Gel particle water adjuvant special for PRRS (porcine reproductive and respiratory syndrome) inactivated vaccine as well as preparation method and application of gel particle water adjuvant

Abstract

The invention relates to the technical field of biological medicine. The invention provides a special gel particle water adjuvant for a PRRS (porcine reproductive and respiratory syndrome) inactivated vaccine as well as a preparation method and application of the special gel particle water adjuvant. The gel particle water adjuvant comprises a conventional water adjuvant and special gel particles. According to the special gel particle water adjuvant for the PRRS inactivated vaccine, the porcine reproductive and respiratory syndrome virus antigen can exist in the form of gel particles at an injection part, degradation is delayed, long-range release is achieved, the existence time of the antigen in tissues is prolonged, the antigen can continuously act on the body, an effective neutralizing antibody is generated, and the immune response of the porcine reproductive and respiratory syndrome virus antigen is improved. The problems that the action time of a conventional PRRS inactivated vaccine and an organism is short, and any antibody cannot be generated are solved, and meanwhile, obvious vaccine adverse reaction is not caused.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a gel particle water adjuvant special for PRRS inactivated seedlings, and a preparation method and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an acute, highly contagious viral infectious disease in pigs characterized by reproductive disorders and respiratory symptoms. PRRS is a special pathogen, and the production of its antibodies depends on a long period of contact and reaction between the antigen and the animal body. If effective neutralizing antibodies are to be produced, it takes longer, which is much longer than viruses such as swine fever and foot-and-mouth disease. After immunization with conventional inactivated vaccines of porcine reproductive and respiratory syndrome, almost no antibodies of any form are produced. Therefore, the inactivated vaccine of porcine reproductive and respiratory syndrome cannot imitate the adjuvant method of inactivated vaccines such as foot-and-mouth disease, and a special adjuvant must be prepared. Under natural infection conditions, the production of effective neutralizing antibodies against the pathogen generally requires continuous infection for about 3 weeks. Therefore, for a long period of time, the immunity of the disease depends on attenuated live vaccines, which produce a certain degree of protection through long-term proliferation infection. Some live vaccine strains have poor infectivity and it is difficult to produce uniform and effective protection. The problem with inactivated vaccines is even more obvious. The effect of using ordinary adjuvants to delay absorption and degradation is limited. After being injected into the body, the antigen is quickly degraded and disabled, which seriously inhibits the effectiveness of the PRRS inactivated vaccine. It can only be compensated by repeated immunizations within a short period of time (1 to 2 weeks), but it is still difficult to achieve the desired effect.

[0003] At present, the adjuvant of inactivated vaccine mainly adopts oil adjuvant, but simply increasing the amount of oil adjuvant to prolong the retention time of antigen in tissue will cause serious local lesions and immune stress. If the immune stress is to be reduced, the proportion of oil adjuvant needs to be reduced. This will lead to accelerated absorption and decomposition of antigen, and ultimately make it difficult for inactivated vaccine to take effect. Therefore, it is necessary to provide a new type of vaccine adjuvant to solve the above problems. Summary of the invention

[0004] The purpose of the present invention is to provide a special gel particle water adjuvant for PRRS inactivated vaccine and its preparation method and application, so that the PRRS antigen in the vaccine can exist in the tissue for a longer time, continue to act on the body, produce effective neutralizing antibodies, and at the same time do not cause obvious adverse reactions to the vaccine.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a gel particle water adjuvant specially used for PRRS inactivated seedlings. The gel particle water adjuvant comprises a conventional water adjuvant and special gel particles.

[0007] Preferably, the conventional water adjuvant is an aluminum gel adjuvant;

[0008] The special gel particles include any one or more of agar particles, agarose particles or dextran particles, and high molecular weight chitosan particles. The diameter of the special gel particles is 1 to 4 mm, and the special gel particles are in a thin gel state.

[0009] Preferably, the gel particle aqueous adjuvant also includes one or more of a polymer, a surfactant, an immunostimulant and an adjuvant enhancing component.

[0010] The invention also provides application of the gel particle water adjuvant in preparing inactivated seedlings.

[0011] Preferably, the type of inactivated vaccine is porcine reproductive and respiratory syndrome inactivated vaccine.

[0012] The present invention also provides an inactivated porcine reproductive and respiratory syndrome vaccine, which comprises the gel particle water adjuvant and inactivated porcine reproductive and respiratory syndrome virus liquid.

[0013] The present invention also provides a method for preparing the porcine reproductive and respiratory syndrome inactivated vaccine, comprising the following steps:

[0014] (1) mixing the inactivated porcine reproductive and respiratory syndrome virus liquid with the gel granules to obtain a mixed solution, and allowing the mixed solution to stand for 50 to 70 minutes;

[0015] (2) The mixture after standing is mixed with a conventional water adjuvant to obtain a porcine reproductive and respiratory syndrome inactivated vaccine.

[0016] Preferably, the titer of the virus in the inactivated porcine reproductive and respiratory syndrome virus solution in step (1) is 1×10 6.0 ~1×10 7.6 TCID 50 / mL, and the mass ratio of the inactivated porcine reproductive and respiratory syndrome virus liquid to the gel granules is 80-120:0.01-0.3.

[0017] Preferably, the volume ratio of the inactivated porcine reproductive and respiratory syndrome virus liquid to the conventional water adjuvant is 80-120:0.3-3.

[0018] Preferably, one or more of polymers, surfactants, immunostimulants and adjuvant enhancing components may be added during the mixing in step (2).

[0019] The present invention provides a gel particle water adjuvant dedicated to a PRRS inactivated vaccine, and a preparation method and application thereof, wherein the gel particle water adjuvant comprises a conventional water adjuvant and special gel particles. The gel particle water adjuvant dedicated to a PRRS inactivated vaccine of the present invention can make porcine reproductive and respiratory syndrome virus antigens exist in the form of gel particles at the injection site, delay degradation, release over a long period of time, prolong the existence time of the antigen in the tissue, enable the antigen to continuously act on the body, and produce effective neutralizing antibodies, thereby avoiding the problem that conventional PRRS inactivated vaccines have a short action time on the body and cannot produce any antibodies, and at the same time do not bring about obvious vaccine adverse reactions. DETAILED DESCRIPTION

[0020] The invention provides a gel particle water adjuvant specially used for PRRS inactivated seedlings. The gel particle water adjuvant comprises a conventional water adjuvant and special gel particles.

[0021] In the present invention, the conventional water adjuvant is preferably an aluminum gel adjuvant;

[0022] The special gel particles preferably include any one or more of agar particles, agarose particles or dextran particles, and high molecular weight chitosan particles. The diameter of the special gel particles is preferably 1 to 4 mm, and more preferably 2 to 3 mm. The special gel particles are preferably in a thin gel state.

[0023] In the present invention, the gel particle aqueous adjuvant preferably further comprises one or more of a polymer, a surfactant, an immunostimulant and an adjuvant enhancing component.

[0024] The invention also provides application of the gel particle water adjuvant in preparing inactivated seedlings.

[0025] In the present invention, the type of inactivated vaccine is porcine reproductive and respiratory syndrome inactivated vaccine.

[0026] The present invention also provides an inactivated porcine reproductive and respiratory syndrome vaccine, which comprises the gel particle water adjuvant and inactivated porcine reproductive and respiratory syndrome virus liquid.

[0027] The present invention also provides a method for preparing the porcine reproductive and respiratory syndrome inactivated vaccine, comprising the following steps:

[0028] (1) mixing the inactivated porcine reproductive and respiratory syndrome virus liquid with the gel granules to obtain a mixed solution, and allowing the mixed solution to stand for 50 to 70 minutes;

[0029] (2) The mixture after standing is mixed with a conventional water adjuvant to obtain a porcine reproductive and respiratory syndrome inactivated vaccine.

[0030] In the present invention, the standing time in step (1) is preferably 60 minutes.

[0031] In the present invention, the titer of the virus in the inactivated porcine reproductive and respiratory syndrome virus solution in step (1) is preferably 1×10 6.0 ~1×10 7.6 TCID 50 / mL, more preferably 1×10 7 TCID 50 / mL, the mass ratio of the inactivated porcine reproductive and respiratory syndrome virus liquid to the gel granules is preferably 80-120:0.01-0.3, and more preferably 100:0.1-0.2.

[0032] In the present invention, the volume ratio of the inactivated porcine reproductive and respiratory syndrome virus solution to the conventional water adjuvant is preferably 80-120:0.3-3, and more preferably 100:1-2.

[0033] In the present invention, during the mixing in step (2), preferably one or more of a polymer, a surfactant, an immunostimulant and an adjuvant enhancing component may be added.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] A method for preparing an inactivated vaccine of porcine reproductive and respiratory syndrome comprises the following steps:

[0037] (1) The porcine reproductive and respiratory syndrome virus liquid was inactivated to obtain a virus titer of 1×10 6.0 TCID 50 / mL of inactivated porcine reproductive and respiratory syndrome virus solution;

[0038] (2) mixing the inactivated porcine reproductive and respiratory syndrome virus solution with agarose particles having a diameter of 1 mm to obtain a mixed solution, and letting it stand for 70 minutes;

[0039] (3) The mixture after standing is mixed with aluminum gel adjuvant to obtain porcine reproductive and respiratory syndrome inactivated vaccine.

[0040] Note: In this example, the mass ratio of the inactivated porcine reproductive and respiratory syndrome virus solution to the agarose particles is 80:0.01;

[0041] The volume ratio of inactivated porcine reproductive and respiratory syndrome virus liquid to aluminum gel adjuvant is 80:2.

[0042] Example 2

[0043] A method for preparing an inactivated vaccine of porcine reproductive and respiratory syndrome comprises the following steps:

[0044] (1) The porcine reproductive and respiratory syndrome virus liquid was inactivated to obtain a virus titer of 1×10 7.6 TCID 50 / mL of inactivated porcine reproductive and respiratory syndrome virus solution;

[0045] (2) mixing the inactivated porcine reproductive and respiratory syndrome virus solution with agar particles having a diameter of 2 mm to obtain a mixed solution, and letting it stand for 50 minutes;

[0046] (3) The mixture after standing is mixed with aluminum gel adjuvant to obtain porcine reproductive and respiratory syndrome inactivated vaccine.

[0047] Note: In this example, the mass ratio of the inactivated porcine reproductive and respiratory syndrome virus solution to the agar particles is 120:0.1;

[0048] The volume ratio of inactivated porcine reproductive and respiratory syndrome virus liquid to aluminum gel adjuvant is 120:1.

[0049] Example 3

[0050] A method for preparing an inactivated vaccine of porcine reproductive and respiratory syndrome comprises the following steps:

[0051] (1) The porcine reproductive and respiratory syndrome virus liquid was inactivated to obtain a virus titer of 1×10 7 TCID 50 / mL of inactivated porcine reproductive and respiratory syndrome virus solution;

[0052] (2) mixing the inactivated porcine reproductive and respiratory syndrome virus solution with agar particles having a diameter of 3 mm to obtain a mixed solution, and letting it stand for 60 minutes;

[0053] (3) The mixture after standing is mixed with aluminum gel adjuvant to obtain porcine reproductive and respiratory syndrome inactivated vaccine.

[0054] Note: In this example, the mass ratio of the inactivated porcine reproductive and respiratory syndrome virus solution to the agar particles is 100:0.2;

[0055] The volume ratio of inactivated porcine reproductive and respiratory syndrome virus liquid to aluminum gel adjuvant is 100:1.

[0056] Test example: immune efficacy test

[0057] 60 14-day-old piglets were randomly divided into 5 groups, 12 in each group, numbered as 1-3 in the experiment, control group and blank control group. Among them, the experimental groups 1-3 were injected with the inactivated porcine reproductive and respiratory syndrome vaccine prepared in Examples 1-3 respectively; the control group was injected with inactivated PRRS virus solution, and the blank control group was injected with normal saline; the injection site was the piglet neck muscle, and the injection volume of each group was 1mL. The piglets were warmed up before use and turned upside down several times. The average body temperature of the piglets in each group was recorded before injection and 12h, 24h, and 36h after injection, and the results are shown in Table 1.

[0058] Table 1 Body temperature of piglets in each group before and after injection (℃)

[0059] Before injection Injection 12h Injection 24h Injection 36h Example 1 39.2 39.3 39.4 39.2 Example 2 39.1 39.5 39.5 39.3 Example 3 39.0 93.5 93.5 3.94 Inactivation of viruses 39.1 39.3 39.3 39.1 Normal saline 39.0 39.1 39.0 39.1

[0060] It can be seen from Table 1 that after the piglets were injected with the porcine reproductive and respiratory syndrome inactivated vaccine prepared by the present invention for immunization, the piglets had a certain stress, but the magnitude was not large, and they were basically close to normal 36 hours after the injection, indicating that the porcine reproductive and respiratory syndrome inactivated vaccine prepared by the present invention has good safety.

[0061] 21 days after the first immunization (i.e., the first injection), the piglets in each treatment group were randomly divided into two groups, each with 6 piglets, and one group of piglets was boosted once, and the injection method was the same as the first injection. At 4 weeks after the first immunization and 4 weeks after the second immunization, the neutralizing antibody titers of the first immunization and booster immunization of each group of piglets were counted, respectively, among which, the piglets injected with the inactivated vaccine prepared in Examples 1 to 3 were neutralized using the CH-1a strain. The results are shown in Table 2.

[0062] Table 2 Statistical results of neutralizing antibody titers in each group of piglets after immunization

[0063] One immunization group Booster group Example 1 <![CDATA[1:2 3.3 ]]> <![CDATA[1:2 4.8 ]]> Example 2 <![CDATA[1:2 3.6 ]]> <![CDATA[1:2 4.8 ]]> Example 3 <![CDATA[1:2 3.6 ]]> <![CDATA[1:2 5.3 ]]> Inactivation of viruses 0 0 Normal saline / /

[0064] As shown in Table 2, the titer of the neutralizing antibody in piglets was 1:2 after 4 weeks of injection of the porcine reproductive and respiratory syndrome inactivated vaccine prepared by the present invention. 3.6 Around 1:2, neutralizing antibodies can be produced and have protective effect. Four weeks after booster immunization, the titer of neutralizing antibodies in piglets is 1:2 5 This indicates that the porcine reproductive and respiratory syndrome inactivated vaccine prepared by the present invention can significantly improve the immune efficacy.

[0065] As can be seen from the above embodiments, the present invention provides a gel particle water adjuvant dedicated to PRRS inactivated vaccine, and a preparation method and application thereof, wherein the gel particle water adjuvant includes conventional water adjuvant and special gel particles. The gel particle water adjuvant dedicated to PRRS inactivated vaccine of the present invention can make porcine reproductive and respiratory syndrome virus antigen exist in the form of gel particles at the injection site, delay degradation, release over a long period of time, prolong the existence time of antigen in tissue, enable antigen to continue to act with the body, produce effective neutralizing antibodies, avoid the problem that conventional PRRS inactivated vaccine has a short action time with the body and cannot produce any antibodies, and at the same time does not bring obvious vaccine adverse reactions.

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gel granule water adjuvant specially used for PRRS inactivated seedlings, characterized in that: The gel particle water adjuvant includes conventional water adjuvant and special gel particles.

2. The gel particle aqueous adjuvant according to claim 1, characterized in that: The conventional water adjuvant is aluminum gel adjuvant; The special gel particles include any one or more of agar particles, agarose particles or dextran particles, and high molecular weight chitosan particles. The diameter of the special gel particles is 1 to 4 mm, and the special gel particles are in a thin gel state.

3. The gel particle aqueous adjuvant according to any one of claims 1 to 2, characterized in that: The gel particle water adjuvant also includes one or more of a polymer, a surfactant, an immunostimulant and an adjuvant enhancing component.

4. Use of the gel particle aqueous adjuvant according to any one of claims 1 to 3 in the preparation of inactivated vaccine.

5. The use according to claim 4, characterized in that: The type of inactivated vaccine is porcine reproductive and respiratory syndrome inactivated vaccine.

6. An inactivated vaccine of porcine reproductive and respiratory syndrome, characterized in that: The porcine reproductive and respiratory syndrome inactivated vaccine comprises the gel particle water adjuvant according to any one of claims 1 to 3 and an inactivated porcine reproductive and respiratory syndrome virus liquid.

7. The method for preparing the porcine reproductive and respiratory syndrome inactivated vaccine according to claim 6, characterized in that: The steps include: (1) mixing the inactivated porcine reproductive and respiratory syndrome virus liquid with the gel granules to obtain a mixed solution, and allowing the mixed solution to stand for 50 to 70 minutes; (2) The mixture after standing is mixed with a conventional water adjuvant to obtain a porcine reproductive and respiratory syndrome inactivated vaccine.

8. The preparation method according to claim 7, characterized in that: The titer of the virus in the inactivated porcine reproductive and respiratory syndrome virus solution in step (1) is 1×10 6.0 ~1×10 7.6 TCID 50 / mL, and the mass ratio of the inactivated porcine reproductive and respiratory syndrome virus liquid to the gel granules is 80-120:0.01-0.

3.

9. The preparation method according to claim 8, characterized in that: The volume ratio of the inactivated porcine reproductive and respiratory syndrome virus liquid to the conventional water adjuvant is 80-120:0.3-3.

10. The preparation method according to claim 8, characterized in that: During the mixing in step (2), one or more of polymers, surfactants, immunostimulants and adjuvant enhancing components may be added.