixing and incubating the nucleic acid immune adjuvant aqueous solution obtained in the step (1) with the PEG-nucleic acid vaccine/ionizable cationic lipid composite nanoparticle solution with the cationic lipid inserted on the surface, and ultrafiltering to remove the free nucleic acid immune adjuvant, thereby obtaining the nucleic acid vaccine loaded by the ionizable cationic lipid in the lipid carrier, wherein the nucleic acid vaccine loaded by the cationic lipid and the nucleic acid immune adjuvant are orderly co-loaded by the nucleic acid immune adjuvant.
Further, the molar ratio of the lipid-PEG to the total lipid in the step (1) is 0.2-1.5. Specifically, the molar ratio is 0.2-0.5, 0.5-0.7, 0.7-1, 1-1.2 or 1.2-1.5. Preferably, the molar ratio is 0.5%.
Further, the concentration of the citric acid buffer solution in the step (1) is 15-25 mM, and the pH is 3.8-4.2. Specifically, the concentration is 15-18, 18-20, 20-22 or 22-25 mM; the pH is 3.8-4 or 4-4.2.
Further, the ratio of nitrogen to phosphorus of the ionizable cationic lipid to the nucleic acid vaccine in the step (2) is 3-30. Specifically, the nitrogen-phosphorus ratio is 3-5, 5-10, 10-15, 15-20, 20-25 or 25-30. Preferably, the nitrogen to phosphorus ratio is 10.
Further, the volume ratio of the lipid ethanol solution to the nucleic acid vaccine aqueous solution in the step (2) is 1: 2-1: 4. specifically, the volume ratio is 1: 2-1: 3 or 1: 3-1: 4.
Further, the mixing mode of the lipid ethanol solution and the nucleic acid vaccine aqueous solution in the step (2) is manual addition mixing or microfluidic mixing.
Further, the dialysis medium in the step (3) is deionized water.
Further, the dialysis temperature in the step (3) is 2-6 ℃. Specifically, the dialysis temperature is 2 to 4℃or 4 to 6 ℃.
Further, the volume ratio of the PEG-nucleic acid vaccine/ionizable cationic lipid composite nanoparticle solution to the cationic lipid absolute ethanol solution after dialysis in the step (4) is 3:1.
Further, the molar ratio of the cationic lipid in the step (4) to the ionizable cationic lipid in the PEG-nucleic acid vaccine/ionizable cationic lipid composite nanoparticle is 0-1. Specifically, the molar ratio is 0.8-1, 0.6-0.8, 0.5-0.6, 0.4-0.5, 0.2-0.4, 0.1-0.2 or 0-0.1. Preferably, the molar ratio is 0.5.
Further, the mass ratio of the nucleic acid immunoadjuvant to the nucleic acid vaccine in the step (5) is 2:1 to 0.02:1. specifically, the mass ratio is 2:1 to 1.5: 1. 1.5:1 to 1:1. 1:1 to 0.5: 1. 0.5:1 to 0.1: 1. 0.1:1 to 0.05:1 or 0.05:1 to 0.02:1. preferably, the mass ratio is 1:1.
Further, the incubation temperature in the step (5) is room temperature.
The invention also provides the use of the aforementioned composition or the aforementioned complex lipid nanoformulation in the preparation of a vaccine product having one or more of the following efficacy:
(1) Inducing cytokine secretion, recruiting and promoting proliferation and maturation of immune-related cells.
(2) The expression and presentation of antigen proteins are improved.
(3) Inducing and enhancing immune responses.
(4) Preventing and treating viral infection.
Further, the cytokine is selected from one or more of IFN-gamma, TNF-a, IL-1, IL-6, MCP-1 and GM-CSF.
Further, the immune-related cells are selected from one or more of dendritic cells, monocytes, macrophages, natural killer cells and T lymphocytes.
Further, the immune response is specific and non-specific.
Further, the virus may be selected from novel coronavirus delta variants.
In some embodiments, the nucleic acid vaccine product is a novel coronavirus nucleic acid vaccine product.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
The nucleotide sequence information used in the present application is as follows:
TTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTAGAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTACGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCGGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCAAACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGGTGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCGTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAAATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTACACA
EXAMPLE 1 construction and characterization of DNA vaccine encoding novel coronavirus S protein
According to the complete genome of the novel coronavirus delta isolate (GenBank: MW 931310), a DNA fragment with the coding S protein gene sequence shown as SEQ ID No.1 is synthesized, a Kozak sequence GCCACCATG and a translation terminator TGA are respectively added at the front end and the rear end of the sequence, the DNA fragment is cloned into a pVAX1 plasmid through SbfI and SalI enzyme cutting sites, and a Spike protein expression plasmid DNA vector (pS) with the total length of 7092bp is constructed, and the structure is shown in figure 1A. The constructed pS plasmid was transformed into competent E.coli, and an appropriate amount of the bacterial liquid was streaked on a solid bacterial medium and cultured overnight in an incubator at 37 ℃. The monoclonal colonies on the plates were picked, transferred to 4mL of liquid bacterial medium, and cultured with shaking in an incubator at 37 ℃. After 6 hours, the bacterial liquid was transferred to 300mL of liquid medium, and cultured with shaking at 37℃for 14 hours. After centrifugation at 5000rpm for 10min to collect the cells, the pS plasmid was extracted according to the instructions using the plasmid extraction kit.
The resulting plasmid was digested simultaneously with EcoRI and Xhol, and agarose gel-electrophoresis was performed, as shown in FIG. 1B, lane 1 was the uncleaved pS plasmid, lane 2 was the EcoRI and Xhol digested sample, and lane M was DNA MARKER. The results show that: the bands obtained by double enzyme digestion of the obtained plasmid are about 3000bp and 4000bp respectively, and are consistent with the theoretical sizes of vector plasmids and inserted S protein coding fragments, so that the plasmid DNA vaccine for coding novel coronavirus S proteins is proved to be successfully constructed.
Example 2 preparation of cationic lipid or ionizable cationic lipid based nucleic acid vaccine/(ionizable) cationic lipid composite nanoparticles
The cationic lipid DOTAP, the ionizable cationic lipid DLin-MC3-DMA (hereinafter abbreviated as MC 3), cholesterol (Chol), distearoyl phosphatidylcholine (DSPC) and mPEG-dimyristoylglycerol (DMG-PEG 2000) were weighed and dissolved in chloroform to prepare a lipid stock solution, which was stored at-20 ℃.
Preparation of cationic lipid DOTAP-based nucleic acid vaccine cationic lipid composite nanoparticles (ps@dlnp): nucleic acid vaccine pS was diluted to 0.04mg/mL with deionized water, and simultaneously a lipid stock solution of DOTAP, chol, DSPC and DMG-PEG2k (molar ratio 50:38.5:10:1.5) was taken separately, chloroform was removed by rotary evaporation under vacuum, and dissolved in absolute ethanol. Fixing ethanol: the volume ratio of the aqueous solution is 1:3, according to the ratio of cationic lipid to N/P of pS plasmid of 3,5,10,20 and 30 respectively, the lipid ethanol solution is added into the aqueous solution of pS plasmid for mixing, vortex oscillating for 15s, standing for 30min at room temperature, placing the obtained solution into a dialysis bag with the molecular weight cut-off of 10kDa, dialyzing overnight in deionized water at 4 ℃ to obtain pS@DLNP based on DOTAP cationic lipid.
Preparation of ionizable cationic lipid MC 3-based nucleic acid vaccine ionizable cationic lipid composite nanoparticles (ps@mlnp): nucleic acid vaccine pS was dissolved in 20mM citric acid buffer (pH 4.0) while an absolute ethanol solution in which MC3, chol, DSPC, DMG-PEG2k (50:38.5:10:1.5) was dissolved was added to the pS aqueous solution in a ratio of ethanol to aqueous phase of 1:3, the N/P ratio of ionizable cationic lipid to pS plasmid was 3,5,10,20 and 30, respectively, mixed with shaking for 15s, left at room temperature for 30min, dialyzed overnight in deionized water at 4℃to remove ethanol and citric acid, allowing the formulation environment to become neutral, yielding pS@MLNP based on ionizable cationic lipid MC 3.
Particle size characterization: the prepared pS@DLNP and pS@MLNP solutions are placed in a sample cell, a laser particle size analyzer (Zetasizer ZS 90) is used for setting the detection temperature to 25 ℃, the scattering angle is 90 DEG, the incident wavelength of He-Ne is 632nm, the refractive index is 1.33, and the particle size and the distribution of the prepared nucleic acid vaccine (ionizable) cationic lipid composite nano-carrier are determined.
Surface potential characterization: 100. Mu.L of the prepared pS@DLNP and pS@MLNP were sampled, 950. Mu.L of 0.1XPBS solution (pH 7.4) was added for dissolution, and after mixing, the mixture was added to a potential cell using a 1mL syringe, and the surface potential was measured by a laser particle sizer (Zetasizer ZS 90) at a detection temperature of 25 ℃.
Gel electrophoresis verification of nucleic acid vaccine loading: 0.3g agarose was taken into a conical flask, 30mL 1 XTAE buffer was added and heated in a microwave oven until the agarose dissolved. When the solution cooled to 50-60 ℃,3 μl GelRed (10000×) dye was added and mixed well. Pouring the agarose solution into a gel preparation tank with a comb inserted for cooling and solidification, carefully pulling out the comb, putting the prepared agarose gel into an electrophoresis tank, and pouring 1 xTAE buffer until the upper surface of the gel is over. mu.L of pS@DLNP or pS@MLNP sample (containing 200ng pS) was added to each gel well, and the control group was a composite nanocarrier sample treated with 1% Triton X-100 (carrier structure was destroyed, pS was released). Electrophoresis was performed for 30min at 120V. After electrophoresis, the gel is observed by a gel imaging system.
Particle size, potential and gel electrophoresis characterization results of pS@DLNP and pS@MLNP are shown in FIG. 2. The particle size of pS@DLNP prepared according to the ratio (N/P) of cationic lipid to pS nitrogen-phosphorus of 3,5,10,20 and 30 is between 95 and 140nm, and is reduced along with the increase of N/P; the potential is between +10mV and +30mV, and the potential is gradually increased; the electrophoresis result shows that the sample which is not treated by 1% Triton membrane rupture has no free DNA band within the range of N/P ratio of 3-30, and obvious plasmid DNA band exists after membrane rupture, which proves that the prepared pS@DLNP lipid nanoparticle can efficiently and stably encapsulate the nucleic acid vaccine. The particle size of pS@MLNP prepared based on the ionizable cationic lipid MC3 is about 150-250nm, the surface potential is electronegative, the nucleic acid vaccine can be well loaded when N/P is more than 3, and the complete loading of the nucleic acid vaccine can be realized when N/P is more than 10.
Example 3 preparation and characterization of cationic lipid-based nucleic acid vaccine and nucleic acid immunoadjuvant ordered co-supported composite lipid nanocarriers
Preparation of cationic lipid-based nucleic acid vaccine and nucleic acid immunoadjuvant ordered co-supported composite lipid nanocarriers (pIC & ps@dlnp): because the surface of the prepared pS@DLNP nano-particle is high in electropositivity, the nano-particle and the nucleic acid immunoadjuvant can be directly incubated to realize electrostatic-effect-based loading, and the nucleic acid vaccine and the nucleic acid immunoadjuvant orderly co-loaded composite lipid nano-carrier is prepared. The specific operation steps are as follows: dissolving a nucleic acid immune adjuvant Poly (I: C) in pure water, rapidly mixing the solution with the prepared pS@DLNPs solution according to a certain ratio of Poly (I: C)/pS (0.02:1, 0.1:1,0.2:1,1:1 and 2:1), and carrying out shaking incubation for 2 hours at 26 ℃ to prepare the nucleic acid vaccine and nucleic acid immune adjuvant orderly co-carried composite lipid nano carrier pIC & pS@DLNP.
Particle size characterization: 400 mu L of the pIC & pS@DLNP solution prepared above is placed in a sample cell, a laser particle size analyzer (Zetasizer ZS 90) is used for setting the detection temperature to 25 ℃, the scattering angle is 90 DEG, the incident light wavelength of He-Ne is 632nm, the refractive index is 1.33, and the particle size and distribution of the prepared composite lipid nano carrier are determined.
Surface potential characterization: 100. Mu.L of the prepared pIC & pS@DLNP sample was taken, and 950. Mu.L of 0.1XPBS solution (pH 7.4) was added for dissolution, and after mixing, the mixture was added to a potential cell by a 1mL syringe, and the surface potential was measured by a laser particle size analyzer (Zetasizer ZS 90) at a detection temperature of 25 ℃.
Gel electrophoresis verification of pS@DLNP vs. nucleic acid immunoadjuvant loading: 0.3g agarose was taken into a conical flask, 30mL 1 XTAE buffer was added and heated in a microwave oven until the agarose dissolved. When the solution cooled to 50-60 ℃,3 μl GelRed (10000×) dye was added and mixed well. Pouring the agarose solution into a gel preparation tank with a comb inserted for cooling and solidification, carefully pulling out the comb, putting the prepared agarose gel into an electrophoresis tank, and pouring 1 xTAE buffer until the upper surface of the gel is over. mu.L of pIC & pS@DLNP prepared from different Poly (I: C)/pS ratios were added to gel wells and electrophoresed for 30min at 120V voltage. After electrophoresis, the gel is observed by a gel imaging system.
The particle size, potential and gel electrophoresis detection results are shown in FIG. 3. For the PEG-containing pS@DLNP, after the PEG-containing pS@DLNP is incubated with poly (I: C), the particle size of the nano particles is not obviously changed, but the surface potential of the obtained composite lipid nano particles is obviously reduced along with the increase of the binding amount of poly (I: C); gel electrophoresis showed that Poly (I: C) was able to be fully bound by pS@DLNP when Poly (I: C)/pS.ltoreq.1. And for pS@DLNP without PEG modification, after incubation with high concentration of Poly (I: C) (i.e., poly (I: C)/pS > 1), the particle size of the obtained composite lipid nanoparticle increases, the potential rises, which may be caused by that pS@DLNP is not wrapped by a PEG hydrophilic layer, and when the Poly (I: C) is excessive, the nanoparticle aggregates and the structure changes; however, gel electrophoresis results prove that the pS@DLNP without PEG modification can load the nucleic acid immune adjuvant Poly (I: C) well.
Example 4 preparation and characterization of ordered loaded composite lipid nanocarriers (pIC & pS@MLNP/D) based on nucleic acid vaccines and nucleic acid immunoadjuvants with inner core of ionizable cationic lipids and surface of cationic lipids
Since the surface of ps@mlnp prepared from example 2 and based on ionizable cationic lipid MC3 is electronegative and cannot be loaded with the same negatively charged nucleic acid immunoadjuvant, we obtained ps@mlnp/D with surface electropositive properties by introducing positively charged cationic lipid DOTAP onto its surface and continued to be used to load nucleic acid immunoadjuvant, the preparation method is as follows: DOTAP/MC3 (molar ratio) 0.1:1,0.2:1,0.5:1 and 1:1, was added to pS@MLNP in a ratio of water to absolute ethanol volume ratio of 3:1 and dialyzed overnight to obtain pS@MLNP with cationic lipids inserted after the surface (pS@MLNP/D). Meanwhile, pS@MLNP with different PEG2000 modification degrees (0.2%, 0.5%,1% and 1.5%) is prepared, and the influence of different PEG contents on the Poly (I: C) load and the physicochemical properties of the preparation is examined according to the molar ratio of DOTAP to MC3 being 1:1.
Preparation of ordered co-carried composite lipid nano-carrier based on nucleic acid vaccine with inner core of nucleic acid vaccine/ionizable cationic lipid and nucleic acid immunoadjuvant/cationic lipid on surface: dissolving a nucleic acid immune adjuvant Poly (I: C) in pure water, rapidly mixing with the prepared pS@DLNP/D solution, and carrying out shaking incubation for 2 hours at 26 ℃ to prepare the nucleic acid vaccine and nucleic acid immune adjuvant orderly co-carried composite lipid nano carrier pIC & pS@MLNP/D.
Particle size and surface potential characterization: the particle size and surface potential of the prepared pS@MLNP/D and pIC & pS@MLNP/D were characterized as described above.
Gel electrophoresis verification of the co-load of nucleic acid vaccine and nucleic acid immunoadjuvant: the nucleic acid immunoadjuvant and nucleic acid vaccine loading of pIC & pS@MLNP/D was verified as described previously.
The results of the relevant characterization are shown in FIG. 4, the post-insertion modification of pS@MLNP by different amounts of cationic lipid DOTAP does not change the particle size, the surface potential rises with the increase of the DOTAP addition amount, and when DOTAP/MC3 is 1, the surface potential reaches +8.1mV, which indicates that the cationic lipid can be inserted into the pS@MLNP lipid membrane and realize the surface electropositivity of the cationic lipid. And the characterization result of post-insertion modification (the molar ratio of DOTAP to MC3 is 1:1) of the cationic lipid on pS@MLNP with different PEG modification degrees (0.2%, 0.5%,1% and 1.5%) shows that pS@MLNP can be prepared when the content of PEG is reduced to 0.2%, and the change amplitude of the surface potential after DOTAP is inserted is larger, so that the cationic lipid can be more easily inserted into the surface of pS@MLNP by reducing the PEG to obtain pS@MLNP/D. And pS@MLNP/D was loaded with Poly (I: C) at a ratio of 1:1 with pS (I: C), the nanoparticle size became larger and the surface potential slightly decreased, indicating that Poly (I: C) was bound to the surface of pS@MLNP/D, resulting in pIC & pS@MLNP/D with ordered co-loading of nucleic acid vaccine and nucleic acid immunoadjuvant. The gel electrophoresis result of pIC & pS@MLNP/D treated by 1% Triton-X100 shows obvious electrophoresis bands of the nucleic acid vaccine and the nucleic acid immunoadjuvant, and the results show that the nucleic acid vaccine with the inner core of the nucleic acid vaccine/ionizable cationic lipid and the nucleic acid vaccine with the surface of the nucleic acid immunoadjuvant/cationic lipid and the nucleic acid immunoadjuvant orderly co-carried composite lipid nano carrier (pIC & pS@MLNP/D) are successfully prepared.
Example 5 release verification of nucleic acid vaccine and nucleic acid immunoadjuvant on ordered Co-supported composite lipid nanocarriers
The pIC & pS@DLNP prepared was treated with 5 XPBS and 1% Triton-X100, respectively, followed by agarose gel electrophoresis and imaging according to the aforementioned electrophoresis protocol, and analyzed for dissociation of the nucleic acid immunoadjuvant from the complex lipid nanocarriers.
The result of electrophoresis is shown in FIG. 5. The treatment of 5 XPBS can destroy the electrostatic effect between the nucleic acid immunoadjuvant and pS@DLNP, so that the nucleic acid immunoadjuvant is released from the composite lipid nano-carrier, and the immunostimulation effect is further exerted; the treatment does not lead to release of the nucleic acid vaccine pS, which means that the structure of pS@DLNP is not affected, and that the vector can be ensured to be electropositive after release of the nucleic acid immunoadjuvant and effectively deliver the nucleic acid vaccine into cells for expression.
EXAMPLE 6 immune activation of macrophages by nucleic acid vaccine and nucleic acid immunoadjuvant ordered co-carried composite lipid nanoformulations
Inoculating RAW264.7 cells in logarithmic growth phase into 12-well plate, 3×10 5 cells per well, and 1mL of complete culture medium; the cells were incubated overnight at 37℃in a 5% CO 2 cell incubator. pIC & pS@DLNP (with and without PEG modification) and pIC & pS@MLNP/D were added to the cell culture broth at a pS concentration of 5. Mu.g/mL, while a blank group, a naked pS group and a naked pIC group were set as controls. After incubation at 37℃for 24h, the culture broth was centrifuged at 2000rpm for 3min to remove cell debris, diluted to appropriate concentrations and the supernatant was assayed for the levels of cytokines IL-6 and TNF- α by ELISA. Collecting cells, adding 200 μl of cell lysate, standing on ice for 10min, repeatedly blowing with a pipette, extracting cell lysate, centrifuging at 3000rpm for 10min, diluting with PBS for 10 times, detecting protein content in lysate with BCA protein content kit, and calculating average release amount of cytokines. Average release of cytokines = (total cytokine content of cell culture supernatant x fold of dilution)/(total protein in cell lysate x fold of dilution).
As shown in FIG. 6, the detection results of cytokines show that the naked nucleic acid vaccine has no immune stimulation basically, and the free poly (I: C) and pIC & pS@DLNP and pIC & pS@MLNP/D containing poly (I: C) both significantly stimulate macrophages to generate high levels of TNF-alpha and IL-6, which indicates that the nucleic acid vaccine and the nucleic acid immune adjuvant ordered co-carried composite lipid nano preparation have activation effect on immune related cells such as macrophages.
Example 7 cell transfection and antigen expression detection of nucleic acid vaccine and nucleic acid immunoadjuvant ordered Co-carried composite lipid nanoformulations
Human alveolar basal epithelial cells A549 cells of lung cancer in logarithmic growth phase are inoculated into 12-well plates, 1X 10 5 cells per well and 1mL of complete culture medium; the cells were incubated overnight at 37℃in a 5% CO 2 cell incubator. Different experimental groups were set up, including untreated cell group, naked pS group, pIC & pS@DLNP (no PEG modification) group, pIC & pS@MLNP/D group. The above formulation was diluted with serum-free cell culture medium and added to the cells such that the final concentration of pS per well was 5. Mu.g/mL. After 6h incubation at 37℃under 5% CO 2, 1mL of complete medium was added to each well. After 48 hours of further culture, the culture broth was removed, 200. Mu.L of cell lysate was added to each well to treat the cells, and the cells were centrifuged at 5000rpm for 10 minutes to remove cell debris. Total protein concentration was detected using BCA assay kit and S protein expression levels were detected using ELISA. Antigen expression amount = concentration of S protein in cell lysate/total protein in cell lysate x fold dilution.
The detection results are shown in fig. 7, compared with the blank control group (treated by simple cell culture solution) and the naked pS group, the cells treated by pIC & ps@dlnp and pIC & ps@mlnp/D have obvious S protein expression, which indicates that the prepared nucleic acid vaccine and nucleic acid immunoadjuvant orderly co-loaded composite lipid nano preparation can effectively deliver the nucleic acid vaccine into the cells and express antigen proteins.
Example 8 stimulation Induction of cytokines after immunization with nucleic acid vaccine and nucleic acid immunoadjuvant ordered Co-carried composite lipid nanoformulations
After the mice are adaptively raised, 4% chloral hydrate is injected into the abdominal cavity according to the volume of 10 mu L/g to anesthetize the mice, then the mice are fixed, the hair on the neck and the forechest is removed by using depilatory cream, the skin on the neck is cut off by surgical scissors after the mice are sterilized by 75% ethanol, and a small opening of about 1cm is formed. The neck muscles were opened with forceps to expose the trachea. Using an insulin syringe inserted into the trachea, 100. Mu.L of formulation (including PBS, pS, poly (I: C), pS@DLNP, pS@MLNP/D, pIC & pS@DLNP, and pIC & pS@MLNP/D groups) was slowly injected into the trachea, and the mice were flicked and rotated vertically to rapidly and uniformly disperse the injection solution into the lungs of the mice. The mice were then sutured to the neck incision with a medical needle and surgical suture, and continued to be observed as awakening, and subsequently fed. On day 14 after the third immunization of the mice, isoflurane anesthetized the mice, 1mL of sterile 1 x PBS (ph=7.4) was slowly pushed in through the trachea with a 5mL syringe, gently massaged for 1min and slowly sucked back into the lungs of the mice, and alveolar lavage fluid (BALF) was recovered at about 0.5-0.7 mL. Centrifuging the collected BALF at 4 ℃ and 2000rpm for 10min, sucking the supernatant into a sterile enzyme-free centrifuge tube, measuring the content of TNF-alpha and IL-6 in the BALF by using an ELISA method, and detecting the total protein content in the BALF by using a BCA method.
The levels of cytokines TNF- α and IL-6 in the alveolar lavage fluid of the experimental mice are shown in FIG. 8, and the nucleic acid vaccine loaded with poly (I: C) and the nucleic acid immunoadjuvant sequentially co-load the composite lipid nanopreparation, including pIC & pS@DLNP and pIC & pS@MLNP/D, with respect to pS@DLNP and pS@MLNP/D loaded with poly (I: C), can significantly stimulate the experimental animals to generate high levels of TNF- α and IL-6, indicating that the nucleic acid immunoadjuvant loaded on the surface of the composite lipid nanopreparation has an immune activation effect.
Example 9 verification of Induction of specific IgA antibody production after Vaccination of nucleic acid vaccine and nucleic acid immunoadjuvant ordered Co-carried composite lipid nanoformulations by tracheal immunization
The experimental mice were immunized as in example 8, alveolar lavage fluid was collected at day 14 after the third immunization, and the content of anti-S protein-specific IgA antibody therein was detected by ELISA.
The level of anti-S protein IgA antibody in the alveolar lavage fluid of the experimental mice after the nucleic acid vaccine and the nucleic acid immunization adjuvant are sequentially co-carried with the composite lipid nano-preparation is shown in FIG. 9. Compared with the blank control group, the pure nucleic acid vaccine group (naked pS group) and the pure nucleic acid immunity adjuvant group (poly (I: C)), the BALF of the mice immunized by pS@DLNP and pS@MLNP/D can detect obvious anti-S protein specific IgA antibodies, and the co-loaded nucleic acid immunity adjuvant poly (I: C) can further improve the level of IgA antibodies generated by nucleic acid vaccines (namely pIC & pS@DLNP and pIC & pS@MLNP/D groups) through mucosal immunity induction.
Example 10 verification of Induction of specific IgG antibody production after tracheal immunization of nucleic acid vaccine and nucleic acid immunoadjuvant ordered Co-carried composite lipid nanoformulations
Experimental mice were immunized as in example 8, peripheral blood was collected on day 14 after the third immunization, and the content of anti-S protein-specific IgG antibodies therein was detected by ELISA.
The level of anti-S protein IgG antibodies in the blood of experimental mice after the nucleic acid vaccine and nucleic acid immunization adjuvant orderly co-carrier composite lipid nano-preparation are immunized is shown in figure 10. Compared with a blank control group, a pure vaccine group (naked pS), a pure nucleic acid immune adjuvant group (naked poly (I: C)) and the like, the pIC & pS@DLNP, pIC & pS@MLNP/D and the like can induce and generate high-level specific IgG antibodies in immune mice, so that the nucleic acid vaccine and the nucleic acid immune adjuvant orderly co-loaded composite lipid nano preparation have good immune induction effect.
Example 11 characterization of the atomization Effect of nucleic acid vaccine and nucleic acid immunoadjuvant ordered Co-carried composite lipid nanoformulations
PIC & pS@DLNP and pIC & pS@MLNP/D were prepared separately, and sodium fluorescein was added to make the concentration of sodium fluorescein 300. Mu.g/mL in the preparation, which was then added to the liquid medicine cup of a micro-grid vibration atomizer, a siliconized coverslip subjected to ethanol impurity removal and dehydration was placed 10cm away from the atomizer spray port, the atomizer was turned on to drop the mist onto the coverslip for 10 seconds, the coverslip was immediately transferred onto the slide with the droplet facing upward, and the droplet morphology and size were observed by a fluorescence microscope.
The atomization result is shown in FIG. 11. The diameters of droplets generated by atomizing pIC & pS@DLNP and pIC & pS@MLNP/D preparations by a micro-grid vibration atomizer are all below 5 μm. The particle size can ensure that the nucleic acid vaccine and the nucleic acid immune adjuvant orderly co-load composite lipid nano preparation are inhaled into the respiratory tract along with respiratory airflow after atomization and deposited in the lung and bronchus, so as to realize the respiratory tract mucosa immunization of the nucleic acid vaccine.
The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.
Claims (10)
1. A composition comprising a nucleic acid vaccine and a nucleic acid immunoadjuvant, wherein the nucleic acid vaccine is a vaccine comprising plasmid DNA or mRNA encoding an antigenic protein.
2. The composition of claim 1, wherein the composition has one or more of the following characteristics:
1) The nucleic acid vaccine comprises a nucleic acid fragment shown as SEQ ID No. 1;
2) The nucleic acid immune adjuvant is selected from one or more of CpG oligonucleotide, poly-ICLC or Poly (I: C) double-stranded RNA.
3. A composite lipid nano-preparation co-carried by a nucleic acid vaccine and a nucleic acid immune adjuvant, which is characterized in that the composite lipid nano-preparation contains the composition as claimed in claim 1 or 2 and a carrier, wherein the carrier of the composite lipid nano-preparation is a lipid membrane formed by self-assembly of lipid molecules.
4. The complex lipid nanoformulation of claim 3, wherein the complex lipid nanoformulation has one or more of the following characteristics:
1) The nucleic acid vaccine and the nucleic acid immune adjuvant are respectively loaded in the carrier and on the surface;
2) The lipid membrane contains one or more of cationic lipids, ionizable cationic lipids, cholesterol, phospholipids or pegylated phospholipids; preferably, the lipid membrane contains a plurality of cationic lipids, cholesterol, phospholipids and pegylated phospholipids, or contains a plurality of cationic lipids, ionizable cationic lipids, cholesterol, phospholipids and pegylated phospholipids; more preferably, the cationic lipid is DOTAP, or the ionizable cationic lipid is Dlin-MC3-DMA;
3) The nucleic acid vaccine is DNA or RNA vaccine for encoding the S protein of the novel coronavirus;
4) Nucleic acid immune adjuvant the nucleic acid immune adjuvant is Poly (I: C) double stranded RNA.
5. The complex lipid nanoformulation of claim 4, wherein the complex lipid nanoformulation has one or more of the following characteristics:
1) When the carrier of the composite lipid nano preparation contains cationic lipid or ionizable cationic lipid, the nitrogen-phosphorus ratio of the nucleic acid vaccine to the cationic lipid or ionizable cationic lipid in the composite lipid nano preparation is 3-30;
2) The mass ratio of the nucleic acid immunoadjuvant to the nucleic acid vaccine in the composite lipid nano preparation is 2:1 to 0.02:1, a step of;
3) The particle size of the composite lipid nano preparation is 80-300 nm;
4) The surface potential of the composite lipid nano preparation is-30 to-5 mV.
6. The method of preparing a complex lipid nanoformulation according to any one of claims 3 to 5, wherein the method of preparing comprises any one of the following methods:
1) When the lipid membrane in the composite lipid nano preparation contains a plurality of cationic lipids, cholesterol, phospholipids and pegylated phospholipids, uniformly mixing and incubating cationic lipids, cholesterol, phospholipids and pegylated phospholipid ethanol solution with nucleic acid vaccine aqueous solution, dialyzing to prepare lipid nano particles internally loaded with nucleic acid vaccine, and mixing and incubating the aqueous solution of the lipid nano particles with nucleic acid immune adjuvant aqueous solution to obtain the composite lipid nano preparation orderly loaded with nucleic acid vaccine and nucleic acid immune adjuvant:
2) When the lipid membrane in the composite lipid nano preparation contains a plurality of cationic lipids, ionizable cationic lipids, cholesterol, phospholipids and pegylated phospholipids, uniformly mixing and incubating an ionizable cationic lipid ethanol solution and a nucleic acid vaccine-citric acid aqueous solution, and dialyzing to remove ethanol and citric acid to prepare the lipid nano particles of the internal load nucleic acid vaccine; mixing the lipid nanoparticle aqueous solution with a cationic lipid ethanol solution, and dialyzing to obtain lipid nanoparticles with cationic lipids on the surfaces; finally, mixing and incubating the lipid nano particles with the cationic lipid on the surfaces with the nucleic acid immunoadjuvant water solution to obtain the nucleic acid vaccine and nucleic acid immunoadjuvant orderly loaded composite lipid nano preparation.
7. The method of manufacture of claim 6, comprising one or more of the following features:
a) The proportion of the polyethylene glycol phospholipid in the lipid membrane in the method 1) is 0 to 1.5 percent;
b) The proportion of the polyethylene glycol phospholipid in the method 2) to the total lipid of the lipid membrane is 0.2-1.5%.
8. The composite lipid nano-preparation prepared by the preparation method according to claim 6 or 7.
9. Use of a composition according to claim 1 or 2, a complex lipid nanoformulation according to any one of claims 3-5 or a complex lipid nanoformulation according to claim 8 in the preparation of a vaccine product.
10. The use according to claim 9, wherein the vaccine product has one or more of the following efficacy:
1) Inducing cytokine secretion, recruiting and promoting proliferation and maturation of immune-related cells;
2) Enhancing expression and presentation of the antigen protein;
3) Inducing and enhancing immune responses;
4) Preventing and treating viral infections; preferably, the virus is a novel coronavirus.
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