CN116115746A - Vaccine adjuvant and vaccine composition containing semen Momordicae total saponin and aluminum salt - Google Patents

Abstract

The application provides a vaccine adjuvant containing semen momordicae total saponin and aluminum salt and a vaccine composition thereof. Compared with the prior art, the vaccine adjuvant can obviously improve the titer of specific antibodies (or neutralizing antibodies) after antigen immunization, and can effectively enhance the immune response activity of rabies vaccines and influenza vaccines. The vaccine adjuvant has the advantages of good immune effect, convenient use and the like, and provides a new adjuvant choice for vaccines.

Description

Vaccine adjuvant and vaccine composition containing semen Momordicae total saponin and aluminum salt

Technical Field

The application belongs to the field of medicines, and particularly relates to a human vaccine adjuvant containing semen momordicae total saponins and aluminum salts and a vaccine composition thereof.

Background

The immunoadjuvant is a substance capable of nonspecifically changing or enhancing specific immune response of an organism to an antigen, and is required to be nontoxic, high in purity, and stable in property with a certain adsorption capacity. The action mechanism of the immunoadjuvant mainly can increase the surface area of an antigen and improve immunogenicity; the slow release effect on the antigen is achieved, and the residence time of the antigen in the tissue is prolonged; promote inflammatory response and stimulate active immune response. Most of the current commercial vaccine adjuvants are aluminum salt adjuvants, but aluminum salt can cause inflammation at injection sites and irritate local erythema, granuloma and subcutaneous nodules, and at the same time, aluminum salt adjuvants can delay the production of neutralizing antibodies of partial vaccines, thus being limited in application.

The traditional Chinese medicine is a traditional Chinese medicine, and modern pharmacology shows that most of the traditional Chinese medicines have the effects of improving immunity, resisting tumors, resisting fatigue and the like. The discovery of new immunologically active natural products has become an important point of research, particularly in the search for new generation vaccine adjuvants. The saponins are important active substances widely existing in traditional Chinese medicines and natural medicines, and in recent years, research discovers that part of saponins have better tonifying effect on immune organs. Meanwhile, in recent years, it has also been found that if a chemical component derived from a Chinese medicine or a Chinese medicine extract is combined with another type of adjuvant as an adjuvant composition, the activity of the above composition may be stronger than that of a single adjuvant. Semen Momordicae is dry mature seed of Momordicae Charantiae of Momordica of Cucurbitaceae, and has effects of resolving hard mass, relieving swelling, removing toxic substance, and treating sore.

Disclosure of Invention

In view of the problems of the prior art, the present application provides a vaccine adjuvant comprising total saponins of momordica cochinchinensis and aluminum salts, and a vaccine composition thereof. Through activity tracking and activity verification, the research discovers that the semen momordicae total saponin (MCS) has good immunoadjuvant activity, and can obviously promote the immune response of experimental individuals to specific vaccines.

In particular, the present application relates to the following:

1. a vaccine adjuvant comprising total saponins of momordica cochinchinensis and aluminum salts.

2. The vaccine adjuvant according to item 1, further comprising physiological saline or water for vaccine injection.

3. Vaccine adjuvant according to claim 1, characterized in that the mass ratio of total saponins of momordica cochinchinensis to aluminium salt is 0.0001-1000:1, preferably 0.1-4:1.

4. The vaccine adjuvant according to item 1, wherein the aluminium salt is selected from one or both of aluminium hydroxide and aluminium phosphate.

5. A vaccine composition comprising the vaccine adjuvant of any one of claims 1-4 and a vaccine antigen or DNA encoding said antigen.

6. The vaccine composition of claim 5, wherein the ratio of vaccine adjuvant to vaccine antigen is (0.1-1000): (0.10-200) μg/IU or (0.1-1000): (0.1-500. Mu.g/g), preferably 33.3-133.3. Mu.g/g or 40-80. Mu.g/IU.

7. The vaccine composition of claim 5, further comprising a pharmaceutical excipient and a second vaccine adjuvant.

8. The vaccine composition of claim 5, wherein the vaccine is a rabies vaccine or an influenza vaccine.

9. The vaccine composition according to claim 5, 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.

10. Use of the vaccine adjuvant of any one of items 1-4 in the preparation of a vaccine formulation, vaccine composition.

Compared with the prior art, the application has the following technical effects:

(1) The active ingredient MCS in the vaccine adjuvant and the vaccine composition is derived from natural plants, has definite sources and rich resources, and has better safety and stability.

(2) The mixture of the MCS and the aluminum salt can obviously improve the titer of specific antibodies (or neutralizing antibodies) after antigen immunization, can effectively enhance the immune response level of an organism to vaccines, has stronger activity than that of a single aluminum salt adjuvant, can promote the proliferation of spleen lymphocytes compared with the single aluminum salt, and has an immune promoting effect.

(3) The vaccine adjuvant is a mixture of MCS and aluminum salt, has the advantages of good immune effect, convenient use and the like, and provides a new adjuvant choice for vaccines.

Drawings

FIG. 1 is the effect of a mixture of MCS and aluminium salts on the level of antibodies to rabies vaccine immunized mice;

FIG. 2 is the effect of dose ratio of MCS to rabies vaccine on the level of rabies vaccine immunized mouse IgG antibodies;

FIG. 3 shows the effect of different MCS to aluminium salt ratios on the level of rabies vaccine immunized mouse IgG antibodies;

FIG. 4 is the effect of a mixture of MCS and aluminum salts on the level of mouse IgG antibodies immunized against influenza vaccine;

FIG. 5 is the effect of dose ratio of MCS to rabies vaccine on the level of influenza vaccine rabies vaccine immunized mouse IgG antibodies;

FIG. 6 is a graph showing the effect of different MCS to aluminum salt ratios on the level of IgG antibodies in influenza vaccine immunized mice.

FIG. 7 is a graph showing the effect of MCS and its proportion to aluminium salt on proliferation of spleen lymphocytes in mice.

Wherein, # represents P <0.05 compared to the negative control group; # represents P <0.01 compared to the negative control group; # # # indicates P <0.005 compared to the negative control group.

Detailed Description

The present application is further illustrated below with reference to examples, it being understood that the examples are for further illustration and explanation of the application only and are not intended to limit the application.

Unless defined otherwise, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the materials and methods are described herein below. In case of conflict, the present specification, including definitions therein, will control and materials, methods, and examples, will control and be in no way limiting. The present application is further illustrated below in conjunction with specific examples, but is not intended to limit the scope of the present application.

As used herein, the term "vaccine" refers to any formulation 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 increases, augments, upregulates, alters, or otherwise promotes an immune response (e.g., 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 a particulate substance (including cells, cell fragments or cell-derived fragments) or a living (usually attenuated) organism or virus. Examples of suitable antigens include (but are not limited to): proteins, glycoproteins, lipoproteins, skin, carbohydrates/polysaccharides, lipopolysaccharides, toxins, viruses, bacteria, fungi and parasites. Other suitable antigens include minimal components of the antigen, such as, but not limited to, antigenic determinants, epitopes or skin. Still other antigens that are suitable include those described in U.S. Pat. No. 5,855,894. The antigen may be naturally occurring (naturally expressed or produced), synthetic, or derived from recombinant DNA methodologies familiar to those skilled in the art.

As used herein, the term "pharmaceutical excipients" refers to substances that have been reasonably evaluated in terms of safety and included in pharmaceutical formulations in order to address the general terms of formability, effectiveness, stability, safety of the formulations when they are added to the formulations, except for the main drug. The pharmaceutical excipients not only form, serve as carriers and improve stability, but also have important functions of solubilization, dissolution assistance, sustained and controlled release and the like, and are important components which can influence the quality, safety and effectiveness of the medicine. The pharmaceutical excipients described herein may be suitable carriers or excipients, emulsifiers, wetting agents, preservatives, stabilizers, antioxidants, adjuvants (e.g., aluminum hydroxide adjuvants, oil adjuvants, freund's complete adjuvants, freund's incomplete adjuvants), and the like.

Momordica segment is a dried mature seed of Momordica cochinchinensis Momordica cochinchinensis (Lour.) of Momordica genus of Cucurbitaceae family. Has effects of detumescence, resolving hard mass, eliminating toxic substance and treating sore, and can be used for treating pyocutaneous disease, acute mastitis, scrofula, hemorrhoid, fistula, tinea, and tinea corporis. Modern research has found that: semen Momordicae has antiinflammatory, analgesic, antiviral, antibacterial, liver fibrosis resisting, and antitumor effects.

As used herein, the term "momordica total saponins" refers to the sum of momordica saponins extracted from momordica seeds, including but not limited to momordica saponin I and momordica saponin II. The total saponins of Momordica cochinchinensis can be prepared by methods known in the art, and can also be obtained by commercial purchase.

The present application also provides a vaccine adjuvant comprising total saponins of momordica cochinchinensis and an aluminum salt.

In a specific embodiment, the total saponins of cochinchina momordica seed are obtained by the preparation method of the total saponins of cochinchina momordica seed.

In a specific embodiment, the aluminum salt is selected from one or both of aluminum phosphate or aluminum hydroxide. For example, the aluminum salt is aluminum phosphate, or aluminum hydroxide, or a mixture of aluminum phosphate and aluminum hydroxide.

In the vaccine adjuvant, the momordica cochinchinensis total saponin and the aluminum salt can be present in any mass ratio.

In a specific embodiment, in the vaccine adjuvant, the mass ratio of the total saponins of cochinchina momordica seed to the aluminum salt is 0.0001-1000:1.

In a specific embodiment, in the vaccine adjuvant, the mass ratio of the total saponins of momordica cochinchinensis to the aluminum salt is 0.1-4:1, for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1.

In a specific embodiment, the vaccine adjuvant may further comprise physiological saline or water for vaccine injection. Wherein, the content of the total saponin and the aluminum salt of the semen momordicae in the vaccine adjuvant can be adjusted according to actual needs.

In a specific embodiment, the vaccine adjuvant consists of total saponins of momordica cochinchinensis with an aluminum salt.

In a specific embodiment, the vaccine adjuvant consists of total saponins of cochinchina momordica seed and aluminum salt, wherein the mass ratio of the total saponins of cochinchina momordica seed to the aluminum salt is 0.1-4:1.

In a specific embodiment, the vaccine adjuvant consists of total saponins of semen Momordicae, aluminum salt and physiological saline or water for vaccine injection.

In a specific embodiment, the vaccine adjuvant consists of total saponins of semen momordicae, aluminum salt and normal saline or water for vaccine injection, wherein the mass ratio of the total saponins of semen momordicae to the aluminum salt is 0.1-4:1.

The present application also provides a vaccine composition comprising a vaccine adjuvant comprising any one of the total saponins and aluminium salts of momordica cochinchinensis in the present application and an antigen or DNA encoding said antigen.

The vaccine adjuvant is used in the vaccine composition in an amount effective to provide a therapeutic effect, the effective amount being an amount that increases, augments, upregulates, alters or otherwise promotes an immune response to the antigen. In particular, 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 will be appreciated by those skilled in the art, the therapeutically effective amount will vary and will be determined on a case-by-case basis, in the present application, the dosages of the vaccine adjuvant and vaccine antigen are not particularly limited, and are appropriately selected according to the method of administration, the subject, the age of the subject, the dosage form, the route of administration, and the like.

In a specific embodiment, the ratio of vaccine adjuvant to vaccine antigen is (0.1 μg to 1000 μg): (0.10 IU to 200 IU) antigen, for example, may be 0.1. Mu.g: 0.10IU, 0.1 μg:200IU, 1000 μg:0.10IU, 1000 μg:200IU; or alternatively

(0.1-1000. Mu.g): (0.1. Mu.g to 500. Mu.g) antigen, for example, may be 0.1. Mu.g: 0.1 μg, 0.1 μg:500 μg, 1000 μg:0.1 μg, 1000 μg:500 μg.

In a specific embodiment, the ratio of the vaccine adjuvant to the vaccine antigen is 33.3 to 133.3 μg/μg.

In a specific embodiment, the ratio of the vaccine adjuvant to the vaccine antigen is 40 to 80 μg/IU.

Those skilled in the art will readily recognize that the therapeutic dosage and length of treatment may vary depending upon the type, weight and condition of the patient to be treated, their individual response to the vaccine composition, and the particular route of administration selected. In some cases, dosage levels below the lower limit of the foregoing range may be therapeutically effective, while in other cases still larger doses may be employed without causing any adverse side effects, provided that such larger doses are first divided into several small doses for administration during the day. The challenge dose is considered ideal whenever secondary stress or exposure is likely to occur.

The vaccine adjuvant and the antigen or the DNA encoding the antigen in the vaccine combination of the present application may be contained together in one composition and may be formulated in separate compositions, where the route of administration of the vaccine adjuvant and the antigen or the DNA encoding the antigen may be the same or different. In this application, the vaccine adjuvant and the antigen or DNA encoding the antigen may be administered simultaneously or differently over time, i.e., the vaccine adjuvant and the antigen or DNA encoding the antigen may be administered simultaneously or separately (e.g., the vaccine adjuvant is administered before or after the vaccine antigen is administered). Vaccine adjuvants and antigens or DNA encoding the antigens may be provided as a kit comprising them. However, from the viewpoint of reducing the burden on the patient, it is preferable that the vaccine adjuvant and the antigen or the DNA encoding the antigen are contained in one composition so that administration can be performed simultaneously. The mode of administration of the vaccine composition, whether co-administered or co-administered, may be any suitable route which delivers the vaccine composition to the host, whether co-administered or co-administered.

In a preferred embodiment, the vaccine adjuvant or vaccine composition of the present application further comprises a pharmaceutical adjuvant and a second vaccine adjuvant.

The medicinal auxiliary materials are as described above.

The vaccine adjuvants of the present application may be administered as part of a vaccine formulation, optionally containing an additional second vaccine adjuvant. The second vaccine adjuvant is a different adjuvant from the total saponin adjuvants of momordica cochinchinensis of the present application, and may be one or two or more, examples of suitable second vaccine adjuvants include those known in the art,

the vaccine adjuvants or vaccine compositions of the present application may further comprise one or more antioxidants selected from the group consisting of: sodium bisulphite, sodium sulphite, sodium metabisulphite, sodium thiosulfate, sodium methylcaseinate, L-ascorbic acid, isoascorbic acid, acetylcysteine, cysteine, monothioglycerol, thioglycolic acid, thiolactic acid, thiovein, dithiothreitol, dithioerythritol, glutathione, ascorbyl eleostearic acid, butylated tibioflavonoid Miao Xiang, butylated light toluene, nordihydroguaiaretic acid, propyl gallate, alpha-tocopherol, and mixtures thereof.

The vaccine adjuvants or vaccine compositions of the present application may further comprise one or more preservatives, examples of suitable preservatives include (but are not limited to): the composition comprises a composition selected from the group consisting of phenylchlorosilyseiul, syringoseiul, benzoic acid, fillyl alcohol, methyl p-light benzoate, ethyl p-light benzoate, propyl p-light benzoate Ding Han, sodium benzoate, phenol, and mixtures thereof. As will be appreciated by those skilled in the art, the presence or absence of the preservative will depend on the antigen. For example, if the antigen is a live bacterial antigen, no preservative need be added.

The vaccine adjuvants or vaccine compositions of the present application are useful for preventing or treating a disease caused by a pathogen, cancer cell or allergen in a human or animal by administering a therapeutically effective amount of the adjuvant composition or vaccine to a human or animal susceptible to the disease.

According to the present application, the pathogen may be any pathogen, including (but not limited to): bacteria, protozoa, worms, viruses and fungi. Diseases in animals caused by the pathogen include (but are not limited to): bovine respiratory disease, swine respiratory disease, pneumonia, pasteurellosis, coccidiosis, anaplasmosis, and infectious keratitis.

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

The vaccine composition of the present application may be various types of vaccine, such as a rabies vaccine or an influenza vaccine, etc.

In a specific embodiment, the mass ratio of total saponins of semen momordicae to aluminium salt is 0.1-4:1 for rabies vaccine.

In a specific embodiment, the mass ratio of the total saponins of momordica cochinchinensis to the aluminum salt is 1-2:1 for the rabies vaccine.

In a specific embodiment, the ratio of total saponins of Momordica cochinchinensis to vaccine is 40-80 μg/IU for rabies vaccine.

In a specific embodiment, the ratio of total saponins of Momordica cochinchinensis to vaccine is 40-60 μg/IU for rabies vaccine.

In a specific embodiment, for influenza vaccine, the mass ratio of total saponin of semen momordicae to aluminum salt is 0.1-4:1.

In a specific embodiment, for influenza vaccine, the mass ratio of total saponin of semen momordicae to aluminum salt is 1-2:1.

In a specific embodiment, for influenza vaccines, the ratio of total saponins of semen Momordicae to vaccine is 33.3 to 133.3 μg/μg.

In a specific embodiment, for influenza vaccines, the ratio of total saponins of semen Momordicae to vaccine is 66.7-100 μg/μg.

The vaccine type of the vaccine composition can be an inactivated virus vaccine, an attenuated vaccine, an inactivated vaccine, a protein vaccine, a DNA vaccine or a polypeptide vaccine, and the like.

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

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

Examples of injectable solutions include emulsions and liposomes comprising an aqueous solution and an oleaginous composition, such as an aqueous solution formulation or an aqueous suspension formulation in which the vaccine adjuvant and the antigen or DNA encoding the antigen are dissolved or dispersed in water, or an oleaginous solution formulation or oleaginous 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 or aqueous suspensions and the like comprising distilled water for injection and optionally comprising buffers, pH adjusting agents, stabilizers, isotonic agents and/or emulsifiers.

The vaccine adjuvants or vaccine compositions of the present application may 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, either as a feed or as a drenching composition. In a preferred embodiment, the vaccine adjuvant or vaccine composition of the present application is injected intramuscularly, intravenously or subcutaneously.

The application also provides application of any one of the vaccine adjuvants in preparation of vaccine preparations and vaccine compositions.

Examples

Experimental materials

Aluminum salt adjuvant: production by Croda company

Example 1

Preparation of semen Momordicae total saponins:

extraction and purification of semen Momordicae total saponins

Pulverizing semen Momordicae, and sieving with 40 mesh sieve. Adding 10-20 times of 80% ethanol, reflux-extracting at 85 ℃ for 2 times each for 2.5 hours, filtering, combining the filtrates, concentrating under reduced pressure to recover ethanol, extracting with petroleum ether and ethyl acetate until the upper liquid is nearly colorless, extracting with water saturated n-butanol until the extract is nearly colorless, combining the n-butanol extracts, and recovering n-butanol under reduced pressure to obtain n-butanol extract.

(1) Purifying the extracted semen Momordicae saponin. Dissolving n-butanol layer extract with methanol, adding excessive ethyl acetate, precipitating large amount of powder with the addition of ethyl acetate, and collecting saponin powder for use.

(2) D101 macroporous resin purification saponin the pretreated D101 macroporous resin wet method is put into a column, and the obtained saponin powder is dissolved by adding water, and the sample amount of the saponin powder is m (the sample amount) =0.1: 1-0.8:1 ratio of D101 type macroporous resin column, wherein the diameter-to-height ratio of the resin column is 8: 15-1:15, and the sample loading flow rate is 1-3 BV/h. After the liquid medicine passes through the resin column, adsorbing for 2-5 hours, eluting with water, 30%, 50% and 70% ethanol water solution in sequence, collecting each fraction eluent, recovering the solvent under reduced pressure, and drying to obtain semen Momordicae total saponins, wherein the content of the saponins is determined to be 81%.

The prepared total saponins of momordica cochinchinensis are used in the following examples of the present application.

Rabies vaccine: liaoning adult organism stock Co.

Animals: female ICR mice, 4-6 weeks old, were purchased from Liaoning long Biotechnology Co., ltd.

Preparation of an "adjuvant-vaccine composition":

MCS "adjuvant-vaccine composition": and respectively measuring total saponin (MCS) of semen Momordicae and a proper amount of diploid inactivated rabies vaccine, dissolving with normal saline to make each milliliter of solution contain 1000 mug of MCS and 2.5IU of rabies vaccine, filtering with a 0.22 mu m microporous filter membrane, and aseptically packaging.

MCS mixed with aluminium salt "adjuvant-vaccine composition": and respectively measuring an appropriate amount of MCS, aluminum salt and diploid inactivated rabies vaccine, dissolving the materials with normal saline, filtering the solution containing 500 mug of MCS, 500 mug of aluminum salt and 2.5IU of rabies vaccine in each milliliter of solution with a 0.22 mu m microporous filter membrane, and carrying out sterile split charging.

"adjuvant-vaccine composition" of positive control aluminium salt: and respectively measuring an appropriate amount of MCS and diploid inactivated rabies vaccine, dissolving the MCS and the diploid inactivated rabies vaccine in normal saline, filtering the solution containing 1000 mug of aluminum salt and 2.5IU of rabies vaccine in each milliliter of solution by using a microporous filter membrane of 0.22 mu m, and carrying out sterile split charging.

Experimental grouping and dose:

MCS adjuvant group: 0.25IU rabies vaccine+100 μg MCS/dose;

MCS and aluminum salt mixed adjuvant group: 0.25IU rabies vaccine+50 μg aluminum salt+50 μg MCS/animal;

aluminum salt positive control group: 0.25IU rabies vaccine+100 μg aluminum salt adjuvant/vehicle;

negative control group: 0.25IU rabies vaccine/animal.

Immunization scheme:

mice were randomly divided into 4 groups of 10 mice each. Intramuscular injection of the above composition was 0.1 ml/dose, and after one week of primary immunization, a second immunization was performed. The serum of each group of mice was collected 14 days after the second immunization, and the neutralizing antibody and IgG specific antibody level in each serum was detected by RFFIT and ELISA methods, respectively.

Results:

as shown in fig. 1, the aluminum salt, MCS and the mixture of MCS and aluminum salt all significantly increased the specific IgG antibody level (P < 0.01) of the rabies vaccinated mice compared to the negative control group, and the adjuvant activity of the mixture of MCS, MCS and aluminum salt was similar (slightly better) to that of aluminum salt at the same total dose.

Example 2: optimal dose of semen Momordicae total saponins (MCS) as rabies vaccine adjuvant

Rabies vaccine: liaoning adult organism stock Co.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Fukang Biotech Co.

Preparation of an "adjuvant-vaccine composition":

"adjuvant-vaccine composition": the MCS and the rabies vaccine are respectively measured with proper amounts, dissolved by normal saline and prepared into five parts of composition solution, so that each milliliter of solution respectively contains 250, 500, 1000, 1500 and 2000 mug of MCS and 25IU of rabies vaccine, and the solution is filtered by a microporous filter membrane with the thickness of 0.22 mu m and packaged in an aseptic manner. At this time, the ratio of MCS adjuvant to influenza vaccine in the composition was 10, 20, 40, 60, 80 (μg: IU), respectively.

"adjuvant-vaccine composition" of positive control aluminium salt: and respectively weighing proper amounts of aluminum salt and rabies vaccine, dissolving the aluminum salt and the rabies vaccine by using normal saline, filtering the solution containing 1000 mug of aluminum salt and 25IU of rabies vaccine in each milliliter of solution by using a 0.22 mu m microporous filter membrane, and carrying out sterile split charging.

Experimental grouping and dose:

MCS adjuvant group 1:2.5IU rabies vaccine+25 μg MCS/dose;

MCS adjuvant group 2:2.5IU rabies vaccine+50 μg MCS/dose;

MCS adjuvant group 3:2.5IU rabies vaccine+100 μg MCS/dose;

MCS adjuvant group 4:2.5IU rabies vaccine+150 μg MCS/dose;

MCS adjuvant group 5:2.5IU rabies vaccine+200 μg MCS/dose;

an aluminum salt positive control group is 2.5IU rabies vaccine+100 mug aluminum salt adjuvant/animal;

negative control group, 2.5IU rabies vaccine/animal.

Immunization scheme:

intramuscular injection of the above composition was 0.1 ml/dose, and after one week of primary immunization, a second immunization was performed. Serum was collected from each group of mice 14 days after the second immunization, and each serum was assayed for IgG-specific antibody levels by ELISA.

Results:

as shown in fig. 2, with different doses of MCS as adjuvant administration, MCS adjuvant groups 3, 4, 5 mice were found to have significantly higher levels of antibodies in vivo than the negative control group, similar to the aluminum salt positive control group, for the rabies vaccine. Although the antibody level in the mice of the MCS adjuvant group 5 is improved to some extent, the antibody level is lower than that of the adjuvant groups 3 and 4, and the dosage of the saponin is larger. In summary, when MCS is used as an adjuvant for rabies vaccine, the effect is better when the ratio of MCS to vaccine dose is 40-80 (μg: IU), and preferably 40-60 (μg: IU).

Example 3: optimal proportion of MCS and aluminium salt as rabies vaccine adjuvant

Rabies vaccine: liaoning adult organism stock Co.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Fukang Biotech Co.

1. Preparing a vaccine composition:

different total doses of MCS and aluminum salt mixed adjuvant vaccine composition: the amounts of MCS, aluminium salt and rabies vaccine were individually measured according to the optimal total adjuvant dose (1000. Mu.g/dose, 0.1mL per dose) in example 3, dissolved in physiological saline, such that the sum of MCS and aluminium salt dose per mL of solution was 1000. Mu.g:

(1) 90.9 μg MCS, 909.1 μg aluminum salt, 25IU rabies vaccine;

(2) 333.3 μg MCS, 666.6 μg aluminium salt and 25IU rabies vaccine;

(3) 500 μg MCS, 500 μg aluminum salt, 25IU rabies vaccine;

(4) 666.6 μg MCS, 333.3 μg aluminium salt and 25IU rabies vaccine;

(5) 800 mug of MCS, 200 mug of aluminium salt and 25IU of rabies vaccine;

filtering with 0.22 μm microporous membrane, and packaging under aseptic condition.

Positive control group-aluminium salt adjuvant vaccine composition: and respectively weighing proper amounts of aluminum salt and rabies vaccine, dissolving the aluminum salt and the rabies vaccine by using normal saline, filtering the solution containing 1000 mug of aluminum salt and 25IU of rabies vaccine in each milliliter of solution by using a 0.22 mu m microporous filter membrane, and carrying out sterile split charging.

Negative control group-vaccine without adjuvant: weighing a proper amount of rabies vaccine, dissolving with normal saline, filtering with a microporous membrane of 0.22 μm to obtain a solution containing 25IU of rabies vaccine, and packaging in sterile condition.

2. Immunization scheme:

mice were randomly divided into 7 groups of 10 mice each. Intramuscular injection of the above composition was 0.1 ml/dose, and after one week of primary immunization, a second immunization was performed. Serum was collected from each group of mice 14 days after the second immunization, and each serum was assayed for IgG-specific antibody levels by ELISA.

Experimental grouping and dose:

MCS mixed with aluminum salt group 1:2.5IU rabies vaccine +

90.9 μg aluminum salt adjuvant+9.1 μg MCS;

MCS and aluminum salt mix group 2:2.5IU rabies vaccine +

66.7 μg aluminum salt adjuvant+33.3 μg MCS;

MCS and aluminum salt mix group 3:2.5IU rabies vaccine +

50 μg aluminum salt adjuvant+50 μg MCS;

MCS and aluminum salt mix group 4:2.5IU rabies vaccine +

33.3 μg aluminum salt adjuvant+66.7 μg MCS;

MCS and aluminum salt mix group 5:2.5IU rabies vaccine +

20 μg aluminum salt adjuvant+80 μg MCS;

positive control group, 2.5IU rabies vaccine+100 mug aluminium salt adjuvant/animal;

negative control group, 2.5IU rabies vaccine/animal.

3. Results:

as shown in FIG. 3, when the MCS and the aluminum salt in different proportions are used as adjuvants, it is found that the antibody level in mice in the mixed group of MCS and aluminum salt is significantly higher than that in the negative control group (namely, the ratio of MCS to the amount of aluminum salt is 0.1-4 (μg: μg)), and the effects of groups 3 and 4 are slightly better than those of the aluminum salt (namely, the ratio of MCS to the amount of aluminum salt is 1-2 (μg: μg)) in the rabies vaccine.

Example 4: adjuvant activity of semen Momordicae total saponins (MCS) on H1N1 influenza vaccine

H1N1 influenza vaccine: liaoning adult organism stock Co.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Fukang Biotech Co.

Preparation of an "adjuvant-vaccine composition":

MCS "adjuvant-vaccine composition": and respectively measuring an appropriate amount of MCS and H1N1 influenza vaccine, dissolving the materials with normal saline, enabling each milliliter of solution to contain 1000 mug of MCS and 30 mug of influenza vaccine, filtering the solution with a 0.22 mug microporous filter membrane, and carrying out sterile split charging.

MCS mixed with aluminium salt "adjuvant-vaccine composition": and (3) respectively measuring an appropriate amount of MCS, aluminum salt and H1N1 influenza vaccine, dissolving the materials in normal saline, enabling each milliliter of solution to contain 500mg of MCS, 500mg of aluminum salt and 30 mug of influenza vaccine, filtering the materials by using a microporous filter membrane with the thickness of 0.22 mu m, and carrying out sterile split charging.

"adjuvant-vaccine composition" of positive control aluminium salt: and respectively weighing proper amounts of aluminum salt and H1N1 influenza vaccine, dissolving the aluminum salt and the H1N1 influenza vaccine in normal saline, filtering the solution containing 1000 mug of aluminum salt and 30 mug of influenza vaccine in each milliliter of solution by using a microporous filter membrane of 0.22 mu m, and carrying out sterile split charging.

Immunization scheme:

mice were randomly divided into 4 groups of 10 mice each. Intramuscular injection of the above composition was 0.1 ml/dose, and 2 weeks after the initial immunization, the 2 nd immunization was performed. ELISA method detects mouse serum IgG specific antibody titer 14 days after the 2 nd immunization.

Results:

as shown in fig. 4, the aluminum salt, MCS and the mixture of MCS and aluminum salt significantly increased the specific IgG antibody level (P < 0.01) of influenza vaccinated mice compared to the negative control group, and the adjuvant activity of the mixture of MCS and aluminum salt was similar to that of aluminum salt at the same total dose.

Example 5: optimal amount of Momordica cochinchinensis Total saponins (MCS) as influenza vaccine adjuvant

Influenza vaccine: liaoning adult organism stock Co.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Fukang Biotech Co.

Preparation of an "adjuvant-vaccine composition":

"adjuvant-vaccine composition": the MCS and the influenza vaccine are respectively measured and proper amounts are respectively measured, dissolved by normal saline and prepared into five parts of composition solutions, so that each milliliter of solution respectively contains 250, 500, 1000, 1500 and 2000 mug of MCS and 15 mug of influenza vaccine, and the solution is filtered by a microporous filter membrane with the thickness of 0.22 mu m and is packaged in an aseptic way. At this time, the ratio of MCS adjuvant to influenza vaccine in the composition was 16.7, 33.3, 66.7, 100, 133.3 (μg: μg), respectively.

"adjuvant-vaccine composition" of positive control aluminium salt: and respectively weighing proper amounts of aluminum salt and influenza vaccine, dissolving with normal saline, enabling each milliliter of solution to contain 1000mg of aluminum salt and 15mg of influenza vaccine, filtering with a microporous filter membrane of 0.22 mu m, and carrying out sterile split charging.

Experimental grouping and dose:

MCS adjuvant group 1:1.5 μg influenza vaccine+25 μg MCS/only;

MCS adjuvant group 2:1.5 μg influenza vaccine+50 μg MCS/only;

MCS adjuvant group 3:1.5 μg influenza vaccine+100 μg MCS/only;

MCS adjuvant group 4:1.5 μg influenza vaccine+150 μg MCS/only;

MCS adjuvant group 5:1.5 μg influenza vaccine+200 μg MCS/only;

an aluminum salt positive control group, 1.5 mug influenza vaccine+100 mug aluminum salt adjuvant/vehicle;

negative control group 1.5 μg influenza vaccine/dose.

Immunization scheme:

intramuscular injection of the above composition was 0.1 ml/dose, and after one week of primary immunization, a second immunization was performed. Serum was collected from each group of mice 14 days after the second immunization, and each serum was assayed for IgG-specific antibody levels by ELISA.

Results:

as shown in fig. 5, with different doses of MCS as adjuvants, it was found that for influenza vaccine, MCS adjuvant groups 2, 3, 4, 5 mice had significantly higher levels of antibodies than the negative control group, similar to the aluminum salt positive control group. Although the antibody level in the mice of the MCS adjuvant group 5 is improved to some extent, the antibody level is lower than that of the adjuvant groups 3 and 4, and the dosage of the saponin is larger. In summary, when MCS is used as an adjuvant for influenza vaccine, the effect is good when the ratio of MCS to vaccine dose is 33.3 to 133.3 (μg: μg), and preferably 66.7 to 100 (μg: μg).

Example 6: optimal ratio of MCS to aluminium salt as influenza vaccine adjuvant

Influenza vaccine: liaoning adult organism stock Co.

Animals: female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Fukang Biotech Co.

1. Preparing a vaccine composition:

different total doses of MCS and aluminum salt mixed adjuvant vaccine composition: the amounts of MCS, aluminium salt and influenza vaccine were measured separately according to the total adjuvant dose (1000. Mu.g/dose, 0.1mL each) in example 3, dissolved in physiological saline, such that the sum of MCS and aluminium salt dose per mL of solution was 1000. Mu.g:

(1) 90.9 μg of MCS, 909.1 μg of aluminum salt, and 15 μg of influenza vaccine;

(2) 333.3. Mu.g of MCS, 666.6. Mu.g of aluminium salt and 15. Mu.g of influenza vaccine;

(3) 500 μg of MCS, 500 μg of aluminum salt, and 15 μg of influenza vaccine;

(4) 666.6 μg of MCS, 333.3 μg of aluminum salt, and 15 μg of influenza vaccine;

(5) 800 μg of MCS, 200 μg of aluminum salt, and 15 μg of influenza vaccine;

filtering with 0.22 μm microporous membrane, and packaging under aseptic condition.

Positive control group-aluminium salt adjuvant vaccine composition: and respectively weighing proper amounts of aluminum salt and influenza vaccine, dissolving with normal saline, enabling each milliliter of solution to contain 1000 mug of aluminum salt and 15 mug of influenza vaccine, filtering with a 0.22 mug microporous filter membrane, and carrying out sterile split charging.

Negative control group-vaccine without adjuvant: weighing a proper amount of influenza vaccine, dissolving with normal saline, filtering with a microporous membrane of 0.22 μm to obtain 15 μg of influenza vaccine in each ml of solution, and packaging in sterile condition.

2. Immunization scheme:

mice were randomly divided into 7 groups of 10 mice each. Intramuscular injection of the above composition was 0.1 ml/dose, and after one week of primary immunization, a second immunization was performed. Serum was collected from each group of mice 14 days after the second immunization, and each serum was assayed for IgG-specific antibody levels by ELISA.

Experimental grouping and dose:

MCS mixed with aluminum salt group 1:1.5 μg influenza vaccine +

90.9 μg aluminum salt adjuvant+9.1 μg MCS;

MCS and aluminum salt mix group 2:1.5 μg influenza vaccine +

66.7 μg aluminum salt adjuvant+33.3 μg MCS;

MCS and aluminum salt mix group 3:1.5 μg influenza vaccine +

50 μg aluminum salt adjuvant+50 μg MCS;

MCS and aluminum salt mix group 4:1.5 μg influenza vaccine +

33.3 μg aluminum salt adjuvant+66.7 μg MCS;

MCS and aluminum salt mix group 5:1.5 μg influenza vaccine +

20 μg aluminum salt adjuvant+80 μg MCS;

positive control group 1.5 μg influenza vaccine+100 μg aluminium salt adjuvant/vehicle;

negative control group 1.5 μg influenza vaccine/dose.

3. Results:

as shown in FIG. 6, when different proportions of MCS and aluminum salt were used as adjuvants, it was found that the antibody levels in mice of the MCS and aluminum salt mixed group 1-5 were significantly higher than those of the negative control group (i.e., the ratio of MCS to aluminum salt used was 0.1-4 (. Mu.g.: mu.g)), and that the effects of groups 3 and 4 were slightly better than those of the aluminum salt (i.e., the ratio of MCS to aluminum salt used was 1-2 (. Mu.g.: mu.g)) for influenza vaccine.

Example 7: promoting effect of MCS and MCS-aluminum salt on cellular immunity of mice

1. Preparing a solution

RPMI1640 complete medium: 90mL of RPM 1640 basal medium (containing double antibody) is added with 10mL of FBS, 100 mu L of glutamine (final concentration of 2 mg/mL) and 5 mu L of 2-mercaptoethanol (final concentration of 50 mu g/mL) to prepare the kit.

ConA solution: 200 mu L of PBS is added into 0.1mg of Canavalia gladiata protein solid to prepare mother solution with the concentration of 0.5mg/mL for later use. 10 mu L of mother solution is taken, 90 mu L of RPMI1640 complete medium is added to prepare 50 mu L/mL of ConA solution, and the mixture is prepared on site.

LPS solution: 1mg of lipopolysaccharide solid powder is taken, 1mL of PBS is added to prepare lipopolysaccharide mother liquor with the concentration of 1mg/mL for standby. The mother liquor 5. Mu.L is added with 95. Mu.L of RPMI1640 complete culture medium to prepare 50. Mu.L/mL solution, and the solution is prepared at present.

2. Preparation of mouse spleen lymphocytes

ICR female mice were sacrificed by cervical vertebra removal, 75% alcohol bath was performed for 3-5 min (low temperature), the mice were removed, placed in a sterile super clean bench, spleens were removed, sheared, placed in a 200 mesh screen petri dish, added with about 3-10 mL Hank's solution, and gently ground with a sterile syringe core until spleens were clear red. Centrifuging the ground cell suspension for 5-15 min at 800-1500 r/min, discarding the supernatant, adding 1-2 mL of erythrocyte lysate, blowing uniformly, centrifuging for 5-15 min at room temperature of 800-1500 r/min, and reversely removing the supernatant. Adding basic culture medium, blowing uniformly, centrifuging at 1500r/min for 5min, and discarding supernatant. 30 to 100. Mu.L of the cell suspension was added with 100. Mu.L of 2% trypan blue staining solution, hank's solution (final concentration of trypan blue: 0.4%) was added, and the mixture was counted by a hemocytometer.

Count 4 large angle cells on the counting plate: total number of cells = total number of 4 large corner cells/4 x 10000 x dilution x suspension volume. Calculating the cell survival rate, and ensuring that the survival rate of spleen lymphocytes is more than 95%; the cells were kept ready for use.

3. Administration and grouping

The CCK-8 method was used to detect cell activity. Diluting lymphocytes according to 1-2.5X10 in 96-well plate ⁶ The culture plates were inoculated with individual/mL. Dissolving semen Momordicae total saponin, semen Momordicae total saponin and aluminum salt mixture and aluminum salt in culture medium to obtain final concentration:

blank control group: adding an equal amount of culture medium;

MCS dosing group 1:100 μg/mL;

MCS dosing group 2:400 μg/mL;

MCS dosing group 3:800 μg/mL;

MCS mixed with aluminum salt group 1:50 mug/mL of total saponins of Momordica cochinchinensis+50 mug/mL of aluminum salt;

MCS and aluminum salt mix group 2:200 mug/mL of total saponins of Momordica cochinchinensis+200 mug/mL of aluminum salt;

MCS and aluminum salt mix group 3:400 mug/mL of total saponins of Momordica cochinchinensis+400 mug/mL of aluminum salt;

aluminum salt dosing group 1:100 μg/mL;

aluminum salt dosing group 2:400 μg/mL;

aluminum salt dosing group 3:800 μg/mL;

the total volume per well was 100. Mu.L. After the sample addition, the mixture is placed at 37 ℃ and 5 percent CO ₂ Incubating in an incubator for 24-48 h. After adding 5. Mu.L of CCK-8 to each well and continuing to culture in an incubator for 2-4 hours, absorbance at 450nm is detected by using an enzyme-labeled instrument.

4. Results

As shown in FIG. 7, MCS and aluminum salt mixtures all promote proliferation of spleen lymphocytes, and aluminum salts have no obvious effect.

Claims (10)

1. A vaccine adjuvant comprising total saponins of momordica cochinchinensis and aluminum salts.

2. The vaccine adjuvant of claim 1, further comprising physiological saline or water for vaccine injection.

3. Vaccine adjuvant according to claim 1, characterized in that the mass ratio of total saponins of momordica cochinchinensis to aluminium salt is 0.0001-1000:1, preferably 0.1-4:1.

4. Vaccine adjuvant according to claim 1, wherein the aluminium salt is selected from one or both of aluminium hydroxide and aluminium phosphate.

5. A vaccine composition comprising the vaccine adjuvant of any one of claims 1-4 and a vaccine antigen or DNA encoding said antigen.

6. The vaccine composition of claim 5, wherein the ratio of vaccine adjuvant to vaccine antigen is (0.1-1000): (0.10-200) μg/IU or (0.1-1000): (0.1-500. Mu.g/g), preferably 33.3-133.3. Mu.g/g or 40-80. Mu.g/IU.

7. The vaccine composition of claim 5, further comprising a pharmaceutical excipient and a second vaccine adjuvant.

8. The vaccine composition of claim 5, wherein the vaccine is a rabies vaccine or an influenza vaccine.

9. The vaccine composition according to claim 5, 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.

10. Use of a vaccine adjuvant according to any one of claims 1-4 in the preparation of a vaccine formulation, a vaccine composition.

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