Special oil adjuvant for inactivated vaccine of porcine reproductive and respiratory syndrome as well as preparation method and application of special oil adjuvant

Abstract

The invention provides a special oil adjuvant for a porcine reproductive and respiratory syndrome inactivated vaccine as well as a preparation method and application of the special oil adjuvant, and belongs to the technical field of biological medicines. The special oil adjuvant for the porcine reproductive and respiratory syndrome inactivated vaccine comprises a mineral oil adjuvant and gel granules. According to the porcine reproductive and respiratory syndrome inactivated vaccine prepared by the invention, the porcine reproductive and respiratory syndrome virus antigen can exist in the form of gel particles at an injection part, so that the required immunizing dose and injection dose are obviously reduced, the existence time of the antigen in tissues is prolonged, the antigen can continuously act on the body, and the immunity of the human body is improved. The problem that a conventional porcine reproductive and respiratory syndrome inactivated vaccine cannot stimulate a body to form an antibody is solved, and a high-titer neutralizing antibody can be generated. Meanwhile, the oil adjuvant special for the porcine reproductive and respiratory syndrome inactivated vaccine, prepared by the invention, also has good stability and safety.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine, 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 is mainly oil adjuvant, which can prevent the absorption and degradation of antigen under appropriate conditions. However, it is not feasible to prolong the retention time of antigen in tissue simply by increasing the dosage of oil adjuvant, which will cause serious local lesions and immune stress. Therefore, when producing PRRS inactivated vaccine, various measures are taken, such as reducing the proportion of oil adjuvant to avoid excessive immune stress. However, this has accelerated the absorption and decomposition of antigen and greatly shortened the action time of the body, resulting in ineffective immunization of inactivated vaccine in many cases.

[0004] Therefore, it is necessary to provide a special oil adjuvant for PRRS inactivated vaccine that can make the PRRS antigen in the vaccine exist in the tissue for a longer time and continue to act on the body without causing obvious adverse reactions to the vaccine, so as to provide new ideas for the control of PRRS. Summary of the invention

[0005] The purpose of the present invention is to provide a special oil adjuvant for porcine reproductive and respiratory syndrome 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.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine, which comprises a mineral oil adjuvant and gel granules.

[0008] Preferably, the mineral oil is white oil; and the gel particles are substances capable of forming porous gels.

[0009] Preferably, the gel particles include any one or more of agar particles, dextran particles, agarose particles, and high molecular weight chitosan particles, and the particle size of the gel particles is 0.5 to 3 mm.

[0010] Preferably, the special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine also includes a non-ionic surfactant and / or an adjuvant enhancing component.

[0011] The invention provides application of the special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine in preparing the porcine reproductive and respiratory syndrome inactivated vaccine.

[0012] The present invention provides a method for preparing an inactivated porcine reproductive and respiratory syndrome vaccine by using the special oil adjuvant for the inactivated porcine reproductive and respiratory syndrome vaccine, comprising the following steps:

[0013] (1) mixing an inactivated porcine reproductive and respiratory syndrome virus solution with gel granules to obtain a mixed solution;

[0014] (2) The mixed solution obtained in step (1) is mixed with a mineral oil adjuvant to obtain an inactivated vaccine of porcine reproductive and respiratory syndrome.

[0015] 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 solution and the gel granules when mixed is 80-120:0.01-0.3.

[0016] Preferably, the mass ratio of the mineral oil adjuvant to the inactivated porcine reproductive and respiratory syndrome virus solution is 12-60:80-120.

[0017] Preferably, a nonionic surfactant and / or adjuvant is added during the mixing in step (2) to enhance the ingredients, and the mixing method is self-emulsifying mixing, the temperature of the self-emulsifying mixing is 35-39° C., the speed is 400-800 rpm, and the time is 15-25 min.

[0018] The invention provides an inactivated porcine reproductive and respiratory syndrome vaccine prepared according to the method.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention provides a special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine, wherein the special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine includes a mineral oil adjuvant and a gel granule. When the porcine reproductive and respiratory syndrome inactivated vaccine is prepared, the gel granule is first mixed with an inactivated porcine reproductive and respiratory syndrome virus solution and allowed to stand, and the porous adsorption property of the gel granule is utilized to fully adsorb the inactivated porcine reproductive and respiratory syndrome virus solution and the gel granule, and then the mineral oil adjuvant is added to prepare the porcine reproductive and respiratory syndrome inactivated vaccine. The porcine reproductive and respiratory syndrome inactivated vaccine prepared by the invention can make the porcine reproductive and respiratory syndrome virus antigen exist in the form of gel particles at the injection site, significantly reduce the required immunization dose and injection dose, prolong the existence time of the antigen in the tissue, enable the antigen to continue to act on the body, avoid the problem that the conventional porcine reproductive and respiratory syndrome inactivated vaccine cannot stimulate the body to form antibodies, and can produce high-titer neutralizing antibodies. At the same time, the special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine prepared by the invention also has good stability and safety. DETAILED DESCRIPTION

[0021] The invention provides a special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine, which comprises a mineral oil adjuvant and gel granules.

[0022] In the present invention, the mineral oil is white oil; and the gel particles are substances that can form porous gel.

[0023] In the present invention, the gel particles include any one or more of agar particles, dextran particles, agarose particles, and high molecular weight chitosan particles, preferably agarose particles; the particle size of the gel particles is 0.5 to 3 mm.

[0024] In the present invention, the special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine also includes a non-ionic surfactant and / or an adjuvant enhancing component.

[0025] In the present invention, the nonionic surfactant includes Span-80 and Tween-80, and the mass ratio of Span-80 to Tween-80 is 1-3:1-3, preferably 1-2:1-2, and more preferably 1:1.

[0026] In the present invention, the adjuvant enhancing component is a substance having an enhanced immunostimulatory effect, including imidazole compounds and polymers.

[0027] The invention provides application of the special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine in preparing the porcine reproductive and respiratory syndrome inactivated vaccine.

[0028] The present invention provides a method for preparing an inactivated porcine reproductive and respiratory syndrome vaccine by using the special oil adjuvant for the inactivated porcine reproductive and respiratory syndrome vaccine, comprising the following steps:

[0029] (1) mixing an inactivated porcine reproductive and respiratory syndrome virus solution with gel granules to obtain a mixed solution;

[0030] (2) The mixed solution obtained in step (1) is mixed with a mineral oil adjuvant to obtain an inactivated vaccine of porcine reproductive and respiratory syndrome.

[0031] In the present invention, 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, preferably 5×10 6.6 ~5×10 7.6 TCID 50 / mL, more preferably 1×10 7.2 TCID 50 / mL, the mass ratio of the inactivated porcine reproductive and respiratory syndrome virus solution and the gel granules when mixed is 80-120:0.01-0.3, preferably 90-110:0.1-0.3, and more preferably 100:0.2.

[0032] In the present invention, the mass ratio of the mineral oil adjuvant to the inactivated porcine reproductive and respiratory syndrome virus solution is 12-60:80-120, preferably 18-30:90-110, and more preferably 20:100.

[0033] In the present invention, a nonionic surfactant and / or an adjuvant is further added to enhance the ingredients during the mixing in step (2), and the mixing method is self-emulsifying mixing. The temperature of the self-emulsifying mixing is 35-39°C, preferably 36-38°C, and more preferably 37°C. The speed is 400-800rpm, preferably 500-700rpm, and more preferably 600rpm. The time is 15-25min, preferably 18-22min, and more preferably 20min.

[0034] In the present invention, the mass ratio of the nonionic surfactant to the porcine reproductive and respiratory syndrome virus antigen solution is 2-6:80-120, preferably 3-5:90-110, and more preferably 4:100.

[0035] The invention provides an inactivated porcine reproductive and respiratory syndrome vaccine prepared according to the method.

[0036] The technical solution provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention. The particle size of the gel particles used in the following examples is 0.5 to 3 mm.

[0037] Example 1

[0038] A method for preparing an inactivated vaccine of porcine reproductive and respiratory syndrome, comprising the following steps:

[0039] (1) Prepare the PRRS virus CH-1a strain to a virus titer of 1×10 7.0 TCID 50 / mL of inactivated PRRS virus solution;

[0040] (2) 100 parts by weight of the inactivated PRRS virus solution prepared in step (1) and 0.3 parts by weight of agarose particles are fully allowed to stand and mix to obtain a mixed solution (the diameter of the virus particles in the obtained mixed solution is about 3 mm, the quality is brittle, and it will deform during injection, and cannot be directly used as an inactivated seedling);

[0041] (3) The mixed solution obtained in step (2) is self-emulsified and mixed with 20 parts by mass of a white oil adjuvant and 4 parts by mass of a non-ionic surfactant at a temperature of 37° C. and a speed of 600 rpm for 20 minutes to prepare a porcine reproductive and respiratory syndrome inactivated vaccine; wherein the non-ionic surfactant is obtained by mixing Span-80 and Tween-80 in a mass ratio of 1:1.

[0042] Example 2

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

[0044] (1) Prepare the PRRS virus CH-1a strain to a virus titer of 1×10 7.2 TCID 50 / mL of inactivated PRRS virus solution;

[0045] (2) 100 parts by weight of the inactivated PRRS virus solution obtained in step (1) and 0.1 parts by weight of agarose particles are fully mixed to obtain a mixed solution (the diameter of the virus particles in the obtained mixed solution is about 1 mm);

[0046] (3) The mixed solution obtained in step (2) is self-emulsified and mixed with 60 parts by mass of a white oil adjuvant and 4 parts by mass of a non-ionic surfactant at a temperature of 37° C. and a speed of 600 rpm for 20 minutes to prepare an inactivated porcine reproductive and respiratory syndrome vaccine; wherein the non-ionic surfactant is obtained by mixing Span-80 and Tween-80 in a mass ratio of 1:1.

[0047] Test example

[0048] 1. Stability test

[0049] 10 mL of the porcine reproductive and respiratory syndrome inactivated vaccine prepared in Examples 1 and 2 were taken respectively, placed in a centrifuge tube, centrifuged at 3000 rpm for 15 min, and the stratification of the porcine reproductive and respiratory syndrome inactivated vaccine in each group of centrifuge tubes was observed. The results showed that the porcine reproductive and respiratory syndrome inactivated vaccine in the centrifuge tubes of Examples 1 and 2 did not show obvious stratification, a small amount of water appeared in the lower layer of the vaccine, but the water phase precipitated at the bottom of the centrifuge tube was less than 0.5 mL, indicating that the porcine reproductive and respiratory syndrome inactivated vaccine prepared in Examples 1 and 2 had good stability and met the requirements of the Chinese Veterinary Pharmacopoeia.

[0050] 2. Immune efficacy test

[0051] 40 14-day-old piglets were randomly divided into 4 groups, 10 in each group. Among them, 2 groups were injected with the porcine reproductive and respiratory syndrome inactivated vaccine prepared in Examples 1-2; the remaining two groups were injected with inactivated PRRS virus solution in one group and physiological saline in the other group; 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 each group of piglets was recorded before injection and 12h, 24h, and 36h after injection, and the results are shown in Table 1.

[0052] Table 1 Body temperature of piglets in each group before and after injection (℃)

[0053] Before injection Injection 12h Injection 24h Injection 36h Example 1 39.1 39.8 39.5 39.2 Example 2 39.0 39.6 39.3 39.3 Inactivation of viruses 39.1 39.2 39.1 39.2 Normal saline 39.1 39.2 39.2 39.1

[0054] As can be seen from Table 1, after the piglets were injected with the porcine reproductive and respiratory syndrome inactivated vaccine prepared by the present invention for immunization, the piglets still had a certain degree of 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.

[0055] 14 days after the first immunization (i.e., the first injection), the piglets in each treatment group were randomly divided into two groups, each with 5 piglets, and one of the groups was boosted once, with the same injection method as the first injection. At the 4th week after the first immunization and the 4th week after the second immunization, the neutralizing antibody titers of the first immunization and booster immunization of each group of piglets were counted, wherein the piglets injected with the inactivated vaccine prepared in Example 1 were subjected to neutralization tests using the CH-1a strain, and the piglets injected with the inactivated vaccine prepared in Example 2 were subjected to neutralization tests using the NADC30 strain. The results are shown in Table 2.

[0056] Table 2 Statistical results of neutralizing antibody titers in each group of piglets after immunization

[0057] One immunization group Booster group Example 1 <![CDATA[1:2 4.1 ]]> <![CDATA[1:2 5.2 ]]> Example 2 <![CDATA[1:2 3.6 ]]> <![CDATA[1:2 5.0 ]]> Inactivation of viruses 0 0 Normal saline / /

[0058] 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. 4 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.

[0059] 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 special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine, characterized in that: The special oil adjuvant for the porcine reproductive and respiratory syndrome inactivated vaccine comprises a mineral oil adjuvant and gel granules.

2. The special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine as claimed in claim 1, characterized in that: The mineral oil is white oil; the gel particles are substances capable of forming porous gel.

3. The special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine as claimed in claim 2, characterized in that: The gel particles include any one or more of agar particles, dextran particles, agarose particles, and high molecular weight chitosan particles, and the particle size of the gel particles is 0.5 to 3 mm.

4. The special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine according to any one of claims 1 to 3, characterized in that: The special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine also includes a non-ionic surfactant and / or an adjuvant enhancing component.

5. Use of the special oil adjuvant for porcine reproductive and respiratory syndrome inactivated vaccine as described in any one of claims 1 to 4 in the preparation of porcine reproductive and respiratory syndrome inactivated vaccine.

6. A method for preparing an inactivated porcine reproductive and respiratory syndrome vaccine using the special oil adjuvant for the inactivated porcine reproductive and respiratory syndrome vaccine according to any one of claims 1 to 4, characterized in that: The steps include: (1) mixing an inactivated porcine reproductive and respiratory syndrome virus solution with gel granules to obtain a mixed solution; (2) The mixed solution obtained in step (1) is mixed with a mineral oil adjuvant to obtain an inactivated vaccine of porcine reproductive and respiratory syndrome.

7. The method according to claim 6, 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 solution and the gel granules when mixed is 80-120:0.01-0.

3.

8. The method according to claim 7, characterized in that The mass ratio of the mineral oil adjuvant to the inactivated porcine reproductive and respiratory syndrome virus solution is 12-60:80-120.

9. The method according to claim 8, characterized in that During the mixing in step (2), a nonionic surfactant and / or adjuvant is added to enhance the ingredients. The mixing method is self-emulsifying mixing. The temperature of the self-emulsifying mixing is 35-39° C., the speed is 400-800 rpm, and the time is 15-25 min.

10. An inactivated porcine reproductive and respiratory syndrome vaccine prepared according to the method according to any one of claims 6 to 9.