Polyethyleneimine modified laminarin as well as preparation method and application thereof

Abstract

The invention relates to the field of immunological adjuvants, in particular to polyethyleneimine modified laminarin (CLam) as well as a preparation method and application thereof. According to the functional modified polysaccharide, laminarin (Lam) is used as a basic framework, polyethyleneimine (PEI) is grafted in the structure of the laminarin through chemical reaction, and a novel adjuvant material is constructed. The polyethyleneimine modified laminarin prepared by the invention is used as a delivery carrier of an H1N1 antigen, can effectively induce the rise of the level of an H1N1 specific antibody IgG and antibody subtypes (IgG1, IgG2a and IgG2b) thereof, and simultaneously induces Th1 and Th2 type mixed immune response. The obtained CLAM can be used as a novel vaccine adjuvant to remarkably improve antigen-specific immune response, so that strong humoral immune response is induced.

Description

Technical Field

[0001] The present invention relates to the field of immunological adjuvants, and in particular to a preparation method and application thereof for preparing an antigen delivery carrier by functionally modifying polysaccharides to improve the activity of vaccine adjuvants. Background Art

[0002] With the risk of interaction and co-epidemic of respiratory infectious diseases such as COVID-19 and influenza, the development and application of new vaccines are particularly important in the prevention and control of infectious diseases. At present, the therapeutic effect of vaccines cannot achieve the expected immune effect due to weak immunogenicity and low delivery efficiency. Therefore, how to enhance the immune response and improve the delivery efficiency is a huge challenge for the future development of vaccines. The emergence of adjuvants has solved this problem to a certain extent. Adjuvants can be used together with antigens or before antigens to immunize the body to improve the body's immune effect on the antigen, thereby more effectively protecting the body. At present, adjuvants including aluminum adjuvants, oil emulsion adjuvants, Freund's adjuvants, nanoparticles and polymers can enhance the efficacy, breadth and durability of immune responses. However, the safety of adjuvant use, the complexity of preparation and the instability of immune enhancement have limited the widespread clinical application of vaccines. Therefore, it is urgent to develop simpler, safer and more effective adjuvants to improve the immune effect of vaccines.

[0003] As a treasure of China, many scholars have conducted in-depth research on the composition and pharmacology of traditional Chinese medicine. Studies have found that traditional Chinese medicine contains a variety of immunomodulatory components, which have the advantages of few adverse reactions, small toxic side effects, and bidirectional regulation, and are expected to be developed into a new vaccine adjuvant. Polysaccharides, as one of the active ingredients of traditional Chinese medicine, have good biocompatibility, low toxicity and safety, and can be used as adjuvants to enhance the immune effect of vaccines, thereby promoting the body's specific immunity and non-specific immunity. At the same time, due to the wide variety of polysaccharides, diverse pharmacological activities and complex components, they have a broad research background in the field of immune adjuvants. Laminarin (Lam) is a natural polymer with a clear molecular structure and has a wide range of pharmacological activities, such as enhancing immunity, anti-oxidation, and anti-tumor pharmacological effects. Studies have found that laminarin can improve the phagocytic function of macrophages, enhance the humoral immunity of mice, and significantly improve the level of cellular immune response in mice. Lentinus edible mushrooms, as a medicinal and edible fungus, have pharmacological activities such as regulating immunity, anti-tumor, and anti-virus. Lentinan (LNT), as one of the main active ingredients in shiitake mushrooms, can inhibit viral replication by stimulating immune cell maturation, differentiation and proliferation, and improving cell response to lymphokines. It can also regulate innate responses and specific immunity. Therefore, the molecular structures of laminarin and lentinan are clear and easy to modify, and have broad application prospects. However, laminarin and lentinan have negative charges on their surfaces and cannot effectively load, encapsulate and deliver antigens in a targeted manner, which limits their research on antigen delivery carriers.

[0004] Functional modification can be used to give specific functions to a polysaccharide by adjusting its composition, size, shape and structure, so as to design new vaccine adjuvants. Functional modification is not only crucial for improving antigen stability and prolonging the half-life of antigens in the body, but also can enhance biological activity to a certain extent. Polyethyleneimine is a cationic polymer that can be used as a non-viral gene carrier and has great potential in antigen delivery and vaccine adjuvants. In addition, polyethyleneimine has primary amine, secondary amine and tertiary amine groups on every two carbon atoms. The rich positive charges on its surface can produce a proton sponge effect, promote lysosomal escape and antigen presentation reactions, and thus promote the level of immune response. In addition, polyethyleneimine modification can improve biocompatibility, degradation and targeting. Most importantly, the surface of polyethyleneimine-modified polysaccharides is positively charged, and there is attraction between the positively charged polysaccharide and the negatively charged cell membrane, which is easy to fuse with the cell membrane, promotes the effective binding of polysaccharides to cells, increases cell internalization, and thus promotes antigen presentation. Therefore, polyethyleneimine can be used to modify polysaccharides. Its rich amino groups react chemically with the hydroxyl or carboxyl groups on the surface of polysaccharides, which can not only solve the poor targeting of polysaccharides and the inability to deliver antigens, but also make them easier to be taken up by cells and enhance the pharmacological activity of polysaccharides, achieving a "win-win" effect. At present, polyethyleneimine has been widely used in the research of cationic nanogels, cationic micelles, and cationic dendrimers, but there are few studies on the adjuvant activity of polyethyleneimine modified natural polysaccharides and their application in antigen delivery carriers.

[0005] Influenza virus belongs to the family of Orthomyxoviridae and can cause acute respiratory diseases in many animals. It has the characteristics of short incubation period, concentrated onset, fast transmission and strong infectivity. With the rapid mutation of influenza virus and the severity of the disease after infection, it has brought great harm to the breeding and economy of livestock and poultry industry. The protective measures against influenza virus are mainly based on vaccination, which can not only reduce the incidence rate, but also reduce the severity after infection. Inactivated vaccine is the main influenza vaccine currently used (such as trivalent inactivated vaccine) due to its mature preparation process, good safety and convenient storage. However, the immunogenicity of inactivated vaccine is weak. How to enhance the immunogenicity of inactivated vaccine, improve the immune efficacy and save vaccine antigens, while developing safe and effective influenza virus vaccine adjuvants is of great significance. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing polyethyleneimine-modified laminarin (CLam) as a novel vaccine adjuvant, which not only ensures the pharmacological activity of laminarin, but also significantly improves the antigen-specific immune response, so as to solve the problems of poor targeting, inability to deliver antigens, rapid metabolism, and large dosage of polysaccharides as vaccine adjuvants in the prior art. On this basis, the application of polyethyleneimine-modified laminarin as a vaccine adjuvant is studied in combination with influenza A inactivated antigen. The present invention has prepared polyethyleneimine-modified Lentinan (CLNT) in the existing technology, and studied it as a model antigen (chicken ovalbumin, OVA) delivery carrier, and found that CLNT can significantly promote the body's humoral immune response and cellular immune response levels. With the prevalence of influenza A disease, and to verify the universality and promotion of the functionalized modified polysaccharide platform, the present invention prepared polyethyleneimine-modified laminarin on the basis of the research on polyethyleneimine-modified Lentinan, and further studied the effect of functionalized modified polysaccharides on the activity of vaccine adjuvants with H1N1 influenza A antigen as the research object. The present invention found that compared with CLNT, CLam can better promote the body's humoral immune response level against H1N1 antigens and is suitable as a new vaccine adjuvant.

[0007] In order to achieve the above invention objectives, the technical solutions provided by the present invention are as follows:

[0008] A polyethyleneimine-modified laminarin, the structural formula of which is shown in Formula I:

[0009]

[0010] Among them, the value range of n is 12 to 18, and the value range of x is 220 to 250.

[0011] In a second aspect, the present invention provides a method for preparing polyethyleneimine-modified laminarin according to the first aspect, wherein polyethyleneimine is chemically bonded to laminarin;

[0012] Wherein, the structural formula of the laminarin is shown in Formula II:

[0013]

[0014] The polyethyleneimine has a weight average molecular weight of 9 to 11 KDa, preferably 10 KDa;

[0015] The weight average molecular weight of the laminarin is 3-6KDa, preferably 5230Da.

[0016] Specifically, the following steps are included:

[0017] (1) Mixing the laminarin with pyridine, adding succinic anhydride and 4-dimethylaminopyridine to react to obtain laminarin-succinic acid; laminarin is a basic skeleton structure. Preferably, the reaction mixture obtained by adding succinic anhydride and 4-dimethylaminopyridine to react is placed in a rotary evaporator to remove most of the solvent, dialyzed with a 1KDa dialysis bag, and freeze-dried to obtain laminarin-succinic acid.

[0018] (2) The laminarin-succinic acid described in step (1) is mixed with dimethyl sulfoxide (DMSO), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and hydroxysuccinimide are added for activation, and then the polyethyleneimine is added for reaction, and polyethyleneimine-modified laminarin is obtained after dialysis and freeze-drying. The polyethyleneimine chemically modifies the laminarin-succinic acid, and the amino groups in the polyethyleneimine react with the hydroxyl groups in the laminarin, so that the laminarin carries a positive charge, thereby obtaining polyethyleneimine-modified laminarin.

[0019] Wherein, in step (1), 38 mg of laminarin is mixed in each mL of pyridine;

[0020] The molar ratio of succinic anhydride to laminarin is 50:1;

[0021] The molar ratio of 4-dimethylaminopyridine to laminarin is 10:1;

[0022] The reaction temperature is 100°C and the reaction time is 5h.

[0023] Wherein, in step (2), the molar ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the laminarin in step (1) is 50:1;

[0024] The molar ratio of the hydroxysuccinimide to the laminarin in step (1) is 50:1;

[0025] The molar ratio of the polyethyleneimine to the laminarin in step (1) is 100:1.

[0026] Wherein, in step (2), the activation is carried out at room temperature for 24 hours; the reaction is carried out at room temperature for 48 hours; and the dialysis is carried out with a dialysis membrane having a molecular retention rate of 20KDa.

[0027] Preferably, in step (2), the amount of DMSO used is the same as the amount of pyridine used in step (1).

[0028] In a third aspect, the present invention provides the use of the polyethyleneimine-modified laminarin described in the first aspect in a vaccine adjuvant.

[0029] In a fourth aspect, the present invention provides a vaccine comprising an antigen and the polyethyleneimine-modified laminarin of the first aspect.

[0030] Wherein, the antigen is an inactivated H1N1 virus A antigen.

[0031] Wherein, in the vaccine, the concentration of polyethyleneimine-modified laminarin is 1-8 mg / mL; the hemagglutination titer of H1N1 influenza A inactivated antigen is 2 6 ~2 8 Preferably, the concentration of polyethyleneimine-modified laminarin is 4 mg / mL; the hemagglutination titer of H1N1 influenza A inactivated antigen is 2 7 .

[0032] Preferably, the preparation method of the vaccine comprises the following steps: dissolving the polyethyleneimine-modified laminarin described in the first aspect in PBS for standby use, wherein the concentration of the polyethyleneimine-modified laminarin is 1 to 8 mg / mL; then preparing an inactivated H1N1 virus A antigen for standby use, wherein the hemagglutination titer of the inactivated H1N1 virus A antigen is 2 6 ~2 8 The polyethyleneimine-modified laminarin and the H1N1 inactivated antigen were mixed at a volume ratio of 1:1, the reaction mixture was placed in a stirrer for mixing, and allowed to stand for a predetermined time to allow it to be fully mixed, thereby obtaining H1N1 / CLam, wherein the stirrer speed was 150 rpm, and the predetermined standing time was 1 hour.

[0033] The polyethyleneimine-modified laminarin can significantly increase the levels of H1N1-specific antibodies and their subtypes, and can also induce mixed Th1 and Th2 immune responses, thus having a good vaccine adjuvant effect.

[0034] A novel vaccine adjuvant, characterized in that laminarin modified with polyethyleneimine is used as a delivery material and an adjuvant material.

[0035] Beneficial effects:

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] (1) The present invention provides a method for preparing polyethyleneimine-modified polysaccharides, which fills the gap in domestic and foreign research. The method of the present invention is simple to operate, has universal applicability, and is easy to promote. The polyethyleneimine-modified laminarin prepared by the method of the present invention has a zeta potential of +22.87 mV and a nitrogen content of 20.5% through zeta potential, elemental analysis, infrared spectroscopy, and nuclear magnetic resonance analysis. It is found that the polyethyleneimine-modified laminarin has not only the primary and secondary amine absorption peaks of polyethyleneimine, but also the nuclear magnetic resonance shift of polyethyleneimine, and polyethyleneimine is successfully bonded to the structure of laminarin.

[0038] (2) There are few reports on the vaccine adjuvant activity of polyethyleneimine-modified laminarin polysaccharide at home and abroad. The present invention proves that after the polyethyleneimine functionalization modification of polysaccharide, polyethyleneimine-modified laminarin polysaccharide can significantly increase the level of H1N1-specific IgG antibody and its different subtype antibody levels (IgG1, IgG2a, IgG2b and IgG3) in mice, and has stronger adjuvant activity.

[0039] (3) The polyethyleneimine-modified laminarin prepared by the present invention can not only maintain the pharmacological activity of laminarin, but also solve the problem that laminarin cannot deliver antigens. As a vaccine adjuvant, it can significantly improve the body's humoral immune response. Not only has a functional polysaccharide vaccine adjuvant platform been designed, but it also has an important application background in solving influenza A diseases.

[0040] The present invention modifies laminarin by polyethyleneimine, in order to give the polysaccharide surface a positive charge and enable it to have the ability of targeted delivery and enhance its adjuvant activity. Based on the research on polyethyleneimine-modified Lentinan loaded with model antigen OVA, the present invention loads H1N1 antigen (H1N1 / CLam and H1N1 / CLNT) on polyethyleneimine-modified laminarin and polyethyleneimine-modified Lentinan, which not only studies the universality of the functional modified polysaccharide platform of this method, but also provides a theoretical basis for the research of influenza A disease vaccine. After the H1N1 / CLam prepared by the present invention was injected subcutaneously into the ventral side of mice, it was found that it could better promote the body's humoral immune response level and induce Th1 and Th2 mixed immune response reactions. Therefore, CLam is more likely to be used as a delivery carrier to enhance the body's immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0042] Figure 1 The synthetic route of polyethyleneimine-modified laminarin.

[0043] Figure 2 is the zeta potential of polyethyleneimine-modified laminarin in Example 1.

[0044] Figure 3 The infrared spectra of polyethyleneimine, laminarin and polyethyleneimine-modified laminarin in Example 1 are shown.

[0045] Figure 4 These are the NMR analysis images of polyethyleneimine, laminarin and polyethyleneimine-modified laminarin in Example 1.

[0046] Figure 5 The zeta potential, particle size and PDI of the polyethyleneimine-modified laminarin and polyethyleneimine-modified lentinan mixed with the H1N1 inactivated antigen in Example 3.

[0047] Figure 6 This is a schematic diagram of the effect of polyethyleneimine-modified laminarin and polyethyleneimine-modified Lentinan on serum IgG antibodies after immunization of mice in Example 3.

[0048] Figure 7 This is a schematic diagram of the effect of polyethyleneimine-modified laminarin and polyethyleneimine-modified Lentinan on serum IgG1 antibodies after immunization of mice in Example 3.

[0049] Figure 8 This is a schematic diagram of the effect of polyethyleneimine-modified laminarin and polyethyleneimine-modified Lentinan on serum IgG2a antibodies after immunization of mice in Example 3.

[0050] Fig. 9 This is a schematic diagram of the effect of polyethyleneimine-modified laminarin and polyethyleneimine-modified Lentinan on serum IgG2b antibodies in mice immunized with the polyethylenimine in Example 3. DETAILED DESCRIPTION

[0051] The specific embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings, but are not intended to be limiting of the present invention. The experimental methods and reagents described in the following examples are all commonly used products in the art unless otherwise specified.

[0052] In the following examples, the PBS, unless otherwise specified, consists of 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.76 mM potassium dihydrogen phosphate, 2.7 mM potassium chloride, and has a pH of 7.2-7.4.

[0053] In the following examples, the laminarin has a structural formula as shown in Formula II:

[0054]

[0055] The laminarin of the present invention has a weight average molecular weight of 5230 Da.

[0056] Example 1 Preparation of polyethyleneimine-modified laminarin

[0057] 1. Preparation method

[0058] Preparation method of polyethyleneimine modified laminarin Figure 1 As shown, the details are as follows:

[0059] (1) 0.38 g of laminarin (Lam) was dissolved in 10 mL of pyridine, and 50 molar equivalents of succinic anhydride and 10 molar equivalents of 4-dimethylaminopyridine were added to the laminarin. After dissolution and clarification, the mixture was reacted at 100° C. for 5 h. After the reaction was completed, most of the solvent was removed by a rotary evaporator, and the residue was dialyzed with a 1 KDa dialysis bag to remove excess raw materials, and lyophilized to obtain laminarin-succinic acid.

[0060] (2) The laminarin-succinic acid obtained in step (1) was dissolved in 10 mL of DMSO, and 50 molar equivalents of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 50 molar equivalents of laminarin were added to hydroxysuccinimide, and activated at room temperature for 24 h. 100 molar equivalents of polyethyleneimine (weight average molecular weight of 10 KDa) was added to laminarin, and the reaction was continued at room temperature for 48 h, and then 10 mL of water was added to quench the reaction. Finally, the remaining raw materials were dialyzed with a 20 KDa dialysis bag, and the polyethyleneimine-modified laminarin (CLam) was obtained by freeze-drying.

[0061] 2. Zeta potential analysis

[0062] Laminarin and polyethyleneimine-modified laminarin (1 mg / mL) were diluted 10 times with distilled water (concentration after dilution was 0.1 mg / mL) and then loaded into a sample cell. The potential was measured at room temperature using a Malvern laser particle size analyzer.

[0063] The results show Figure 2 As shown, the potentials of laminarin and polyethyleneimine-modified laminarin are -8.68 mV and +22.87 mV, respectively, which preliminarily indicates that laminarin has been successfully positively charged.

[0064] 3. Elemental analysis

[0065] 2-3 mg of polyethyleneimine, laminarin and polyethyleneimine-modified laminarin were weighed respectively for nitrogen determination, and the nitrogen content was calculated.

[0066] The results showed (Table 1) that the nitrogen contents of laminarin and polyethyleneimine-modified laminarin were 0% and 20.5%, respectively, indicating that laminarin contained the structure of polyethyleneimine.

[0067] Table 1 Nitrogen content in each sample

[0068]

[0069] 4. Infrared spectroscopy analysis

[0070] 2 mg of polyethyleneimine, laminarin and polyethyleneimine-modified laminarin were weighed and added to agate together with 100 mg of dry potassium bromide. After grinding, they were pressed into thin slices. The infrared spectra were measured by Fourier transform infrared spectrometer with a wavelength range of 4000-400 cm -1 .

[0071] The results show that Figure 3 ), polyethyleneimine-modified laminarin at 1700 cm -1 and 1600cm -1 There are NH bending vibration peaks of primary and secondary amines at 1400-1000 cm -1 The CN stretching vibration peak at further confirmed the presence of primary and secondary amines. These results indicate that polyethyleneimine has been successfully bonded into the structure of laminarin.

[0072] 5. NMR analysis

[0073] 10 mg of polyethyleneimine, laminarin and polyethyleneimine-modified laminarin were weighed respectively, dissolved in heavy water (D2O), and then placed in a nuclear magnetic resonance tube, and measured using a nuclear magnetic resonance instrument at room temperature.

[0074] The results show that Figure 4 ), the characteristic peak shift of hydroxyl in laminarin is δ3.0~5.0ppm, while in polyethyleneimine-modified laminarin, not only the characteristic peak shift of hydroxyl is present, but also a new proton peak appears at δ2.0~3.0ppm, which is the proton peak of CH2- of -NHCH2 in polyethyleneimine, indicating that polyethyleneimine is successfully bonded to the skeleton of laminarin.

[0075] The results of zeta potential, elemental analysis, infrared spectrum analysis and nuclear magnetic resonance analysis showed that polyethyleneimine-modified laminarin had been successfully prepared.

[0076] Example 2 Preparation of H1N1 Inactivated Antigen

[0077] (1) Seed virus amplification:

[0078] Select 10-day-old SPF chicken embryos and add H1N1 virus stock solution (hemagglutination titer of 2 8 )10-fold serial dilution (10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 ) and then inoculated into the allantoic cavity of chicken embryos, 0.1 mL was inoculated into each chicken embryo, and the holes were sealed with candles and placed in an incubator at 37°C. After 48 hours, the chicken embryo allantoic fluid with the highest dilution and the highest hemagglutination titer was harvested as the virus antigen solution.

[0079] (2) Inactivation of viral antigens:

[0080] Add β-propiolactone to the harvested virus antigen liquid according to the ratio (virus antigen liquid: β-propiolactone is 1:2000 v / v), place it at 4°C, shake it continuously, and collect the inactivated antigen liquid after 24 to 48 hours.

[0081] (3) Determination of HA:

[0082] The inactivated antigen solution prepared in step (2) was diluted with PBS in multiples (the dilution multiples were 2, 4, 8, 16, 32 ... 4096, respectively), and then 1% chicken red blood cell suspension was added to all wells. After mixing, the agglutination effect was observed within 30 minutes to determine the hemagglutination titer of the H1N1 inactivated antigen.

[0083] The results showed that the hemagglutination titer of H1N1 inactivated antigen was 2 7 .

[0084] Example 3 Effect of polyethyleneimine-modified laminarin on humoral immune response of mice immunized with H1N1 1. Preparation method:

[0085] In order to verify the universality of the functional modified polysaccharide platform, polyethyleneimine-modified laminarin (CLam, 4 mg / mL, solvent: PBS), polyethyleneimine-modified lentinan (CLNT, 4 mg / mL, solvent: PBS), laminarin (4 mg / mL, solvent: PBS), and lentinan (4 mg / mL, solvent: PBS) were added to the H1N1 inactivated antigen solution (hemagglutination titer: 2) prepared in Example 2. 7 ) in a centrifuge tube with a volume ratio of 1:1, placed in a stirrer for mixing (150rpm) and then allowed to stand for 1h to obtain polyethyleneimine-modified laminarin vaccine (H1N1 / CLam), polyethyleneimine-modified lentinan vaccine (H1N1 / CLNT), laminarin vaccine (H1N1 / Lam) and lentinan vaccine (H1N1 / LNT), and the zeta potential, particle size and PDI thereof were measured by a Malvern particle size analyzer. The preparation method of the polyethyleneimine-modified lentinan is described in the reference (A General Strategy toward Self-assembled Nanovaccine Based on Cationic Lentinanto Induce Potent Humoral and Cellular Immune Responses)

[0086] The results show that Figure 5), the zeta potential of H1N1 / Lam was -9.65mV, the particle size was 217.0nm, and the PDI was 0.397; the zeta potential of H1N1 / CLam became +13.7mV, the particle size was 487.3nm, and the PDI was 0.345.

[0087] The zeta potential of H1N1 / LNT is -9.35mV, the particle size is 408.5nm, and the PDI is 0.39; the zeta potential of H1N1 / CLNT becomes +14.2mV, the particle size is 358.3nm, and the PDI is 0.3. The results show that after PEI modification, the potentials of H1N1 / CLam and H1N1 / CLNT both become positively charged, the particle size of H1N1 / CLam increases, and the particle size of H1N1 / CLNT decreases, but the PDI of both decreases, and the system tends to be stable.

[0088] 2. Animal grouping and immunization procedure

[0089] Six-week-old BALB / C mice (16-20 g) were randomly divided into six groups as follows:

[0090] Blank group (Control): injection of 200 μL PBS;

[0091] Antigen group (H1N1): Inject 100 μL of H1N1 inactivated antigen (hemagglutination titer 2 7 ) and 100 μL PBS;

[0092] Laminarin group (H1N1 / Lam): 100 μL H1N1 inactivated antigen (hemagglutination titer 2 7 ) and 100 μL laminarin (4 mg / mL, solvent: PBS);

[0093] Polyethyleneimine-modified laminarin group (H1N1 / CLam): 100 μL of H1N1 inactivated antigen (hemagglutination titer of 2 7 ) and 100 μL polyethyleneimine-modified laminarin (4 mg / mL, solvent: PBS);

[0094] Aluminum adjuvant group (H1N1 / Alum): injection of 100 μL H1N1 inactivated antigen (hemagglutination titer of 2 7 ) and 100 μL aluminum adjuvant (2.5 mg / mL, solvent is PBS);

[0095] Polyethyleneimine-modified Lentinan group (H1N1 / CLNT): 100 μL of H1N1 inactivated antigen (hemagglutination titer of 2 7 ) and 100 μL polyethyleneimine-modified Lentinan (4 mg / mL, solvent: PBS);

[0096] The blank group (Control) was used as the negative control, and the aluminum adjuvant (Alum) was used as the positive control. All the above injection methods were ventral subcutaneous injections, and the injection volume was 200 μL. The day of injection was taken as day 0, and the second immunization was performed on the 14th day. Blood was collected from the mouse eyeballs on the 21st, 28th, 35th, 42nd and 49th days to detect the level of specific antibodies. At the same time, the H1N1 / CLNT group was set up for comparison at a uniform level in order to compare the adjuvant activity of the polysaccharide modified with polyethyleneimine.

[0097] 3. ELISA method to determine the level of H1N1-specific antibodies in serum samples

[0098] Dilute H1N1 influenza antigen (hemagglutination titer 2) with coating buffer 2 ) and add it to a 96-well plate at 100 μL / well, and coat overnight at 4°C. Discard the coating solution, wash repeatedly with PBST washing solution (PBS containing 0.05% Tween20, pH 7.4) for 3 times, and discard the waste solution. Add BSA blocking solution (10 μg / mL) to each well for blocking (37°C, 2h), discard the blocking solution, wash repeatedly with PBST washing solution for 3 times, and discard the waste solution. Dilute the collected mouse serum with PBS buffer and add it to the 96-well plate at 100 μL / well, and incubate at 37°C for 2h. Wash each well with PBST washing solution 3 times, and discard the waste solution. Add detection antibodies IgG, IgG1, IgG2a and IgG2b (100 μL / well, dilution ratio of 1:5000) to each well and incubate at 37°C for 1h. Each well was washed three times with PBST solution, and the waste solution was discarded. Then, the color developing solution and the stop solution were added, and the absorbance value (450 nm) was detected on an enzyme reader.

[0099] The results show that Figure 6), within 21 to 49 days after the first immunization, compared with the Control, H1N1, and H1N1 / LNT groups, H1N1 / CLNT can significantly increase the level of IgG antibodies in mouse serum, and its effect is also significantly higher than that of H1N1 / Alum (p<0.001). It is worth noting that with the increase of time, the antibody level of H1N1 / CLNT is also gradually increased, and the IgG antibody level of H1N1 / CLNT reaches the highest on the 35th day after immunization. Compared with the Control, H1N1, and H1N1 / Lam groups, H1N1 / CLam can significantly increase the level of IgG antibodies in mouse serum, and its effect is also significantly higher than that of H1N1 / Alum (p<0.001). With the increase of time, the antibody level of H1N1 / CLNT is also gradually increased, and the IgG antibody level of H1N1 / CLam reaches the highest on the 35th day after immunization. Compared with H1N1 / CLNT, the IgG antibody level of H1N1 / CLam was significantly higher than that of H1N1 / CLNT within 49 days (p<0.001), and the IgG antibody level of H1N1 / CLam was the highest, indicating that H1N1 / CLam can better promote the secretion of efficient and long-lasting H1N1-specific antibodies.

[0100] Note: *p<0.05, **p<0.01, ***p<0.001, ns means no significant difference.

[0101] The results showed that for IgG1 antibodies ( Figure 7 ), within 21 to 49 days after the first immunization, H1N1 / CLNT can significantly increase the level of IgG1 antibody in mouse serum compared with the Control, H1N1, and H1N1 / LNT groups (p<0.001). At 21 and 28 days after the first immunization, the IgG1 antibody level of H1N1 / CLNT was not significantly different from that of H1N1 / Alum (p>0.05). On the 35th day after immunization, the IgG1 antibody level of H1N1 / CLNT rose rapidly and was significantly higher than that of H1N1 / Alum (p<0.001). Compared with the Control, H1N1, and H1N1 / Lam groups, H1N1 / CLam can significantly increase the level of IgG1 antibody in mouse serum, and its effect is also significantly higher than that of H1N1 / Alum (p<0.001). Compared with H1N1 / CLNT, the IgG1 antibody level of H1N1 / CLam was significantly higher than that of H1N1 / CLNT within 49 days (p<0.001), and the IgG1 antibody level reached the highest on the 28th day after immunization, indicating that H1N1 / CLam can more quickly promote the secretion of H1N1-specific IgG1 antibodies.

[0102] About IgG2a antibody ( Figure 8), 21 days after the first immunization, there was no significant difference in the IgG2a antibody levels between H1N1 / CLNT and H1N1 / CLam (p>0.05). On the 35th day after immunization, the IgG2a antibody level of H1N1 / CLam increased rapidly and was significantly higher than that of H1N1 / CLNT (p<0.001), indicating that H1N1 / CLam can better induce the secretion of high levels of H1N1-specific IgG2a antibodies.

[0103] About IgG2b antibody ( Fig. 9 ), within 21 to 49 days after the first immunization, the IgG2b antibody level of H1N1 / CLam was significantly higher than that of H1N1 / CLNT (p<0.001), and on the 49th day after immunization, the IgG2b antibody level of H1N1 / CLNT was low, while the IgG2b antibody level of H1N1 / CLam was still at a high level, indicating that H1N1 / CLam is more capable of inducing persistent H1N1-specific IgG2b antibody secretion.

[0104] The above results show that both polyethyleneimine-modified Lentinan and polyethyleneimine-modified laminarin can promote mixed Th1 and Th2 immune responses, but polyethyleneimine-modified laminarin can better activate the body's humoral immune response.

[0105] Note: *p<0.05, **p<0.01, ***p<0.001, ns means no significant difference.

[0106] According to the above animal test results, it can be concluded that polyethyleneimine-modified laminarin can significantly increase the levels of IgG, IgG1, IgG2a and IgG2b antibodies compared to polyethyleneimine-modified Lentinan, and promote sustained and lasting humoral immune response. At the same time, polyethyleneimine-modified laminarin enhances the secretion of Th1 and Th2 cytokines, and can be used as an adjuvant to enhance the immune effect of vaccines.

[0107] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

[0108] The present invention provides a polyethyleneimine-modified laminarin and a method for preparing the same and a method for using the same as a vaccine adjuvant. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be noted that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be considered as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A polyethyleneimine-modified laminarin, the structural formula of which is shown in Formula I: in, The value range of n is 12 to 18, and the value range of x is 220 to 250.

2. The method for preparing polyethyleneimine-modified laminarin according to claim 1, characterized in that: Polyethyleneimine is chemically linked to laminarin; Wherein, the structural formula of the laminarin is shown in Formula II: The polyethyleneimine has a weight average molecular weight of 9 to 11 KDa; The weight average molecular weight of the laminarin is 3-6KDa.

3. The preparation method according to claim 2, characterized in that: The steps include: (1) mixing the laminarin with pyridine, adding succinic anhydride and 4-dimethylaminopyridine to react to obtain laminarin-succinic acid; (2) The laminarin-succinic acid described in step (1) is mixed with dimethyl sulfoxide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and hydroxysuccinimide are added for activation, and then the polyethyleneimine is added for reaction, and polyethyleneimine-modified laminarin is obtained by dialysis and freeze-drying.

4. The preparation method according to claim 3, characterized in that: In step (1), 38 mg of laminarin is mixed in each mL of pyridine; The molar ratio of succinic anhydride to laminarin is 50:1; The molar ratio of 4-dimethylaminopyridine to laminarin is 10:1; The reaction temperature is 100°C and the reaction time is 5h.

5. The preparation method according to claim 3, characterized in that: In step (2), the molar ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the laminarin in step (1) is 50:1; The molar ratio of the hydroxysuccinimide to the laminarin in step (1) is 50:1; The molar ratio of the polyethyleneimine to the laminarin in step (1) is 100:

1.

6. The preparation method according to claim 3, characterized in that: In step (2), the activation is carried out at room temperature for 24 hours; the reaction is carried out at room temperature for 48 hours; and the dialysis is carried out with a dialysis membrane having a molecular retention rate of 20 KDa.

7. Use of the polyethyleneimine-modified laminarin according to claim 1 in a vaccine adjuvant.

8. A vaccine comprising an antigen and the polyethyleneimine-modified laminarin according to claim 1.

9. The vaccine according to claim 8, characterized in that The antigen is an inactivated H1N1 virus antigen.

10. The vaccine according to claim 9, characterized in that In the vaccine, the concentration of polyethyleneimine-modified laminarin is 1-8 mg / mL; the hemagglutination titer of H1N1 influenza A inactivated antigen is 2 6 ~2 8 .

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