WO2024040979A1

WO2024040979A1 - Ginseng acidic polysaccharide vaccine adjuvant, vaccine composition, and use thereof

Info

Publication number: WO2024040979A1
Application number: PCT/CN2023/087301
Authority: WO
Inventors: 翟健秀; 殷军; 周荔葆
Original assignee: 沈阳药科大学
Priority date: 2022-08-23
Filing date: 2023-04-10
Publication date: 2024-02-29
Also published as: CN116036262A

Abstract

Provided is a vaccine adjuvant comprising ginseng acidic polysaccharide (GAPS), which can significantly improve the titer of a specific antibody (or a neutralizing antibody) after antigen immunization and can effectively enhance the immune response activity of a rabies vaccine, an influenza vaccine, a hepatitis B vaccine, a hepatitis A vaccine, a hepatitis C vaccine, a hand-foot-and-mouth vaccine, an HPV vaccine, and a novel coronavirus vaccine.

Description

A kind of ginseng acidic polysaccharide vaccine adjuvant, vaccine composition and application thereof

Technical field

This application relates to the field of medical technology, specifically to a ginseng acidic polysaccharide (GAPS) vaccine adjuvant, vaccine composition and application thereof.

Background technique

Vaccines can activate humoral immune responses in organisms to produce antibodies, or activate cytotoxic T cells and other lymphocytes through cellular immune responses to resist invading foreign pathogens and prevent disease (Cavallo Fet al., Vaccination for treatment and prevention of cancer animal models.Adv Immunol.2006.90:175-213.Review). Although the vaccine has the effect of activating the immune system, in clinical use it is often found that it cannot exert its due effect on certain groups whose immune systems are too weak, such as the elderly and children. Therefore, the addition of appropriate amounts of vaccine adjuvants is necessary. .

Vaccine adjuvants refer to substances that can non-specifically change or enhance the body's specific immune response to antigens. They are required to be non-toxic, high in purity, and have certain adsorption capacity and stable properties. The main mechanism of action of vaccine adjuvants is to increase the surface area of the antigen and improve immunogenicity; to slow-release the antigen and prolong the residence time of the antigen in tissues; to promote inflammatory response and stimulate active immune response.

The functions of adjuvants can usually be divided into two categories. The first is to adsorb antigens and assist the antigens to be engulfed by cells, such as aluminum salts and M59 emulsifiers (O'Hagan D T, Wack A, Podda A. MF59 is a safe and potent vaccine adjuvant for flu vaccines in humans: what did we learn during its development? Clin Pharmacol Ther. 2007 Dec; 82(6):740-4; 4. Clapp T, Siebert P, Chen D, Jones Braun L. Vaccines with aluminum-containing adjuvants: optimizing vaccine efficacy and thermal stability. J Pharm Sci. 2011Feb; 100(2):388-401); the other is immunomodulatory factors, such as Freund's adjuvant containing mycobacteria (CFA-mycobacteria) (Hoft DF, Blazevic A, Abate G, Hanekom WA, KaplanG, Soler JH, Weichold F, Geiter L, Sadoff JC, Horwitz MA. A new recombinantbacille Calmette-Guérin vaccine safely induces significantly enhanced tuberculosis-specific immunity in human volunteers. J Infect Dis.2008Nov15;198(10):1491-501). Most vaccine adjuvants currently on the market are aluminum salt adjuvants, but aluminum salts can cause inflammation at the injection site and stimulate local erythema, granulomas, and subcutaneous nodules. At the same time, aluminum salt adjuvants may delay the development of neutralizing antibodies in some vaccines. are produced and subject to various limitations in application. Therefore, there is an urgent need to develop novel adjuvants to improve the antigen specificity of vaccines or their anti-tumor and anti-infection capabilities.

Contents of the invention

This application provides a new vaccine adjuvant, ginseng acidic polysaccharide (GAPS), which has good activity and can improve the body's response to rabies vaccine, influenza vaccine, hepatitis B vaccine, hepatitis A vaccine, hepatitis C vaccine, hand, foot and mouth vaccine, HPV vaccine, new Coronavirus vaccine-specific antibody (or neutralizing antibody) titer levels.

Specifically, this application adopts the following technical solutions

1. A vaccine adjuvant comprising ginseng acidic polysaccharide (GAPS).

2. The vaccine adjuvant according to item 1 also includes physiological saline or water for vaccine injection or pharmaceutical excipients. Further preferably, the vaccine adjuvant is composed of ginseng acidic polysaccharide (GAPS) and physiological saline or vaccine injection water or pharmaceutical excipients. For example, the vaccine adjuvant is composed of ginseng acidic polysaccharide (GAPS) and physiological saline, such as, The vaccine adjuvant is composed of ginseng acidic polysaccharide (GAPS) and vaccine water for injection. For example, the vaccine adjuvant is composed of ginseng acidic polysaccharide (GAPS) and pharmaceutical excipients.

3. The vaccine adjuvant according to item 1 or 2, wherein the ginseng acidic polysaccharide is extracted from ginseng.

4. The vaccine adjuvant according to item 3, the extraction process includes,

Take ginseng and extract total ginseng polysaccharides.

Dissolve the extracted ginseng total polysaccharides in deionized water.

After performing column chromatography, elute with eluent.

Dialyze the eluate with distilled water,

After freeze-drying, ginseng acidic polysaccharide was obtained.

5. The vaccine adjuvant according to item 4, the column of the column chromatography is a DEAE cellulose column or a macroporous resin column, preferably a DEAE cellulose column, and further preferably the column chromatography is performed at 2 to 8 mL/min. Loading.

6. The vaccine adjuvant according to item 4 or 5, the eluent is water and an alkali solution, a borax solution or a salt solution, the salt solution is preferably a NaCl solution, and further preferably the concentration of the eluent is 0.3～0.7mol/L, and further preferably the elution flow rate is 0.5～2mL/min.

7. The vaccine adjuvant according to any one of items 4 to 6, before dialyzing the eluate with distilled water, it also includes detecting A ₄₉₀ of the eluate using the phenol sulfuric acid method, collecting the absorption peaks, and then dialyzing, Preferably, the dialysis time is 24 to 72 hours.

8. A vaccine composition comprising the vaccine adjuvant described in any one of items 1-7 and an antigen or DNA encoding the antigen.

9. The vaccine composition according to item 8, the dosage ratio of the GAPS to the antigen is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is:

10. The vaccine composition according to item 8 or 9, which further includes pharmaceutical excipients and a second vaccine adjuvant.

11. The vaccine composition according to any one of items 8-10, which is a rabies vaccine, an influenza vaccine, a hepatitis B vaccine, a hepatitis A vaccine, a hepatitis C vaccine, a hand, foot and mouth vaccine, an HPV vaccine or a new coronavirus vaccine .

12. The vaccine composition according to any one of items 8-11, wherein the vaccine type is an inactivated virus vaccine, an attenuated vaccine, an inactivated vaccine, a protein vaccine, a DNA vaccine or a polypeptide vaccine.

13. Use of the vaccine adjuvant described in any one of items 1 to 7 or the ginseng acidic polysaccharide (GAPS) related to any one of items 1 to 7 in the preparation of vaccine preparations, vaccine compositions or antibodies.

14. A method for preventing and/or treating diseases, comprising administering the vaccine composition described in any one of items 8 to 12 to a subject.

15. The method according to item 14, wherein the disease is selected from rabies, influenza, hepatitis B, hepatitis A, hepatitis C, hand, foot and mouth disease, HPV disease or novel coronavirus disease.

Invention effect

(1) The active ingredient ginseng acidic polysaccharide GAPS disclosed in this application is used as a vaccine adjuvant and has high adjuvant activity. Due to its high content of sulfate radicals and carboxylate radicals, acidic polysaccharides have a high negative charge density and may participate in a variety of enzymatic reactions and immune activities in the body (for example, heparin, which is widely found in animals, is a small molecule acidic polysaccharide. ), acidic polysaccharides are widely derived from animals, plants and microorganisms. Their monosaccharide composition, the type and number of acidic groups on the sugar chain vary with the source species. However, acidic polysaccharides derived from plants have a macromolecular structure and are mostly used as water-soluble dietary fiber. Research on adjuvant activity is very limited. This application has undergone a large number of screening tests. During the activity screening process, it was found that ginseng acidic polysaccharides have high adjuvant activity and can effectively enhance the immune response activity of various vaccines. In particular, ginseng acidic polysaccharides come from natural plants with clear sources and resources. Rich, and the ingredients have good safety and stability.

(2) The ginseng acidic polysaccharide GAPS of the present application can significantly increase the specific antibody (or neutralizing antibody) titer after antigen immunization, and can effectively enhance rabies vaccine, influenza vaccine, hepatitis B vaccine, hepatitis A vaccine, hepatitis C vaccine, hand, foot and mouth vaccine Immune response activity of vaccines, HPV vaccines, and novel coronavirus vaccines. And its activity is significantly higher than that of currently commonly used vaccine adjuvants.

(3) The ginseng acidic polysaccharide GAPS vaccine adjuvant of the present application has the advantages of good immune effect and easy use, and provides a new adjuvant option for vaccines.

Description of drawings

The accompanying drawings are used for a better understanding of the present application and do not constitute an undue limitation of the present application. in:

Figure 1 is the elution-absorbance curve of ginseng polysaccharide;

Figure 2 is the absorbance-glucose content standard curve;

Figure 3 is the absorbance-protein content standard curve;

Figure 4 shows the effects of GAPS and GPS on IgG antibody levels in mice immunized with rabies vaccine;

Figure 5 shows the effects of GAPS and GPS on neutralizing antibody titers in mice immunized with rabies vaccine;

Figure 6 shows the effects of GAPS and GPS on neutralizing antibody levels in mice immunized with influenza vaccine;

Figure 7 shows the effects of different doses of GAPS on neutralizing antibody levels in mice immunized with influenza vaccine;

Figure 8 shows the effects of GAPS and GPS on IgG antibody titers in mice immunized with hepatitis B vaccine;

Figure 9 shows the effects of GAPS and GPS on neutralizing antibody levels in mice immunized with hepatitis A vaccine;

Figure 10 shows the effects of different doses of GAPS on neutralizing antibody levels in mice immunized with hepatitis A vaccine;

Figure 11 shows the effects of GAPS and GPS on IgG antibody levels in mice immunized with hepatitis C vaccine;

Figure 12 shows the effects of GAPS and GPS on neutralizing antibody levels in mice immunized with hand, foot and mouth vaccine;

Figure 13 shows the effects of GAPS and GPS on IgG antibody levels in mice immunized with hand, foot and mouth vaccine;

Figure 14 shows the effects of different doses of GAPS on neutralizing antibody levels in mice immunized with hand, foot and mouth vaccine;

Figure 15 shows the effects of GAPS and GPS on IgG antibody titers in mice immunized with HPV vaccine;

Figure 16 shows the effect of different doses of GAPS on IgG antibody titers in mice immunized with HPV vaccine;

Figure 17 shows the effects of GAPS and GPS on IgG antibody levels in mice immunized with the new coronavirus vaccine;

Figure 18 Effects of different doses of GAPS on IgG antibody levels in mice immunized with the new coronavirus vaccine.

Detailed ways

Exemplary embodiments of the present application are described below, including various details of the embodiments of the present application to facilitate understanding, and they should be considered to be exemplary only. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the application. Also, descriptions of well-known functions and constructions are omitted from the following description for clarity and conciseness.

The present application discloses a vaccine adjuvant, including ginseng acidic polysaccharide (GAPS).

In a preferred embodiment, the vaccine adjuvant further includes physiological saline or water for vaccine injection or pharmaceutical excipients.

As used herein, the term "vaccine" refers to any preparation of an antigen or immunogenic substance suitable for stimulating active immunity in an animal or human.

As used herein, the term "adjuvant" refers to any substance or mixture of substances that enhances, increases, up-regulates, alters, or otherwise promotes an immune response (eg, a humoral or cellular immune response) to an antigen in an animal.

As used herein, the term "antigen" refers to any substance that, when introduced into an immunocompetent human or animal, stimulates a humoral and/or cell-mediated immune response. The antigen may be a pure substance, a mixture of substances or particulate matter (including cells, cell fragments or cell-derived fragments) or a live (usually attenuated) organism or virus. Examples of suitable antigens include, but are not limited to, proteins, glycoproteins, lipoproteins, peptides, carbohydrates/polysaccharides, lipopolysaccharides, toxins, viruses, bacteria, fungi, and parasites. Other suitable antigens include minimal components of the antigen, such as, but not limited to, epitopes, epitopes or peptides. Still other suitable antigens include those described in U.S. Patent No. 5,855,894. Antigens may be natural (naturally expressed or produced), synthetic, or derived by recombinant DNA methodologies familiar to those skilled in the art.

As used in this article, the term "pharmaceutical excipients" refers to the collective name for all pharmaceutical materials other than the main drug that are added to the prescription to improve the formability, effectiveness, stability, and safety of the preparation during the production of drugs and preparation of prescriptions. Substances that have been reasonably evaluated for their safety and are included in pharmaceutical preparations. In addition to excipients, serving as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, dissolution, and sustained and controlled release. They are important ingredients that may affect the quality, safety, and effectiveness of drugs. The pharmaceutical excipients described in this application can be appropriate carriers or excipients, emulsifiers, wetting agents, preservatives, stabilizers, antioxidants, adjuvants (such as aluminum hydroxide adjuvant, oil adjuvant, Freund's Complete adjuvant and Freund's incomplete adjuvant), etc.

As used herein, the term "ginseng acidic polysaccharide" refers to acidic polysaccharides extracted from the Araliaceae plant ginseng. The extraction from Araliaceae plant ginseng can be extracted from original ginseng, or it can be extracted from processed ginseng, such as from secondary developed products of ginseng, such as ginseng total polysaccharides extracted from ginseng. Extraction from ginseng, such as extraction from other parts remaining after the total ginseng polysaccharide is extracted from ginseng, such as extraction from ginseng extraction waste, such as extraction from ginseng-containing medicinal residues, etc., as long as it contains ginseng acidic polysaccharide, any ginseng product The ginseng acidic polysaccharide extracted from can be used as an adjuvant for the vaccine of this application.

In a preferred embodiment, the ginseng acidic polysaccharide is purchased. In a preferred embodiment, the ginseng acidic polysaccharide is extracted from ginseng. In a preferred embodiment, the ginseng acidic polysaccharide is Acidic polysaccharides are extracted from ginseng polysaccharides. In a preferred embodiment, the ginseng acidic polysaccharides are extracted in the following manner:

Take ginseng and extract total ginseng polysaccharides.

Dissolve the extracted ginseng total polysaccharides in deionized water.

After performing column chromatography, elute with eluent.

Dialyze the eluate with distilled water,

After freeze-drying, ginseng acidic polysaccharide was obtained.

As used herein, the term "ginseng polysaccharide" refers to the total polysaccharides extracted from the Araliaceae plant ginseng. In this application, the term "ginseng total polysaccharide" has the same meaning and can be used interchangeably. The method for extracting ginseng total polysaccharide from ginseng can be any method in the art that can extract ginseng total polysaccharide from ginseng. It can also be extracted through the following methods:

Extract ginseng with water to obtain the first extract,

Add protease to the first extract, incubate it, and then extract to obtain the second extract,

Extract the second extract through ethanol and water to obtain ginseng crude polysaccharide,

Dissolve ginseng crude polysaccharide and then dialyze, and remove the protein in the retentate.

The protein-removed retentate was extracted with ethanol to obtain total ginseng polysaccharides.

In a specific embodiment, the step of obtaining the first extract includes:

Crush 1kg of dried ginseng roots to 10-20 mesh, add 8 times the amount of water (g:mL), boil in a boiling water bath for 8 hours, filter and collect the filtrate; continue to add 10 times the amount of water (g:mL) to the filter residue, and boil in a boiling water bath Heating for 8h. Combine the two filtrates and centrifuge at 6000 rpm for 10 min. The supernatant is evaporated to dryness to obtain the first extract.

In a specific embodiment, in the step of obtaining the second extract, the protease culture temperature is 30°C to 40°C, and the culture time is 1 to 3 hours.

In a specific embodiment, the step of obtaining the second extract includes:

Neutral protease was added to the first extract, incubated at 40°C for 3 hours, and then inactivated at 100°C for 30 minutes. The mixed solution was centrifuged at 10,000 rpm to obtain the supernatant, which was evaporated to dryness to obtain the second extract.

In a specific embodiment, the steps of obtaining ginseng crude polysaccharide include:

The second extract was soaked in 10 times the volume of 95% ethanol at low temperature for 2.0 h. Filter, take the filter residue, add 2 times the volume of distilled water to dissolve, extract with boiling water reflux 3 times, 1.0h each time, filter with 120 mesh nylon cloth, combine the extracts, concentrate to 500mL, centrifuge for 20 minutes (5000rpm), discard the precipitate. Add 95% ethanol to the supernatant until the final concentration of ethanol is 90%, and let it stand for 1 hour. Centrifuge for 20 minutes (5000 rpm), collect the precipitate, add 400 mL of distilled water to the precipitate, then add 95% ethanol to the supernatant to a final concentration of 80%, let stand for 1 hour, and centrifuge for 20 minutes (5000 rpm) , let stand at room temperature overnight, and collect the precipitate. The next day, the precipitate was washed twice with absolute ethanol and 95% ethanol. And freeze-dried to obtain ginseng crude polysaccharide,

In a specific embodiment, the molecular weight cutoff during dialysis is 800-2500kDa, for example, it can be 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 220 0kDa , 2300kDa, 2400kDa, preferably 1000-1500kDa.

In a specific embodiment, Sevag reagent is used to remove proteins in the retentate.

In a specific embodiment, the steps of dissolving crude ginseng polysaccharide and then dialyzing it, and removing the protein in the retentate include:

Prepare a 30% aqueous solution of ginseng crude polysaccharide, add a 1000kDa dialysis bag for dialysis, and let it stand overnight. Take the liquid in the dialysis bag, add 1/4 volume of Sevag reagent to it, and let it sit Centrifuge to remove the gelatinous precipitate. Repeat the above operation 5 times.

In a specific embodiment, the protein-removed retentate is extracted with ethanol, and the steps of obtaining total ginseng polysaccharides include:

Combine the protein-removed supernatants and concentrate under reduced pressure to remove organic reagents. Add 95% ethanol to a final concentration of 80%, let it stand at 4°C overnight, centrifuge, and remove the supernatant. The precipitate was washed twice with 95% ethanol and absolute ethanol. Total ginseng polysaccharides were obtained by freeze-drying.

In the process of extracting ginseng acidic polysaccharide of the present application, when performing column chromatography and then using eluent to elute, the column can be any column in the field that can realize column chromatography, which is a preferred embodiment. The column of the column chromatography is a DEAE cellulose column or a macroporous resin column, preferably a DEAE cellulose column. It is further preferred that the column chromatography is loaded at 2 to 8 mL/min, for example, it can be 3 mL/min or 4 mL/min. ,5mL/min,6mL/min,7mL/min.

The eluent can be any one or more than two solutions in the art that can achieve the purpose of elution. In a preferred embodiment, the eluent is water and an alkali solution, or water and borax. solution, or water and salt solution. In a further preferred implementation method, the eluent is water and a salt solution. In a further preferred implementation method, the eluent is water and NaCl solution.

The concentration of the eluent can be any concentration in the art that can achieve the purpose of elution. In a preferred embodiment, the concentration of the eluent is 0.3-0.7 mol/L, for example, it can be 0.4 mol/L, 0.5mol/L, 0.6mol/L.

The elution flow rate can be any flow rate in the art that can achieve the purpose of elution. In a preferred embodiment, the elution flow rate is 0.5-2mL/min, for example, it can be 0.6mL/min, 0.7mL /min, 0.8mL/min, 0.9mL/min, 1.0mL/min, 1.1mL/min, 1.2mL/min, 1.3mL/min, 1.4mL/min, 1.5mL/min, 1.6mL/min, 1.7mL /min, 1.8mL/min, 1.9mL/min.

In a preferred embodiment, before dialyzing the eluate with distilled water, the method further includes detecting A ₄₉₀ of the eluate using the phenol sulfuric acid method, collecting absorption peaks, and then performing dialysis. Preferably, the dialysis time is 24 to 72 hours. , for example, it can be 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours.

The present application further provides a vaccine composition, including a vaccine adjuvant containing any one of the ginseng acidic polysaccharides in the present application and an antigen or DNA encoding the antigen.

The dosage of the vaccine adjuvant in the vaccine composition is an effective dosage to achieve a therapeutic effect, and the dosage is An effective amount is an amount that enhances, increases, upregulates, alters, or otherwise promotes the immune response to an antigen. Specifically, a therapeutically effective amount is an amount that induces immunity in an animal susceptible to a disease caused by a pathogen, cancer cell, or allergen. As those skilled in the art will appreciate, therapeutically effective amounts will vary and be determined on a case-by-case basis. In this application, the doses of vaccine adjuvants and vaccine antigens are not particularly limited and will vary depending on the method of administration, subject The patient, age of the subject, dosage form, administration route, etc. should be selected appropriately. In a preferred embodiment, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: For example, the dosage ratio can be 0.005, 0.05, 0.5, 1, 1.25, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 ,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,160,166.7,170,180,200,500,1000,10000 (μg/IU or μg:μg), preferably, the dosage ratio is: Preferably, the dosage ratio is 1.25-125 (μg/IU or μg:μg); preferably, the dosage ratio is 1.25-50 (μg/IU or μg:μg); preferably, the dosage ratio is 1.25～25 (μg/IU or μg:μg); Preferably, the dosage ratio is 1.25～12.5 (μg/IU or μg:μg); Preferably, the dosage ratio is 1.25～10 (μg/IU or μg:μg); Preferably, the dosage ratio is 1.25 to 5 (μg/IU or μg:μg); Preferably, the dosage ratio is 5 to 166.7 (μg/IU or μg:μg); Preferably, The dosage ratio is 5 to 125 (μg/IU or μg:μg); preferably, the dosage ratio is 5 to 50 (μg/IU or μg:μg); preferably, the dosage ratio is 5 to 25 (μg/IU or μg:μg); Preferably, the dosage ratio is 5 to 12.5 (μg/IU or μg:μg); Preferably, the dosage ratio is 5 to 10 (μg/IU or μg:μg ); Preferably, the dosage ratio is 10 to 166.7 (μg/IU or μg:μg); Preferably, the dosage ratio is 10 to 125 (μg/IU or μg:μg); Preferably, the dosage The dosage ratio is 10 to 50 (μg/IU or μg:μg); preferably, the dosage ratio is 10 to 25 (μg/IU or μg:μg); preferably, the dosage ratio is 10 to 12.5 (μg/ IU or μg:μg); preferably, the dosage ratio is 12.5-166.7 (μg/IU or μg:μg); preferably, the dosage ratio is 12.5-125 (μg/IU or μg:μg); preferably Preferably, the dosage ratio is 12.5-50 (μg/IU or μg:μg); preferably, the dosage ratio is 12.5-25 (μg/IU or μg:μg); preferably, the dosage ratio is 25 ~166.7 (μg/IU or μg:μg); Preferably, the dosage ratio is 25~125 (μg/IU or μg:μg); Preferably, the dosage ratio is 25~50 (μg/IU or μg :μg); Preferably, the dosage ratio is 12.5 to 25 (μg/IU or μg:μg); Preferably, the dosage ratio is 50 to 166.7 (μg/IU or μg:μg); Preferably, the dosage ratio is The dosage ratio is 50-125 (μg/IU or μg:μg); preferably, the dosage ratio is 125-166.7 (μg/IU or μg:μg).

In a preferred embodiment, when the vaccine composition provided in this application is used for influenza vaccine, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: For example, it can be 17 (μg:μg), 20 (μg:μg), 25 (μg:μg), 30 (μg:μg), 35 (μg:μg), 40 (μg:μg), 45 (μg:μg ), 50(μg:μg), 55(μg:μg), 60(μg:μg), 65(μg:μg), 70(μg:μg), 75(μg:μg), 80(μg:μg) , 90(μg:μg), 100(μg:μg), 110(μg:μg), 120(μg:μg), 130(μg:μg), 140(μg:μg), 150(μg:μg), 160(μg:μg), with better effect.

In a preferred embodiment, when the vaccine composition provided in this application is used in a hepatitis A vaccine, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: For example, it can be 6 (μg:IU), 7 (μg:IU), 8 (μg:IU), 9 (μg:IU), 10 (μg:IU), 15 (μg:IU), 20 (μg:IU ), 25(μg:IU), 30(μg:IU), 35(μg:IU), 40(μg:IU), 45(μg:IU), 49(μg:IU), have better effects.

In a preferred embodiment, when the vaccine composition provided in this application is used for hand, foot and mouth vaccine, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: For example, it can be 1.5(μg:IU), 2(μg:IU), 2.5(μg:IU), 3(μg:IU), 4(μg:IU), 5(μg:IU), 6(μg:IU ), 7(μg:IU), 8(μg:IU), 9(μg:IU), 10(μg:IU), 15(μg:IU), 16(μg:IU), 17(μg:IU) , 18(μg:IU), 19(μg:IU), 20(μg:IU), 21(μg:IU), 22(μg:IU), 23(μg:IU), 24(μg:IU), Have better results.

In a preferred embodiment, when the vaccine composition provided in this application is used for HPV vaccine, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: For example, it can be 13(μg:μg), 14(μg:μg), 15(μg:μg), 16(μg:μg), 17(μg:μg), 18(μg:μg), 19(μg:μg ), 20(μg:μg), 25(μg:μg), 30(μg:μg), 35(μg:μg), 40(μg:μg), 50(μg:μg), 60(μg:μg) , 70(μg:μg), 80(μg:μg), 90(μg:μg), 100(μg:μg), 110(μg:μg), 120(μg:μg), 121(μg:μg), 124(μg:μg), with better effect.

In a preferred embodiment, the vaccine composition provided in this application is used in a new coronavirus vaccine, and the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: For example, it can be 11(μg:μg), 12(μg:μg), 13(μg:μg), 14(μg:μg), 15(μg:μg), 16(μg:μg), 17(μg:μg ), 18(μg:μg), 19(μg:μg), 20(μg:μg), 25(μg:μg), 30(μg:μg), 35(μg:μg), 40(μg:μg) , 45(μg:μg), 46(μg:μg), 47(μg:μg), 48(μg:μg), 49(μg:μg), have better effects.

Those skilled in the art will readily recognize that the dosage and length of treatment may vary depending on the type, weight, and condition of the patient to be treated, their individual response to the vaccine composition, and the particular route of administration chosen. In some cases, dosage levels below the lower end of the foregoing ranges may be therapeutically effective, while in other cases, still larger dosages may be employed without causing any deleterious side effects, provided such larger dosages are First divide into several small doses to be administered throughout the day. Whenever secondary stress or exposure is likely to occur, a challenge dose is considered ideal.

The vaccine adjuvant and the antigen or the DNA encoding the antigen in the vaccine combination of the present application can be included together in one composition and can be formulated in separate compositions. When formulated in separate compositions, the vaccine adjuvant and The route of administration of the antigen or the DNA encoding the antigen may be the same or different. In the present application, the vaccine adjuvant and the antigen or DNA encoding the antigen may be administered simultaneously or with a temporal difference, i.e., the vaccine adjuvant and the antigen or DNA encoding the antigen may be administered simultaneously or separately (e.g., vaccine The adjuvant is administered before or after the vaccine antigen is administered). Vaccine adjuvants and antigens or DNA encoding said antigens may be provided as kits containing them. However, from the perspective of reducing patient burden, it is preferable that the vaccine adjuvant and the antigen or DNA encoding the antigen are included in one composition so that they can be administered simultaneously. Whether co-administered or simultaneously administered, the mode of administration of the vaccine composition can be any suitable route that delivers the vaccine composition to Host.

In a preferred embodiment, the vaccine adjuvant or vaccine composition of the present application also includes pharmaceutical auxiliary materials and a second vaccine adjuvant.

The pharmaceutical excipients are as described above.

The vaccine adjuvants of the present application can be administered as part of a vaccine formulation, optionally containing an additional second vaccine adjuvant. The second vaccine adjuvant is other adjuvant different from the ginseng acidic polysaccharide adjuvant of the present application, and may be one or more than two. Examples of appropriate second vaccine adjuvants include those known in the art. adjuvants,

The vaccine adjuvant or vaccine composition of the present application may further comprise one or more antioxidants selected from the group consisting of: sodium bisulfite, sodium sulfite, metabisulfite Sodium, sodium thiosulfate, sodium methoxysulfite, L-ascorbic acid, erythorbic acid, acetylcysteine, cysteine, monothioglycerol, mercaptoacetic acid, thiolactic acid, sulfide, dithiol Thiothreitol, dithioerythritol, glutathione, ascorbyl palmitolic acid, butylated hydroxytoluene, butylated hydroxytoluene, nordihydroguaiaretic acid, propyl gallate Tocopherol, a-tocopherol, and mixtures thereof.

The vaccine adjuvant or vaccine composition of the present application may further comprise one or more preservatives. Examples of suitable preservatives include (but are not limited to): benzalkonium chloride, ethyl alcohol, benzoic acid, ethyl alcohol, p- Formaben, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, sodium benzoate, phenol, and mixtures thereof. As one skilled in the art will appreciate, the presence or absence of preservatives will depend on the antigen. For example, if the antigen is a live bacterial antigen, no preservatives are needed.

The vaccine adjuvant or vaccine composition of the present application can be used to prevent or treat diseases caused by pathogens, cancer cells or allergens in humans or animals by administering a therapeutically effective amount of the adjuvant to humans or animals susceptible to the disease. composition or vaccine.

According to the present application, the pathogen can be any pathogen, including but not limited to: bacteria, protozoa, helminths, viruses and fungi. Diseases in animals caused by such pathogens include, but are not limited to: bovine respiratory disease, porcine respiratory disease, pneumonia, pasteurellosis, coccidiosis, anaplasmosis, and infectious keratitis.

According to the present application, the cancer cells can be any type of cancer cells in the art. According to this application, the allergen may be any allergen known in the art.

For example, the vaccine composition of the present application can be a rabies vaccine, an influenza vaccine, a hepatitis B vaccine, a hepatitis A vaccine, a hepatitis C vaccine, a hand, foot and mouth vaccine, an HPV vaccine or a new coronavirus vaccine, etc.

For example, the vaccine type of the vaccine composition of the present application can be an inactivated virus vaccine, an attenuated vaccine, an inactivated vaccine, a protein vaccine, a DNA vaccine or a polypeptide vaccine, etc.

The vaccine adjuvant or vaccine composition of the present application can be used to protect or treat humans and non-human animals such as livestock and livestock animals, including (but not limited to) cattle, horses, sheep, pigs, goats, rabbits, cats, dogs and other animals in need. Other mammals treated. Preferably, the vaccine adjuvant or vaccine composition of the present application is used to protect or treat humans. As will be appreciated by those skilled in the art, the vaccine adjuvant or vaccine composition of the present application to be administered can be selected based on the patient to be protected or treated.

The compositions of the present application can be prepared by general methods, in which one or more pharmaceutically acceptable diluents or carriers are added, for example, in an oral pharmaceutical form, such as tablets, capsules, granules, powders, and lozenges. , syrups, emulsions, suspensions, etc., or parenteral drugs, such as topical drugs, suppositories, injections, eye drops, intranasal agents, transpulmonary agents, etc. Preferred examples of formulations include injectable or intranasal solutions, or lyophilized formulations prepared by lyophilizing said solutions.

Examples of injectable solutions include emulsions and liposomes containing aqueous solutions and oleaginous compositions, such as aqueous solutions in which a vaccine adjuvant and an antigen or DNA encoding the antigen are dissolved or dispersed in water. A solution formulation or an aqueous suspension formulation, or an oily solution formulation or an oily suspension formulation in which the vaccine adjuvant and the antigen or DNA encoding the antigen are dissolved or dispersed in oil.

Examples of aqueous solutions, aqueous solution formulations or aqueous suspension formulations include aqueous solutions containing distilled water for injection and optionally containing buffers, pH adjusters, stabilizers, isotonic agents and/or emulsifiers, or Aqueous suspension, etc.

The vaccine adjuvant or vaccine composition of the present application can be administered by oral, intramuscular, intravenous, subcutaneous, intraocular, parenteral, topical, intravaginal or rectal routes. For administration to cattle, pigs or other livestock, the adjuvant composition or vaccine adjuvant may be administered orally in feed or as a gavage composition. In a preferred embodiment, the vaccine adjuvant or vaccine composition of the present application is injected intramuscularly, intravenously or subcutaneously.

The ginseng acidic polysaccharide vaccine adjuvant provided by this application can significantly increase the specific antibody (or neutralizing antibody) titer after antigen immunization, and can be used for rabies vaccine, influenza vaccine, hepatitis B vaccine, hepatitis A vaccine, hepatitis C vaccine, and hand, foot and mouth vaccine. , HPV vaccine, new coronavirus vaccine and other vaccines, can effectively enhance immune response activity. As the experimental results of this application show, compared with commonly used vaccine adjuvants in the prior art, such as aluminum salt vaccine adjuvants, in equivalent experiments Under the conditions, ginseng acidic polysaccharide adjuvant can significantly increase the level of neutralizing antibodies in the blood of vaccinated mice (P< 0.01); for some vaccines, such as hepatitis B vaccine, the antibody levels in experimental mice are more significantly improved (P<0.001).

At the same time, this application has gone through a large number of tests to explore the impact of using different doses of ginseng polysaccharide vaccine adjuvants on the adjuvant activity of the vaccine. It was found that different doses have a certain impact on the adjuvant activity, and also have effects on different types of vaccines. Different effects. After many attempts, the dosage of ginseng polysaccharide vaccine adjuvant with better effect has been optimized. In addition, this application has further optimized the dosage of vaccine adjuvant with specific and better effects for different vaccines, such as The results of the examples in this application show that for influenza vaccines, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: has a better effect; for hepatitis A vaccine, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: When used for hand, foot and mouth vaccine, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: When used for HPV vaccine, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: has better results; For use in the new coronavirus vaccine, the dosage ratio of the ginseng acidic polysaccharide to the vaccine antigen is: has better results.

Example

Experimental Materials

Aluminum salt adjuvant: produced by Croda Company

Preparation of ginseng polysaccharide:

Take 1kg of dried ginseng roots and crush it to 10-20 mesh, add 8 times the amount of water (g:mL), cook in a boiling water bath for 8 hours, filter and collect the filtrate; continue to add 10 times the amount of water (g:mL) to the filter residue, boiling water The bath is heated for 8 hours. Combine the two filtrates and centrifuge at 6000 rpm for 10 min. The supernatant was evaporated to dryness, neutral protease (Neutrase, enzyme concentration 800U/g _extract ) was added to the extract, incubated at 40°C for 3 hours, and then inactivated at 100°C for 30 minutes. The mixed solution was centrifuged at 10,000 rpm to obtain the supernatant, which was evaporated. Dry.

Soak in 10 times the volume of 95% ethanol at low temperature for 2.0 hours. Filter, take the filter residue, add 2 times the volume of distilled water to dissolve, extract with boiling water reflux 3 times, 1 hour each time, filter with 120 mesh nylon cloth, combine the extracts, concentrate to 500mL, centrifuge for 20 minutes (5000rpm), discard the precipitate. Add 95% ethanol to the supernatant until the final concentration of ethanol is 90%, and let it stand for 1 hour. Centrifuge for 20 minutes (5000 rpm), collect the precipitate, add 400 mL of distilled water to the precipitate, then add 95% ethanol to the supernatant to a final concentration of 80%, let stand for 1 hour, and centrifuge for 20 minutes (5000 rpm) , let stand at room temperature overnight, and collect the precipitate.

The next day, the precipitate was washed twice with absolute ethanol and 95% ethanol. The ginseng crude polysaccharide was freeze-dried and prepared into a 30% aqueous solution, added to a 1000kDa dialysis bag for dialysis, and left to stand overnight. Take the liquid in the dialysis bag, add 1/4 volume of Sevag reagent to it, let it stand and centrifuge to remove the gel-like precipitate. Repeat the above operation 5 times, combine the supernatants, concentrate under reduced pressure to remove organic reagents, add 95% ethanol to a final concentration of 80%, let stand at 4°C overnight, centrifuge, and remove the supernatant. The precipitate was washed twice with 95% ethanol and absolute ethanol. Freeze-dried ginseng polysaccharide.

Preparation of ginseng acidic polysaccharide:

Weigh 50g of the ginseng polysaccharide prepared above, dissolve it in 500mL of deionized water, and perform DEAE cellulose column chromatography. The specific steps are as follows:

Pretreatment: Add distilled water to prevent bubbles into the column tube, add DEAE packing to the column tube, and let it stand for 24 hours; rinse 1 column volume with 0.5M hydrochloric acid; then balance 4 column volumes with ultrapure water;

Take total ginseng polysaccharides, dissolve them in distilled water, load the sample at a speed of 4 mL/min, and elute with water and 0.3 mol/L NaCl solution respectively. The flow rate is set to 0.5 mL/min. The eluate was tested for A ₄₉₀ using the phenol sulfuric acid method, the absorption peaks were collected, dialyzed against distilled water for 72 hours, and freeze-dried. Ginseng acidic polysaccharide was obtained, and the elution curve is shown in Figure 1.

Determination of ginseng acidic polysaccharide content

The phenol-sulfuric acid method was used to determine the content of ginseng acidic polysaccharides. Using different concentrations of glucose as the abscissa and the absorbance value at OD490nm as the ordinate, draw a standard curve, as shown in Figure 2. Calculate the polysaccharide content based on the glucose standard curve and sample absorbance. The content of ginseng acidic polysaccharides is 97.3%.

Determination of protein content in ginseng acidic polysaccharide

The protein content in ginseng acidic polysaccharide was determined using the Bradford method. Taking bovine serum albumin as the abscissa and OD595nm as the ordinate, draw a standard curve, as shown in Figure 3. Calculate the protein content based on the bovine serum albumin standard curve and sample absorbance. The protein content of ginseng acidic polysaccharide is 0.3%.

Example 1: Immune adjuvant activity of ginseng acidic polysaccharide (GAPS) to rabies vaccine

Diploid inactivated rabies vaccine: produced by Liaoning Chengda Biological Co., Ltd.

Animals: Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.

1. Preparation of vaccine composition:

High-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and diploid inactivated rabies vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GPS and 2.5 IU of rabies vaccine, and add 0.22 Filter with μm microporous membrane and pack aseptically.

High-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and diploid inactivated rabies vaccine respectively, and dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GAPS and 2.5 IU of rabies vaccine. Filter through 0.22μm microporous membrane and pack aseptically.

Low-dose GPS adjuvant vaccine composition: Measure appropriate amounts of GPS and diploid inactivated rabies vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 2.5 IU of rabies vaccine, filter with 0.22 μm micropores Membrane filtration, sterile packaging.

Low-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of GAPS and diploid inactivated rabies vaccine respectively, dissolve them in physiological saline so that each milliliter of the solution contains 250 μg of GAPS and 2.5 IU of rabies vaccine, filter with 0.22 μm micropores Membrane filtration, sterile packaging.

Positive control group - aluminum salt adjuvant vaccine composition: measure appropriate amounts of aluminum salt and diploid inactivated rabies vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of aluminum salt and 2.5 IU of rabies vaccine. Filter through 0.22μm microporous membrane and pack aseptically.

Negative control group - vaccine without adjuvant: Dissolve an appropriate amount of diploid inactivated rabies vaccine in physiological saline so that each milliliter of the solution contains 2.5IU of rabies vaccine, filter it with a 0.22μm microporous filter, and sterilize Pack.

2. Immunization plan:

Mice were randomly divided into 6 groups, with 10 mice in each group. Inject 0.1 ml/animal of the vaccine composition prepared above intramuscularly. One week after the first immunization, the second immunization is performed. Sera from mice in each group were collected 14 days after the second immunization, and the neutralizing antibodies and IgG-specific antibody titers in each serum were detected by RFFIT and ELISA.

Experimental groups and doses:

GPS high-dose adjuvant group: 0.25IU rabies vaccine + 250μg GPS/animal;

GAPS high-dose adjuvant group: 0.25IU rabies vaccine + 250μg GAPS/animal;

GPS low-dose adjuvant group: 0.25IU rabies vaccine + 25μg GPS/animal;

GAPS low-dose adjuvant group: 0.25IU rabies vaccine + 25μg GAPS/animal;

Positive control group: 0.25IU rabies vaccine + 25μg aluminum salt adjuvant/animal;

Negative control group: 0.25IU rabies vaccine/animal.

3. Result:

As shown in Figures 4 and 5, compared with the negative control group, aluminum salts and high and low doses of GAPS can increase the levels of IgG antibodies and neutralizing antibodies in mice vaccinated with rabies. Compared with the aluminum salt positive control group, high and low doses of GAPS (250 μg, 25 μg) can significantly increase the IgG antibody level and neutralizing antibody level in rabies vaccine mice (P<0.01).

Example 2: Immune adjuvant activity of ginseng acidic polysaccharide (GAPS) for influenza vaccine

Influenza vaccine: produced by Liaoning Chengda Biological Co., Ltd.

1. Preparation of vaccine composition:

High-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and influenza vaccine respectively, dissolve them in physiological saline so that each milliliter of the solution contains 2500 μg of GPS and 15 μg of influenza vaccine, and filter with a 0.22 μm microporous membrane. Passed, sterile aliquots.

High-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and influenza vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GPS and 15 μg of influenza vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Low-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and influenza vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 15 μg of influenza vaccine, and filter with a 0.22 μm microporous membrane. Passed, sterile aliquots.

Low-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and influenza vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 15 μg of influenza vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Positive control group-aluminum salt adjuvant vaccine composition: measure appropriate amounts of aluminum salt and influenza vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of aluminum salt and 15 μg of influenza vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Negative control group - vaccine without adjuvant: Dissolve an appropriate amount of influenza vaccine in physiological saline so that each milliliter of the solution contains 15 μg of influenza vaccine, filter it with a 0.22 μm microporous filter, and pack aseptically.

2. Immunization plan:

Mice were randomly divided into 6 groups, with 10 mice in each group. Inject 0.1ml/animal of the above composition intramuscularly, and perform the second immunization one week after the first immunization. On the 14th day after the second immunization, the blood of each mouse was collected, and the neutralizing antibody level of the mouse serum influenza vaccine was detected by hemagglutination inhibition test.

Experimental groups and doses:

GPS high-dose adjuvant group: 1.5μg influenza vaccine + 250μg GPS/bird;

GAPS high-dose adjuvant group: 1.5μg influenza vaccine + 250μg GAPS/bird;

GPS low-dose adjuvant group: 1.5μg influenza vaccine + 25μg GPS/bird;

GAPS low-dose adjuvant group: 1.5μg influenza vaccine + 25μg GAPS/bird;

Positive control group: 1.5 μg influenza vaccine + 25 μg aluminum salt adjuvant/animal;

Negative control group: 1.5μg influenza vaccine/animal.

3. Result:

As shown in Figure 6, compared with the negative control group, both low and high doses of GAPS can significantly increase the antibody levels in influenza vaccinated mice (P<0.01); compared with the positive control group (aluminum salt adjuvant) , both low and high doses of GAPS can significantly increase the level of neutralizing antibodies in the blood of influenza vaccinated mice (P<0.01).

Example 3: Immune adjuvant activity of different doses of ginseng acidic polysaccharide (GAPS) for influenza vaccine 1. Preparation of vaccine composition: Measure appropriate amounts of GAPS and influenza vaccine respectively, dissolve them with physiological saline, and prepare five composition solutions respectively. GAPS adjuvant group 1, GAPS adjuvant group 2, GAPS adjuvant group 3, GAPS adjuvant group 4, and GAPS adjuvant group 5, so that each ml of solution in each group contains 50, 250, 1250, 2500, and 5000 μg respectively. GAPS and 15 μg of influenza vaccine are filtered through a 0.22 μm microporous membrane and packed aseptically.

At this time, the ratios of GAPS adjuvant to influenza vaccine in each group of vaccine compositions were 3.3, 16.7, 83.3, 166.7, and 333.3 (μg:μg) respectively.

2. Immunization plan:

Mice were randomly divided into 7 groups, with 10 mice in each group. Inject 0.1ml/animal of the above composition intramuscularly, and perform the second immunization one week after the first immunization. On the 14th day after the second immunization, the blood of each mouse was collected, and the neutralizing antibody level of the mouse serum influenza vaccine was detected by hemagglutination inhibition test.

Experimental groups and doses:

GAPS adjuvant group 1: 1.5μg influenza vaccine + 5μg GAPS/bird;

GAPS adjuvant group 2: 1.5μg influenza vaccine + 25μg GAPS/bird;

GAPS adjuvant group 3: 1.5μg influenza vaccine + 125μg GAPS/bird;

GAPS adjuvant group 4: 1.5μg influenza vaccine + 250μg GAPS/bird;

GAPS adjuvant group 5: 1.5μg influenza vaccine + 500μg GAPS/bird;

Negative control group: 1.5μg influenza vaccine/animal.

3. Result:

As shown in Figure 7, using different doses of GAPS as an adjuvant, it can be found that for influenza vaccines, the antibody levels in mice in GAPS adjuvant groups 2, 3, 4, and 5 were significantly higher than those in the negative control group and the positive control group. . However, the antibody levels in mice in GAPS adjuvant group 5 were significantly lower than those in adjuvant group 4, and the amount of polysaccharide used was greater. There was no significant difference in the antibody levels of mice in GAPS adjuvant group 1 with the positive control group and the negative control group.

Example 4: Immune adjuvant activity of ginseng acidic polysaccharide (GAPS) for hepatitis B vaccine

Hepatitis B vaccine: produced by Liaoning Chengda Biological Co., Ltd.

1. Preparation of vaccine composition:

High-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and hepatitis B vaccine respectively, dissolve them in physiological saline so that each milliliter of the solution contains 2500 μg of GPS and 25 μg of hepatitis B vaccine, and filter with a 0.22 μm microporous membrane. Passed, sterile aliquots.

High-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and hepatitis B vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GPS and 25 g of hepatitis B vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Low-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and hepatitis B vaccine respectively, dissolve them in physiological saline so that each milliliter of the solution contains 250 μg of GPS and 25 μg of hepatitis B vaccine, and filter with a 0.22 μm microporous membrane. Passed, sterile aliquots.

Low-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and hepatitis B vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 25 μg of hepatitis B vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Positive control group - aluminum salt adjuvant vaccine composition: measure appropriate amounts of aluminum salt and hepatitis B vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of aluminum salt and 25 μg of hepatitis B vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Negative control group - vaccine without adjuvant: Dissolve an appropriate amount of hepatitis B vaccine in physiological saline so that each milliliter of solution contains 25 μg of hepatitis B vaccine, filter it with a 0.22 μm microporous filter, and pack aseptically.

2. Immunization plan:

Mice were randomly divided into 6 groups, with 10 mice in each group. Inject 0.1ml/animal of the above composition intramuscularly, and perform the second immunization 2 weeks after the first immunization. On the 14th day after the second immunization, the blood of the mice was collected, and the serum hepatitis B antibody levels of the mice were detected by ELISA experiment.

Experimental groups and doses:

GPS high-dose adjuvant group: 2.5μg hepatitis B vaccine + 250μg GPS/animal;

GAPS high-dose adjuvant group: 2.5 μg hepatitis B vaccine + 250 μg GAPS/bird;

GPS low-dose adjuvant group: 2.5μg hepatitis B vaccine + 25μg GPS/animal;

GAPS low-dose adjuvant group: 2.5 μg hepatitis B vaccine + 25 μg GAPS/bird;

Positive control group: 2.5 μg hepatitis B vaccine + 25 μg aluminum salt adjuvant/animal;

Negative control group: 2.5μg hepatitis B vaccine/animal.

3. Result:

As shown in Figure 8, compared with the negative control group, aluminum salts and high and low doses of GAPS can significantly increase the antibody levels in mice immunized with hepatitis B vaccine (P<0.001); compared with the positive control group (aluminum salt adjuvant) In comparison, high-dose GAPS (250 μg/mouse) can significantly increase the antibody level in mice immunized with hepatitis B vaccine (P<0.001). The adjuvant activity of low-dose GAPS (25 μg/mouse) for hepatitis B vaccine is similar to that of aluminum salts. .

Example 5: Adjuvant activity of ginseng acidic polysaccharide (GAPS) for hepatitis A vaccine

Diploid hepatitis A vaccine: produced by Liaoning Chengda Biological Co., Ltd.

1. Preparation of vaccine composition:

High-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and hepatitis A vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GPS and 50 IU of hepatitis A vaccine, and filter with a 0.22 μm microporous membrane. Passed, sterile aliquots.

High-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and hepatitis A vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GPS and 50 IU of hepatitis A vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Low-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and hepatitis A vaccine respectively, dissolve them in physiological saline so that each milliliter of the solution contains 250 μg of GPS and 50 IU of hepatitis A vaccine, and filter with a 0.22 μm microporous membrane. Passed, sterile aliquots.

Low-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and hepatitis A vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 50 IU of hepatitis A vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Positive control group - aluminum salt adjuvant vaccine composition: measure appropriate amounts of aluminum salt and hepatitis A vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of aluminum salt and 50 IU of hepatitis A vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Negative control group - vaccine without adjuvant: Dissolve an appropriate amount of influenza vaccine in physiological saline so that each milliliter of the solution contains 50IU of hepatitis A vaccine, filter it with a 0.22μm microporous filter, and pack aseptically.

2. Immunization plan:

Mice were randomly divided into 4 groups, with 10 mice in each group. 0.1 ml of the above composition was injected intramuscularly per animal. Two weeks after the first immunization, the second immunization was performed. On the 14th day after the second immunization, the blood of each mouse was collected, and the neutralizing antibody level in the serum was detected.

Experimental groups and doses:

GPS high-dose adjuvant group: 5IU hepatitis A vaccine + 250 μg GPS/bird;

GAPS high-dose adjuvant group: 5IU hepatitis A vaccine + 250 μg GAPS/bird;

GPS low-dose adjuvant group: 5IU hepatitis A vaccine + 25μg GPS/animal;

GAPS low-dose adjuvant group: 5IU hepatitis A vaccine + 25 μg GAPS/bird;

Positive control group: 5IU hepatitis A vaccine + 25 μg aluminum salt adjuvant/animal;

Negative control group: 5IU hepatitis A vaccine/animal.

3. Result:

As shown in Figure 9, compared with the negative control group, aluminum salts and high and low doses of GAPS can significantly increase the antibody levels in mice immunized with hepatitis A vaccine (P<0.05); compared with the positive control group (aluminum salt adjuvant) In comparison, both low and high doses of GAPS can significantly increase the antibody levels in mice immunized with hepatitis A vaccine (P<0.05).

Example 6: Adjuvant activity of different doses of ginseng acidic polysaccharide (GAPS) for hepatitis A vaccine

1. Preparation of vaccine composition:

Measure appropriate amounts of GAPS and hepatitis A vaccine respectively, dissolve them in physiological saline, and prepare five composition solutions: GAPS adjuvant group 1, GAPS adjuvant group 2, GAPS adjuvant group 3, GAPS adjuvant group 4, and GAPS adjuvant. Group 5, make each group contain 125, 250, 1250, 1250, 2500 and 5000 μg of GAPS and 50 IU of hepatitis A vaccine are filtered through a 0.22 μm microporous membrane and packed aseptically. At this time, the ratios of GAPS adjuvant and hepatitis A vaccine in each group of vaccine compositions were 2.5, 5, 25, 50, and 100 (μg:IU) respectively.

2. Immunization plan:

Experimental groups and doses:

GAPS adjuvant group: 5IU hepatitis A vaccine + 12.5μg GAPS/bird;

GAPS adjuvant group 2: 5IU hepatitis A vaccine + 25μg GAPS/bird;

GAPS adjuvant group 3: 5IU hepatitis A vaccine + 125 μg GAPS/bird;

GAPS adjuvant group 4: 5IU hepatitis A vaccine + 250 μg GAPS/bird;

GAPS adjuvant group 5: 5IU hepatitis A vaccine + 500 μg GAPS/bird;

Negative control group: 5IU hepatitis A vaccine/animal.

3. Result:

As shown in Figure 10, using different doses of GAPS as an adjuvant, it can be found that for hepatitis A vaccine, the antibody levels in mice in GAPS adjuvant groups 2, 3, and 4 were significantly higher than those in the negative control group and the positive control group. Compared with the positive control group, although the antibody levels in the mice of GAPS adjuvant group 5 have increased to a certain extent, the effect is weaker than that of adjuvant group 4, and the dosage of polysaccharide is larger.

Example 7: Adjuvant activity of ginseng acidic polysaccharide (GAPS) for hepatitis C vaccine

Hepatitis C vaccine: produced by Liaoning Chengda Biological Co., Ltd.

1. Preparation of vaccine composition:

High-dose GPS adjuvant vaccine composition: measure appropriate amounts of ginseng polysaccharide (GPS) and hepatitis C vaccine respectively, Dissolve in physiological saline so that each milliliter of the solution contains 2500 μg of GPS and 50 μg of hepatitis C vaccine. Filter with a 0.22 μm microporous membrane and pack aseptically.

High-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and hepatitis C vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GPS and 50 μg of hepatitis C vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Low-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and hepatitis C vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 50 μg of hepatitis C vaccine, and filter with a 0.22 μm microporous membrane. Passed, sterile aliquots.

Low-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and hepatitis C vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 50 μg of hepatitis C vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Positive control group - aluminum salt adjuvant vaccine composition: measure appropriate amounts of aluminum salt and hepatitis C vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of aluminum salt and 50 μg of hepatitis C vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Negative control group - vaccine without adjuvant: Dissolve an appropriate amount of hepatitis C vaccine in physiological saline so that each milliliter of solution contains 50 μg of hepatitis C vaccine, filter it with a 0.22 μm microporous filter, and pack aseptically.

2. Immunization plan:

Mice were randomly divided into 6 groups, with 10 mice in each group. 0.1 ml of the above composition was injected intramuscularly per animal. Two weeks after the first immunization, the second immunization was performed. On the 14th day after the second immunization, the blood of each mouse was collected, and the level of specific antibody IgG in the serum was detected.

Experimental grouping:

GPS high-dose adjuvant group: 5μg hepatitis C vaccine + 500μg GPS/animal;

GAPS high-dose adjuvant group: 5 μg hepatitis C vaccine + 500 μg GAPS/bird;

GPS low-dose adjuvant group: 5μg hepatitis C vaccine + 50μg GPS/animal;

GAPS low-dose adjuvant group: 5 μg hepatitis C vaccine + 50 μg GAPS/bird;

Positive control group: 5 μg hepatitis C vaccine + 50 μg aluminum salt adjuvant/animal;

Negative control group: 5 μg hepatitis C vaccine/animal.

3. Result:

As shown in Figure 11, compared with the negative control group, aluminum salts and high and low doses of GAPS can significantly increase the antibody levels in mice immunized with hepatitis C vaccine (P<0.001); compared with the positive control group (aluminum salt adjuvant) In comparison, both high and low doses of GAPS could significantly increase the antibody levels in mice immunized with hepatitis C vaccine (P<0.01).

Example 8: Adjuvant activity of ginseng acidic polysaccharide (GAPS) for hand, foot and mouth vaccine

Hand, foot and mouth vaccine: produced by Liaoning Chengda Biological Co., Ltd.

1. Preparation of vaccine composition:

High-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and hand, foot and mouth vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GPS and 200 IU of hand, foot and mouth vaccine, filter with 0.22 μm micropores Membrane filtration, sterile packaging.

High-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and hand, foot and mouth vaccine respectively, dissolve them in physiological saline, so that each milliliter of solution contains 2500 μg of GPS and 200 IU of hand, foot and mouth vaccine, and use 0.22 μm micropores Filter through membrane and aliquot aseptically.

Low-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and hand, foot and mouth vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 200 IU of hand, foot and mouth vaccine, filter with 0.22 μm micropores Membrane filtration, sterile packaging.

Low-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and hand, foot and mouth vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 200 IU of hand, foot and mouth vaccine, and use 0.22 μm micropores Filter through membrane and aliquot aseptically.

Positive control group - aluminum salt adjuvant vaccine composition: measure appropriate amounts of aluminum salt and hand, foot and mouth vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of aluminum salt and 200 IU of hand, foot and mouth vaccine, and use 0.22 μm micropores Filter through membrane and aliquot aseptically.

Negative control group - vaccine without adjuvant: Dissolve an appropriate amount of hand, foot and mouth vaccine in physiological saline so that each milliliter of solution contains 200IU of hand, foot and mouth vaccine, filter it with a 0.22 μm microporous filter, and pack aseptically.

2. Immunization scheme: Mice were randomly divided into 6 groups, 10 mice in each group. Inject 0.1ml/mouse of the above composition intramuscularly. 2 weeks after the first immunization, perform the second immunization. On the 14th day after the second immunization, collect the blood of each mouse and detect serum neutralizing antibodies and specific antibody IgG. level.

Experimental grouping:

GPS high-dose adjuvant group: 20IU hand, foot and mouth vaccine + 250 μg GPS/bird;

GAPS high-dose adjuvant group: 20IU hand, foot and mouth vaccine + 250 μg GAPS/bird;

GPS low-dose adjuvant group: 20IU hand, foot and mouth vaccine + 25 μg GPS/bird;

GAPS low-dose adjuvant group: 20IU hand, foot and mouth vaccine + 25 μg GAPS/bird;

Positive control group: 20IU hand, foot and mouth vaccine + 25 μg aluminum salt adjuvant/animal;

Negative control group: 20IU hand, foot and mouth vaccine/animal.

3. Result:

As shown in Figures 12 and 13, compared with the negative control group, aluminum salts and high and low doses of GAPS can significantly increase the antibody levels in mice immunized with hand, foot and mouth vaccine (P<0.05); compared with the aluminum salt positive control group In comparison, both high and low doses of GAPS could significantly increase the levels of neutralizing antibodies and IgG antibodies in mice vaccinated with hand, foot and mouth vaccination (P<0.01).

Example 9: Adjuvant activity of different doses of ginseng acidic polysaccharide (GAPS) for hand, foot and mouth vaccine

1. Prepare vaccine composition: measure appropriate amounts of GPS and hand, foot and mouth vaccine respectively, dissolve them in physiological saline, and prepare five composition solutions: GAPS adjuvant group 1, GAPS adjuvant group 2, GAPS adjuvant group 3, GAPS Adjuvant group 4, GAPS adjuvant group 5, each group contains 125, 250, 1250, 2500, 5000μg of GPS and 200IU of hand, foot and mouth vaccine per milliliter of solution, filtered with a 0.22μm microporous membrane, sterile Packaging. At this time, the ratios of GPS adjuvant and hand, foot and mouth vaccine in the composition were 0.625, 1.25, 6.25, 12.5, and 25 (μg:IU) respectively.

2. Immunization scheme: Mice were randomly divided into 7 groups, 10 mice in each group. 0.1 ml of the above composition was injected intramuscularly per animal. Two weeks after the first immunization, the second immunization was performed. On the 14th day after the second immunization, the blood of each mouse was collected, and the serum neutralizing antibody level was detected.

Experimental groups and doses:

GAPS adjuvant group 1: 20IU hand, foot and mouth vaccine + 12.5μg GPS/bird;

GAPS adjuvant group 2: 20IU hand, foot and mouth vaccine + 25 μg GPS/bird;

GAPS adjuvant group 3: 20IU hand, foot and mouth vaccine + 125 μg GPS/bird;

GAPS adjuvant group 4: 20IU hand, foot and mouth vaccine + 250 μg GPS/animal;

GAPS adjuvant group 5: 20IU hand, foot and mouth vaccine + 500 μg GPS/bird;

Negative control group: 20IU hand, foot and mouth vaccine/animal.

3. Result:

As shown in Figure 14, using different doses of GAPS as an adjuvant, it can be found that for hand, foot and mouth vaccine, the antibody levels in mice in GAPS adjuvant groups 2, 3, 4, and 5 were significantly higher than those in the negative control group and the positive control Group. Compared with the negative control group, although the antibody level in mice in GAPS adjuvant group 1 increased to a certain extent, it was significantly lower than that in adjuvant groups 2 to 5.

Example 10: Adjuvant activity of ginseng acidic polysaccharide (GAPS) to HPV vaccine

HPV vaccine: produced by Liaoning Chengda Biological Co., Ltd.

1. Preparation of vaccine composition:

High-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and HPV vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GPS and 20 μg of HPV vaccine, and filter with a 0.22 μm microporous membrane. Passed, sterile aliquots.

High-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and HPV vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 2500 μg of GPS and 20 μg of HPV vaccine, and filter with a 0.22 μm microporous membrane. Filter and aliquot aseptically.

Low-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and HPV vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 20 μg of HPV vaccine, and filter with a 0.22 μm microporous membrane. Passed, sterile aliquots.

Low-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and HPV vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of GPS and 20 μg of HPV vaccine, and filter with a 0.22 μm microporous membrane. Filter and aliquot aseptically.

Positive control group-aluminum salt adjuvant vaccine composition: measure appropriate amounts of aluminum salt and HPV vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of aluminum salt and 20 μg of HPV vaccine, and filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

Negative control group - vaccine without adjuvant: Dissolve an appropriate amount of HPV vaccine in physiological saline so that each milliliter of solution contains 20 μg of HPV vaccine, filter it with a 0.22 μm microporous filter, and pack aseptically.

2. Immunization scheme: Mice were randomly divided into 6 groups, 10 mice in each group. 0.1 ml of the above composition was injected intramuscularly per animal. Two weeks after the first immunization, the second immunization was performed. On the 14th day after the second immunization, the blood of each mouse was collected, and the level of specific antibody IgG in the serum was detected.

Experimental groups and doses:

GPS high-dose adjuvant group: 2μg HPV vaccine + 250μg GPS/bird;

GAPS high-dose adjuvant group: 2μg HPV vaccine + 250μg GAPS/bird;

GPS low-dose adjuvant group: 2μg HPV vaccine + 25μg GPS/bird;

GAPS low-dose adjuvant group: 2μg HPV vaccine + 25μg GAPS/bird;

Positive control group: 2μg HPV vaccine + 25μg aluminum salt adjuvant/animal;

Negative control group: 2μg HPV vaccine/animal.

3. Result:

As shown in Figure 15, compared with the negative control group, aluminum salts and high and low doses of GAPS can significantly increase the antibody levels in mice immunized with HPV vaccine (P<0.001); compared with the aluminum salt positive control group, high-dose GAPS , low-dose GAPS can significantly increase the level of IgG antibodies in mice vaccinated with HPV (P<0.05).

Example 11: Adjuvant activity of different doses of ginseng acidic polysaccharide (GAPS) for HPV vaccine

1. Prepare the vaccine composition: measure appropriate amounts of GAPS and HPV vaccines respectively, dissolve them in physiological saline, and prepare five composition solutions: GAPS adjuvant group 1, GAPS adjuvant group 2, GAPS adjuvant group 3, GAPS adjuvant Dosage group 4, GAPS adjuvant group 5, each group contains 125, 250, 1250, 2500, 5000 μg of GAPS and 20 μg of HPV vaccine per milliliter of solution, filtered with a 0.22 μm microporous filter, and sterilely packed. . At this time, the ratios of GAPS adjuvant and HPV vaccine in each group of vaccine compositions were 6.25, 12.5, 62.5, 125, and 250 (μg:μg) respectively.

2. Immunization scheme: Mice were randomly divided into 7 groups, 10 mice in each group. Inject 0.1 of the above composition intramuscularly ml/mouse, 2 weeks after the first immunization, the second immunization was performed. On the 14th day after the second immunization, the blood of each mouse was collected, and the level of specific antibody IgG in the serum was detected.

Experimental groups and doses:

GAPS adjuvant group 1: 2μg HPV vaccine + 12.5μg GAPS/bird;

GAPS adjuvant group 2: 2μg HPV vaccine + 25μg GAPS/bird;

GAPS adjuvant group 3: 2μg HPV vaccine + 125μg GAPS/bird;

GAPS adjuvant group 4: 2μg HPV vaccine + 250μg GAPS/bird;

GAPS adjuvant group 5: 2μg HPV vaccine + 500μg GAPS/bird;

Positive control group: 2μg HPV vaccine + 25μg aluminum salt adjuvant/animal;

Negative control group: 2μg HPV vaccine/animal.

result:

As shown in Figure 16, using different doses of GAPS as an adjuvant, it can be found that for the HPV vaccine, the antibody levels in mice in the GAPS adjuvant groups 2, 3, and 4 were significantly higher than the negative control group and the positive control group. Although the antibody levels in mice in GAPS adjuvant groups 1 and 5 increased to a certain extent, they were significantly lower than those in adjuvant groups 2 to 4, and were not significantly different from the positive control group.

Example 12: Adjuvant activity of ginseng acidic polysaccharide (GAPS) on novel coronavirus vaccine (COVID-19 vaccine)

COVID-19 vaccine: produced by Liaoning Chengda Biological Co., Ltd.

1. Preparation of vaccine composition:

High-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and new coronavirus vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 5000 μg of GPS and 50 μg of new coronavirus vaccine, and filter with 0.22 μm micropores. Membrane filtration, sterile packaging.

High-dose GAPS adjuvant vaccine composition: Measure appropriate amounts of ginseng acidic polysaccharide (GAPS) and new coronavirus vaccine respectively, dissolve them in physiological saline, so that each milliliter of solution contains 5000 μg of GPS and 50 μg of new coronavirus vaccine, and use 0.22 μm micropores Filter through membrane and aliquot aseptically.

Low-dose GPS adjuvant vaccine composition: Measure appropriate amounts of ginseng polysaccharide (GPS) and new coronavirus vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 500 μg of GPS and 50 μg of new coronavirus vaccine, and filter with 0.22 μm micropores. Membrane filtration, sterile packaging.

Low-dose GAPS adjuvant vaccine composition: measure ginseng acidic polysaccharide (GAPS) and COVID-19 Dissolve an appropriate amount of coronavirus vaccine in physiological saline so that each milliliter of the solution contains 500 μg of GPS and 50 μg of COVID-19 vaccine. Filter it with a 0.22 μm microporous filter and pack aseptically.

Positive control group - Aluminum salt adjuvant vaccine composition: Measure appropriate amounts of aluminum salt and new coronavirus vaccine respectively, dissolve them in physiological saline, so that each milliliter of solution contains 500 μg of aluminum salt and 50 μg of new coronavirus vaccine, and use 0.22 μm micropores Filter through membrane and aliquot aseptically.

Negative control group - vaccine without adjuvant: Dissolve an appropriate amount of the new coronavirus vaccine in physiological saline so that each milliliter of the solution contains 50 μg of the new coronavirus vaccine, filter it with a 0.22 μm microporous filter, and pack aseptically.

Experimental groups and doses:

GPS high-dose group: 5μg COVID-19 vaccine + 500μg GPS/animal;

GAPS high-dose group: 5μg COVID-19 vaccine + 500μg GAPS/bird;

GPS low-dose group: 5μg COVID-19 vaccine + 50μg GPS/animal;

GAPS low-dose group: 5μg COVID-19 vaccine + 50μg GAPS/bird;

Positive control group: 5 μg COVID-19 vaccine + 50 μg aluminum salt adjuvant/animal;

Negative control group: 5μg new coronavirus vaccine/animal.

3. Result:

As shown in Figure 17, compared with the negative control group, aluminum salts and high and low doses of GAPS can significantly increase the antibody levels in mice immunized with the new coronavirus vaccine (P<0.01); compared with the aluminum salt positive control group, high-dose GAPS , Low-dose GAPS can significantly increase the level of IgG antibodies in mice vaccinated with the new coronavirus vaccine (P<0.05);.

Example 13: Adjuvant activity of different doses of ginseng acidic polysaccharide (GAPS) on novel coronavirus vaccine (COVID-19 vaccine)

1. Prepare the vaccine composition: measure appropriate amounts of GPS and COVID-19 vaccines respectively, dissolve them in physiological saline, and prepare five composition solutions: GAPS adjuvant group 1, GAPS adjuvant group 2, GAPS adjuvant group 3, GAPS adjuvant Dosage group 4, GAPS adjuvant group 5, each group contains 250, 500, 2500, 5000, 10000 μg of GPS and 50 μg of new coronavirus vaccine per milliliter of solution, with 0.22 Filter with μm microporous membrane and pack aseptically. At this time, the ratios of GPS adjuvant to COVID-19 vaccine in each group of vaccine compositions were 6.25, 12.5, 62.5, 125, and 250 (μg:μg) respectively.

Positive control group - Aluminum salt adjuvant vaccine composition: Measure appropriate amounts of aluminum salt and COVID-19 vaccine respectively, dissolve them in physiological saline so that each milliliter of solution contains 250 μg of aluminum salt and 50 μg of COVID-19 vaccine, filter with a 0.22 μm microporous membrane Filter and aliquot aseptically.

2. Immunization scheme: Mice were randomly divided into 7 groups, 10 mice in each group. 0.1 ml of the above composition was injected intramuscularly per animal. Two weeks after the first immunization, the second immunization was performed. On the 14th day after the second immunization, the blood of each mouse was collected, and the level of specific antibody IgG in the serum was detected.

Experimental groups and doses:

GAPS adjuvant group 1: 5μg COVID-19 vaccine + 25μg GPS/bird;

GAPS adjuvant group 2: 5μg COVID-19 vaccine + 50μg GPS/bird;

GAPS adjuvant group 3: 5μg COVID-19 vaccine + 250μg GPS/bird;

GAPS adjuvant group 4: 5μg COVID-19 vaccine + 500μg GPS/bird;

GAPS adjuvant group 5: 5μg COVID-19 vaccine + 1000μg GPS/bird;

Positive control group: 5 μg COVID-19 vaccine + 25 μg aluminum salt adjuvant/animal;

Negative control group: 5μg new coronavirus vaccine/animal.

3. Result:

As shown in Figure 18, using different doses of GAPS as an adjuvant, it can be found that for the new coronavirus vaccine, the antibody levels in the mice in the GAPS adjuvant groups 2, 3, and 4 were significantly higher than the negative control group and the positive control group. Compared with the negative control group, although the antibody levels in mice in GAPS adjuvant groups 1 and 5 increased to a certain extent, there was no significant difference from the positive control group.

Although the embodiments of the present application have been described above, the present application is not limited to the above-mentioned specific embodiments and application fields. The above-mentioned specific embodiments are only illustrative and instructive, rather than restrictive. Under the inspiration of this description and without departing from the scope of protection of the claims of this application, those of ordinary skill in the art can also make many forms, which all belong to the scope of protection of this application.

Claims

A vaccine adjuvant is characterized by including ginseng acidic polysaccharide (GAPS).
The vaccine adjuvant according to claim 1, further comprising physiological saline or water for vaccine injection or pharmaceutical excipients.
The vaccine adjuvant according to claim 1 or 2, wherein the ginseng acidic polysaccharide is extracted from ginseng.
The vaccine adjuvant according to claim 3, wherein the extraction process includes:

Take ginseng and extract total ginseng polysaccharides.

Dissolve the extracted ginseng total polysaccharides in deionized water.

After performing column chromatography, elute with eluent.

Dialyze the eluate with distilled water,

After freeze-drying, ginseng acidic polysaccharide was obtained.
The vaccine adjuvant according to claim 4, characterized in that the column of the column chromatography is a DEAE cellulose column or a macroporous resin column, preferably a DEAE cellulose column, and further preferably the column chromatography is performed with 2 to Load sample at 8mL/min.
The vaccine adjuvant according to claim 4 or 5, characterized in that the eluent is water and an alkali solution, a borax solution or a salt solution, the salt solution is preferably a NaCl solution, and the eluent is further preferably The concentration is 0.3~0.7mol/L, and it is further preferred that the elution flow rate is 0.5~2mL/min.
The vaccine adjuvant according to any one of claims 4 to 6, characterized in that before dialyzing the eluate with distilled water, it also includes detecting A 490 of the eluate using the phenol sulfuric acid method, collecting the absorption peaks, and then Dialysis is performed, preferably, the dialysis time is 24 to 72 hours.
A vaccine composition, characterized by comprising the vaccine adjuvant according to any one of claims 1-7 and an antigen or DNA encoding the antigen.
The vaccine composition according to claim 8, wherein the dosage ratio of the GAPS to the antigen is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is: Preferably, the dosage ratio is:
The vaccine composition according to claim 8 or 9, characterized in that the vaccine composition further includes pharmaceutical auxiliary materials and a second vaccine adjuvant.
The vaccine composition according to any one of claims 8-10, characterized in that the vaccine composition is a rabies vaccine, an influenza vaccine, a hepatitis B vaccine, a hepatitis A vaccine, a hepatitis C vaccine, a hand, foot and mouth vaccine, an HPV vaccine or a new type of vaccine. Coronavirus vaccine.
The vaccine composition according to any one of claims 8-11, characterized in that the vaccine type is an inactivated virus vaccine, an attenuated vaccine, an inactivated vaccine, a protein vaccine, a DNA vaccine or a polypeptide vaccine.
Use of the vaccine adjuvant according to any one of claims 1 to 7 or the ginseng acidic polysaccharide (GAPS) related to any one of claims 1 to 7 in the preparation of vaccine preparations, vaccine compositions or antibodies.
A method of preventing and/or treating disease, comprising administering the vaccine composition of any one of claims 8-12 to a subject.
The method according to claim 14, wherein the disease is selected from rabies, influenza, hepatitis B, hepatitis A, hepatitis C, hand, foot and mouth disease, HPV disease or novel coronavirus disease.