WO2022036885A1 - Temperature-sensitive hydrogel adjuvant for veterinary vaccines, preparation method and use thereof - Google Patents

Abstract

A temperature-sensitive hydrogel adjuvant for veterinary vaccines, a preparation method and the use thereof, which relate to the field of veterinary vaccine adjuvants. The temperature-sensitive hydrogel adjuvant for veterinary vaccines consists of the following components in percentages by mass: 15-50% of a polymer micelle substance, and 0.1-5% of a non-ionic cellulose ether, with the balance being water. The preparation method of a temperature-sensitive hydrogel adjuvant of the veterinary vaccine comprises the specific steps of: weighing out the components, mixing, and swelling into a uniform system at 2-10°C to obtain the temperature-sensitive hydrogel adjuvant. The temperature-sensitive hydrogel adjuvant has simple and readily available raw materials, is low in price, small in viscosity, has few side effects and is capable of prolonging the immune duration.

Description

Thermosensitive hydrogel adjuvant for veterinary vaccine, preparation method and application thereof technical field

The invention relates to the field of veterinary vaccine adjuvants, in particular to a thermosensitive hydrogel adjuvant for veterinary vaccines, a preparation method and applications thereof.

Background technique

Adjuvants generally refer to substances that non-specifically enhance the body's specific immune response to an antigen. According to their different mechanisms of action, adjuvants can be divided into two categories: one is immunostimulatory adjuvants, such as LPS, cytokines, etc., which directly stimulate or activate the active cells of the immune system; the other is antigenic Presentation system (also known as vaccine delivery system, vaccine delivery system, VDS), such as aluminum glue, mineral oil, liposomes, etc., can protect vaccine antigens and prolong the retention time of antigens in the body, thereby forming continuous immune stimulation and Efficient immune response.

At present, mineral oil adjuvant is still widely used in animal vaccines. However, due to the high proportion of white oil and surfactant in the vaccine, the animals have a greater stress response after injection, and even cause aseptic purulent damage and inoculation. Granulomas near the site have adverse effects on the quality of livestock and poultry products; and traditional water-in-oil vaccines are usually viscous and difficult to inject, which seriously affects the immune effect of the vaccine and the effective prevention and control of epidemic diseases.

Thermosensitive hydrogel is a kind of hydrogel, which has been applied in drug release, tissue engineering, cell engineering and so on. When the temperature of the system is lower than the critical transition temperature, the temperature-sensitive matrix can combine with water molecules to form hydrogen bonds, so that the gel matrix can be dissolved in water, and the system is a flowing liquid phase. When the temperature rises to the critical transition temperature, the hydrogen bond is destroyed, the polymer chain shrinks under the action of hydrophobicity, the gel-forming matrix is separated from the water phase, and the system undergoes a phase transition and becomes a gel state. The gel system has a special network cross-linked structure, which can encapsulate the drug or other active ingredients in the three-dimensional network structure, and release the drug or other active substances slowly with the erosion of the gel, which can prevent rapid degradation or loss. Live, prolong the action time of the drug in the body, and then achieve the purpose of sustained release and controlled release.

However, there is a lack of thermosensitive hydrogel adjuvants suitable for veterinary vaccines in the prior art, with low viscosity, small side effects and prolonged immunity duration.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a temperature-sensitive hydrogel adjuvant for veterinary vaccines, which is simple and easy to obtain, low in price, low in viscosity, small in side effects and can significantly prolong the duration of immunity.

Another object of the present invention is to provide a method for preparing the thermosensitive hydrogel adjuvant of the above-mentioned veterinary vaccine.

Another object of the present invention is to provide the application of the thermosensitive hydrogel adjuvant for veterinary vaccines.

The purpose of the present invention adopts following technical scheme to realize:

A thermosensitive hydrogel adjuvant for veterinary vaccines is composed of the following components according to the mass percentage:

Polymeric micellar substance 15%-50%,

Nonionic cellulose ether 0.1%-5%,

The remainder is water.

In the present invention, the polymer micellar substance is one or a mixture of Poloxamer 407, Poloxamer 188, Soluplus and Carbomer.

In the present invention, the nonionic cellulose ether is hydroxypropyl methylcellulose.

In the preferred technical scheme, the thermosensitive hydrogel adjuvant of the veterinary vaccine is composed of the following components according to the mass percentage:

In the present invention, the poloxamers are poloxamer 407 and poloxamer 188, the mass percentage of poloxamer 407 is 14-30%, and the mass percentage of poloxamer 188 is 1-5%.

The present invention also provides a method for preparing a temperature-sensitive hydrogel adjuvant for the veterinary vaccine, the specific steps comprising: weighing each component, mixing, and swelling into a uniform system at 2-10° C. to obtain a temperature-sensitive hydrogel adjuvant. type of hydrogel adjuvant.

In the present invention, the mixing is carried out at 0-10°C.

The present invention also provides a vaccine containing the temperature-sensitive hydrogel adjuvant of the veterinary vaccine.

In the present invention, the thermosensitive hydrogel vaccine comprises a thermosensitive hydrogel adjuvant and an aqueous antigen solution in a mass ratio of 0.5-4:1.

In the present invention, the antigen is porcine circovirus or porcine parvovirus.

Beneficial effects of the present invention:

(1) The temperature-sensitive hydrogel adjuvant of the veterinary vaccine of the present invention has simple components, readily available raw materials, low price, and convenient use; the vaccine prepared by combining with the adjuvant has good acupuncture and low viscosity and is easy to use The injection significantly reduces the workload during vaccination; it is easy to store, has few side effects, and can significantly prolong the duration of immunity. The biosafety and in vivo degradability of the selected raw materials of the present invention are both good. After gelation, the gel forms a cross-linked network three-dimensional structure, which can load the antigen in the cross-linked pores, and then the semi-solid gel is eroded in vivo to achieve the slow release effect of the antigen.

(2) The preparation method of the thermosensitive hydrogel adjuvant of the present invention is simple and easy to operate and has low cost.

Description of drawings

Figure 1. Observation of tissue sections at different vaccine injection sites. (1), (2), (3), (4), (5), (6), (7), (8) correspond to thermosensitive hydrogel vaccine PCV2-1, thermosensitive hydrogel vaccine, respectively Glue vaccine PCV2-2, thermosensitive hydrogel vaccine PCV2-3, thermosensitive hydrogel vaccine PCV2-4, thermosensitive hydrogel vaccine PCV2-5, thermosensitive hydrogel vaccine PCV2-6, Temperature-sensitive hydrogel vaccine PCV2-7 and temperature-sensitive hydrogel vaccine PCV2-8 immunized mice; (9) mice in the control vaccine PCV2-1 immunization group; (10) a blank control injected with normal saline group of mice.

detailed description

The present invention will be described in detail through the following examples, but the present invention does not only include the following examples, but also includes other examples based on the invention.

The sources of each material in the present invention are as follows:

Poloxamer 407 and Poloxamer 188 were purchased from Sigma Company.

Carbomer was purchased from Lubrizol Corporation.

Soluplus, purchased from Shanghai BASF Company, is a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer composed of N-vinyl-ε-caprolactam, vinyl acetate and polyethylene glycol (57:30:13 ) copolymerized with a molecular weight of 90,000 to 140,000 g/mol.

Hydroxypropyl methylcellulose was purchased from sigma.

Example 1 Screening of thermosensitive hydrogel adjuvants

Thermosensitive hydrogel adjuvant 1, according to mass percentage, consists of the following components:

Thermosensitive hydrogel adjuvant 2, according to mass percentage, consists of the following components:

Thermosensitive hydrogel adjuvant 3, according to mass percentage, is composed of the following components:

Thermosensitive hydrogel adjuvant 4, according to mass percentage, consists of the following components:

Thermosensitive hydrogel adjuvant 5, according to mass percentage, consists of the following components:

Thermosensitive hydrogel adjuvant 6, according to mass percentage, consists of the following components:

Thermosensitive hydrogel adjuvant 7, according to mass percentage, consists of the following components:

Thermosensitive hydrogel adjuvant 8, according to mass percentage, consists of the following components:

According to the components and contents of each thermosensitive hydrogel adjuvant, each substance was weighed, added to water, stirred in an ice-water bath, left standing at 4°C for 12 hours to swell into a homogeneous system, and then used 0.22 The temperature-sensitive hydrogel adjuvants 1-8 were obtained by filtration and sterilization through a μm filter.

Using the inversion method (a thermosensitive gel matrix and its preparation method and application, patent number ZL201610243442.8), the above-mentioned hydrogels prepared with different formulas were placed in a vial, put into a water bath, and the temperature of the water bath began to rise from 30 ° C , control the heating rate at 0.5°C/min, and maintain each temperature point for 5-10min. The vial was tilted 60 degrees, and the gelling time of each sample at different temperatures was recorded on the basis that the sample did not flow within 30 seconds.

As a result, it was found that the above-mentioned 8 thermosensitive hydrogel adjuvants could not be gelled below 32°C, and the system maintained fluidity. When the temperature rises to 34 °C, the viscosity of the sample increases sharply, the fluidity of the system drops sharply, and the system gradually gels.

The gelation time of each of the above thermosensitive hydrogel adjuvants at 36-40° C. (a common body temperature range for humans or animals) was investigated, and the results are shown in Table 1. It can be seen from Table 1 that the temperature-sensitive hydrogel adjuvants 1-8 can form gels at 36-40°C, and with the increase of temperature, the gel-forming time gradually shortens.

Table 1 Gel-forming time of different hydrogels at 36-40 °C

group 36℃ 36.5℃ 37℃ 37.5℃ 38℃ 38.5℃ 39℃ 39.5℃ 40℃ Recipe 1 4'21 4'11 3'19 3'16 3'04 2'55 2'27 1'55 1'15 Recipe 2 3'47 3'44 3'02 2'23 2'05 1'41 1'27 1'05 0'53

Recipe 3 3'35 3'26 3'12 2'49 2'33 2'08 1'40 1'24 1'08 Recipe 4 9'50 9'23 9'01 8'40 8'18 8'02 7'36 7'11 6'42 Recipe 5 2'48 2'19 2'00 1'47 1'38 1'25 1'00 0'50 0'37 Recipe 6 5'15 4'53 4'41 4'22 4'01 3'50 3'36 3'11 2'49 Recipe 7 3'45 3'31 3'22 3'09 2'53 2'38 2'14 2'04 1'48 Recipe 8 2'35 2'18 2'09 2'00 1'49 1'34 1'27 1'05 0'35

Note: 4'21 in Table 1 means 4 minutes and 21 seconds, and so on.

Example 2 Preparation, property evaluation and application of thermosensitive hydrogel vaccine

(1) Preparation of porcine circovirus type 2 inactivated virus solution

Porcine circovirus type 2 inactivated virus solution (DBN-SX07 strain) was provided by Chengdu Tianbang Biotechnology Co., Ltd., and the virus content per milliliter of virus solution before inactivation was 10 ⁶ TCID ₅₀ . The inactivated virus liquid complies with the relevant requirements of "Compilation of Quality Standards for Veterinary Biological Products" (2011).

(2) Preparation of porcine circovirus type 2 thermosensitive hydrogel inactivated vaccine

The thermosensitive hydrogel adjuvants 1-8 prepared in Example 1 were respectively mixed with porcine circovirus type 2 inactivated virus liquid (DBN-SX07 strain) at a mass ratio of 3:1 to prepare corresponding thermosensitive Thermosensitive hydrogel vaccine PCV2-1, thermosensitive hydrogel vaccine PCV2-2, thermosensitive hydrogel vaccine PCV2-3, thermosensitive hydrogel vaccine PCV2-4, thermosensitive hydrogel vaccine PCV2 -5. Thermosensitive hydrogel vaccine PCV2-6, thermosensitive hydrogel vaccine PCV2-7, thermosensitive hydrogel vaccine PCV2-8. The numbering of the vaccine is consistent with that of the thermosensitive hydrogel adjuvant.

Preparation of control vaccine PCV2-1: refer to invention patent ZL201610243442.8, weigh 30 g of Soluplus, add 50 mM phosphate buffer to a total mass of 100 g, disperse at 4°C, and prepare a control thermosensitive gel matrix. The control thermosensitive gel matrix and the porcine circovirus type 2 inactivated virus solution were mixed uniformly at a mass ratio of 3:1 to prepare a control vaccine PCV2-1.

(3) Detection of physical properties of vaccines

The above thermosensitive hydrogel vaccines 1-8 and the control vaccine PCV2-1 were stored at 4°C and 37°C, respectively, and the appearance changes were observed, and the measurements were carried out according to the rotational viscometer in the appendix of the current "Chinese Veterinary Pharmacopoeia". The viscosity was measured, and the specific results are shown in Table 2.

Table 2 Comparison of physical properties of each

vaccine Appearance (4℃) Appearance (37℃) Viscosity (cP, 25℃) Thermosensitive hydrogel vaccine PCV2-1 clear solution translucent gel 2.08±0.25

Thermosensitive hydrogel vaccine PCV2-2 clear solution translucent gel 3.76±0.61 Thermosensitive hydrogel vaccine PCV2-3 clear solution translucent gel 2.03±0.75 Thermosensitive hydrogel vaccine PCV2-4 clear solution translucent gel 12.38±0.72 Thermosensitive hydrogel vaccine PCV2-5 clear solution translucent gel 26.04±0.53 Thermosensitive hydrogel vaccine PCV2-6 clear solution translucent gel 13.09±0.31 Thermosensitive hydrogel vaccine PCV2-7 clear solution translucent gel 3.99±0.43 Thermosensitive hydrogel vaccine PCV2-8 clear solution translucent gel 23.01±0.91 Control vaccine PCV2-1 clear solution translucent gel 63.97±4.06

According to the requirements of the Chinese Veterinary Pharmacopoeia, the viscosity of all injectable vaccines should not exceed 200cP. The vaccines prepared by using the thermosensitive hydrogel adjuvant of the present invention all meet the requirements, and it can be seen from Table 2 that the viscosity of the vaccine prepared by using the thermosensitive hydrogel adjuvant is significantly lower than that of the control vaccine PCV2-1, which is convenient for Clinical injection use.

(4) Mouse safety evaluation of thermosensitive hydrogel vaccine

100 Balb/c female mice (18-22 g) were randomly divided into 10 groups of 10 mice each. A group of mice were immunized with each of the above thermosensitive hydrogel vaccines and the control vaccine PCV2-1. The immunization method was as follows: mice were immunized subcutaneously on the back of the neck, each mouse was immunized with 0.2 mL of the vaccine, and a blank control group that was only injected with normal saline was set. After immunization, the conditions of the mice were observed for 14 consecutive days, including feed intake, water intake, mental state, changes at the injection site and abnormal behavior, and tissue sections were used to evaluate the pathological changes of mice in different experimental groups. The tissue section results are shown in Figure 1. The results showed that the mice in the control vaccine PCV2-1 test group had mild stress reactions such as licking the injection site continuously after the injection, and there was still slight hair loss near the injection site 14 days later, and a small amount of inflammatory cells could be seen from the slice results. infiltration. However, the mice in the other thermosensitive hydrogel vaccine groups had no obvious stress response, the mice were in good mental state, and there was no abnormal behavior change. After injection, there was no skin damage or hair loss at the injection site in each group. See obvious inflammatory cells appear.

(4) Evaluation of immune efficacy of thermosensitive hydrogel vaccine

One hundred 14-day-old piglets with negative porcine circovirus type 2 antigen and antibody were selected and randomly divided into 10 groups with 10 pigs in each group. Each thermosensitive hydrogel vaccine and the control vaccine PCV2-1 were immunized to a group of piglets respectively, and a blank control group was not immunized at the same time. The immunization program was as follows: the first immunization at 14 days old, and the second immunization after an interval of 21 days, and the dose of each immunization was 1.0ml/head. At 1 month, 2 months, 3 months, 4 months and 6 months after the last immunization, blood was collected to separate serum, and porcine circovirus type 2 antibody ELISA detection kit (Wuhan Keqian Biological Products Co., Ltd.) was used for testing. Antibody detection. The PCV2 antibody titer of each piglet in the blank control group should be negative (not higher than 1:40), and the PCV2 antibody titer of the immunized pigs ≥ 1:40 was judged as antibody positive, and the antibody positive rate was counted.

Table 3 Comparison of immune efficacy of each vaccine

Note: The same column in Table 3 does not contain the same letter, indicating a significant difference, p<0.05.

According to the requirements of "Compilation of Quality Standards for Veterinary Biological Products (2010)", the antibody positive rate ≥ 80% is qualified. It can be seen from Table 3 that the immune duration of the vaccine prepared with thermosensitive hydrogel adjuvant 1-3 can reach 6 months, and the immunity duration of other vaccines does not exceed 3 months. Therefore, the thermosensitive hydrogel The immune efficacy of the vaccine prepared by adjuvant 1-3 was significantly better than that of the thermosensitive hydrogel adjuvant 4-8 and the vaccine prepared by the control thermosensitive gel matrix. The vaccine prepared by using the thermosensitive hydrogel adjuvant 1-3 fully meets the immune and disease prevention requirements of commercial pigs, and has very good application value.

(5) Piglet safety evaluation of thermosensitive hydrogel vaccine

In the title (4) of this example, the body temperature of the piglets was detected for 7 consecutive days after the first immunization at the age of 14. The specific results are shown in Table 4. As can be seen from Table 4, the body temperature of pigs after immunization with each thermosensitive hydrogel vaccine was within the normal range of 39.0-40.0 °C, and there was no fever symptoms, while the control vaccine PCV2-1 was immunized 1-5 days after the first immunization. Pig body temperature is greater than 40.0 degrees. At the same time, the macroscopic observation of the injection site was performed on the pigs after the first immunization, and it was found that the pigs injected with each thermosensitive hydrogel vaccine did not find abnormal phenomena such as injection site damage and ulcers; while the pigs immunized with the control vaccine PCV2-1 were not found to be abnormal. , 2 had lumps at the injection site, which disappeared in about 7 days. After the second immunization, there were no visible adverse reactions at the injection site of the piglets in each group. The above results show that the vaccine prepared by using thermosensitive hydrogel adjuvant 1-8 has little side reaction after immunizing piglets and is safe for piglets.

Table 4 The average body temperature change (℃) of piglets in each group 7 days after immunization

group 0 days 1 day 2 days 3 days 4 days 5 days 6 days 7 days Thermosensitive hydrogel vaccine PCV2-1 39.5 39.7 39.5 39.4 39.8 39.8 39.6 39.7

Thermosensitive hydrogel vaccine PCV2-2 39.7 39.7 39.3 39.4 39.3 39.5 39.6 39.6 Thermosensitive hydrogel vaccine PCV2-3 39.7 39.7 39.6 39.7 39.8 39.7 39.7 39.6 Thermosensitive hydrogel vaccine PCV2-4 39.5 39.5 39.5 39.7 39.5 39.5 39.4 39.5 Thermosensitive hydrogel vaccine PCV2-5 39.7 39.7 39.8 39.7 39.5 39.6 39.1 39.7 Thermosensitive hydrogel vaccine PCV2-6 39.6 39.5 39.5 39.6 39.7 39.7 39.6 39.9 Thermosensitive hydrogel vaccine PCV2-7 39.6 39.7 39.7 39.7 39.5 39.5 39.8 39.7 Thermosensitive hydrogel vaccine PCV2-8 39.6 39.5 39.5 39.7 39.7 39.6 39.6 39.4 Control vaccine PCV2-1 39.8 40.1 40.1 40.4 40.3 40.1 39.7 39.7 blank 39.8 39.5 39.7 39.7 39.6 39.4 39.7 39.4

Example 3 Preparation and application of thermosensitive hydrogel inactivated vaccine for porcine parvovirus disease

(1) Porcine parvovirus (PPV BJ-2 strain) inactivated antigen

The inactivated virus solution of porcine parvovirus BJ-2 strain was provided by Jinyu Baoling Bio-Pharmaceutical Co., Ltd. The virus content per milliliter of virus solution before inactivation was not less than 10 ⁶ TCID ₅₀ . The porcine parvovirus inactivated virus liquid complies with the relevant requirements of the Compilation of Quality Standards for Veterinary Biological Products (2012).

(2) Preparation of thermosensitive hydrogel inactivated vaccine for porcine parvovirus disease

The thermosensitive hydrogel adjuvants 1-8 in Example 1 were respectively mixed with the inactivated virus liquid of the porcine parvovirus BJ-2 strain at a mass ratio of 3:1 to obtain the thermosensitive hydrogel vaccine PPV-1, warm Sensitive hydrogel vaccine PPV-2, thermosensitive hydrogel vaccine PPV-3, thermosensitive hydrogel vaccine PPV-4, thermosensitive hydrogel vaccine PPV-5, thermosensitive hydrogel vaccine PPV-6, thermosensitive hydrogel vaccine PPV-7, thermosensitive hydrogel vaccine PPV-8. The vaccine number is the same as that of the thermosensitive hydrogel adjuvant.

Preparation of the control vaccine PPV-1: According to the invention patent ZL201610243442.8, weigh 30g of Soluplus, add 50mM phosphate buffer to a total mass of 100g, disperse at 4°C, and prepare a control temperature-sensitive gel matrix. The control thermosensitive gel matrix and the inactivated virus solution of the porcine parvovirus BJ-2 strain were evenly mixed at a mass ratio of 3:1 to obtain a control vaccine PPV-1.

(3) Detection of physical properties of vaccines

The thermosensitive hydrogel vaccine PPV-1 to thermosensitive hydrogel vaccine PPV-8 and the control vaccine PPV-1 in the title (2) of this example were stored at 4°C and 37°C respectively, and the appearance changes were observed. And according to the current "Chinese Veterinary Pharmacopoeia" appendix rotational viscometer measurement method for viscosity measurement, the specific results are shown in Table 5.

Table 5 Comparison of physical properties of various porcine parvovirus vaccines

vaccine Appearance (4℃) Appearance (37℃) Viscosity (cP, 25℃) Thermosensitive hydrogel vaccine PPV-1 clear solution translucent gel 3.11±0.33 Thermosensitive hydrogel vaccine PPV-2 clear solution translucent gel 4.32±0.11 Thermosensitive hydrogel vaccine PPV-3 clear solution translucent gel 3.03±0.15 Thermosensitive hydrogel vaccine PPV-4 clear solution translucent gel 14.38±0.27 Thermosensitive hydrogel vaccine PPV-5 clear solution translucent gel 27.04±0.93 Thermosensitive hydrogel vaccine PPV-6 clear solution translucent gel 15.91±0.23 Thermosensitive hydrogel vaccine PPV-7 clear solution translucent gel 5.32±0.19 Thermosensitive hydrogel vaccine PPV-8 clear solution translucent gel 33.27±0.21 Control vaccine PPV-1 clear solution translucent gel 78.79±5.78

It can be seen from Table 5 that the viscosity of the vaccine prepared by using thermosensitive hydrogel adjuvant 1-8 is significantly lower than 200cP, which meets the viscosity requirements of the "Chinese Veterinary Pharmacopoeia" for injection vaccines, and the viscosity is significantly lower than that of the control. The vaccine PPV-1 is more convenient for clinical injection and reduces labor intensity.

(4) Safety evaluation

Referring to the relevant requirements of "Compilation of Quality Standards for Veterinary Biological Products (2012)", the safety evaluation of piglets and the safety evaluation of suckling mice were carried out respectively.

Safety evaluation of piglets: The thermosensitive hydrogel vaccine PPV-1 to thermosensitive hydrogel vaccine PPV-8 and the control vaccine PPV-1 were inoculated with 4-6 week old healthy susceptible piglets (porcine parvovirus hemagglutination). The inhibitory antibody (PPV HI) titer is not higher than 1:8), 2 heads of each vaccine are immunized, and 4 ml of the vaccine is injected intramuscularly in each head and neck for 21 days. Results: There were no local or systemic adverse reactions caused by the vaccine in each group.

Safety evaluation of suckling mice: The temperature-sensitive hydrogel vaccine PPV-1 to the temperature-sensitive hydrogel vaccine PPV-8 and the control vaccine PPV-1 were respectively inoculated into 3-5 day old suckling mice, and each vaccine was immunized for 5 days. Only, each subcutaneous injection of 0.1 ml of the vaccine, continuous observation for 10 days. Results: All groups were healthy and alive, and there were no local or systemic adverse reactions caused by the vaccine.

(5) Evaluation of immune efficacy

The thermosensitive hydrogel vaccine PPV-1 to thermosensitive hydrogel vaccine PPV-8 and the control vaccine PPV-1 were inoculated with 350-400 g healthy guinea pigs (porcine parvovirus hemagglutination inhibitory antibody (PPV HI) titers respectively Not higher than 1:8), 5 mice were immunized with each vaccine, and each guinea pig was intramuscularly injected with 0.5 ml of the vaccine. The blank control group consisted of 5 guinea pigs, which were not immunized. On 28 days, 2 months, 3 months, 4 months, 5 months, and 6 months after immunization, blood was collected from guinea pigs in each group, serum was separated, and the titer of porcine parvovirus hemagglutination inhibitory antibody was detected (the detection method was according to the Chinese Veterinary Pharmacopoeia appendix requirements).

Table 6 Comparison of immune efficacy of each vaccine

According to the requirements of "Compilation of Quality Standards for Veterinary Biological Products (2012)", at least 4 immunized guinea pigs should show antibody positive, and the titer should not be lower than 1:64 (PPV HI≥6, log2), control guinea pig hemagglutination inhibitory antibody The titers should not be higher than 1:8 (PPV HI≤3, log2), and it is judged as qualified. As can be seen from Table 6, the immunization duration after immunizing guinea pigs with the vaccine prepared by using thermosensitive hydrogel adjuvant 1-3 can reach more than 6 months, and using thermosensitive hydrogel adjuvant 4-8 and control For the vaccine prepared with thermosensitive gel matrix, the level of hemagglutination inhibitory antibody decreased significantly 5 months after immunization, which was lower than 1:64 (PPV HI ≥ 6, log2), and the immunization duration was only 4 months, and the level was significantly lower. Antibody titers after immunization with vaccines prepared with thermosensitive hydrogel adjuvants 1-3. Therefore, the immune efficacy of the vaccine prepared by thermosensitive hydrogel adjuvant 1-3 was significantly better than that of the vaccine prepared by thermosensitive hydrogel adjuvant 4-8 and control thermosensitive gel matrix. The vaccine prepared by using thermosensitive hydrogel adjuvant 1-3 fully meets the quality standards of veterinary biological products, meets the immune disease prevention requirements of commercial pigs, and has very good application value.

Claims (10)

A thermosensitive hydrogel adjuvant for veterinary vaccine, characterized in that, according to mass percentage, it is composed of the following components: Polymeric micellar substance 15%-50%, Nonionic cellulose ether 0.1%-5%, The remainder is water. The thermosensitive hydrogel adjuvant for veterinary vaccines according to claim 1, wherein the macromolecular micellar substance is one of Poloxamer 407, Poloxamer 188, Soluplus, and Carbomer. one or a mixture of several. The thermosensitive hydrogel adjuvant for veterinary vaccines according to claim 1, wherein the nonionic cellulose ether is hydroxypropyl methylcellulose. The thermosensitive hydrogel adjuvant of veterinary vaccine according to claim 3, is characterized in that, according to mass percentage composition, is made up of the following components:

The thermosensitive hydrogel adjuvant for veterinary vaccine according to claim 4, wherein the poloxamer is poloxamer 407 and poloxamer 188, and the mass percentage of poloxamer 407 is The content is 14-30%, and the mass percentage of Poloxamer 188 is 1-5%. The method for preparing a thermosensitive hydrogel adjuvant for a veterinary vaccine according to claim 1, wherein the specific steps include: weighing each component, mixing, and swelling into a homogeneous system at 2-10°C, That is, a thermosensitive hydrogel adjuvant is obtained. The method for preparing a thermosensitive hydrogel adjuvant for a veterinary vaccine according to claim 6, wherein the mixing is carried out at 0-10°C. A vaccine containing the thermosensitive hydrogel adjuvant of the veterinary vaccine according to any one of claims 1 to 6. The thermosensitive hydrogel vaccine of the veterinary vaccine according to claim 8, wherein the thermosensitive hydrogel vaccine comprises a thermosensitive hydrogel adjuvant with a mass ratio of 0.5-4:1 and Antigen aqueous solution. The thermosensitive hydrogel vaccine according to claim 9, wherein the antigen is porcine circovirus or porcine parvovirus.

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