AU2020463769B2

AU2020463769B2 - Temperature-sensitive hydrogel adjuvant for veterinary vaccines, preparation method and use thereof

Info

Publication number: AU2020463769B2
Application number: AU2020463769A
Authority: AU
Inventors: Bihua DENG; Yu Lu; Fang Ma; Jinfeng MIAO; Wenzhu YIN; Jinqiu Zhang; Mingxu Zhou
Original assignee: Jiangsu Yanjiang Agricultural Science Research Institute
Current assignee: Jiangsu Yanjiang Agricultural Science Research Institute
Priority date: 2020-08-21
Filing date: 2020-11-11
Publication date: 2023-11-09
Anticipated expiration: 2040-11-11
Also published as: ZA202203353B; WO2022036885A1; CN111840214B; CN111840214A; AU2020463769A1

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

THERMO-SENSITIVE TYPE HYDROGEL ADJUVANT FOR VETERINARY VACCINE, PREPARATION METHOD, AND APPLICATION THEREOF

TECHNICAL FIELD The present invention relates to the field of adjuvants for veterinary vaccines, in particular to a thermo-sensitive type hydrogel adjuvant for a veterinary vaccine, a preparation method and application thereof.

BACKGROUNDART An adjuvant, generally, refers to a substance that can strengthen a specific immune response of a living body to an antigen nonspecifically. Based on different mechanisms of action, the adjuvant can be divided into two categories: immunostimulatory adjuvants, such as LPS, and cytokines, which achieve an effect of a direct stimulation or activation to active cells of an immune system, and an antigen presentation system (also known as a vaccine delivery system (VDS)), such as alumina gel, mineral oil, and liposome, which can protect a vaccine antigen, and prolong retention time of the antigen in the living body, thereby forming a continuous immunostimulation and an efficient immunoreaction. At present, the mineral oil adjuvants are still used in animal vaccines widely, however, as white oil and a surfactant have high proportions in the vaccine, animals have large stress reactions after being injected with the vaccines, and even, aseptic suppuration injury and granulomas adjacent to vaccination sites are caused, which pose an adverse impact on a quality of animal products. In addition, the traditional water-in-oil vaccine is, generally, high in viscosity, and not easy to inject, which severely affects an immune effect of the vaccine and an effective prevention and control of epidemic diseases. A thermo-sensitive type hydrogel belongs to one kind of hydrogels, which has been applied in sustained drug release, tissue engineering, cell engineering, and other sections. A system is a flowing liquid phase, and when a system temperature is lower than a critical transformation temperature, a thermo-sensitive type substrate can be bonded with a water molecule to form a hydrogen bond, by which a gelatinized substrate is dissolved in water. When the temperature is elevated to the critical transformation temperature, the hydrogen bond is destroyed; a polymer chain retracts under hydrophobic effect, the gelatinized substrate is separated from a water phase, and the system undergoes phase transformation to be a gel phase. The gel system has a special net cross-linked structure, in which drugs or other active components can be wrapped in a three-dimensional net structure; and with a denudation of gel, the drugs or other active substances are released slowly, which avoid rapid degradation or deactivation and prolong action time of the drugs in the living body, thereby fulfilling an objective of controlled release. However, the thermo-sensitive type hydrogel adjuvant which is low in viscosity and less in side effect, is able to prolong a duration of immunity, and is suitable for veterinary vaccines hasn't been found yet in the prior art.

SUMMARY OF THE INVENTION The present invention aims to provide a thermo-sensitive type hydrogel adjuvant for a veterinary vaccine. The adjuvant is readily available in raw materials, low in costs, low in viscosity, and less in a side effect, and can prolong a duration of immunity significantly. The present invention further aims to provide a preparation method of the thermo-sensitive type hydrogel adjuvant for the veterinary vaccine. The present invention further aims to provide application of the thermo-sensitive type hydrogel adjuvant for the veterinary vaccine. The objectives of the present invention are fulfilled by the following technical solutions: The thermo-sensitive type hydrogel adjuvant for the veterinary vaccine consists of the following components in percentage by mass:

15% to 50% of polymer micelle substance, 0.1% to 5% of nonionic cellulose ether, and the balance of water. In the present invention, the polymer micelle substance is one of poloxamer 407, poloxamer 188, Soluplus, and Carbomer or a mixture of more of them. In the present invention, the nonionic cellulose ether is hydroxypropyl methyl cellulose. In the preferred technical solution, the thermo-sensitive type hydrogel adjuvant for the veterinary vaccine consists of the following components in percentage by mass: The poloxamer 15%-35%, Soluplus 0.1-10%, The Carbomer 0.01%-0.2%, The hydroxypropyl methyl cellulose 0.1%-5%, the balance of water. In the present invention, the poloxamer is the poloxamer 407 and the poloxamer 188, where a content of the poloxamer 407 is 14% to 30% in percentage by mass, and a content of the poloxamer 188 is 1% to 5% in percentage by mass. The present invention further provides a preparation method of the thermo-sensitive type hydrogel adjuvant for the veterinary vaccine, which includes steps: weighing each component, mixing, swelling at 2C to 10°C to form a homogeneous system, and obtaining the thermo-sensitive type hydrogel adjuvant. In present invention, the mixing is conducted at 0°C to 10°C. The present invention further provides a vaccine containing the thermo-sensitive type hydrogel adjuvant for the veterinary vaccine. In the present invention, the thermo-sensitive type hydrogel vaccine includes the thermo-sensitive type hydrogel adjuvant and an aqueous solution of an antigen, where a mass ratio of the adjuvant to the aqueous solution of the antigen is 0.5-4: 1. In the present invention, the antigen is a porcine circovirus virus or porcine parvovirus. The present invention has the following beneficial effects: (1) The thermo-sensitive type hydrogel adjuvant for the veterinary vaccine of the present invention is simple in components, readily available in the raw materials, low in the costs, and easy to use; the vaccine prepared in combination with the adjuvant is high in syringeability, low in viscosity, and easy to inject, which relieves the workload significantly when animals get vaccinated; and the vaccine is easy to store and less in side effect, and is able to prolong the duration of immunity significantly. The raw materials in the present invention are high in biosecurity and excellent in in-vivo degradability. Gel shows a three-dimensional net cross-linked structure after being gelatinized, in which the antigen can be loaded in a crosslinking hole, and then, achieves slow-release effect along with a denudation of the semi-solid gel in the body. (2) The preparation method of the thermo-sensitive type hydrogel adjuvant is easy to operate, and low in the costs.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an observation of tissue slices at different vaccine injection sites. (1), (2), (3), (4), (5), (6), (7), and (8) correspond to mice of immunization groups, injected with a thermo-sensitive type hydrogel vaccine PCV2-1, a thermo-sensitive type hydrogel vaccine PCV2-2, a thermo-sensitive type hydrogel vaccine PCV2-3, a thermo-sensitive type hydrogel vaccine PCV2-4, a thermo-sensitive type hydrogel vaccine PCV2-5, a thermo-sensitive type hydrogel vaccine PCV2-6, a thermo-sensitive type hydrogel vaccine PCV2-7, a thermo-sensitive type hydrogel vaccine PCV2-8 respectively; (9) is mice of an immunization group, injected with a control vaccine PCV2-1; and (10) is the mice of a blank control group , injected with normal saline.

DETAILED DESCRIPTION OF THE INVENTION The present invention will be explained detailedly with reference to embodiments, but the present invention not only includes the following embodiments, but also includes other embodiments based on the present invention. In the present invention, materials are as follows: Poloxamer 407 and poloxamer 188 are purchased from Sigma Company. Carbomer is purchased from Lubrizol Company. Soluplus, purchased from Shanghai BASF Company, is a graft copolymer of polyethylene caprolactam-polyvinyl acetate-polyethylene glycol, which is formed by a copolymerization of N-vinyl-epsilon-caprolactam, vinyl acetate, and polyethylene glycol (57: 30: 13); and a molecular weight ranges from 90,000 to 140,000 g/mol. The hydroxypropyl methyl cellulose is purchased from the sigma Company. Embodiment 1 Screening of thermo-sensitive type hydrogel adjuvants A thermo-sensitive type hydrogel adjuvant 1 consists of the following components in percentage by mass: Poloxamer 407 21.5%, Poloxamer 188 1.25%, Soluplus 2.5%, Carbomer 940 0.01%, Hydroxypropyl methyl cellulose (Item No. of sigma Company: H8384) 0.25%, the balance of water. A thermo-sensitive type hydrogel adjuvant 2 consists of the following components in percentage by mass: Poloxamer 407 17.78%,

Poloxamer 188 3.22%, Soluplus 7.8%, Carbomer 943 0.02%, Hydroxypropyl methyl cellulose (Item No. of sigma Company: 423238) 2.9%, the balance of water. A thermo-sensitive type hydrogel adjuvant 3 consists of the following components in percentage by mass: The poloxamer 407 28.18%, The poloxamer 188 1.18%, Soluplus 0.25%, Carbomer 971 0.1%, Hydroxypropyl methyl cellulose (Item No. of sigma Company: H9262) 0.20%, the balance of water. A thermo-sensitive type hydrogel adjuvant 4 consists of the following components in percentage by mass: The poloxamer 188 1.25%, Soluplus 12.5%, Carbomer 971 0.5%, Hydroxypropyl methyl cellulose (Item No. of sigma Company: H7509) 0.33%, the balance of water. A thermo-sensitive type hydrogel adjuvant 5 consists of the following components in percentage by mass: The poloxamer 407 17.5%, Soluplus 10.25%, Carbomer 940 0.2%, Hydroxypropyl methyl cellulose (Item No. of sigma Company: H8384) 2%, the balance of water. A thermo-sensitive type hydrogel adjuvant 6 consists of the following components in percentage by mass: The poloxamer 407 24.43%, The poloxamer 188 1.43%, Carbomer 1342 0.1% Hydroxypropyl methyl cellulose (Item No. of sigma Company: 423238) 4.36%, the balance of water. A thermo-sensitive type hydrogel adjuvant 7 consists of the following components in percentage by mass: The poloxamer 407 18.2%, The poloxamer 188 0.65%, Soluplus 6.3%, Hydroxypropyl methyl cellulose (Item No. of sigma Company: H8384) 1.2%, the balance of water. A thermo-sensitive type hydrogel adjuvant 8 consists of the following components in percentage by mass: The poloxamer 407 28.05%, The poloxamer 188 0.8%, Soluplus 1.2%, The Carbomer 943 0.1%, the balance of water. After being weighed based on the component and the content of each thermo-sensitive type hydrogel adjuvant, each substance was added into the water and stirred in an ice-water bath, and a mixture was stood at 4C for 12 h for swelling until a homogeneous system was formed; and then, after the system was filtered with a 0.22 pm filter membrane for sterilizing, the thermo-sensitive type hydrogel adjuvants 1-8 were obtained respectively.

Hydrogels prepared according to different formulas were put into a penicillin bottle through a turn technique (a thermo-sensitive gel substrate and a preparation method thereof and application thereof, with a patent number of ZL201610243442.8), the penicillin bottle was put into a water bath, and then, the water bath was heated from 30°C; and a temperature rise rate was controlled at 0.5°C/min, and time was maintained for 5 to 10 min at each temperature point. The penicillin bottle inclined at an angle of 60 degrees, gelation time of each sample at different temperatures was recorded based on a gelation decision criterion that each sample kept in a non-flowing state within 30s. Results showed that a system kept in the flowing state, with the above 8 thermo-sensitive type hydrogel adjuvants failing to be gelatinized at below 32°C in the system. When the temperature elevated to 34C, a viscosity of the sample was increased sharply, and a system liquidity declined dramatically, and the system was gelatinized gradually. The gelation time of each thermo-sensitive type hydrogel adjuvant at 36C to 40°C (a common body temperature range for humans or animals) was investigated, with results shown in Table 1. Table 1 showed that the thermo-sensitive type hydrogel adjuvants 1-8 can be gelatinized at 36C to 40°C, and there was a trend of gradually reducing the gelation time with temperature increasing. Table 1 Gelation time of different hydrogels at 36C to 40°C Group 36°C 36.5°C 37°C 37.5°C 38°C 38.5°C 39°C 39.5°C 40°C Formula 1 4'21 4'11 3'19 3'16 3'04 2'55 2'27 1'55 1'15 Formula 2 3'47 3'44 3'02 2'23 2'05 1'41 1'27 1'05 0'53 Formula 3 3'35 3'26 3'12 2'49 2'33 2'08 1'40 1'24 1'08 Formula 4 9'50 9'23 9'01 8'40 8'18 8'02 7'36 7'11 6'42 Formula 5 2'48 2'19 2'00 1'47 1'38 1'25 1'00 0'50 0'37 Formula 6 5'15 4'53 4'41 4'22 4'01 3'50 3'36 3'11 2'49 Formula 7 3'45 3'31 3'22 3'09 2'53 2'38 2'14 2'04 1'48

Formula 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 indicated 4 min 21 s, and so forth. Embodiment 2 Preparation, property evaluation, and application of thermo-sensitive type hydrogel vaccine (1) Preparation of porcine circovirus type 2 inactivated virus fluid The porcine circovirus type 2 inactivated virus fluid (a DBN-SX07 strain) is provided by Chengdu Tecbond Biotechnology Co., Ltd., and before inactivation, a virus content in the virus fluid per milliliter is 106TCID50. The inactivated virus fluid meets relevant requirements of Compilation of Quality Standards for Veterinary Biological Products (2011 ). (2) Preparation of porcine circovirus type 2 thermo-sensitive type hydrogel inactivated vaccine The thermo-sensitive type hydrogel adjuvants 1-8 prepared in the embodiment 1 were mixed with the porcine circovirus type 2 inactivated virus fluid (the DBN-SX07 strain) at a mass ratio of 3: 1 respectively and uniformly, and then, a thermo-sensitive type hydrogel vaccine PCV2-1, a thermo-sensitive type hydrogel vaccine PCV2-2, a thermo-sensitive type hydrogel vaccine PCV2-3, a thermo-sensitive type hydrogel vaccine PCV2-4, a thermo-sensitive type hydrogel vaccine PCV2-5, a thermo-sensitive type hydrogel vaccine PCV2-6, a thermo-sensitive type hydrogel vaccine PCV2-7, and a thermo-sensitive type hydrogel vaccine PCV2-8 were prepared. Numerical numbers of the vaccines kept in consistence with numeral numbers of the thermo-sensitive type hydrogel adjuvants. A preparation of a control vaccine PCV2-1: with reference to a patent for the invention ZL201610243442.8, 30 g of Soluplus was weighed, 50 mM of phosphate buffer was added until a total mass was 100 g, the Soluplus was dispersed at 4C, and a control thermo-sensitive type gel substrate was obtained. The control thermo-sensitive type gel substrate was mixed with the porcine circovirus type 2 inactivated virus fluid at the mass ratio of 3: 1 uniformly, and the control vaccine PCV2-1 was prepared.

(3) Testing of physical properties of vaccines The thermo-sensitive type hydrogel vaccines 1-8 and the control vaccine PCV2-1 were stored at 4C and 37C respectively, changes in appearances were observed, and a viscosity was tested by the rotary viscosimeter method for determination in an Appendix of the current Chinese Veterinary Pharmacopoeia, with results shown in Table 2. Table 2 Comparison of physical properties of vaccines

Vaccine Appearance Appearance Viscosity (cP, (4°C) (37°C) and 25°C) Thermo-sensitive type Transparent Semi-transparent 2.08±0.25 hydrogel vaccine PCV2-1 solution gel Thermo-sensitive type Transparent Semi-transparent 3.76±0.61 hydrogel vaccine PCV2-2 solution gel Thermo-sensitive type Transparent Semi-transparent 2.03±0.75 hydrogel vaccine PCV2-3 solution gel Thermo-sensitive type Transparent Semi-transparent 12.38±0.72 hydrogel vaccine PCV2-4 solution gel Thermo-sensitive type Transparent Semi-transparent 26.04±0.53 hydrogel vaccine PCV2-5 solution gel Thermo-sensitive type Transparent Semi-transparent 13.09±0.31 hydrogel vaccine PCV2-6 solution gel Thermo-sensitive type Transparent Semi-transparent 3.99±0.43 hydrogel vaccine PCV2-7 solution gel Thermo-sensitive type Transparent Semi-transparent 23.01±0.91 hydrogel vaccine PCV2-8 solution gel

Control vaccine PCV2-1 Transparent Semi-transparent 63.97±4.06 solution gel The viscosities of all vaccines for injection shall not go beyond 200 cP according to the requirements of Chinese Veterinary Pharmacopoeia. The vaccines prepared from the thermo-sensitive type hydrogel adjuvants of the present invention meet the requirements. Table 2 showed that the viscosities of the vaccines were significantly lower than that of the control vaccine PCV2-1, making the vaccines convenient to be applied as clinical injections. (4) Safety evaluation of thermo-sensitive type hydrogel vaccines in mice 100 Balb/c female mice (18 g to 22 g) were divided into 10 groups randomly, each of which included 10 mice. The thermo-sensitive type hydrogel vaccines and the control vaccine PCV2-1 were used for immunizing a group of mice respectively. An immune method was as follows: the mice were immunized subcutaneously at scruffs, each of which was immunized with 0.2 mL of vaccine, and meanwhile, a blank control group, which was only injected with normal saline, was set. After the immunization, the mice were observed for 14 consecutive days, including feed intake, water intake, mental state, changes in injection sites, and abnormal behaviors, and pathological changes in the mice of different test groups were evaluated through tissue slices. Results of the tissue slices were shown in Fig. 1. The results showed that the mice in the test group, injected with the control vaccine PCV2-1 had the constant licking of the injection sites, and other minor stress reactions after being injected, and there were cases with slightunhairing nearby the injection sites after 14 days. In addition, the results of the slices showed a mild inflammatory cell infiltration. The mice in other thermo-sensitive type hydrogel vaccine groups showed no obvious stress reactions, and were in the good mental state without changes in the abnormal behaviors; and after the injection, there was no any skin damage or unhairing at the injection sites in each group. The results of the slices showed no obvious inflammatory cells. (4) Evaluation of immune efficiency of thermo-sensitive type hydrogel vaccines Hundred 14-day-old piglets with negative porcine circovirus type 2 antigens and antibodies were selected, and the piglets were divided into 10 groups randomly, each of which included 10 piglets. The thermo-sensitive type hydrogel vaccines and the control vaccine PCV2-1 were used for immunizing a group of piglets respectively, and meanwhile, a blank control group was set for not immunization. An immune procedure was as follows: the 14-day-old piglets were immunized for the first time, and were immunized for the second time at intervals of 21 days, with an immunizing dose of 1.0 ml/piglet per time. Blood sampling and the separation of serum were conducted sequentially, and the antibodies were tested with an ELISA assay kit for the porcine circovirus type 2 antibodies (Wuhan Keqian Biological Products Co., Ltd.) within 1 month, 2 months, 3 months, 4 months, and 6 months after the last immunization. A valence of a PCV2 antibody of each piglet in the blank control group should be negative (not higher than 1:40); the valence of the PCV2 antibody of each immunized piglet, which was larger than or equal to 1:40, indicated antibody positive; and antibody positive rates were counted. Table 3 Comparison of immune efficiency of each vaccine Antibody positive rates at different time after the immunization (positive Group number/total number) 1 2 3 4 6 month month month month month Immunization group with thermo-sensitive type hydrogel 10/1oa 10/1oa 10/1oa 9 / 10 b 9 / 10 b vaccine PCV2-1 Immunization group with thermo-sensitive type hydrogel 10/1oa 10/1oa 10/1oa 10/1oa 10/1oa vaccine PCV2-2 Immunization group with thermo-sensitive type hydrogel 10/1oa 10/1oa 10/1oa 10/1oa 9 / 10 b vaccine PCV2-3 Immunization group with thermo-sensitive type hydrogel 9 /1 0 b 8/10c 7 / 10 d 5/10 4/ 10d vaccine PCV2-4 Immunization group with thermo-sensitive type hydrogel 10/1oa / 9 10 b 8/10c 6 /11 0 d 5/10c vaccine PCV2-5 Immunization group with thermo-sensitive type hydrogel 10/1oa 9 /10 b 9 / 10 b 7/10c 5/10C vaccine PCV2-6 Immunization group with thermo-sensitive type hydrogel 9 /10 b 8/10C 8/10C 7/10C 4 / 10 d vaccine PCV2-7 Immunization group with thermo-sensitive type hydrogel 9 /10 b 9 /10b 8/10C 7/10C 5/10C vaccine PCV2-8 Immunization group with control /1 0 b 8/10C 8/10C 9 7/10C 5/10C vaccine PCV2-1 Blank control group 0/10 0/10 0/10 0/10 0/10 Note: letters were not the same in the same column in Table 3, indicating that a difference was significant, that is, p<0.05. According to the requirements of Compilation of Quality Standards for Veterinary Biological Products (2010), if the antibody positive rates were larger than or equal to 80%, the vaccines were deemed to be accepted. Table 3 showed that a duration of immunity of the vaccines prepared from the thermo-sensitive type hydrogel adjuvants 1-3 could be up to 6 months, and the duration of immunity of other vaccines didn't exceed 3 months. Therefore, the immune efficiency of the vaccines prepared from the thermo-sensitive type hydrogel adjuvants 1-3 was significantly superior to that of the vaccines prepared from the thermo-sensitive type hydrogel adjuvants 4-8 and the control thermo-sensitive type gel substrate. The vaccines prepared from the thermo-sensitive type hydrogel adjuvants 1-3 met the requirements of immunization of commercial piglets against diseases completely, which had a good application value. (5) Safety evaluation of thermo-sensitive type hydrogel vaccines in piglets In the title (4) of the embodiment, the 14-day-old piglets were immunized for the first time, and then, a body temperature was monitored for 7 consecutive days, with results shown in Table 4. Table 4 showed that after the immunization with each thermo-sensitive type hydrogel vaccine, the body temperature of the piglets fell within a normal range from 39.0°C to 40.0°C, and no fever symptoms were found; while the body temperature of the piglets was higher than 40.0°C within 1 to 5 d after being immunized for the first time with the control vaccine PCV2-1. Meanwhile, after an observation of the injection sites of the piglets which were immunized for the first time with naked eyes, no any injury, ulcer, and other abnormal phenomena at the injection sites were found in the piglets injected with each thermo-sensitive type hydrogel vaccine; and of the piglets injected with the immune control vaccine PCV2-1, there were 2 piglets which presented bumps at the injection sites, and the bumps would disappear after about 7 days. After the second immunization, no adverse reactions which were visible to the naked eyes were found at the injection sites of the piglets in each group. The above results indicated that after the vaccines which were prepared from the thermo-sensitive type hydrogel adjuvants 1-8 were used for immunizing the piglets, the side reaction was very small, and the piglets were safe. Table 4 Average body temperature changes (C) of piglets in each group within

7 days after immunization

Day Day Day Day Day Day Day Day Group 0 1 2 3 4 5 6 7 Thermo-sensitivetype hydrogel vaccine 39.5 39.7 39.5 39.4 39.8 39.8 39.6 39.7 PCV2-1 Thermo-sensitive type hydrogel vaccine 39.7 39.7 39.3 39.4 39.3 39.5 39.6 39.6 PCV2-2 Thermo-sensitive type hydrogel vaccine 39.7 39.7 39.6 39.7 39.8 39.7 39.7 39.6 PCV2-3 Thermo-sensitive type hydrogel vaccine 39.5 39.5 39.5 39.7 39.5 39.5 39.4 39.5 PCV2-4 Thermo-sensitive type hydrogel vaccine 39.7 39.7 39.8 39.7 39.5 39.6 39.1 39.7 PCV2-5 Thermo-sensitive type hydrogel vaccine 39.6 39.5 39.5 39.6 39.7 39.7 39.6 39.9 PCV2-6 Thermo-sensitive type hydrogel vaccine 39.6 39.7 39.7 39.7 39.5 39.5 39.8 39.7 PCV2-7 Thermo-sensitive type hydrogel vaccine 39.6 39.5 39.5 39.7 39.7 39.6 39.6 39.4 PCV2-8 Control vaccine PCV2-1 39.8 40.1 40.1 40.4 40.3 40.1 39.7 39.7 Blank control group 39.8 39.5 39.7 39.7 39.6 39.4 39.7 39.4 Embodiment 3 Preparation and application of thermo-sensitive type hydrogel inactivated vaccines for porcine parvovirus disease (1) Porcine parvovirus (PPV BJ-2 strain) inactivated antigen A porcine circovirus (BJ-2 strain) inactivated virus fluid is provided by the Spirit Jinyu Biological Pharmaceutical Co., Ltd., and before inactivation, a content of viruses in the virus fluid per milliliter is 106TCID50. The porcine parvovirus inactivated virus fluid meets relevant requirements of Compilation of Quality Standards for Veterinary Biological Products (2012). (2) Preparation of thermo-sensitive type hydrogel inactivated vaccines for porcine parvovirus disease The thermo-sensitive type hydrogel adjuvants 1-8 in the embodiment 1 were mixed with the porcine circovirus (BJ-2 strain) inactivated virus fluid respectively and uniformly at a mass ratio of 3: 1, and then, a thermo-sensitive type hydrogel vaccine PPV-1, a thermo-sensitive type hydrogel vaccine PPV-2, a thermo-sensitive type hydrogel vaccine PPV-3, a thermo-sensitive type hydrogel vaccine PPV-4, a thermo-sensitive type hydrogel vaccine PPV-5, a thermo-sensitive type hydrogel vaccine PPV-6, a thermo-sensitive type hydrogel vaccine PPV-7, and a thermo-sensitive type hydrogel vaccine PPV-8 were obtained. Serial numbers of the vaccines kept in consistence with serial numbers of the thermo-sensitive type hydrogel adjuvants. A preparation of a control vaccine PPV-1: with reference to a patent for the present invention ZL201610243442.8, 30 g of Soluplus was weighed, 50 mM of phosphate buffer was added until a total mass was 100 g, the Soluplus was dispersed at 4C, and a control thermo-sensitive type gel substrate was obtained. The control thermo-sensitive type gel substrate was mixed with the porcine circovirus (BJ-2 strain) inactivated virus fluid uniformly at the mass ratio of 3: 1, and the control vaccine PPV-1 was obtained. (3) Testing of physical properties of vaccines The thermo-sensitive type hydrogel vaccines PPV-1 to PPV-8 and the control vaccine PPV-1 in the title (2) of the embodiment were stored at 4C and 37°C respectively, changes in appearances were observed, and a viscosity was tested by the rotary viscosimeter method for determination in an Appendix of the current Chinese Veterinary Pharmacopoeia, with results shown in Table 5. Table 5 Comparison of physical properties of vaccines for porcine parvovirus disease

Vaccine Appearance Appearance Viscosity (cP, and (4°C) (37°C) 25°C) Thermo-sensitive type Transparent Semi-transparent 3.11±0.33 hydrogel vaccine PPV-1 solution gel Thermo-sensitive type Transparent Semi-transparent 4.32±0.11 hydrogel vaccine PPV-2 solution gel Thermo-sensitive type Transparent Semi-transparent 3.03±0.15 hydrogel vaccine PPV-3 solution gel Thermo-sensitive type Transparent Semi-transparent 14.38±0.27 hydrogel vaccine PPV-4 solution gel Thermo-sensitive type Transparent Semi-transparent 27.04±0.93 hydrogel vaccine PPV-5 solution gel Thermo-sensitive type Transparent Semi-transparent 15.91±0.23 hydrogel vaccine PPV-6 solution gel Thermo-sensitive type Transparent Semi-transparent 5.32±0.19 hydrogel vaccine PPV-7 solution gel Thermo-sensitive type Transparent Semi-transparent 33.27±0.21 hydrogel vaccine PPV-8 solution gel

Control vaccine PPV-1 Transparent Semi-transparent 78.79±5.78 solution gel Table 5 showed that the viscosities prepared from the thermo-sensitive type hydrogel adjuvants 1-8 were significantly lower than 200 cP, which met the requirements on the vaccine viscosity in Chinese Veterinary Pharmacopoeia; and the viscosities were significantly lower than that of the control vaccine PPV-1, and thus, a labor intensity was lessened, which makes the vaccines more convenient in a clinical injection. (4) Safety evaluation The safety evaluations of piglets and suckling mice were conducted respectively with reference to the relevant requirements of Compilation of Quality Standards for Veterinary Biological Products (2012). The safety evaluation of the piglets: 4-to-6-week-old healthy susceptible piglets (a valence of a hemagglutination inhibiting antibody (PPV HI) of porcine parvovirus was not higher than 1: 8) got vaccinated respectively by the thermo-sensitive type hydrogel vaccines PPV-1 to PPV-8 and the control vaccine PPV-1, each vaccine was used for immunizing 2 piglets, and each piglet was injected with 4 mL of vaccine intramuscularly at a neck, and then, was observed for consecutive 21 days. Results: no local or systemic adverse reactions arising from the vaccines were found in each group. The safety evaluation of the suckling mice: 3-to-5-day-old suckling mice got vaccinated respectively by the thermo-sensitive type hydrogel vaccines PPV-1 to PPV-8 and the control vaccine PPV-1, each vaccine was used for immunizing 5 suckling mice, and each suckling mouse was injected with 0.1 mL of vaccine subcutaneously, and then, was observed for consecutive 10 days. Results: each group was healthy and lively, and no local or systemic adverse reactions arising from the vaccines were found in each group. (5) Evaluation of immune efficiency Healthy guinea pigs with the body weight ranged from 350 g to 400 g (the valence of the hemagglutination inhibiting antibody (PPV HI) of the porcine parvovirus was not higher than 1: 8) got vaccinated respectively by the thermo-sensitive type hydrogel vaccines PPV-1 to PPV-8 and the control vaccine PPV-1, each vaccine was used for immunizing 5 guinea pigs, and each guinea pig was injected with 0.5 mL of vaccine intramuscularly. There were 5 guinea pigs in the blank control group, which were not immunized. Blood sampling and the separation of serum were conducted on the guinea pigs of each group sequentially, and the valence of the hemagglutination inhibiting antibody of the porcine parvovirus was tested (a testing method is based on the requirements of the Appendix of the Chinese Veterinary Pharmacopoeia) within 28 days, 2 months, 3 months, 4 months, 5 months, and 6 months after the immunization. Table 6 Comparison of immune efficiency of each vaccine Mean value (log2) of PPV HI at different time after immunization Group Day 2 3 4 5 6 28 month month month month month Immunization group with thermo-sensitive type 11.1 11.3 12.2 11.8 11.4 10.8 hydrogel vaccine PPV-1

Immunization group with thermo-sensitive type 11.0 11.2 11.8 11.0 10.5 10.2 hydrogel vaccine PPV-2 Immunization group with thermo-sensitive type 11.0 11.2 11.7 11.5 11.3 11.0 hydrogel vaccine PPV-3 Immunization group with thermo-sensitive type 9.4 8.6 7.9 7.0 5.9 5.1 hydrogel vaccine PPV-4 Immunization group with thermo-sensitive type 9.2 8.2 7.7 6.6 5.3 4.7 hydrogel vaccine PPV-5 Immunization group with thermo-sensitive type 9.0 8.8 8.6 7.4 5.9 5.1 hydrogel vaccine PPV-6 Immunization group with thermo-sensitive type 9.0 8.2 7.7 7.4 5.7 5.2 hydrogel vaccine PPV-7 Immunization group with thermo-sensitive type 8.8 8.6 7.9 7.6 5.7 5.0 hydrogel vaccine PPV-8 Immunization group with 9.0 9.0 8.8 7.6 5.4 5.0 control vaccine PPV- 1 Blank control group 2.0 2.0 2.0 2.0 2.0 2.0

According to the requirements of Compilation of Quality Standards for Veterinary Biological Products (2012), the at least 4 immunized guinea pigs should show the antibody positive, and the valence should not be lower than 1: 64 (PPV HI 6, and log2); and the valences of the hemagglutination inhibiting antibodies of the control guinea pigs should not be higher than 1: 8 (PPV Hl3, and log2), indicating that the vaccine was deemed to be accepted. Table 6 showed that the duration of immunity of the guinea pigs immunized by the vaccines prepared from the thermo-sensitive type hydrogel adjuvants 1-3 could be up to more than 6 months; and after the immunization with the vaccines prepared from the thermo-sensitive type hydrogel adjuvants 4-8 and the control thermo-sensitive type gel substrate, a level of the hemagglutination inhibiting antibody was reduced significantly within 5 months after immunization, which was lower than 1: 64 (PPV HI 6, and log2), the duration of immunity was only 4 months, and the valence of the antibody was significantly lower than that of the antibody after the immunization with the vaccines prepared from the thermo-sensitive type hydrogel adjuvants 1-3.

Therefore, the immune efficiency of the vaccines prepared from the thermo-sensitive type hydrogel adjuvants 1-3 was significantly superior to that of the vaccines prepared from the thermo-sensitive type hydrogel adjuvants 4-8 and the control thermo-sensitive type gel substrate. The vaccines prepared from the thermo-sensitive type hydrogel adjuvants 1-3 complied with the quality standards for veterinary biological products completely, and met the requirements of the immunization of commercial piglets against diseasescompletely, which had a good application value.

Claims

CLAIMS:

1. A thermo-sensitive type hydrogel adjuvant for a veterinary vaccine, wherein, the thermo-sensitive type hydrogel adjuvant consists of the following components in percentage by mass: The poloxamer 15%-35%, Soluplus 0.1-10%, The Carbomer 0.01%-0.2%, The hydroxypropyl methyl cellulose 0.1%-5%, the balance of water.

2. The thermo-sensitive type hydrogel adjuvant according to claim 1, wherein, the poloxamer is poloxamer 407 and poloxamer 188, wherein a content of the poloxamer 407 is 14% to 30% in percentage by mass, and a content of the poloxamer 188 is 1% to 5% in percentage by mass.

3. A preparation method of the thermo-sensitive type hydrogel adjuvant according to claim 1, wherein, the methodcomprises steps: weighing each component, mixing, swelling at 2C to 10°C to form a homogeneous system, and obtaining the thermo-sensitive type hydrogel adjuvant.

4. The preparation method according to claim 3, wherein, the mixing is conducted at 0°C to 10°C.

5. A vaccine containing the thermo-sensitive type hydrogel adjuvantaccording to claim 1 or 2.

6. The vaccine according to claim 5, wherein,, the vaccine includes the thermo-sensitive type hydrogel adjuvant and an aqueous solution of an antigen, where a mass ratio of the thermo-sensitive type hydrogel adjuvant to the aqueous solution of the antigen is 0.5-4: 1.

7. The vaccine according to claim 6, wherein, the antigen is a porcine circovirus virus or porcine parvovirus.

(1) 2)

(5) (6 (7) 8)

(9) (10)

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