Cationic polysaccharide copolymer adjuvant, and vaccine

Abstract

A latex polymerization product useful as a vaccine adjuvant material is obtained by polymerizing an olefin compound in water by using a cationic derivative of a polysaccharide and adjusting a latex solution of a surfactant, and obtaining a non-viral vector for feeding deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) into a cell. The latex solution is composed of a polymer obtained by polymerizing an olefin compound in water with a cationic derivative of a polysaccharide. The complex is obtained by reacting various inactivated viruses, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) with the latex solution. In contrast to vectors from dangerous viruses, the use of such a non-viral vector is safe and, because the vector is synthetic, stable. The selectivity of the cell membrane is important for enhancing the transformation efficiency as a vector. In order to further enhance the transformation efficiency, it is necessary to have a hydrophobic-hydrophilic domain, specifically, in order to increase the effectiveness of the vaccine adjuvant material, it has been found that a latex composed of a copolymer material of a cationic polysaccharide and a vinyl monomer has excellent endocytosis and TLR7 agonist properties.

Description

Cationic polysaccharide copolymer adjuvants and vaccines.

[0001] The water-soluble linear polysaccharide cationic derivative-olefin-containing monomer graft copolymer of the present invention is produced by graft polymerizing a water-soluble linear polysaccharide cationic derivative with an olefin-containing monomer in water to produce a latex polymerization product useful as a vaccine adjuvant material. The present invention suggests that any water-soluble cationic derivative of a linear polysaccharide with a hydroxyl group can be graft polymerized with an olefin-containing monomer in water to produce a latex polymerization product useful as a vaccine adjuvant material. An adjuvant is an auxiliary agent for a main ingredient and is used in combination with the main ingredient to support, enhance, or improve the inherent activity of the active ingredient. In the field of immunology, adjuvants are also called antigenic enhancers and are substances injected together with antigens to enhance their antigenicity. The present invention improves vaccines by incorporating a TLR7 agonist possessed by the water-soluble linear polysaccharide cationic derivative-olefin of the present invention into the vaccine.

[0002] Currently, global development of COVID-19 vaccines is focused primarily on viral vector vaccines and mRNA vaccines. Clinical trials have begun at Oxford University / AstraZeneca in the UK, Pfizer / BioNTech in Germany, and Moderna in the US. The vectors used are adeno-associated virus vectors (AAV vectors) and lipid nanoparticle liposome vectors (lipofection), respectively. Viral vectors are generally suspected of pathogenicity, and repeated administration is contraindicated due to the generation of neutralizing antibodies due to their immunogenicity. Transfection using lipid liposomes involves many unstable elements, such as critical micelles, making in vivo use difficult. Non-viral vectors also pose stability challenges in vivo. While most viral vectors are integrated into host chromosomes and mRNA is stably expressed in transfected cell lines, there are concerns about excessive acquired immunity due to repeated administration. Vaccines are generally biological products that are effective in preventing infectious diseases when administered. By inactivating and injecting weakened pathogens into the body, the body produces antibodies, making the target infectious disease less susceptible. Vaccines can be broadly divided into live vaccines, inactivated vaccines, and toxoids. Recently, vaccines using pathogen messenger RNA and DNA have been developed. The overall issues with vaccines remain unchanged, and key design factors include form, manufacturing method, adjuvant, and administration method. Administration methods include subcutaneous, oral, and intradermal injection, and are specified for each vaccine. Simultaneous administration of multiple live and inactivated vaccines is prohibited as a general rule. The interval between multiple doses is specified as at least four weeks for live vaccines and at least one week for inactivated vaccines, raising concerns about optimal administration (though there is a global trend toward allowing simultaneous administration of multiple vaccines). The difficulty of producing high-potency vaccines with RNA and viral vaccines, such as those for currently rampant viral infections (COVID-19), poses a challenge, as few vaccines offer satisfactory efficacy. Therefore, the development of adjuvants to enhance potency is essential. In drug delivery systems (DDS), the particle size can be increased by adjusting the balance of water solubility and lipid solubility and the molecular weight, making it possible to achieve the EPR effect and RES suppression effect.In the EPR effect, drugs penetrate angiogenic blood vessels with a diameter of 10 to 200 nm and accumulate in tumor tissue. In RES inhibition, Kupffer cells in the liver and macrophage cells in the adrenal gland phagocytose particles larger than 400 nm as foreign bodies and eliminate them. Drugs are degraded by drug metabolism in the liver, and particles smaller than 5 nm are excreted by filtration in the glomeruli of the kidney. By optimizing particle size, it is possible to maintain drug concentrations in the body for long periods of time. This method, known as prodrug formation, improves the convenience of drugs. However, there are issues such as the need for a positive charge relative to the negatively charged cancer cell surface for more efficient uptake by cancer cells and the possibility of heat sterilization of DDS for safety reasons. While latex polymerization products have traditionally been produced as immunoassay materials, most have been produced by emulsion polymerization in aqueous solutions in the presence of surfactants. Therefore, soapless products free of surfactants are desirable. This is because surfactants present in aqueous solutions affect the function of latex diagnostic agents. To address this issue, a water-soluble linear polysaccharide cationic derivative-olefin monomer graft copolymer has been produced as a soapless latex polymerization product useful as an immunoassay material by graft polymerizing a water-soluble linear polysaccharide cationic derivative with an olefin monomer in water using a redox initiator. This soapless water-soluble linear polysaccharide cationic derivative-olefin monomer graft copolymer latex and latex diagnostic reagent have already been patented. This latex is used in antibody-adsorbed latex diagnostic reagents. Emulsion polymerization is a polymerization method in which olefin monomers are suspended in an aqueous solution and emulsified, usually with a surfactant. Specifically, the polymer chains grow by interaction with the monomer or growing chains at the water-solvent interface through hydrogen bonding, Coulomb forces, charge-transfer interactions, van der Waals forces, etc., resulting in the formation of microparticles in the aqueous solution. The polymerization product typically exists as a mixture of the polymerized monomer and surfactant. This surfactant, which is considered to be an impurity, can cause problems when used in latex diagnostic reagents.We have now unexpectedly discovered that this technique for producing soapless latex can be used to form supramolecular compounds that may have substrate selectivity as artificial enzymes, and to produce latex polymerization products that are useful as drug delivery systems (DDS) for nucleic acids and drugs. [Patent Document 1] Patent Application No. 248476, 1984, Patent No. 2126650, Patent No. 2620180, Patent No. 4650605.

[0003] Problems to be Solved by the Invention: Most currently available drug delivery systems (DDS) have difficulty controlling the balance of water- and lipid-solubility and molecular weight, and have safety issues such as heat sterilization. Furthermore, for efficient uptake by cancer cells using a DDS, they must be positively charged relative to the negatively charged cancer cell surface, and there are safety issues such as the possibility of autoclaving the DDS for safety reasons. The invention of Patent No. 4650605 clarifies the safety and potential for autoclaving, and not only suppresses side effects associated with drug delivery systems, expands drug convenience, and enhances efficacy, but also demonstrates the possibility of substrate selectivity as an artificial enzyme in a supramolecular anticancer drug complex. Furthermore, it demonstrates the enhancement of anticancer efficacy by immobilizing cancer targeting substances, anticancer drug resistance overcomers, or chemotherapy enhancers to anticancer drugs using a novel immobilization method. More recently, DEAE-dextran grafted with methyl methacrylate (MMA) copolymer (DDMC) has not only improved transfection efficiency but also its cytotoxicity and in vivo retention time from 1 to 25 days compared to DEAE-dextran. This suggests that DEAE-dextran is a more effective adjuvant for inactivated virus vaccines.

[0004] Solution to the Problem: In innate immunity, Toll-like receptors (TLRs) are pattern recognition receptors that recognize viral and bacterial components and transmit defense signals to lymphocytes through the induction of type I interferon (IFN) and inflammatory cytokine production and dendritic cell maturation. There are 10 types of defense signals in humans, but the signals induced by TLR activation are determined by the combination of each TLR and downstream adaptor molecules, resulting in different final cellular responses. Inducing the desired lymphocyte response requires the selection of optimal TLR ligands, which is a strategy for adjuvant development. In recent years, it has become clear that TLRs recognize not only microbial components but also endogenous molecules, drawing attention to their role in autoimmune and inflammatory diseases. This invention considers the function of TLRs as a link between innate and adaptive immunity. Both DEAE-D and the improved DDMC, compounds with α1-6 sugar chains, are TLR7 agonists, which are thought to lead to dendritic cell activation. Furthermore, DDMC has a high endocytosis effect, making it a TLR7 agonist. Furthermore, due to the RES effect, the antigen presentation period is as long as 25 days (compared to half a day for DEAE-D). We believe that the development of adjuvants is crucial in vaccine development. The main reason for this is the TLR7 agonist properties of the cationic derivative-olefin of water-soluble linear polysaccharide of the present invention, which specifically activates TLR7. Currently, known drug delivery methods include a formulation in which paclitaxel is encapsulated in a cationic liposome (Patent Document 2 JP, 2006-517594, A), and a drug-encapsulating polymeric micelle in which a drug such as an anticancer drug is encapsulated in a polymeric micelle made of a block copolymer having a hydrophilic region and a hydrophobic region (Patent Documents 3 to 5).

[0005] Prior Art Literature Patent Document 2: JP-A-2006-517594 Patent Document 3: JP-A-2001-226294 Patent Document 4: JP-A-2005-336402 Patent Document 5: JP-A-2007-023023 These target anionically charged endothelial cells of tumor blood vessels, inhibiting angiogenesis and demonstrating antitumor effects, but side effects are not completely reduced. In genetic engineering, when a gene is transplanted into another organism, a vehicle, or a carrier, is required to carry the gene, which is called a vector. Currently, various viruses, bacterial plasmids, and bacterial infective phages are used as vectors. By attaching the gene to these vectors and infecting them, the gene can be delivered to the bacteria. Using a complex of a cationic polymer and nucleic acid (DNA, RNA) instead of a plasmid or phage allows the nucleic acid of the complex to be delivered directly to pre-prepared cells. Unlike potentially dangerous viral vectors, these non-viral vectors are safe and reliable because they are artificial. In genetic engineering, when a gene is transferred to another organism, a carrier, or a vehicle, is required to deliver the gene; this is called a vector. By using taxanes instead of nucleic acids and complexing them with cationic polymers, the taxane complex can be delivered directly to pre-prepared cells. Cationic polysaccharides are promising cationic polymers because the complex must be able to pass through the cell membrane. This ability depends on the reaction between the positive charge of the complex resulting from the cationic polysaccharide and the negative charge on the cell membrane surface, as well as the interaction between the polysaccharide and the complex on the cell membrane surface. Polymer biocompatibility is important for the permeability of cell membranes as drug delivery systems (DDS) materials. Furthermore, to confer biocompatibility, the cationic polymer drug delivery (DDS) material used must have hydrophobic and hydrophilic domains. Specifically, it is important to form a latex consisting of a copolymer of a cationic polysaccharide such as DEAE-dextran and a vinyl monomer, so that it has both a hydrophobic portion due to the polymerized portion of the vinyl monomer and a hydrophilic portion due to the cationic polysaccharide.That is, the biocompatibility of the resulting complex latex with hydrophobic / hydrophilic domains is important, but we unexpectedly discovered that by further copolymerizing cationic polysaccharides with vinyl monomers, the supramolecular reaction with nucleic acids and taxanes can be enhanced, improving the low uptake rate and efficacy of nucleic acids and taxanes into cells of cationic polysaccharide drug delivery (DDS) materials.

[0006] Effect of the Invention: Currently, global development of COVID-19 vaccines is focused on viral vectors and mRNA vaccines. The first institutions to begin clinical trials are Oxford University and Moderna in the United States. The vectors used are adeno-associated viral vectors (AAV vectors) and LNP liposome vectors (lipofection), respectively. Viral vectors are generally suspected of pathogenicity, and their immunogenicity leads to the generation of neutralizing antibodies, making repeated administration contraindicated. Transfection using lipid liposomes involves many unstable factors, such as limiting micelles, making in vivo use difficult. Non-viral vectors also pose stability challenges in vivo. While most viral vectors integrate into host chromosomes and mRNA is stably expressed in transfected cell lines, they induce acquired immunity through sensitization, making them unsuitable for in vivo use. A copolymer (DDMC) grafted with DEAE-dextran and methyl methacrylate (MMA) not only exhibits improved transfection efficiency, but also reduced cytotoxicity and improved in vivo residence time from 1 to 25 days compared to DEAE-dextran. This indicates that DEAE-dextran is an improved and more effective adjuvant for inactivated virus vaccines. For example, Figure 1 shows a study of DEAE-dextran in rhesus monkeys administered a Venezuelan equine encephalitis (VEE) inactivated virus vaccine (IVEE) with and without the addition of DEAE-dextran. Antibody titers were determined by plaque reduction neutralization tests, and the geometric mean titer (y-axis) was maintained 400-fold when DEAE-dextran was added. In other words, with 5 mg / DEAE-D, the maximum peak was maintained for 72 days after a single vaccination (10 days without DEAE-D). When DEAE-dextran-MMA copolymer (DDMC) was used, the titer was maintained about 4-fold (Table 1). Figure 2 shows the IgM-IgG pattern in this case. (A) shows the results of inoculations with IVEEE alone, and (B) with IVEEE and DEAE-D, respectively, and the titers of serum antibodies, IgM, and IgG, were examined. IgM rises first, followed by IgG, but with IVEEE alone, the titer is relatively low and biphasic; the IgG that rises after IgM also quickly declines. In contrast, the titers are relatively high in the inoculations with IVEEE and DEAE-D, and the IgG that rises maintains a high titer. DEAE-dextran was an excellent adjuvant, but unfortunately, its cytotoxicity was not improved and it was not put to practical use. Compared to DEAE-dextran, DDMC has improved cytotoxicity, and its long residence time is expected to provide an adjuvant effect four times greater than DEAE-dextran. The present invention is a product obtained by grafting an olefin-containing monomer in water onto a cationic derivative of a water-soluble linear polysaccharide containing hydroxyl groups. The structures of the corresponding olefin-containing monomer growing chain and the resulting copolymer chain are shown as chemical structural formulas (Chemical Formula 3), (Chemical Formula 2), and (Chemical Formula 1), respectively. It is clear that the respective bonding relationships are covalent bonds formed by chain transfer of the olefin monomer double bond, which is initiated by radical generation due to abstraction of the hydrogen atom (as a proton by oxidation of the initiator) from the hydroxyl group of the water-soluble linear polysaccharide cationic derivative. This was carried out using tetravalent cerium ions as an initiator, without the use of any surfactants, making it extremely useful for antibody-adsorbed latex diagnostic reagents, etc., without interference. The purpose of the water-soluble linear polysaccharide cationic derivative-olefin monomer graft copolymer latex of the present invention is to be used as an antibody-adsorbing latex diagnostic agent, as is clear from the claims of Patent Application No. 248476 of 1984. That is, the main and essential part of the invention is to produce a latex polymerization product useful as an immunoassay diagnostic material by graft polymerizing an olefin-containing monomer onto a water-soluble polymer having hydroxyl groups in water, and the same purpose can also be achieved by graft copolymers of olefin-containing monomers such as polysaccharides.These graft copolymers, which grow at the interface between a solvent and a solute, are known as "soap-free" materials and are useful for a wide variety of applications. They have attracted particular attention as immunoassay materials, filtration membranes, and biomaterials. Their hydrophilic properties have led to their potential applications in artificial kidney membranes, components, vascular grafts, contact lenses, and gene carriers. However, the interfacial activity of the hydrophobic and hydrophilic domains produced by these latex polymerization products is particularly important for their affinity and permeability to cell membrane surfaces. Furthermore, they enhance supramolecular reactions with compounds such as taxanes, making them unexpectedly promising materials for drug delivery systems (DDS).

[0007] Figure 1 shows differential scanning calorimetry (DSC) curves for a complex of DEAE (diethylaminoethyl)-dextran-MMA copolymer and paclitaxel, and for paclitaxel. Figure 2 is a graph showing infrared absorption spectra for a complex of DEAE (diethylaminoethyl)-dextran-MMA copolymer and paclitaxel, and for paclitaxel. Figure 3 shows the results of administering Venezuelan equine encephalitis (VEE) inactivated virus vaccine (IVEE) to rhesus monkeys with and without the addition of DEAE-dextran. Figure 4 shows the immunoglobulin M-immunoglobulin G pattern analysis under the conditions shown in Figure 3.

[0008] The cationic derivative of linear polysaccharide-olefin monomer graft copolymer of this invention is described in detail below. The cationic derivative of linear polysaccharide-olefin monomer graft copolymer is obtained via the following step (2). It has been found that this polymer forms a complex with an anticancer drug via step (3), which is then taken up by cancer cells and causes cell death. (1) Preparation of a cationic derivative of linear polysaccharide. When present in solid form, the unit formula of a cationic derivative of linear polysaccharide is represented by the following formula: [Chemical Formula 4] (Chemical Formula 4). The hydroxyl groups of this cationic derivative of linear polysaccharide may be partially substituted with acidic groups such as carboxymethyl groups or sulfate ester groups via ether bonds, or partially substituted with alkyl groups, and may contain a cationic functional group represented by X in the formula (4). These cationic derivatives of linear polysaccharides are typically obtained by the Schotten-Baumann reaction in an alkaline solution between the hydroxyl groups of the linear polysaccharide and a chlorine compound of the above cationic substituent, represented by XCl. The linear polysaccharides referred to here are those that can be industrially produced by fermentation, such as dextran and pullulan. The olefin-containing monomers that can be graft-polymerized are those whose general formula is represented by the following repeating unit at the time of polymerization: [Chemical Formula 3] Specifically, alkyl esters of α,β-unsaturated acids such as acrylic acid and methacrylic acid, lower alkyl-substituted cyclohexyl esters such as cyclohexyl esters, 2-hydroxyethyl esters, 2-hydroxypropyl esters, 2-hydroxybutyl esters; acrylamide, methacrylamide, acryl- or methacryl-dimethylamide, C-substituted α,β-unsaturated acids such as acrylic acid and methacrylic acid, and the like. １ ~C ３ Aminoalkyl esters of C １ ~C ３dialkylaminoalkyl esters, glycidyl esters, tetrahydrofurfuryl esters, benzyl esters, polyethylene glycol monoesters; nitrile groups of α,β-unsaturated acids such as acrylonitrile and methacrylonitrile; vinyl alcohol, methyl vinyl alcohol, dimethyl vinyl alcohol; vinyl alcohols such as vinyl acetate, vinyl propionate, and vinyl butyrate, and methyl-substituted vinyl alcohols thereof; １ ~C ３Possible materials include alkyl esters; styrene, vinyltoluene; vinylpyrrolidone; and vinylmethylpyrrolidone. (2) Preparation of Graft Copolymers: The reaction is typically carried out in an aqueous solution. The olefin-containing monomer is added to an aqueous solution of a cationic derivative of linear polysaccharide, followed by the addition of an initiator. Tetravalent cerium salts, tetravalent manganese salts, and ferric salts in hydrogen peroxide are commonly used as initiators, but radical initiators such as potassium persulfate (KPS), azobisisobutylnitrile (AIBN), and benzoyl peroxide (BPO) are also used. The reaction temperature can be selected from a wide range, from room temperature to 80°C. If necessary, the reaction can be continued with nitrogen substitution. The bond relationship between the two is a covalent bond formed by chain transfer of the double bond of the olefin-containing monomer, which is initiated by radical generation due to proton abstraction from the water-soluble cationic derivative of linear polysaccharide. This reaction produces a latex product. This latex polymer can generally be easily formed into a film by casting with water, alcohol, or an organic solvent such as acetone or tetrahydrofuran. Alternatively, after adding an excess of an insoluble solvent such as alcohol to obtain a precipitate, a molded product can be easily produced by heat pressing or other methods. For the above-mentioned purposes, the ratio of backbone polymer to graft polymer in the graft polymer or the polymerization ratio can be selected in various ways depending on the purpose. The polymerization rate of graft polymerization is determined by the graft ratio (%), which is defined as: graft ratio (%) = (amount of graft-polymerized monomer / amount of backbone polymer in the graft copolymer) × 100. In the present invention, the olefin compound forms the graft chain, and a graft ratio in the range of 2% to 5000% is considered appropriate. It has been repeatedly stated that the present invention is a product obtained by graft polymerizing a monomer having an olefin in water onto a cationic derivative of a water-soluble linear polysaccharide having a hydroxyl group, and the structure of the resulting copolymer chain is represented by formula [Chemical Formula 1], which is composed of formulas (Chemical Formula 2) and (Chemical Formula 3), as set forth as a chemical structural formula in the claims. The respective bond relationships are covalent bonds formed by the addition of radicals to the double bonds of the monomer having an olefin, generated by abstraction of a proton from the hydroxyl group of the water-soluble linear polysaccharide cationic derivative.(3) Complexes of Cationic Polysaccharide Copolymers and Nucleic Acids (DNA, RNA) In a gene delivery system using the cationic polysaccharide copolymer of the present invention as a vector, the first step is the formation of a complex between the cationic polysaccharide copolymer of the present invention and a nucleic acid. Specifically, the formation of a complex between a nucleic acid and a copolymer obtained by grafting an olefin monomer onto a cationic partially substituted linear polysaccharide is an important first step in the gene delivery system. Specifically, the complex is a complex between a nucleic acid and a copolymer obtained by grafting an olefin monomer onto a cationic partially substituted linear polysaccharide, characterized by reacting it with deoxyribonucleic acid (DNA) having deoxyribonucleotides as repeating units represented by Chemical Formula 5. The complex is also a complex between a nucleic acid and a copolymer obtained by grafting an olefin monomer onto a cationic partially substituted linear polysaccharide, characterized by reacting it with ribonucleic acid (RNA) having ribonucleotides as repeating units represented by Chemical Formula 6. Formulas (5) and (6) show the structure of a nucleotide, consisting of a purine or pyrimidine base represented by B in the formula, a sugar, and phosphate. Specifically, B in the formula refers to two purine bases, adenine and guanine, and three pyrimidine bases, cytosine, uracil, and thymine. For deoxyribonucleotides, the repeating unit of DNA, adenine and guanine are selected, and cytosine and thymine are selected as pyrimidine bases. For ribonucleotides, the repeating unit of RNA, adenine and guanine are selected, and cytosine and uracil are selected as pyrimidine bases. The sugars constituting deoxyribonucleotides are deoxyribose, and for ribonucleotides, ribose. The cationic polysaccharide copolymer of the present invention and the phosphate moiety of nucleic acids (DNA, RNA) whose repeating units are the nucleotides represented by formulas (5) and (6) easily bind via electrostatic Coulomb force to form a cationic polysaccharide copolymer-nucleic acid complex (PIC). This complex formation is the critical first step in the gene delivery system.For this reason, it is believed that the cationic polymer vector used must have a hydrophobic / hydrophilic domain, and specifically, it is believed to be important to form a latex consisting of a copolymer of a cationic polysaccharide such as DEAE-dextran and a vinyl monomer, which has both a hydrophobic portion due to the polymerized portion of the vinyl monomer and a hydrophilic portion due to the cationic polysaccharide. This is thought to enhance the reaction with nucleic acids and increase the probability of easy introduction into cells by endocytosis and uptake into endosomes (transport endoplasmic reticulum), thereby improving the low DNA and RNA transfer rates of cationic polysaccharide vectors such as DEAE-dextran into cells and cell nuclei. Three types of DEAE (diethylaminoethyl)-dextran-MMA copolymers, Examples 1, 2, and 3, were prepared using the same procedure as in Example 1 for the hydrochloride of DEAE (diethylaminoethyl)-dextran copolymer. Specifically, 2 g of DEAE (diethylaminoethyl)-dextran hydrochloride, which had a nitrogen content of 3% and was based on dextran with an average molecular weight of 500,000, was dissolved in 50 ml of water. Then, to Examples 1, 2, and 3, 3 ml, 4 ml, and 6 ml of methyl methacrylate (MMA) were added, respectively. After thoroughly purging the air in the reaction solution and reaction vessel with nitrogen gas, 100 mg of ceric ammonium nitrate dissolved in 15 ml of 0.1 N nitric acid, in which the dissolved air had been purged with nitrogen gas, was added with vigorous stirring to initiate the reaction. The reaction was carried out at 30° C. for 2 hours, producing a latex. The reaction was terminated using 3 ml of a 1% hydroquinone solution as a terminator, followed by dialysis in water to remove unreacted materials and the initiator, yielding a DEAE-dextran-MMA copolymer latex. This product is extremely useful as a recombinant vector, and test results are shown below. The test method was performed according to (4) Protocol B in the "Best Mode for Carrying Out the Invention" section. 293 cells (human embryonic kidney cells) were transformed with the pCMV-β-Gal plasmid (Invitrogen) and incubated at 37°C for 50 hours, after which expression efficiency was examined. Gene expression was confirmed by β-galactosidase staining (X-gal staining).Transfection was evaluated based on the area of ​​the stained portion. The DEAE-dextran-MMA copolymer in Example 1 with a 150% weight gain showed a value of 3, and the DEAE-dextran-MMA copolymer in Example 2 with a 200% weight gain showed a value of 3, with the starting DEAE-dextran hydrochloride being taken as 1. Here, the weight gain is the ratio of the weight of DEAE-dextran used to the weight of MMA added. That is, weight gain = weight of MMA added / weight of DEAE-dextran hydrochloride used. As in Example 3, when a solution of cationic polysaccharide copolymer was added to a solution of DNA derived from salmon sperm, complete precipitation occurred, resulting in the formation of a complex of cationic polysaccharide copolymer and DNA. When a similar procedure was performed with cationic polysaccharide, complete precipitation took a much longer time than with cationic polysaccharide copolymer. Specifically, the precipitation time for the DEAE-dextran-MMA copolymer / DNA complex was 0.5 hours at a weight gain of 300%, 1 hour at a weight gain of 200%, and 2 hours at a weight gain of 150%. On the other hand, when a similar procedure was performed using the raw material DEAE (diethylaminoethyl)-dextran hydrochloride salt from Example 1, complete precipitation required 96 hours. Similarly, as in the previous example, when a solution of cationic polysaccharide copolymer was added to a yeast-derived RNA solution, complete precipitation occurred, yielding a cationic polysaccharide copolymer / RNA complex. In this case, a similar procedure using cationic polysaccharides required a much longer time for complete precipitation compared to cationic polysaccharide copolymers. These findings indicate that cationic polysaccharide copolymers have a higher reactivity with nucleic acids than cationic polysaccharides. This complex permeates the cell membrane, is easily introduced into cells by endocytosis, and is taken up into endosomes (transport endoplasmic reticulum). The complex is then released from the endosome into the cell chamber, where it induces RNA interference (RNA) and transcription / gene expression. In eukaryotic cells, it eventually penetrates the nuclear membrane to reach the nucleus, where it accumulates as a complex. Within the nucleus, nucleic acids (DNA, RNA) are separated from the complex, facilitating transcription / gene expression. (4) Transfection with Cationic Polysaccharide Copolymer Vectors Protocol A 1. Culture the transformed cells in a 100 mm Petri dish one day before transfection.The cell density of the transformed cells in a 100 mm culture dish was 8 × 10 . ５ In this study, COS-1 cells (African green monkey kidney cells transformed with SV40) were cultured in DMEM medium (containing 10% fetal bovine serum) at 37°C and 5% CO ２2. Prepare a wash solution: 1x PBS (phosphate-buffered saline (Dulbecco & Vogt (1954))). Heat this wash solution, 1x PBS, and a cationic polysaccharide copolymer solution, such as a DEAE-dextran copolymer solution, to 37°C. 3. Dilute to 1x PBS using 10x PBS. Prepare a transfection solution using the following procedure: Using a 100 mm culture dish, dilute 20 μg of a plasmid encoding luciferase (pGL3-Control (Promega, Madison, WI)) as recombinant DNA into a sterile tube with 1x PBS to a total volume of 540 μl. Then, add 28 μl of cationic polysaccharide copolymer solution (10 mg / ml cationic polysaccharide). Tap the sterile tube with your fingers to mix well. 4. Remove the culture medium from the culture dish containing the transformed COS-1 cells. For 100 mm culture dishes, wash the transformed cells twice with 10 ml of 1x PBS (wash solution). 5. Add the DNA-cationic polysaccharide copolymer complex solution prepared in step 3 to the transformed cells. Stir the transformed cells in the culture dish to ensure thorough distribution. 6. Incubate the culture dish at 37°C for 30 minutes. Rock the culture dish occasionally. 7. For 100 mm culture dishes, add 6 ml of growth medium (DMEM medium) to the culture dish. Incubate the culture dish at 37°C for 2 hours and 30 minutes to allow cytotoxicity to develop. The growth medium was replaced and the cells were further incubated for 48-72 hours at 37° C. 8. Expression Efficiency The expression efficiency of COS-1 cell transformation was determined by the incorporated expressed luciferase activity.Specifically, a luciferase assay kit (Promega, Madison, Wis.) was used to determine the TLU value (Turner light units (TLU)) using a Turner modol TD-20e luminometer (Turner Designs, Sunnyvale, Calif.), and the TLU value for each sample was set to 1, relative to that for DEAE-dextran hydrochloride (Mw 500,000, nitrogen content 5%). Protocol B: 1. Culture the transformed cells in a 35-mm petri dish one day before transfection. The cell density of the transformed cells in the 35-mm culture dish was 8 x 10. ５ 293 cells (human fetal kidney cells) were cultured in DMEM medium (containing 10% fetal bovine serum) under 5% CO ２1. Prepare a washing solution: 1x PBS (phosphate-buffered saline (Dulbecco & Vogt (1954))). Heat this washing solution, 1x PBS solution, and the basic polysaccharide copolymer solution to 37°C. 2. Dilute to 1x PBS solution using 10x PBS solution. Prepare the transfection solution using the following procedure. Using a 35mm culture dish, dilute 10µg of pCMV-β-Gal plasmid (Invitrogen) as recombinant DNA into a sterile tube with 1x PBS solution to make 270µl. Then add 14µl of each basic polysaccharide copolymer solution (10mg / ml as basic polysaccharide). Tap the sterile tube with your fingers to mix well. 4. Remove the culture medium from the culture dish containing the transformed 293 cells (human embryonic kidney cells). For 35 mm culture dishes, wash the transformed cells twice with 2 ml of 1x PBS (washing solution). 5.3. Add the DNA-basic polysaccharide copolymer solution prepared in step 3 to the transformed cells. Stir the transformed cells in the culture dish to ensure thorough distribution. 6. Incubate the culture dish at 37°C for 30 minutes. Occasionally shake the culture dish. 7. For 35 mm culture dishes, add 3 ml of growth medium (DMEM medium) to the culture dish. Incubate the culture dish at 37°C for 2 hours and 30 minutes to allow cytotoxicity to be expressed. Replace the growth medium and incubate at 37°C for a further 48-72 hours. 8. Expression Efficiency: Confirm gene expression by β-galactosidase staining (X-gal staining). Transfection was evaluated based on the area of ​​the stained portion, with DEAE-dextran hydrochloride set at 1. (4) Cationic Polysaccharide Copolymer Adjuvant for Inactivated Virus Vaccines. A copolymer (DDMC) grafted with methyl methacrylate (MMA) onto DEAE-dextran not only exhibited high transfection efficiency, but also reduced cytotoxicity and an improved in vivo retention time of 1 to 25 days compared to DEAE-dextran. This indicates that DEAE-dextran is an improved and effective adjuvant for inactivated virus vaccines.For example, in a study using DEAE-dextran, Figure 1 shows the results of administering an inactivated Venezuelan equine encephalitis (VEE) virus vaccine (IVEE) to rhesus monkeys with and without the addition of DEAE-dextran. Antibody titers were measured using a plaque reduction neutralization test. The geometric mean titer (y-axis) was maintained 400-fold when DEAE-dextran was added. In other words, with 5 mg / mL DEAE-D, the maximum peak was maintained for 72 days after a single vaccination (10 days without DEAE-D). With DDMC, the titer was maintained at more than four times the normal level.

[0009] 2 g of DEAE (diethylaminoethyl)-dextran hydrochloride with a nitrogen content of 5% and an average molecular weight (Mw) of 500,000 was dissolved in 50 ml of water, followed by the addition of 8 ml of methyl methacrylate (MMA). After thoroughly purging the air in the reaction solution and reaction vessel with nitrogen gas, 100 mg of ceric ammonium nitrate dissolved in 15 ml of 0.1 N nitric acid (in which the dissolved air had been purged with nitrogen gas) was added to initiate the reaction. The reaction was carried out at 30°C for 2 hours, resulting in the production of a latex. The reaction was terminated using 3 ml of a 1% hydroquinone solution as a terminator. The reaction solution was then poured into a three-fold volume of methanol to obtain a precipitate. This precipitate was thoroughly washed with hot water, centrifuged, and dried under reduced pressure at 50°C. The dried product was then placed in a Soxhlet extractor and subjected to acetone extraction for 24 hours, yielding 1.5 g of DEAE (diethylaminoethyl)-dextran-MMA copolymer hydrochloride. Nitrogen content: 1.7%, grafting rate: 200%, yield relative to DEAE-dextran: 25%. This product is insoluble in water, a good solvent for DEAE-dextran hydrochloride, and in acetone, a good solvent for polymethyl methacrylate. The infrared absorption spectrum of this product shows a carbonyl group absorption at a wavenumber of 1730 cm, which is not seen in DEAE-dextran hydrochloride. −１ It can be seen nearby.

[0010] After carrying out the same reaction as in Example 1, the latex reaction solution was not poured into methanol, but was instead dialyzed in water to remove unreacted materials and the initiator, yielding a DEAE-dextran-MMA copolymer latex. This product is useful as a recombinant vector, and the test results are shown below. The test method was performed according to (4) Protocol A in the "Best Mode for Carrying Out the Invention" section. After incubating COS-1 cells at 37°C for 50 hours, the expression efficiency was examined. Specifically, the expression efficiency of the transformation, which determines the effectiveness of the vector, was determined by the expressed luciferase activity of the COS-1 cells. When compared to the sample in Example 2, which had a 5% nitrogen content and an average molecular weight of 500,000, the expressed luciferase activity was five times higher.

[0011] The DEAE (diethylaminoethyl)-dextran-MMA copolymer latex obtained in Example 2 was adjusted to a 10 mg / ml solution in terms of DEAE (diethylaminoethyl)-dextran. When 2 ml of this solution was added to 1 ml of a salmon sperm-derived DNA solution (20 mg / ml), complete precipitation occurred in 0.4 hours, yielding a complex of 20 mg of DEAE (diethylaminoethyl)-dextran-MMA copolymer and DNA. When a similar procedure was performed with the raw material DEAE (diethylaminoethyl)-dextran hydrochloride, complete precipitation took 96 hours. Figure 1 shows the infrared absorption spectrum of the product itself. Wavenumber 1000 cm −１ From 1100cm −１ The absorption of the pyranose ring derived from DEAE (diethylaminoethyl)-dextran is observed around 1220 cm −１ Absorption due to the stretching vibration of P-0 derived from DNA is observed around 1730 cm −１ Near this point, absorption of the carbonyl group C=0 derived from MMA is observed.

[0012] The DEAE (diethylaminoethyl)-dextran-MMA copolymer latex obtained in Example 2 was adjusted to a 10 mg / ml solution in terms of DEAE (diethylaminoethyl)-dextran. When 2 ml of this solution was added to 1 ml of yeast-derived RNA solution (20 mg / ml), complete precipitation occurred within 4 hours, yielding a complex of 10 mg of DEAE (diethylaminoethyl)-dextran-MMA copolymer and RNA. When a similar procedure was performed using the raw material DEAE (diethylaminoethyl)-dextran hydrochloride, complete precipitation took 144 hours. Figure 2 shows the infrared absorption spectrum of the product itself. Wavenumber 1000 cm −１ From 1100cm −１ The absorption of the pyranose ring derived from DEAE (diethylaminoethyl)-dextran is observed around 1230 cm −１ The absorption due to the stretching vibration of P-0 derived from RNA is observed around 1730 cm −１ Near this point, absorption of the carbonyl group C=0 derived from MMA is observed.

[0013] 4 g of DEAE (diethylaminoethyl)-pullulan hydrochloride with a nitrogen content of 4% and an average molecular weight (Mw) of 200,000 was dissolved in 80 ml of water, followed by the addition of 10 ml of methanol and 35 ml of styrene monomer. The reaction solution and the air in the reaction vessel were thoroughly purged with nitrogen gas, and then 200 mg of ceric ammonium nitrate dissolved in 30 ml of 0.1 N nitric acid, in which the dissolved air had been purged with nitrogen gas, was added to initiate the reaction while stirring vigorously. The reaction was carried out at room temperature for 1 hour, resulting in the formation of a latex. The reaction was terminated using 3 ml of a 1% hydroquinone solution as a terminator. Subsequent purification and drying steps were carried out in the same manner as in Example 1, yielding 7 g of DEAE (diethylaminoethyl)-pullulan-styrene copolymer hydrochloride. Nitrogen content: 0.92%, grafting rate: 350%, yield relative to DEAE-pullulan: 38%.

[0014] After carrying out the same reaction as in Example 5, the latex reaction solution was not poured into methanol, but was instead dialyzed in water to remove unreacted materials and the initiator, yielding a DEAE-pullulan-styrene copolymer latex. This was useful as a recombinant vector. Following the same procedure as in Example 2, the expressed luciferase activity of the latex solution was 1.5 times that of the DEAE-dextran hydrochloride in Example 2.

[0015] The DEAE (diethylaminoethyl)-pullulan-styrene copolymer latex obtained in Example 6 was prepared into a 10 mg / ml solution in terms of DEAE (diethylaminoethyl)-pullulan. 2 ml of this solution was added to 1 ml of a salmon sperm DNA solution (20 mg / ml), resulting in complete precipitation within 2.5 hours, yielding 12 mg of a complex of DEAE (diethylaminoethyl)-pullulan-styrene copolymer and DNA.

[0016] The DEAE (diethylaminoethyl)-pullulan-styrene copolymer latex obtained in Example 6 was prepared into a 10 mg / ml solution in terms of DEAE (diethylaminoethyl)-pullulan. 2 ml of this solution was added to 1 ml of a yeast-derived RNA solution (20 mg / ml), resulting in complete precipitation within 5 hours, yielding 9 mg of a complex of DEAE (diethylaminoethyl)-pullulan-styrene copolymer and RNA.

[0017] 4 g of AE (aminoethyl)-dextran hydrochloride with a nitrogen content of 5% and an average molecular weight (Mw) of 40,000 was dissolved in 90 ml of water, followed by the addition of 5 ml of methanol and 20 ml of butyl methacrylate. The air in the reaction solution and the reaction vessel was thoroughly purged with nitrogen gas, and then 50 mg of ceric ammonium nitrate dissolved in 15 ml of 0.1 N nitric acid, in which the dissolved air had been purged with nitrogen gas, was added while stirring to initiate the reaction. The reaction was carried out at room temperature for 30 minutes, resulting in the formation of a latex. The reaction was terminated with 3 ml of a 1% hydroquinone solution as a terminator. Subsequent purification and drying steps were carried out as in Example 1, yielding 6 g of AE (aminoethyl)-dextran-butyl methacrylate copolymer hydrochloride. Nitrogen content: 1.3%, grafting rate: 300%, yield relative to AE-dextran: 38%. This product is insoluble in water, a good solvent for AE-dextran hydrochloride, and in acetone, a good solvent for polybutyl methacrylate.

[0018] After carrying out the same reaction as in Example 9, the latex reaction solution was not poured into methanol but was instead dialyzed in water to remove unreacted materials and the initiator, yielding an AE (aminoethyl)-dextran-butyl methacrylate copolymer latex. This was useful as a recombinant vector. Following the same procedure as in Example 2, the expressed luciferase activity of the latex solution was 1.5 times that of the DEAE-dextran hydrochloride in Example 2.

[0019] The AE (aminoethyl)-dextran-butyl methacrylate copolymer latex obtained in Example 10 was prepared into a solution of 10 mg / ml AE (aminoethyl)-dextran. 2 ml of this solution was added to 1 ml of a salmon sperm-derived DNA solution (20 mg / ml), resulting in complete precipitation within 3 hours, yielding 12 mg of a complex of AE (aminoethyl)-dextran-butyl methacrylate copolymer and DNA.

[0020] The AE (aminoethyl)-dextran-butyl methacrylate copolymer latex obtained in Example 10 was prepared into a solution of 10 mg / ml AE (aminoethyl)-dextran. 2 ml of this solution was added to 1 ml of a yeast-derived RNA solution (20 mg / ml). Complete precipitation occurred within 5 hours, yielding 10 mg of a complex of AE (aminoethyl)-dextran-butyl methacrylate copolymer and RNA.

[0021] 4 g of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan hydrochloride with a nitrogen content of 3% and an average molecular weight (Mw) of 30,000 was dissolved in 100 ml of water, followed by the addition of 30 ml of methyl acrylate monomer. After thoroughly purging the air in the reaction solution and reaction vessel with nitrogen gas, 200 mg of ceric ammonium nitrate dissolved in 20 ml of 0.1 N nitric acid in which the dissolved air had been purged with nitrogen gas was added to initiate the reaction. The reaction was carried out at room temperature for 1 hour, resulting in the formation of a latex. The reaction was terminated with 4 ml of a 1% solution of hydroquinone as a terminator. Subsequent purification and drying steps were carried out in the same manner as in Example 1, yielding 2 g of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer hydrochloride. Nitrogen content: 1.2%, grafting rate: 150%, yield relative to HPTMA-pullulan: 20%.

[0022] After carrying out the same reaction as in Example 13, the unreacted materials and initiator were removed by dialysis in water to obtain a HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer latex. This was useful as a recombinant vector. Following the same procedure as in Example 2, the expressed luciferase activity of the latex solution was 1.1 times that of the DEAE-dextran hydrochloride in Example 2.

[0023] The HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer latex obtained in Example 14 was prepared into a 10 mg / ml solution in terms of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan. 2 ml of this solution was added to 1 ml of a salmon sperm DNA solution (20 mg / ml), resulting in complete precipitation within 5 hours, yielding 10 mg of a complex of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer and DNA.

[0024] The HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer latex obtained in Example 14 was prepared into a 10 mg / ml solution calculated as HPTMA (2-hydroxypropyltrimethylammonium)-pullulan. 2 ml of this solution was added to 1 ml of a yeast-derived RNA solution (20 mg / ml). Complete precipitation occurred within 6 hours, yielding 9 mg of a complex of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer and RNA.

[0025] 2 g of TEAE (triethylaminoethyl)-dextran hydrochloride with a nitrogen content of 2% and an average molecular weight (Mw) of 300,000 was dissolved in 50 ml of water, 15 ml of methyl acrylate (MA) was added, and the air in the reaction solution and reaction vessel was thoroughly purged with nitrogen gas. Then, while stirring vigorously, 250 mg of ceric ammonium nitrate dissolved in 15 ml of 0.1 N nitric acid in which the dissolved air had been purged with nitrogen gas was added to initiate the reaction. The reaction was carried out at 30°C for 2 hours to produce latex. The reaction was terminated using 3 ml of a 1% hydroquinone solution as a terminator. The reaction solution was then poured into a three-fold volume of methanol to obtain a precipitate. This precipitate was thoroughly washed with hot water, centrifuged, and dried under reduced pressure at 50°C. The dried product was then placed in a Soxhlet extractor and subjected to acetone extraction for 24 hours to obtain 2 g of TEAE (triethylaminoethyl)-dextran-MA copolymer hydrochloride. Nitrogen content: 0.7% Grafting rate: 185% Yield relative to TEAE-dextran: 35% This product is insoluble in water, which is a good solvent for TEAE-dextran hydrochloride, and in acetone, which is a good solvent for methyl acrylate.

[0026] After carrying out the same reaction as in Example 17, the latex reaction solution was not poured into methanol, but was instead dialyzed in water to remove unreacted materials and the initiator, yielding a TEAE-dextran-MMA copolymer latex. This product is useful as a recombinant vector, and the test results are shown below. The test method was performed according to the procedure in (4) of the "Best Mode for Carrying Out the Invention" section. That is, the expression efficiency of transformation, which determines the effectiveness of the vector, was determined by the expressed luciferase activity in COS-1 cells. When compared to the sample of Example 18, which had a value of 1 for DEAE-dextran hydrochloride with a nitrogen content of 5% and an average molecular weight of 500,000, the expressed luciferase activity was three times higher.

[0027] The TEAE (triethylaminoethyl)-dextran-MA copolymer latex obtained in Example 18 was adjusted to a 10 mg / ml solution in terms of TEAE (diethylaminoethyl)-dextran. 2 ml of this solution was added to 1 ml of a salmon sperm-derived DNA solution (20 mg / ml), resulting in complete precipitation within 3 hours, yielding 15 mg of a complex of TEAE (triethylaminoethyl)-dextran-MA copolymer and DNA.

[0028] The TEAE (triethylaminoethyl)-dextran-MA copolymer latex obtained in Example 18 was adjusted to a 10 mg / ml solution in terms of TEAE (triethylaminoethyl)-dextran. 2 ml of this solution was added to 1 ml of a yeast-derived RNA solution (20 mg / ml). Complete precipitation occurred within 5 hours, yielding 8 mg of a complex of TEAE (triethylaminoethyl)-dextran-MA copolymer and RNA.

[0029] The DEAE (diethylaminoethyl)-dextran-MMA copolymer latex obtained in Example 2 was prepared into a 10 mg / ml solution in terms of DEAE (diethylaminoethyl)-dextran. This solution was administered at a concentration of 5 mg / kg in terms of DEAE-D, and Venezuelan equine encephalitis (VEE) inactivated virus vaccine (IVEE) was administered to rhesus monkeys under the same conditions as those shown in Figure 1. The effectiveness of the vaccine was examined under the same conditions as those shown in Figure 1 for the addition of DEAE-dextran. The results were as shown in Table 1.

[0030] Most drug delivery materials currently in practical use have poor cationicity and are difficult to autoclave, resulting in problems with targeting and safety. Drug delivery materials such as the cationic polysaccharide copolymer of the present invention are easily autoclaved, allowing for stable use. The cationic polysaccharide copolymer of the present invention easily binds to the hydrophobic moiety of inactivated viruses or nucleic acids through hydrophobic binding forces, forming a supramolecular cationic polysaccharide copolymer-nucleic acid complex. This supramolecular complex formation is the critical first step in the delivery system for inactivated viruses or nucleic acids. The hydrophobic and hydrophilic domains enhance the supramolecular reaction with inactivated viruses or nucleic acids, and the cationic nature of the copolymer facilitates its introduction into cells via endocytosis, increasing the probability of incorporation into endosomes (transport vesicles). It also exhibits the property of being a TLR7 agonist, specifically activating TLR7 during endocytosis. Above all, the cationic polysaccharide copolymer of the present invention is chemically stable. For example, the solution is sufficiently resistant to autoclaving at 120°C for 15 minutes. To advance drug delivery materials to the industrial level, excellent reproducibility, low cost, and especially chemical stability are important. Additionally, while formalin-inactivation of viral vaccines significantly reduces the toxicity to the host associated with administering live, attenuated vaccines, unfortunately, this process often also reduces the vaccine's antigenicity and protective efficacy. Various methods for compensating for toxicity and efficacy, including the use of adjuvants, are used to enhance immunogenicity. The availability of new, safe, and effective adjuvants is valuable for improving the immunogenicity of weak but potentially useful antigens. The anion-exchange resin DEAE-dextran has been established as an effective adjuvant for inactivated foot-and-mouth disease virus for immunization of guinea pigs and pigs. Due to its stable DDS function, intracellular endocytosis, and long residence time in the body, DDMC is thought to be useful as an adjuvant or carrier for novel coronavirus vaccines, particularly mRNA vaccines.

[0031]

Claims

1. A vaccine adjuvant material comprising a copolymer obtained by graft polymerization of a monomer having an olefin onto a cationic partial substitute of a polysaccharide based on a water-soluble linear polysaccharide represented by the above (chemical formula 1), the formula of the unit of the water-soluble linear polysaccharide cationic derivative being represented by [chemical formula 2], the formula of the unit of the polymer starting from the double bond of the olefin compound being represented by [chemical formula 3], the copolymer being composed of (chemical formula 2) having a graft rate of 2% to 5000% and (chemical formula 3), the polymer having a cationic derivative of a water-soluble linear polysaccharide as the backbone polymer and a compound having an olefin as the graft chain, the copolymer being composed of (chemical formula 2) having a graft rate of 2% to 5000% and (chemical formula 3).

2. A method for producing an adjuvant material for vaccines, comprising: a copolymer obtained by graft polymerization of a monomer having an olefin onto a cationic partial substitute of a polysaccharide based on a water-soluble linear polysaccharide represented by the above (chemical formula 1), the formula of the unit of the water-soluble linear polysaccharide cationic derivative being represented by [chemical formula 2], the formula of the unit of the polymer starting from the double bond of the olefin compound being represented by [chemical formula 3], the copolymer being composed of (chemical formula 2) having a graft rate of 2% to 5000% and (chemical formula 3), the copolymer being composed of (chemical formula 2) and (chemical formula 3) having a graft rate of 2% to 5000%, the monomer having an olefin being the cationic partial substitute of a polysaccharide based on a water-soluble linear polysaccharide represented by the above (chemical formula 1).

3. A vaccine comprising a copolymer adjuvant material obtained by graft polymerization of a monomer having an olefin onto a cationic partially substituted linear polysaccharide based on the polysaccharide represented by the above (chemical formula 1), the formula of the unit of the water-soluble linear polysaccharide cationic derivative being represented by [chemical formula 2], the formula of the unit of the polymer starting from the double bond of the olefin compound being represented by [chemical formula 3], the water-soluble linear polysaccharide cationic derivative being the backbone polymer and a compound having an olefin being the graft chain, the graft rate being in the range of 2% to 5000%, and the copolymer adjuvant material being composed of (chemical formula 2) and (chemical formula 3).

4. A vaccine of a complex consisting of a copolymer obtained by grafting an olefin monomer and deoxyribonucleic acid, the copolymer being composed of (chemical formula 2) and (chemical formula 3), in which the formula of the unit of the water-soluble linear polysaccharide cationic derivative in the general formula [chemical formula 1] is represented by [chemical formula 2], and the formula of the unit of the polymer starting from the double bond of the olefin compound in the formula is represented by [chemical formula 3], and the water-soluble linear polysaccharide cationic derivative is the backbone polymer and a compound having an olefin is the graft chain, and the copolymer is obtained by graft polymerizing a monomer having an olefin onto the cationic partial substitute of a linear polysaccharide based on the linear polysaccharide represented by the above (chemical formula 1) and the cationic partial substitute of the linear polysaccharide based on the linear polysaccharide, the copolymer being composed of (chemical formula 2) and (chemical formula 3) with a graft rate of 2% to 5000%, the copolymer being obtained by graft polymerizing a monomer having an olefin onto the cationic partial substitute of the linear polysaccharide based on the linear polysaccharide, the copolymer being obtained by adding deoxyribonucleic acid (DNA) having deoxyribonucleotides as repeating units represented by the formula [chemical formula 5] to an aqueous solution of the cationic partial substitute of the linear polysaccharide based on the linear polysaccharide.

5. A vaccine of a complex consisting of a copolymer obtained by grafting an olefin monomer and ribonucleic acid, the copolymer being composed of (Chemical formula 2) and (Chemical formula 3), in which the formula of the unit of the water-soluble linear polysaccharide cationic derivative in the general formula [Chemical formula 1] is represented by [Chemical formula 2], the formula of the unit of the polymer starting from the double bond of the olefin compound in the formula is represented by [Chemical formula 3], the water-soluble linear polysaccharide cationic derivative being the backbone polymer and a compound having an olefin being the graft chain, and having a graft rate of 2% to 5000%, and being composed of (Chemical formula 2) and (Chemical formula 3), the copolymer being obtained by graft polymerizing a monomer having an olefin onto a cationic partial substitute of a linear polysaccharide based on the linear polysaccharide represented by the above (Chemical formula 1), the copolymer ... obtained by adding ribonucleic acid (RNA) having ribonucleotides as repeating units represented by the formula [Chemical formula 6] to an aqueous solution of a cationic partial substitute of a linear polysaccharide based on the linear polysaccharide.

6. A vaccine delivery system (DDS) characterized in that the first step is the formation of a complex as claimed in claims 4 and 5, which comprises a copolymer obtained by graft polymerizing a monomer having an olefin onto a cationic partially substituted linear polysaccharide based on the linear polysaccharide represented by (Chemical formula 1) above, the copolymer being composed of (Chemical formula 2) having a graft ratio of 2% to 5000% and (Chemical formula 3), the cationic derivative of the water-soluble linear polysaccharide of claim 1 being the backbone polymer and a compound having an olefin being the graft chain, and a nucleic acid.

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