Double-adjuvant composition and application thereof in preparation of inactivated rabies vaccines for dogs and cats

Abstract

The invention discloses a double-adjuvant composition and application thereof in preparation of inactivated vaccines for rabies of dogs and cats. The dual-adjuvant composition is composed of an adjuvant 1 and an adjuvant 2, the adjuvant 1 is composed of aluminum hydroxide gel and gelatin, and the weight fraction ratio of the aluminum hydroxide gel to the gelatin is (0.2-5): (0.1-2); the adjuvant 2 is composed of propolis and interleukin-2, and the weight ratio of the propolis to the interleukin-2 is (0.1-8): (0.01-0.1). The dual-adjuvant composition is used for preparing inactivated vaccines for rabies of dogs and cats, and can achieve a good protective effect by causing better cellular immune response and antigen slow release effects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a double adjuvant composition and application thereof in preparing inactivated rabies vaccines for dogs and cats. Background Art

[0002] Rabies is an acute zoonotic infectious disease caused by rabies virus infection. Rabies virus is a non-segmented single-stranded RNA virus and an important member of the Rhabdoviridae family. Rabies virus infects the central nervous system of humans and warm-blooded animals, causing fatal encephalomyelitis and paralysis. Human infection with rabies virus mainly comes from scratches or attacks by animals infected with rabies virus. Dogs and cats, as the most common pets in cities, are both carriers of rabies virus. In 2018, the number of pet cats in my country reached 40.64 million, and the number of cat owners was 22.58 million. About 94% of human rabies in my country is caused by domestic dogs, 6% by cats, and a few pigs and mice. Until now, vaccination is the best way to prevent rabies. The development of a rabies vaccine with good immune effect is of great significance to the prevention and control of rabies in my country. A vaccine with good immune effect is supplemented with a good adjuvant to cooperate with the anti-infection properties of the vaccine. Although the classic aluminum gel adjuvant is an adjuvant currently approved for use, the pet industry and the vaccine manufacturing industry have developed rapidly. The aluminum gel adjuvant used in conjunction with the current commercial rabies vaccine often delivers antigens by attaching to the cell membrane surface. It does not enter the cell itself and cannot participate in the intracellular processing and presentation of the antigen. The antigen is finally processed by the lysosomal pathway and presented by the major histocompatibility complex II, triggering a humoral immune response, while the cellular immune response is poor. It is precisely because of this drawback of aluminum adjuvants that it is difficult to provide comprehensive protection to the body when using aluminum adjuvants alone in practical applications, and problems such as large irritation to the injection site and long immune cycle have gradually become prominent, which cannot meet the growing demand for vaccine adjuvants. In view of this, the present invention is specially proposed. Summary of the invention

[0003] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a double adjuvant composition which improves the immunogenicity of the vaccine, has no obvious side effects, has no irritation to the injection site, has a short immunization cycle, and has a strong sustained-release effect, and its application in a high-efficiency double adjuvant inactivated vaccine for dogs and cats.

[0004] In order to achieve the object of the present invention, the technical solution of the present invention is as follows:

[0005] The invention provides a double adjuvant composition, which consists of an adjuvant 1 and an adjuvant 2, wherein the adjuvant 1 consists of aluminum hydroxide gel and gelatin, wherein the weight fraction ratio of the aluminum hydroxide gel to the gelatin is (0.2-5):(0.1-2); and the adjuvant 2 consists of propolis and interleukin-2, wherein the weight ratio of the propolis to the interleukin-2 is (0.1-8):(0.01-0.1).

[0006] Preferably, the weight fraction ratio of the aluminum hydroxide gel and gelatin is (0.5-2):(0.2-1); the adjuvant 2 is composed of propolis and interleukin-2, wherein the weight ratio of propolis to interleukin-2 is (0.5-5):(0.04-0.08).

[0007] The use of the double adjuvant composition in the preparation of inactivated rabies vaccine for dogs or cats also falls within the protection scope of the present invention.

[0008] The present invention also provides a method for preparing the double-adjuvant rabies inactivated vaccine, comprising the following steps:

[0009] 1) adding adjuvant 1 in the adjuvant composition of claim 1 or 2 to the purified and inactivated virus solution, stirring thoroughly, adsorbing and allowing to stand for precipitation, and discarding 10%-50% of the supernatant;

[0010] 2) adding adjuvant 2 in the dual adjuvant composition according to any one of claims 1 to 3, and emulsifying to obtain an inactivated vaccine;

[0011] Among them, in adjuvant 1, the final concentration of aluminum hydroxide gel is 0.2-5 mg / ml, and the final concentration of gelatin is 0.1-2 mg / ml; in adjuvant 2, the final concentration of propolis adjuvant is 0.1-8 mg / ml, and the final concentration of interleukin-2 is 10-100 μg / ml.

[0012] Preferably, in the adjuvant 1, the final concentration of aluminum hydroxide gel is 0.5-2 mg / ml, and the final concentration of gelatin is 0.2-1 mg / ml; the final concentration of propolis adjuvant 2 is 0.5-5 mg / m, and the final concentration of interleukin-2 is 40-80 μg / ml.

[0013] In the step 1), the ratio of discarding part of the supernatant is 30%.

[0014] The double adjuvant inactivated rabies vaccine prepared by the above method also belongs to the protection scope of the present invention.

[0015] The double adjuvant inactivated rabies vaccine can be an inactivated rabies vaccine for dogs and cats, including an inactivated rabies vaccine for dogs and an inactivated rabies vaccine for cats.

[0016] In the above method, the virus solution can be rabies G52 strain, VP12 strain, SAD strain, PV strain, and the concentration of the virus solution should reach 10 5.5 LD 50 / 0.03ml, inoculate the virus seed (production virus seed) into the suspension cells at an inoculum of 0.1%, culture at 33°C, 60rpm, pH 7.2, dissolved oxygen 30%, and harvest the virus solution after 96-192 hours of culture. Determine the virus titer by indirect immunofluorescence method.

[0017] The double adjuvant provided in the embodiment of the present invention can induce a good cellular immune response and antigen sustained release effect for the inactivated rabies vaccine, thereby achieving a good protective effect. In addition, the prepared inactivated rabies vaccine with double adjuvant has less irritation to the injection site and is not prone to allergies, and the prepared vaccine is safe and highly effective.

[0018] (III) Beneficial effects

[0019] The immunoadjuvants in this method are aluminum salt and propolis. First, aluminum salt is used to adsorb protein to form an aluminum protein antigen in an aggregated state, thereby increasing the surface area of ​​the antigen, and then propolis and interleukin-2 are added. During the emulsification process, the aluminum protein antigen is a water-soluble substance and is wrapped in the center of the emulsified particles. The surface of the mixed liquid of propolis and interleukin-2 has lipids, which exist in the lipophilic base of the emulsifier and the oil phase, thus forming a unit-like structure surrounded by a lipid bilayer-lipid body. This double adjuvant antigen delays destruction and release under the protection of the oil phase, can exist in the body for a long time, and slowly releases antigens to play its role as a "warehouse" for long-lasting stimulation, ensuring that the immune system is stimulated with an appropriate amount of antigens so that it is not easy to produce immune tolerance, so that the body can produce antibodies in a long-lasting and efficient manner. At the same time, the granular liposomes can attract a large number of macrophages to engulf them, thereby enhancing antigenicity and improving the immune effect. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The dual adjuvant composition provided by the present invention is composed of adjuvant 1 and adjuvant 2, wherein adjuvant 1 is composed of aluminum hydroxide gel and gelatin, wherein the weight fraction ratio of aluminum hydroxide gel to gelatin is (0.2-5): (0.1-2); and adjuvant 2 is composed of propolis and interleukin-2, wherein the weight ratio of propolis to interleukin-2 is (0.1-8): (0.01-0.1).

[0023] The components used and their functions are as follows:

[0024] The aluminum hydroxide gel in the adjuvant 1 is the only adjuvant approved by the FDA for human vaccines, and its safety can be guaranteed. After the aluminum adjuvant adsorbs the antigen, it increases the surface area of ​​the antigen and changes the configuration of the active gene, thereby enhancing the immunogenicity of the antigen. For small molecular weight soluble antigens or haptens, the antibody production can be increased after adsorption by colloidal particles. After vaccination, it strongly attracts the recognition and endocytosis of antigen-presenting cells such as macrophages, dendritic cells, and Langerhans cells, promotes T cell immunity, and strengthens the synergy between T cells and B cells; on the other hand, the adjuvant can cause local granulomas after injection, so that the antigen is stored in the local tissue, slowly releases the antigen, and prolongs the time for the interaction between antigen-presenting cells and T lymphocytes, thereby improving the antibody response and enhancing humoral immunity. In addition, studies have shown that aluminum hydroxide gel can activate complement and increase the expression of MHC-II class molecules and co-stimulatory molecules. These co-stimulatory molecules can increase the mRNA level of 1L-4 and the expression of IL-1, TNF and IL-6 on peripheral monocytes. It can increase the antibody titer of rabies vaccine and enhance the immune effect of the vaccine.

[0025] The adjuvant activity of aluminum hydroxide gel is closely related to its quality. High-quality aluminum gel has fine molecules, good and stable colloidal state, and strong adsorption. The gelatin in the adjuvant 1 is a colloid, the main component of which is a water-soluble protein (non-uniform polypeptide compound) with a molecular weight of 10,000 to 460,000 derived from partial hydrolysis of animal collagen. It is non-immunogenic in itself and is often used as an emulsifier, stabilizer, thickener, etc. It is widely used in the food industry and the pharmaceutical industry and has good safety. Some studies have shown that gelatin as a vaccine adjuvant may protect antigens from the acid-base environment during the vaccine preparation process, and may have antigen carrier and sustained-release effects, which can effectively enhance the immunogenicity of inactivated vaccines.

[0026] The propolis in the adjuvant 2 is a gelatinous solid with an aromatic odor, which is formed by mixing the secretions of the bees' digestive glands and wax glands with plant buds during the process of collecting honey. There are dozens of biologically active substances in propolis, such as vitamins (VB, VB, niacin, pantothenic acid, etc., amino acids (histidine, lysine, valine, etc., minerals (calcium, phosphorus, potassium, selenium, manganese, cobalt, etc.), polysaccharides, enzymes, etc., which have multiple functions such as antioxidant, antiviral, anti-tumor, anti-inflammatory, hypoglycemic, and hypolipidemic. In addition, propolis also contains a variety of immune active substances, which can not only improve the activity of phagocytes, but also promote the increase in the number of white blood cells and antibodies in the body and stimulate the production of cytokines and specific lectins, which have the effects of enhancing the body's immune function and promoting tissue regeneration. It has a broad spectrum of biological activity and can comprehensively enhance the body's immune function. Compared with conventional adjuvants, propolis adjuvants have the advantages of being pure natural, non-toxic and side-effect-free, no residue, safe, efficient, long-lasting immune effect, and rapid increase in antibody levels. In addition, propolis vaccines have strong heat and cold resistance and a long protection period, which can effectively solve the problems of long immunization cycle and lack of cellular immunity of inactivated rabies vaccines.

[0027] IL-2 in the adjuvant 2 is a cytokine with an immune adjuvant effect. It is a type of immunomodulatory substance that produces responsiveness after the body's immune system is activated by antigens and various immune adjuvants, and has a significant immune adjuvant effect. They are slowly and persistently released in vivo, and can enhance the protective effect of viruses, bacteria, and parasite vaccines. IL-2 can act on various cells such as T, B lymphocytes, macrophages, and NK cells, promote the stimulation of Th1 cells, thereby producing a cell-mediated immune response to intracellular pathogens, and has a wide range of upregulation effects on immune responses.

[0028] The double adjuvant composition is used to prepare inactivated rabies vaccines for dogs and cats, which can induce better cellular immune response and antigen sustained release, thereby achieving good protective effect.

[0029] The rabies virus for production is concentrated 5 times using a 300KD pore size hollow fiber membrane column to obtain a purified virus solution.

[0030] Example 1

[0031] This embodiment provides a double adjuvant inactivated rabies vaccine for dogs and cats. The purified inactivated virus solution (rabies inactivated vaccine PV / BHK-21 strain) is diluted to 10% with PBS at pH 7.4 and 0.01 mol / L. 5.5 LD 50 / 0.03ml) was added to prepare adjuvant 1 and adjuvant 2, the amount of aluminum hydroxide gel in adjuvant 1 was 0.5mg / ml, and the amount of gelatin was 0.2mg / ml; the amount of propolis adjuvant in adjuvant 2 was 0.5mg / ml, and the amount of interleukin-2 was 40μg / ml.

[0032] The preparation method of the above-mentioned adjuvant 1 is as follows: aluminum hydroxide gel is prepared according to the preparation method of aluminum hydroxide gel in physiological saline dilution specified in Part III of the Veterinary Pharmacopoeia, that is, 100 mg of aluminum hydroxide gel is dissolved in 100 ml of 0.85% physiological saline and stirred thoroughly to obtain 1 mg / ml aluminum hydroxide gel, 40 mg of gelatin is added to the prepared 100 ml of 1 mg / ml aluminum hydroxide gel, and the mixture is stirred thoroughly to obtain 2X adjuvant 1.

[0033] The preparation method of the adjuvant 2 is as follows: 100 mg of propolis extracted with ethanol and 8 mg of interleukin-2 are fully mixed to pH 7.4 and 0.01 mol / L 100 ml PBS and stirred to obtain 2X adjuvant 2.

[0034] 100 ml of the above 2X adjuvant 1 and 100 ml of the purified 5.5 LD 50 / 0.03ml of inactivated rabies vaccine PV / BHK-21 virus liquid is fully stirred, adsorbed and allowed to stand for precipitation, 30% of the supernatant is discarded, 100ml of the remaining mixed liquid is taken and 2X 100ml of the above 2X adjuvant 2 is added to emulsify to obtain the inactivated vaccine.

[0035] Example 2

[0036] This embodiment provides a double adjuvant inactivated rabies vaccine for dogs and cats. The purified inactivated virus solution (rabies inactivated vaccine PV / BHK-21 strain) is diluted to 10% with PBS at pH 7.4 and 0.01 mol / L. 5.5 LD 50 / 0.03ml) was added to prepare adjuvant 1 and adjuvant 2, the final concentration of adjuvant 1 aluminum hydroxide gel was 2mg / ml, and the amount of gelatin was 1mg / ml; the amount of adjuvant 2 propolis adjuvant was 5mg / ml, and the amount of interleukin-2 was 80μg / ml.

[0037] The preparation method of the above-mentioned adjuvant 1 is as follows: aluminum hydroxide gel is prepared according to the preparation method of aluminum hydroxide gel saline dilution specified in Part III of the Veterinary Pharmacopoeia, that is, 400 mg of aluminum hydroxide gel is dissolved in 100 ml of 0.85% saline and stirred thoroughly to obtain 4 mg / ml aluminum hydroxide gel, 200 mg of gelatin is added to the prepared 100 ml of 4 mg / ml aluminum hydroxide gel, and the mixture is stirred thoroughly to obtain 2X adjuvant 1.

[0038] The preparation method of the adjuvant 2 is as follows: 1000 mg of propolis extracted with ethanol and 16 mg of interleukin-2 are fully mixed to pH 7.4 and stirred in 100 ml of 0.01 mol / L PBS to obtain 2X adjuvant 2.

[0039] 100 ml of the above 2X adjuvant 1 and 100 ml of the purified 5.5 LD 50 / 0.03ml of inactivated rabies vaccine PV / BHK-21 virus liquid is fully stirred, adsorbed and allowed to stand for precipitation, 30% of the supernatant is discarded, 100ml of the remaining mixed liquid is taken and 2X 100ml of the above 2X adjuvant 2 is added to emulsify to obtain the inactivated vaccine.

[0040] Example 3

[0041] This embodiment provides a double adjuvant inactivated rabies vaccine for dogs and cats. The purified inactivated virus solution (rabies inactivated vaccine PV / BHK-21 strain) is diluted to 10% with PBS at pH 7.4 and 0.01 mol / L. 5.5 LD 50 / 0.03ml) was added to prepare adjuvant 1 and adjuvant 2, the final concentration of adjuvant 1 aluminum hydroxide gel was 1mg / ml, and the amount of gelatin was 0.5mg / ml; the amount of adjuvant 2 propolis adjuvant was 2mg / ml, and the amount of interleukin-2 was 50μg / ml.

[0042] The preparation method of the above-mentioned adjuvant 1 is as follows: aluminum hydroxide gel is prepared according to the preparation method of aluminum hydroxide gel saline dilution specified in Part III of the Veterinary Pharmacopoeia, that is, 200 mg of aluminum hydroxide gel is dissolved in 100 ml of 0.85% saline and stirred thoroughly to obtain 2 mg / ml aluminum hydroxide gel, 100 mg of gelatin is added to the prepared 100 ml of 2 mg / ml aluminum hydroxide gel, and the mixture is stirred thoroughly to obtain 2X adjuvant 1.

[0043] The preparation method of the adjuvant 2 is as follows: 400 mg of propolis extracted with ethanol and 10 mg of interleukin-2 are fully mixed to pH 7.4 and 0.01 mol / L 100 ml PBS and stirred to obtain 2X adjuvant 2.

[0044] 100 ml of the above 2X adjuvant 1 and 100 ml of the purified 5.5 LD 50 / 0.03ml of inactivated rabies vaccine PV / BHK-21 virus liquid is fully stirred, adsorbed and allowed to stand for precipitation, 30% of the supernatant is discarded, 100ml of the remaining mixed liquid is taken and 2X 100ml of the above 2X adjuvant 2 is added to emulsify to obtain the inactivated vaccine.

[0045] Comparative Example 1

[0046] This comparative example provides a commercially available inactivated rabies vaccine.

[0047] Comparative Example 2

[0048] This comparative example provides an inactivated rabies vaccine, which, except for not containing the aluminum gel component, has the same other components and dosages as those of Example 1.

[0049] Comparative Example 3

[0050] This comparative example provides an inactivated rabies vaccine, which, except for not containing gelatin, has the same other ingredients and dosages as those of Example 1.

[0051] Comparative Example 4

[0052] This comparative example provides an inactivated rabies vaccine, which, except for not containing the propolis component, has the same other components and dosages as those of Example 1.

[0053] Comparative Example 5

[0054] This comparative example provides an inactivated rabies vaccine, which, except for not containing the IL-2 component, has the same other components and dosages as those of Example 1.

[0055] Comparative Example 6

[0056] This comparative example provides an inactivated rabies vaccine, which, except for not containing propolis and IL-2, has the same other ingredients and dosages as those of Example 1.

[0057] Comparative Example 7

[0058] This comparative example provides an inactivated rabies vaccine, which, except for not containing aluminum gelatin and gelatin, has the same other ingredients and dosages as those in Example 1.

[0059] The rabies inactivated vaccines provided in the above Example 1 and Comparative Examples 1 to 5 were respectively tested for antigen titer by NIH method, and the titer test results are shown in Table 1. Subsequently, 5 dogs and cats aged 3 months were immunized, each with 1 ml, and 5 dogs and cats were not immunized as a blank control group. After immunization, the reactions and clinical symptoms of the dogs and cats were observed, and the observation indicators and contents are shown in Table 2, and the observation results are shown in Table 3; in addition, blood samples were collected from these dogs and cats 7 days, 14 days, 21 days, and 6 months after immunization, and antibodies were tested by ELISA method, and the test results are shown in Table 4.

[0060] Table 1 NIH test results of antigen titer in each group

[0061] Antigen titer (IU) Example 1 9.6 Example 2 9.4 Example 3 10.5 Comparative Example 1 7.4 Comparative Example 2 8.2 Comparative Example 3 9.3 Comparative Example 4 8.4 Comparative Example 5 8.8 Comparative Example 6 7.2 Comparative Example 7 7.6

[0062] From the results in Table 1, it can be seen that the antigen titer test results between the embodiment and the control example are quite different, indicating that the combination of the embodiment formula can better adsorb antigens and produce higher titers.

[0063] Table 2 Clinical test observation table

[0064]

[0065]

[0066] Table 3 Reaction and clinical symptoms of dogs and cats after injection

[0067]

[0068]

[0069] Note: “-” indicates no local or systemic adverse reactions were observed

[0070] From the results in Table 3, it can be seen that no clinical symptoms occurred in the Examples and Comparative Examples 2-5 containing the ingredients in the Examples, indicating that the ingredients in the Examples are safe.

[0071] Table 4 Antibody test results after injection in dogs and cats (unit: EU / ml)

[0072]

[0073]

[0074]

[0075] Note: ≥0.5EU / ml indicates a positive test result;

[0076] From the above experimental data, it can be seen from Table 1 and Table 4 that the double adjuvant inactivated vaccine in this example can increase the antigen titer, and produce antibodies quickly, for a long time, and without side effects.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual adjuvant composition, consisting of adjuvant 1 and adjuvant 2, characterized in that: The adjuvant 1 is composed of aluminum hydroxide gel and gelatin, wherein the weight fraction ratio of aluminum hydroxide gel to gelatin is (0.2-5):(0.1-2); the adjuvant 2 is composed of propolis and interleukin-2, wherein the weight ratio of propolis to interleukin-2 is (0.1-8):(0.01-0.1).

2. The dual adjuvant composition according to claim 1, characterized in that: The weight fraction ratio of the aluminum hydroxide gel and gelatin is (0.5-2): (0.2-1); the adjuvant 2 is composed of propolis and interleukin-2, wherein the weight ratio of propolis to interleukin-2 is (0.5-5): (0.04-0.08).

3. The dual adjuvant composition according to claim 1, characterized in that: The mass content of aluminum in the aluminum hydroxide gel is 1.2% to 2.2% calculated as aluminum oxide, preferably 1.4% to 1.5%.

4. Use of the double adjuvant composition according to any one of claims 1 to 3 in the preparation of an inactivated rabies vaccine for dogs or cats.

5. A method for preparing a double adjuvant inactivated rabies vaccine, comprising the following steps: 1) adding adjuvant 1 in the adjuvant composition of claim 1 or 2 to the purified and inactivated virus solution, stirring and adsorbing the solution, and allowing the solution to settle, and discarding 10%-50% of the supernatant; 2) adding adjuvant 2 in the dual adjuvant composition according to any one of claims 1 to 3, and emulsifying to obtain an inactivated vaccine; in, In adjuvant 1, the final concentration of aluminum hydroxide gel is 0.2-5 mg / ml, and the final concentration of gelatin is 0.1-2 mg / ml; in adjuvant 2, the final concentration of propolis adjuvant is 0.1-8 mg / ml, and the final concentration of interleukin-2 is 10-100 μg / ml.

6. The method according to claim 5, characterized in that: In the adjuvant 1, the final concentration of aluminum hydroxide gel is 0.5-2 mg / ml, and the final concentration of gelatin is 0.2-1 mg / ml; the final concentration of propolis adjuvant 2 is 0.5-5 mg / ml, and the final concentration of interleukin-2 is 40-80 μg / ml.

7. The method according to claim 5, characterized in that: In the step 1), the ratio of discarding part of the supernatant is 30%.

8. A double adjuvant inactivated rabies vaccine, which is prepared by adding purified inactivated virus liquid to the double adjuvant composition of claim 1 or 2.

9. The inactivated rabies vaccine according to claim 8, characterized in that: The inactivated rabies vaccine is prepared by the method described in any one of claims 5 to 7.

Images