CN113797328A - Compound adjuvant for animal vaccine, preparation method and vaccine - Google Patents

Abstract

The invention relates to a composite adjuvant for an animal vaccine, a preparation method and the vaccine. In one embodiment, the composite adjuvant is made by emulsifying a composition of squalene, SPAN85 and TWEEN 80. In another embodiment, the composite adjuvant is prepared by emulsifying a composition of squalene, SPAN85, TWEEN 80 and saponin. The method of the invention can be used with antigen in a higher proportion, and the animal vaccine adjuvant has good biocompatibility and stability.

Description

Compound adjuvant for animal vaccine, preparation method and vaccine

Technical Field

The invention relates to the field of preparation of animal vaccines, in particular to a compound adjuvant for animal vaccines, a preparation method and a vaccine.

Background

Vaccine adjuvants are substances that can nonspecifically alter or enhance the body's specific immune response to an antigen, exerting an adjuvant effect. The adjuvant can induce the organism to generate long-term and efficient specific immune reaction, improve the protective capability of the organism, reduce the dosage of immune substances and reduce the production cost of the vaccine. In recent years, in order to meet the requirements of novel vaccines, adjuvants have been developed from a traditional, single form to a novel, multiple form, and adjuvant research particularly for mucosal vaccines, DNA vaccines and tumor vaccines has become a hotspot.

At present, in animal vaccines, a more common adjuvant is applied as a mineral oil adjuvant, and the volume ratio of the common antigen to the oil is 1-3: 1. in the production and emulsification process, the upper limit of the proportion of the antigen needs to be strictly controlled in order to stabilize the preparation form; meanwhile, the antigen is used as a water phase, and the viscosity of the emulsified product is increased along with the increase of the addition amount of the water phase, so that the injection experience of the finished product and the absorption of the adjuvant are influenced.

Therefore, there is still a need in the art for an animal vaccine adjuvant that can be used in combination with an antigen at a higher ratio and has good biocompatibility and stability.

Disclosure of Invention

Aiming at the defects in the prior art, one of the purposes of the invention is to provide a composite adjuvant for animal vaccines, wherein the main components of the composite adjuvant are mineral oil, animal tissue extracts, plant extracts and the like, and the composite adjuvant has the effects of good biocompatibility, high antigen loading capacity, low cost and the like.

The above object of the present invention is achieved by the following technical solutions:

in a first aspect, the present invention provides a composite adjuvant for an animal vaccine, which is prepared by emulsifying a composition consisting of squalene, SPAN85 and TWEEN 80.

By adopting the technical scheme, the composition consisting of the three components can be used for preparing the oil phase which is transparent in color and is not layered. When the oil phase is prepared into animal vaccines for use (such as injection), local immune stimulation environment can be induced at the injection site, and the contents of chemokines and cytokines can be increased. In addition, the ability of dendritic cells to take up antigen can be enhanced. In addition, the adjuvant has good biocompatibility, so that the adjuvant has better use safety. In particular, when combined with an antigen (e.g. an artificially inactivated bacterium or virus), the adjuvant can be loaded with a higher proportion of antigen, thereby reducing vaccine usage and increasing vaccine efficiency.

In a preferred example, the technical solution according to the first aspect may be further configured to: the weight ratio of the squalene, the SPAN85 and the TWEEN 80 is (120-140): (15-30): (30-50), preferably 134:23: 43.

In a second aspect, the present invention provides a composite adjuvant for animal vaccines, which is prepared by emulsifying a composition consisting of squalene, SPAN85, TWEEN 80 and saponin.

In a preferred example, the technical solution according to the first aspect may be further configured to: the weight ratio of squalene, SPAN85, TWEEN 80 and saponin is (5-20): (1-4): 2-10): 0.5-3, preferably 12:2:5: 1.

In a preferred example, the technical solution according to the first aspect may be further configured to: the saponin is selected from ginsenoside or saponin. For example, ginsenoside re (re) extracted from ginseng has the effect of improving the immune function of the body and shows good immune adjuvant activity.

In a third aspect, the present invention provides a method of preparing a vaccine using the composite adjuvant according to the first aspect, the method comprising the steps of:

(1) preparing an oil phase: mixing squalene, SPAN85 and TWEEN 80 at the above weight ratio, stirring at medium and low speed for 20-50min, standing to obtain transparent solution;

(2) preparing an aqueous phase: diluting the antigen with PBS with appropriate volume to obtain water phase;

(3) re-mixing the solution obtained in step (1) and then mixing the solution with an aqueous phase: adding the oil phase into the water phase at a volume ratio of 5:1 to 8:1, emulsifying under stirring, and centrifuging to obtain the stable oil-in-water vaccine.

In a fourth aspect, the present invention provides a method for preparing a vaccine using the composite adjuvant according to the second aspect, the method comprising the steps of:

preparing an oil phase: mixing squalene, SPAN85, TWEEN 80 and saponin at the above weight ratio, stirring at medium and low speed for 20-50min, standing to obtain transparent solution;

preparing an aqueous phase: diluting the antigen with PBS with appropriate volume to obtain water phase;

re-mixing the solution obtained in step (1) and then mixing the solution with an aqueous phase: adding the oil phase into the water phase at a volume ratio of 5:1 to 8:1, emulsifying under stirring, and centrifuging to obtain the stable oil-in-water vaccine.

In a preferred example, the technical solution according to the first aspect may be further configured to: the medium-low speed stirring is magnetic stirring, and the rotating speed is 80-160 r/min.

By utilizing the scheme, the preparation of the vaccine can be realized at a lower stirring rotating speed, so that adverse effects on the immune antigen caused by emulsification at a higher temperature generated at a higher rotating speed are avoided.

In a fifth aspect, the present invention provides a vaccine comprising a composite adjuvant according to the first or second aspects described above or made using a method according to the third or fourth aspects.

By utilizing the technical scheme, the vaccine with high water phase/oil phase ratio can be obtained, so that the antigen content of the vaccine is greatly improved.

In summary, the invention includes at least one of the following beneficial technical effects:

1. for animal vaccine emulsification, the antigen can be added in an amount which is 3 times higher than that of the existing conventional adjuvant (such as white oil, Tween 80, span 80 and aluminum stearate), even when the ratio of the antigen to the antigen is as high as 8: 1: stable oil-in-water emulsified small particles can be formed under the volume ratio of the adjuvant;

2. the ratio of the components is adjusted to realize the balance of the ratio of the hydrophilicity to the lipophilicity of the emulsion, so that the emulsification can be realized by using medium and low speed shearing, thereby avoiding the influence of heat generated by high-temperature emulsification on immune antigen;

3. the vaccine adjuvant prepared according to the invention can be used for preparing an oil-in-water type vaccine with high antigen capacity, and the viscosity of the vaccine adjuvant cannot be increased along with the increase of the addition amount of the water phase, so that the problem of viscosity increase caused by the increase of the addition amount of the water phase in the water-in-oil type vaccine is avoided;

4. the loaded antigen is released in the receptor rapidly and continuously, the expression of specific antibody with high concentration is induced rapidly, and humoral immune response is stimulated.

Drawings

FIG. 1 is a schematic of the particle size distribution of a vaccine emulsion prepared according to example 1;

FIG. 2 is a schematic of the particle size distribution of the vaccine emulsion prepared according to example 2;

FIG. 3 is a photograph of a vaccine emulsion prepared according to example 1 after 28 days of storage at 37 ℃;

FIG. 4 is a photograph of a vaccine emulsion prepared according to example 2 after 28 days of storage at 37 ℃;

FIG. 5 is a photograph of the vaccine emulsion prepared according to comparative example 1 after being stored at 37 ℃ for 12 hours;

FIG. 6 is a photograph of the vaccine emulsion prepared according to comparative example 2 after being stored at 37 ℃ for 7 days.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

The content of the antigen in the vaccine has important influence on the final effect of the vaccine. If the antigen content in the vaccine is low, the problems that the virus is easy to inactivate, the activity of the vaccine is greatly influenced by individual difference, the success rate of vaccination is low and the like are easily caused. In vaccines currently in routine use, the volume ratio of antigen to oil is typically 1: 1-3: 1, if the antigen content is too high, problems such as delamination and emulsion instability are likely to occur.

After extensive practice, the inventors of the present application have found that a composition of squalene, SPAN85 and TWEEN 80 (and saponins) in a specific ratio can form a small particle with hydrophilic surface at a suitable temperature according to a certain formulation and mixing process. After self-emulsification or homogenization, the small particles can be combined with an aqueous antigen to form stable 250-350nm lipid particles with good color permeability. The composite adjuvant can be used for preparing vaccines for greatly improving the antigen capacity. Moreover, the composite adjuvant can be prepared under mild stirring conditions, so that the problems of antigen activity reduction caused by heat due to high-speed stirring or shearing and the like are avoided.

Based on the above findings, the present inventors have made the present invention. In one embodiment, the invention provides a composite adjuvant for an animal vaccine, which is prepared by emulsifying a composition comprising squalene, SPAN85 and TWEEN 80. In another embodiment, the present invention provides a composite adjuvant for animal vaccines, which is prepared by emulsifying a composition comprising squalene, SPAN85, TWEEN 80 and saponin.

The present invention will be described in detail below with reference to examples so that those skilled in the art can better understand the present invention. However, those of ordinary skill in the art will appreciate that the examples are provided for illustrative purposes only and are not intended to limit the invention in any way.

Example 1

(1) Mixing squalene, SPAN85 and TWEEN 80 at a weight ratio of 134:23:43, stirring at 150rpm for 30min, standing to obtain transparent solution;

(2) preparing an aqueous phase: the inactivated PCV2 cell culture qualified by aseptic detection is a water phase;

(3) re-mixing the solution obtained in step (1) and then mixing the solution with an aqueous phase: adding the oil phase into the water phase at a volume ratio of 6:1, emulsifying under stirring at 150rpm, and standing to obtain oil-in-water type vaccine. Sucking a small amount of the product and dripping on the surface of cold water to form cloud-like dispersion, wherein the product is oil-in-water type; centrifuging 10ml of finished product at 3000r/min for 15 minutes, and separating out an anhydrous phase at the bottom of the tube; the vaccine is stored for 28 days at 4 ℃ and 37 ℃ respectively, no layering phenomenon occurs, and the obtained vaccine is stable emulsion.

(4) The viscosity of the final product was measured to be 6.3cP using a rotary viscometer.

(5) Preparing 800ml of oil phase according to the step (1), penetrating a PES membrane, layering after autoclaving, mixing uniformly after sterilization, emulsifying the antigen 1:6, and then centrifuged, the emulsion was still homogeneous and stored at 4 ℃ and 37 ℃ for 28 days, respectively, without delamination (see FIG. 3).

Example 2

(1) Mixing squalene, SPAN85, TWEEN 80 and saponin at weight ratio of 12:2:5:1, stirring at 150rpm for 40min, standing to obtain transparent solution;

(2) preparing an aqueous phase: the inactivated PCV2 cell culture qualified by aseptic detection is a water phase;

(3) re-mixing the solution obtained in step (1) and then mixing the solution with an aqueous phase: adding the oil phase into the water phase at a volume ratio of 8:1, emulsifying under stirring at 150rpm, and standing to obtain oil-in-water type vaccine. Sucking a small amount of the product and dripping on the surface of cold water to form cloud-like dispersion, wherein the product is oil-in-water type; centrifuging 10ml of finished product at 3000r/min for 15 minutes, and separating out an anhydrous phase at the bottom of the tube; the obtained vaccine is stable emulsion, and can be stored at 4 deg.C and 37 deg.C for 28 days without layering.

(4) The viscosity of the final product was measured to be 5.8cP using a rotary viscometer.

(5) Preparing 800ml of oil phase according to the step (1), penetrating a PES membrane, layering after autoclaving, mixing uniformly after sterilization, emulsifying the antigen 1:8, and then centrifuged, the emulsion was still homogeneous and stored at 4 ℃ and 37 ℃ for 28 days, respectively, without delamination (see FIG. 4).

Comparative example 1

(1) Preparing an aqueous phase: the inactivated PCV2 cell culture qualified by aseptic detection is a water phase;

(2) using 206 adjuvant as oil phase, preheating water phase and oil phase to 31 deg.C, then using water phase: adding the water phase into the oil phase at the volume ratio of 1:1, stirring at 350rpm for 50 minutes, and standing to obtain the water-in-oil-in-water type vaccine. A small amount of finished products are absorbed and dropped on the surface of cold water and spread in a cloud form to form water-in-oil-in-water type; centrifuging 10ml of finished product at 3000r/min for 15 minutes, and obviously separating out a water phase at the bottom of the tube, wherein the separation amount is more than 0.5 ml; after 12 hours of storage at 37 ℃, the emulsion broke and divided into three layers, the upper and middle layers were both oil and the lower layer was water (see fig. 5).

(3) The viscosity of the final product was measured to be 7.3cP using a rotary viscometer.

Comparative example 2

(1) Preparing an aqueous phase: adding 2% Tween-80 into the inactivated PCV2 cell culture qualified by aseptic detection, and uniformly mixing to obtain a water phase;

(2) adding Marcol52 white oil into Span-80, mixing uniformly to obtain an oil phase, and mixing the oil phase with a water phase: adding the water phase prepared in the step (1) into the oil phase in a volume ratio of 2:1, emulsifying under high-speed stirring, and standing to obtain the oil-in-water type vaccine. A small amount of finished product is absorbed and dripped on the surface of cold water, and the finished product is not diffused except the No. 1 drip and is water-in-oil type; centrifuging 10ml of finished product at 3000r/min for 15 min, and separating out an upper oil phase of the tube, wherein the separated-out amount is about 0.5 ml; storing at 37 deg.C for 21 days, and separating to obtain 5mm white oil phase; the bottom of the lower tube was light and uneven (see FIG. 6).

(3) The viscosity of the final product was measured to be 61.4cP using a rotary viscometer.

Example 3: mouse immunization test

First, test materials

Table 1: vaccine information

Table 2: kit information

3. Test mice: purchased from Hangzhou university and about 30 days old. Of these, 11 male mice and 14 female mice were used. The male mice are divided into 1 group of 5 mice in total, and the rest 1 mouse is used as a control group; female mice were divided into 1 group of 5 mice in total 2 groups, and the remaining 4 mice served as control groups.

II, immunization program: selecting a group (5 mice/group) of immune Yuanjing vaccine (0.2 ml/mouse) from male mice and female mice respectively; the remaining two groups (5/group) were immunized with HS1010 emulsion vaccine, 0.2 ml/group; the control group was injected with 0.2 ml/mouse of physiological saline.

And selecting 2 round clean vaccine immune groups and HS1010 emulsion vaccine immune groups respectively 28 days after the first immunization, performing the second immunization, and collecting blood 7 days after the second immunization, killing and taking livers and spleens for PCR detection.

Thirdly, a sample collection scheme: collecting blood before immunization, separating serum, and storing in refrigerator at-20 deg.C; after immunization, performing tail breaking blood collection and serum separation on each experimental group at intervals of 7 days; at 21 days after the first immunization, except for 2 groups of 4 mice to be immunized twice and one control mouse, the remaining mice were dissected, killed, and liver and spleen were frozen.

Fourthly, test results:

table 3: ELISA detection result (OD value) of mouse serum ring antibody

The 'male' in the graph represents a male mouse; the female parent represents the female mouse.

As can be seen from table 3:

(1) the OD value of the control group is always kept stable and is at a negative level;

(2) the antibody level of the round Jing Nuo vaccine immunization group is increased slowly compared with that of the HS1010 group, and the antibody levels of the two groups gradually tend to be consistent 28 days after immunization, which indicates that the new adjuvant group can stimulate an organism to generate specific antibodies quickly;

(3) the difference of the detected OD values of the round neat vaccine immune group and the same group is larger than that of the HS1010 group;

(4) in the mice immunized twice, the detection value continues to increase 7 days after the round Jing Nuo vaccine immunization group, and the HS1010 group is kept at a stable level.

Claims (9)

1. A composite adjuvant for animal vaccines is characterized in that the composite adjuvant is prepared by emulsifying a composition consisting of squalene, SPAN85 and TWEEN 80.

2. The composite adjuvant according to claim 1, wherein the weight ratio of squalene, SPAN85 and TWEEN 80 is (120-140): (15-30): (30-50), preferably 134:23: 43.

3. The compound adjuvant for animal vaccines is characterized in that the compound adjuvant is prepared by emulsifying a composition consisting of squalene, SPAN85, TWEEN 80 and saponin.

4. The composite adjuvant according to claim 3, wherein the weight ratio of the composition of squalene, SPAN85, TWEEN 80 and saponin is (5-20): 1-4): 2-10): 0.5-3, preferably 12:2:5: 1.

5. The composite adjuvant of claim 4, wherein the saponin is selected from the group consisting of ginsenosides and saponins.

6. A method for preparing a vaccine using the composite adjuvant according to claim 1 or 2, the method comprising the steps of:

(1) preparing an oil phase: mixing squalene, SPAN85 and TWEEN 80 at a certain weight ratio, stirring at medium and low speed for 20-50min, and standing to obtain light yellow clear solution;

(2) preparing an aqueous phase: diluting the antigen with an appropriate volume of PBS to form an aqueous phase;

(3) re-mixing the solution obtained in step (1) and then mixing the solution with an aqueous phase: and adding the water phase into the oil phase =5:1 to 8:1 in volume ratio, emulsifying under medium and low speed magnetic stirring, and standing for 30-45min to obtain the stable oil-in-water type vaccine.

7. A method of preparing a vaccine using the composite adjuvant according to any one of claims 3 to 5, the method comprising the steps of:

(1) preparing an oil phase: mixing squalene, SPAN85, TWEEN 80 and saponin at a weight ratio, stirring at medium and low speed for 20-50min, and standing to obtain light yellow clear solution;

(2) preparing an aqueous phase: diluting the antigen with PBS with appropriate volume to obtain water phase;

(3) re-mixing the solution obtained in step (1) and then mixing the solution with an aqueous phase: and adding the water phase into the oil phase =5:1 to 8:1 in volume ratio, emulsifying under medium and low speed magnetic stirring, and standing for 30-45min to obtain the stable oil-in-water type vaccine.

8. The method according to claim 6 or 7, wherein the medium-low speed stirring is magnetic stirring, and the rotating speed is 80-160 r/min.

9. A vaccine comprising the composite adjuvant according to any one of claims 1 to 5 or made using the method according to any one of claims 6-8.

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