AU2021100723A4

AU2021100723A4 - Preparation method of bivalent inactivated vaccine for bluetongue virus

Info

Publication number: AU2021100723A4
Application number: AU2021100723A
Authority: AU
Inventors: Meiling KOU; Huachun LI; Le Li; Defang LIAO; Haisheng MIAO; Jiarui XIE
Original assignee: Yunnan Acad Of Animal Husbandry And Veterinary Sciences
Current assignee: Yunnan Academy Of Animal Husbandry And Veterinary Sciences
Priority date: 2021-02-05
Filing date: 2021-02-05
Publication date: 2021-04-22
Anticipated expiration: 2029-02-05

Abstract

Austracy The present invention belongs to the technical field of research and development of preparation processes of biological products, and discloses a preparation method of a bivalent inactivated vaccine for Bluetongue virus. The preparation method includes the steps of virus culture, inactivator preparation, inactivation operation, concentration and purification, antigen emulsification and vaccine proportioning. In the present invention, ruminants are efficiently induced in combination with adjuvants, excipients and inactivated antigen suspension. Meanwhile, immune response to the Bluetongue virus is produced; and interference of non-structural proteins is effectively removed, thereby discriminably identifying vaccine immunization and wild strain infection. 1 Drawings of Description 7 5 2 -U 1 0 0 3 6 9 12 15 18 21 24 27 30 Inactivation time (hour) FIG. 1 1

Description

Austracy

The present invention belongs to the technical field of research and development of preparation processes of biological products, and discloses a preparation method of a bivalent inactivated vaccine for Bluetongue virus. The preparation method includes the steps of virus culture, inactivator preparation, inactivation operation, concentration and purification, antigen emulsification and vaccine proportioning. In the present invention, ruminants are efficiently induced in combination with adjuvants, excipients and inactivated antigen suspension. Meanwhile, immune response to the Bluetongue virus is produced; and interference of non-structural proteins is effectively removed, thereby discriminably identifying vaccine immunization and wild strain infection.

Drawings of Description

7

-U2 1

0 3 6 9 12 15 18 21 24 27 30 Inactivation time (hour)

FIG. 1

Description

PREPARATION METHOD OF BIVALENT INACTIVATED VACCINE FOR BLUETONGUE VIRUS

Technical Field

The present invention relates to the technical field of research and development of preparation processes of biological products, and particularly discloses a bivalent inactivated vaccine for Bluetongue virus that can enable ruminants to produce protective immune response and discriminate vaccine immunization and wild strain infection.

Background

Bluetongue (BT) is a non-contact viral infectious disease caused by Bluetongue virus (BTV) and propagated by taking arthropod insects as media, and is mainly distributed in tropical, subtropical and temperate regions. Ruminants such as sheep are mainly infected by the BT. Most of the infected animals are recessively poisoned, and have no clinical symptom. If pregnant animals are infected with the BT, symptoms such as fetal anomaly, stillbirth and abortion may occur. At present, there are at least 27 serotypes discovered from the BTV, wherein major prevalent strains are type 1 and type 16 in China. In recent 10 years, because of various factors such as global warming and sea level rise, the range of activity of various vector insects is increasingly widened and shows a spread trend towards higher-altitude and colder northern regions, thereby bringing a serious challenge to disease prevention and control in China. Mass outbreak of Bluetongue virus 8 (BTV-8) first occurred in Europe in 2006, and serious economic losses were brought to cattle and sheep breeding in countries such as France, Britain and Belgium, and animal husbandry production and human health were severely threatened. It is estimated that, global economic losses caused by the BT may be up to 3 billion dollars per year.

Description

However, the development and research of national BT vaccines are relatively backward. Researches on BTV attenuated vaccines, inactivated vaccines and recombinant vaccines were conducted in sequence over the last 50 years, but were slow in progress and not deep enough. BTV1 and BTV16 type attenuated vaccines that were successfully developed in the 1980s in China have achieved excellent immune protection effects when used for emergency prevention in Xiangfan, Hubei and Wuxi, Chongqing. Although the attenuated vaccines have excellent immunization and protection effects, an attenuated mechanism of the virus is unclear. Thus, risk of virulence return and gene rearrangement with the prevalent strains may exist after use, and vaccine applications are greatly limited. Therefore, the inactivated vaccines are still the most important methods of preventing and controlling the BT prevalence. In China, the BTV was successfully inactivated with hydroxylamine by Yunnan Animal Husbandry and Veterinary Medicine Institute in 1990s. However, certain damage was brought to major immunogenic epitopes during inactivation, and an inactivated antigen immunization effect was poor. There is a foreign report on inactivation of the BTV with p-propiolactone, but the effect of repeating the study was not ideal, and an expected inactivation effect was not achieved. Meanwhile, during mass survey of BTV antibodies, antibodies produced by wild virus infection and vaccine immunization cannot be discriminated by a method for detecting non-structural protein antibodies; and the production process urgently needs to be improved. There are also research reports on the recombinant vaccines, but no marketable product is launched. Binary ethylenimine (BEA) is an important virus inactivator, and is cyclized into BEI in presence of sodium hydroxide. The BEI directly acts on a viral nucleic acid to remove the replication capacity of the virus and maintain the major immunogenicity of the virus. Therefore, the BEA is widely applied to inactivation of foot-and-mouth disease virus and other animal viruses. Due to structural particularity, the BTV is difficult to be completely

Description

inactivated by conventional BEI formulas and inactivation processes, or the immunogenicity is changed after inactivation, which brings a poor immunization effect. Therefore, the preparation process of the BT inactivated vaccine urgently needs to be improved. Therefore, how to provide a preparation method of a bivalent inactivated vaccine for Bluetongue virus that can effectively remove non-structural protein interference and discriminate vaccine immunization and wild strain infection is a technical problem that urgently needs to be solved by those skilled in the art.

Summary

In view of this, the present invention establishes a unique process for BTV culture, inactivation, concentration and purification and strain proportioning without significantly increasing production cost. A production process of a bivalent inactivated vaccine for BTV is obtained by improving a combination of adjuvants, excipients and inactivated antigen suspension, thereby solving the problems that a Bluetongue inactivated vaccine is poor in immunization effect and vaccine immunization and wild strain infection cannot be discriminated. To achieve the above purpose, technical solutions of the present invention are as follows: A preparation method of a bivalent inactivated vaccine for BTV includes the following steps: (1) virus culture: respectively inoculating BHK-21 cells with single serotype Bluetongue seed viruses; culturing the cells by taking serum-free MEM cell culture fluid as culture fluid; slowly dumping the virus culture fluid after 60-80% of the cells have significant lesions; then adding the serum-free MEM cell culture fluid; scraping non-exfoliated cells; collecting the suspension; performing ultrasonic disruption; and centrifuging the suspension to remove a precipitate to obtain the supernatant, that is, BTV suspension;

Description

(2) inactivator preparation: adding binary ethylenimine into a 0.5-0.7% of sodium hydroxide solution to prepare a binary ethylenimine solution having a concentration of 0.1 mol/L; standing the solution at 37°C for 1-2 h to obtain an inactivator; and preserving the inactivator at 4°C for later use; (3) inactivation operation: adding the inactivator into the BTV suspension to inactivate the virus; adding sodium thiosulfate to be uniformly mixed; preparing inactivated Bluetongue antigen suspension after the inactivation; and preserving the inactivated Bluetongue antigen suspension at 4°C for later use; (4) concentration and purification: adding PEG6000 and sodium chloride into the inactivated Bluetongue antigen suspension; stirring the suspension at 40 C until the added materials are fully dissolved; then centrifuging the suspension; removing the supernatant; and resuspending the precipitate, wherein the obtained resuspended matter is a concentrated and purified virus suspension; (5) antigen emulsification: uniformly mixing the concentrated and purified virus suspension with a mixed solution of white oil and Span-80; ultrasonically emulsifying the solution to obtain emulsion; additionally taking the concentrated and purified virus suspension; adding Tween-80 and thimerosal into the suspension; fully and uniformly mixing the materials to prepare outer virus suspension; adding the outer virus suspension into the emulsion for ultrasonic emulsification again; preparing a single Bluetongue inactivated vaccine after the emulsification; and preserving the single Bluetongue inactivated vaccine at 4 0C for later use; (6) vaccine proportioning: respectively emulsifying the preferred type 1 BTV and type 16 BTV inactivated antigens under the same condition according to the step (5) to obtain respective single serotype vaccines; uniformly mixing the type 1 Bluetongue single inactivated vaccine and the type 16 Bluetongue single inactivated vaccine according to a ratio of 1:(2-3) (v/v), thereby obtaining the bivalent inactivated vaccine for BTV.

Description

Binary ethylenimine (BEA) is an important virus inactivator, and is cyclized into BEI in presence of sodium hydroxide. The BEI directly acts on a viral nucleic acid to remove the replication capacity of the virus and maintain the major immunogenicity of the virus. Therefore, the BEA is widely applied to inactivation of foot-and-mouth disease virus and other animal viruses. Due to structural particularity of the BTV, the virus can be inactivated on the basis of maintaining major immunogenic antigen stability of the BTV in the present invention. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, the Bluetongue seed viruses in the step (1) include type 1, type 2, type 3, type 4, type 5, type 7, type 9, type 10, type 12, type 15, type 16, type 21 and type 24 of the BTV; a viral titer of the Bluetongue seed viruses is10--10-7/100 pL;

and an inoculum size is 1-5% (v/v) of the BHK-21 cells. In the present invention, the virus culture, inactivation, concentration and purification methods can be applied to inactivated vaccines of totally 13 serotype strains such as the type 1, type 2, type 3, type 4, type 5, type 7, type 9, type 10, type 12, type 15, type 16, type 21 and type 24 of the BTV. Since only these serotypes are discovered in China at present and other serotypes are not tested, due to similarity of basic structures among the different serotypes of the BTV, the methods are not limited to application in culture, inactivation and purification of these strains. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, in the step (1), a culture temperature is 37°C; culture time is 3-4 days; and after the virus culture fluid is dumped, an addition amount of the serum-free MEM cell culture fluid is 1/10 of the volume of the culture fluid. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, ultrasonic disruption time is 3-5 min; centrifuging time is 20 min; and a rotation speed is 3000 rpm.

rN

Description

Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, in the step (3), an addition amount of the inactivator is 6% (v/v) of the volume of the BTV suspension; a concentration of the inactivator is 0.1 mol/L; the inactivator is stirred in an incubator of 37°C for inactivating for 30 h; and a stirring rate is 100-200 rpm/min. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, in the step (3), an addition amount of the sodium thiosulfate is 0.6% (v/v) of the volume of the BTV suspension; and a molar concentration of the sodium thiosulfate is 1 mol/L. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, in the step (3), the inactivation process includes the following steps: adding an inactivator; then placing the suspension in an incubator at 37°C; stirring the suspension for inactivation for 15 hours; replacing a container after the operation is ended; continuously placing the suspension in the incubator at 37°C; stirring the suspension for inactivation for 10 hours; replacing another container; continuously placing the suspension in the incubator at 37°C; stirring the suspension for inactivation for 5 hours; within totally 30 hours after inactivation, adding the sodium thiosulfate to be uniformly mixed; and terminating an inactivated reaction. In the present invention, by accurately determining the inactivator dose (6 mmol/L) in the virus suspension and the transfer and replacement time and frequency of the inactivation container, the virus can be inactivated on the basis of maintaining major immunogenic antigen stability of the BTV. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, in the step (4), an addition amount of the PEG6000 is 7% of the weight of the inactivated BT antigen suspension; and the addition amount of the sodium chloride is 2.22% of the weight of the inactivated BT antigen suspension. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, in the step (4), the operation of resuspending the precipitate is as follows:

I;

Description

1/10 (v/v) of serum-free MEM cell culture fluid or 0.01 M of tris(hydroxymethyl)aminomethane hydrochloric acid with a pH of 7.2-7.4 is added into the precipitate. The addition amount of the serum-free MEM cell culture fluid or the 0.01 M of tris(hydroxymethyl)aminomethane hydrochloric acid is determined when the precipitate can be completely dissolved in a suck-and-blow manner of the shaken fluid; or the suspension may be diluted at different concentrations according to needs. Polyethylene glycol 6000 (PEG6000) has excellent water solubility. Ether bonds on the molecular surface of the PEG6000 have slight negative charge in a liquid medium and may be linked with positively charged proteins on the virus surface, thereby facilitating stability of antigens on the surface of virus particles. The virus particles linked with the antigens can be precipitated by centrifugation; and the precipitate can be resuspended in the liquid after the liquid is added into the precipitate for shaking. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, in the step (4), the centrifugation is as follows: the solution is centrifuged in a centrifuge at centrifugal force of 1500 g for 1 h, or centrifuged at centrifugal force of 2500 g for 30 min; and in a centrifugal force range of 1500-2500 g, centrifugation time may be appropriately shortened or prolonged when the centrifugal force is increased or decreased. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, in the step (5), the mixed solution of white oil and Span-80 is prepared by uniformly mixing white oil and Span-80 according to a volume ratio of 47:3 and sterilizing the mixture at a high pressure for 15 min. Preferably, in the preparation method of the bivalent inactivated vaccine for BTV, the step (5) includes: uniformly mixing 1 part of the concentrated and purified virus suspension and 2 parts of the mixed solution of white oil and

Description

Span-80; ultrasonically emulsifying the mixture to obtain emulsion; adding 6% of Tween-80 (v/v) and 0.01% of thimerosal (w/v) into 3 parts of the concentrated and purified virus suspension; fully and uniformly mixing the materials to prepare outer virus suspension; adding the outer virus suspension into the emulsion for ultrasonic emulsification again; preparing a single Bluetongue inactivated vaccine after the emulsification; and preserving the single Bluetongue inactivated vaccine at 4°C for later use. In the present invention, a combination of the white oil, the Span-80, the Tween-80 and the thimerosal serves as the adjuvant, the excipient and the activator for vaccine preparation, thereby facilitating the formula and the emulsification process that can increase the immunization effect of the BTV. Through the above technical solutions, compared with the prior art, the present invention discloses and provides the preparation method of the bivalent inactivated vaccine for BTV, and has the beneficial effects as follows: (1) According to the process in the present invention, antibodies to two serotype strains and closed serotype strains can be produced by one-time immunization, so that the immunization efficiency and effect of the BT inactivated vaccine are significantly increased. After single inoculation of sheep, a positive rate of corresponding strains may be more than 90%; and virus attack can be effectively resisted when the positive rate of the antibodies in the sheep is 70% or higher. (2) According to characteristics of the BTV, the vaccine immunization and wild strain infection can be discriminably identified and diagnosed by establishing novel virus culture, concentration and purification manners. (3) The inactivation time of the BTV is shortened; active antigen loss is decreased during inactivation; and by optimizing and improving the inactivation process in the present invention, the BTV antigen can be completely inactivated within 30 hours, thereby avoiding risk of disease spread caused by incomplete

Description

inactivation during vaccine preparation and effectively reducing the antigen loss during inactivation. (4) The vaccine preparation method is simple, feasible, low in cost and high in practicality; requirements for the production and preparation process and facilities and equipment are low in the present invention; the materials are readily available; the operation is simple and convenient; and the preparation method meets the needs of large-scale production.

Description of Drawings

To more clearly describe the technical solutions in the embodiments of the present invention or in the prior art, the drawings required to be used in the description of the embodiments or the prior art will be simply presented below. Apparently, the drawings in the following description are merely some embodiments of the present invention, and for those ordinary skilled in the art, other drawings can also be obtained according to these drawings without contributing creative labor. Fig. 1 is a dynamic curve of BTV inactivation in inactivation operation.

Detailed Description

Technical solutions in the embodiments of the present invention will be described clearly and fully below. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention. The present invention proposes a novel BTV culture, inactivation, purification, concentration and proportioning process. Ruminants are efficiently induced in combination with adjuvants, excipients and inactivated antigen suspension.

Description

Meanwhile, immune response to type 1 and type 16 of the Bluetongue virus is produced; and interference of non-structural proteins is effectively removed, thereby discriminably identifying vaccine immunization and wild strain infection. It shall be understood that the method described in the present invention is not limited to the specific methods and experimental conditions in the present invention because the methods and the conditions can be changed. Further, terms used in the present invention are merely used for describing specific embodiments, rather than a limitation. Partial terms are described below: Inactivation: "inactivation" in the present invention means to enable infectious microorganisms to lose infectivity and simultaneously maintain immunogenicity, which can be understood by those skilled in the art. Immunogenicity: antigens can stimulate specific immune cells of the body after entering the body, and thus the immune cells activate, proliferate and differentiate, and finally the characteristics of immune effector antibodies and sensitized lymphocytes specified at the antigens are produced. Ruminants: the ruminants are any kind of mammals of Artiodactyla that generally have horns; stomachs of the ruminants are divided into four parts; and these animals include sheep, goats, cattle, giraffes and deer. Inoculation: "inoculation" in the present invention means to inject inactivated vaccines into subcutaneous tissues or muscles of the animals by a syringe, which can be understood by those ordinary skilled in the art. The technical solutions of the present invention are further described below through specific embodiments. However, the technical solutions of the present invention are not limited by the embodiments. 1, Culture of BTV of type 1 and type 16 BHK-21 cells grew all over cell culture bottles on a single layer; clean cell culture fluid was dumped; the bottles were completely cleaned once with an appropriate amount of serum-free MEM cell culture fluid; the cell culture fluid was

1 (

Description

completely dumped; each bottle was inoculated with 2 ml of a BT seed virus (type 1 or type 16) having a viral titer of about 10-6 and an appropriate amount of the serum-free MEM cell culture fluid; the cells were cultured at 37°C for 3-4 days; the virus culture fluid was slowly dumped after about 70% of the cells had significant lesions; the serum-free MEM cell culture fluid accounting for 1/10 of the amount of the culture fluid was added; non-exfoliated cells were scraped by a long-handle sterile scraper; the suspension was collected and subjected to ultrasonic disruption for 3-5 minutes; and the suspension was centrifuged in a centrifuge bottle at 3000 rpm for 20 minutes; cell residues were removed; and the obtained supernatant was the BTV suspension. The viral titer of the virus suspension was determined. The virus suspension was diluted to be 10-6/100 pL with the serum-free MEM cell culture fluid and

preserved at 4°C for later use. The BTV suspension of the type 1 and the type 16 had the same preparation method. The BTV of the type 1 was a Y853 strain, and the BTV of the type 16 was a DH strain. The BTV strains were collected in Yunnan key laboratory of tropical and subtropical animal virus diseases. 2, Inactivation of BTV of type 1 and type 16 1) Preparation of 0.1 M of BEI inactivator: 0.175 M of sodium hydroxide (NaOH) solution was prepared; 0.1 M of BEA was added into the solution; the solution was placed at 37°C for 2 hours to complete a cyclization reaction of the BEA; and 0.1 M of BEI solution was formed and preserved at 4°C for later use. The prepared reagent shall be effective only when used within 72 hours. 2) 6% of the above inactivator was added into the BTV suspension, i.e., a final concentration of the inactivator in the virus suspension was 6 mM; 4 million units/L of penicillin sodium, 1 million units/L of streptomycin and 0.1 mg/L of amphotericin B were added into the virus suspension; the materials were fully and uniformly mixed; the mixture was stirred in an incubator of 37°C at a rate of 200 revolutions per minute for inactivation; sterile inactivated containers were

Description

respectively replaced within 15 and 25 hours after inactivation; 0.6% of 1M sodium thiosulfate was added to be fully and uniformly mixed within 30 hours; and after inactivation was completed, the virus suspension was preserved at 4°C. In addition, by optimizing and improving the inactivation process in the present invention, the BTV antigen can be completely inactivated within 30 hours, thereby avoiding risk of disease spread caused by incomplete inactivation during vaccine preparation and effectively reducing the antigen loss during inactivation. Specifically, related tests are conducted on inactivation time of the BTV suspension in the present invention. A dynamic curve of BTV inactivation is shown as Fig. 1. It can be discovered that, the BTV suspension can be completely inactivated within 27 hours. To ensure incomplete inactivation caused by errors, the inactivation time is prolonged to 30 hours. 3, Virus concentration and purification and emulsification proportioning 1) Analytically pure polyethylene glycol 6000 (PEG6000) was added into the inactivated BTV suspension in the step 1 according to a weight ratio of 7%; analytically pure sodium chloride was added into the above inactivated BTV antigen suspension according to a weight ratio of 2.22%; and the PEG6000 and the sodium chloride were fully dissolved in a space at 4°C in manners of stirring, oscillating or shaking within not less than 1 hour and preferably not more than but not limited to 12 hours. 2) The suspension in the step 1) was centrifuged in a centrifuge at centrifugal force of 1500 g for 1 hour, or centrifuged at centrifugal force of 2500 g for 30 min; and in a centrifugal force range of 1500-2500 g, centrifugation time may be appropriately shortened or prolonged when the centrifugal force is increased or decreased. 3) The supernatant was slightly dumped after centrifugation was ended; the precipitate was reserved; the precipitate was resuspended with a small amount of serum-free MEM cell culture fluid or 0.01 M of tris(hydroxymethyl)aminomethane

11)

Description

(Tris) having a pH value of 7.2-7.4 in a physical oscillation or liquid suck-and-blow manner; and the resuspended matter was the purified virus suspension. The addition amount of the serum-free MEM cell culture fluid or the 0.01 M of tris(hydroxymethyl)aminomethane is determined when the precipitate can be completely dissolved in a suck-and-blow manner of the shaken fluid; and the suspension may be diluted at different concentrations with the MEM culture fluid according to needs. In the present embodiment, the antigens were quantified according to a BCA protein quantification method and cELISA quantification method. 1Ig, 5pg, 10pg and 50 pg/animal groups were set; and a blank control group was set. 4, 470 ml of white oil and 30 ml of Span-80 were uniformly mixed; the mixture was sterilized at a high pressure for 15 minutes to form a mixed solution of the white oil and Span-80. Each part was a volume unit. 2 parts of the mixed solution of the white oil and Span-80 and 1 part of the purified inactivated virus suspension were uniformly mixed and then ultrasonically emulsified. Another 3 parts of the inactivated virus suspension were added into 8% of Tween-80 (volume ratio) and 1% of thimerosal; after the materials were dissolved and uniformly mixed, the mixture was added into the emulsion; then ultrasonic emulsification was performed again; the emulsified suspension was preserved at 4°C, that is, the prepared BT inactivated vaccine. The antigens of different content were respectivelyprepared. The emulsified liquids of the BTV of type 1 and the BTV of type 16 were uniformly mixed according to a ratio of 1:2 (v/v), that is, the prepared BT inactivated vaccine. The vaccine was inoculated to muscles or subcutaneous tissues of the ruminants by 2 ml per animal; and antibodies that can resist the BTV infection are produced within 1 week. Vaccination and challenge experiment

Description

30 sheep (at an age of 6-12 months) were randomly selected as experimental animals; after 7 days, when adapting to a new breeding environment, the experimental animals were vaccinated with the inactivated vaccine. The 30 sheep were totally divided into 4 groups, i.e., a test group 1 (10 sheep), a test group 2 (10 sheep), a control group 1 (5 sheep) and a control group 2 (5 sheep) respectively. 2 ml of the inactivated vaccine was injected into muscles of each sheep in the test groups, while 2 ml of the MEM culture fluid was injected into muscles of each sheep in the control groups. A challenge experiment was conducted within 21 days after inoculation of the inactivated vaccine. Challenge strains were as follows: the BTV of type 1 was the Y863 strain (having the viral titer of 10- CEID50); the BTV of type 16 was the DH strain (having the viral titer of 10-4 CEID50); and both the BTV of type 1 and the BTV of type 16 were sheep blood viruses and prepared and collected in Yunnan key laboratory of tropical and subtropical animal virus diseases. The challenge strains were injected via jugular vein by 1 ml per animal; the BTV of type 1 inoculated in the test group 1 and the control group 1 was the Y863 strain; and the BTV of type 16 inoculated in the test group 2 and the control group 2 was the DH strain. Immune effect evaluation of the vaccine (1) Antibody effect evaluation Blood was collected once every 7 days before and after inoculation of the inactivated vaccine; serum was separated; and serum antibodies were detected by a BTV competitive enzyme-linked immunosorbent assay antibody detection method (BTV cELISA). The method is a detection method recommended by World Organization for Animal Health (OIE); and operating methods and criteria refer to national standards of Diagnostic Techniques for Bluetongue (GB/T 18636-2017). Moreover, state changes of the sheep were observed at fixed time every day.

1Ad

Description

The antibody results showed that, the positive rates of the antibodies in both the test group 1 and the test group 2 were 100%; a vaccine immunization effect was excellent; the positive rates of the antibodies in both the control group 1 and the control group 2 were 0%; and the control was normal. All the sheep in the test groups and the immunization group had no obvious clinical symptom. Table 1 cELISA antibody detection and clinical observation results of the BTV inactivated vaccine test Antbod ihibtio rte PI,%)Positive rate Clinical Group Animal No. Aofantibody observation 0 week 1 week 2 weeks 3 weeks 1 12 22 73 71 Normal 2 8 25 62 68 Normal 3 0 16 57 59 Normal 4 0 31 63 66 Normal Test 5 5 15 64 69 100% Normal group 1 6 0 33 56 52 Normal 7 9 14 73 77 Normal 8 0 12 65 69 Normal 9 0 0 63 60 Normal 10 0 0 70 76 Normal 11 0 28 62 78 Normal 12 7 37 75 72 Normal 13 4 31 64 69 Normal 14 0 26 68 83 Normal Test 15 0 18 51 62 Normal group 2 16 6 21 66 69 100% Normal 17 11 14 57 59 Normal 18 0 27 64 66 Normal 19 13 62 62 65 Normal 20 4 55 64 68 Normal 21 8 0 5 2 Normal 22 6 3 7 3 Normal Control 23 0 7 8 7 0% Normal group 1 24 0 2 5 3 Normal 25 0 0 0 0 Normal 26 0 5 0 0 Normal 27 4 0 0 6 Normal Control 28 0 2 9 0 0% Normal group 2 29 2 0 11 8 Normal 30 0 0 0 0 Normal

Description

Notes: the antibody inhibition rate of >50% judges that BTV antibody is positive. (1) Clinical symptom and nucleic acid detection after challenge Clinical symptoms were observed and recorded; whole blood of the sheep collected within 0 week, 1 week and 2 weeks after challenge was detected by real-time RT-PCR. The results showed that, respectively 7 sheep were completely protected in the test group 1 (inoculated with the BTV of type 1) and the test group 2 (inoculated with the BTV of type 16), no nucleic acid was detected in the blood, and the sheep had no clinical symptom. The nucleic acid was detected in the blood of 3 sheep in each of the two groups, and the sheep had clinical symptoms at different degrees, but the symptoms were improved. Table 2 Nucleic acid detection and clinical symptom observation results in the challenge experiment Nucleic acid Ct value of BTV after clIndex score of Animal No. challenge chmial syptoms of Group 0 week 1 week 2 weeks 0 1 N N N 0 2 N N N 0 3 N N N 0 4 N N N 0 Test group 1 5 N N N 0 (inoculated with 6 N 31.33 N 0.5 the BTV of type 1) 7 N 29.35 31.5 1 8 N N N 0 9 N N N 0 10 N 28.76 33.77 0.5 11 N N N 0 12 N N N 0 13 N N N 0 Test group 2 14 N N N 0 (inoculated with 15 N N N 0 the BTV of type 16 N N N 0 16) 17 N N N 0 18 N N N 0 19 N 27.68 33.89 0.5 20 N 29.27 31.68 1 Control group 1 21 N N 30.23 1.5 (inoculated with 22 N N 33.15 1

Description the BTV of type 1) 23 N N 32.35 1 24 N 28.56 30.96 2 25 N N 30.05 2 26 N N 30.09 1.5 Control group 2 27 N N Dead 10 (inoculated with 28 N 28.37 31.01 2 theBTVoftype 29 N 29.32 Dead 10 30 N 30.9 29.83 3

Notes: the Ct value of <34 judges that, the BTV nucleic acid is positive, otherwise negative, labeled as N; the BTV symptoms are scored by complex typical symptoms within 1-2 weeks after virus inoculation, and include increased body temperature, red visible mucosa of the mouth, nose and feet, mental depression and death; score 0 represents zero symptom; the higher the score is, the more obvious the symptoms are; and score 10 represents death. It should be indicated that, in the present invention, the immunization effect is tested by monitoring the cellular immunity and specific humoral immunity produced by the body inoculated with the vaccine. The tests are known to those skilled in the art. Or, a challenge experiment may be conducted, i.e., after the antibodies are induced by the antigens or T cell response occurs within about 1-2 weeks after vaccination for general susceptible animals, virus infection challenge tests are conducted on the vaccinated susceptible animals and unvaccinated susceptible animals with corresponding infectious viruses. Symptom situations related to virus infection or death situations of the animals are monitored. Differences between the vaccinated susceptible animals and the unvaccinated susceptible animals are compared, e.g., significant decrease of the symptoms of the vaccinated susceptible animals related to virus infection, or decrease of viremia, or decrease of the quantity or severity of damage related to virus infection, or decrease of death. The serum may be collected for monitoring levels of antibodies produced by vaccine injection, or the antibody levels may be measured by other methods known to those skilled in the art.

Description

The preparation method of the BT inactivated vaccine provided by the present invention is described in detail above. The principles and embodiments of the present invention are illustrated herein through specific embodiments, and the above illustration is only used to help to understand the method and core idea of the present invention. It should be indicated that for those ordinary skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. Each embodiment in the description is described in a progressive way. The difference of each embodiment from each other is the focus of explanation. The same and similar parts among all of the embodiments can be referred to each other. For the solution disclosed by the embodiments, because the solution corresponds to a method disclosed by the embodiments, the device is simply described. Refer to the description of the method part for the related part.

Claims

1. A preparation method of a bivalent inactivated vaccine for Bluetongue virus (BTV), comprising the following steps: (1) virus culture: respectively inoculating BHK-21 cells with single serotype Bluetongue seed viruses; culturing the cells by serum-free MEM cell culture fluid; slowly dumping the virus culture fluid after 60-80% of the cells have significant lesions; adding a proper amount of the serum-free MEM cell culture fluid; scraping non-exfoliated cells; collecting the suspension; performing ultrasonic disruption; and then centrifuging the suspension to remove a precipitate to obtain the supernatant, that is, BTV suspension; (2) inactivator preparation: adding binary ethylenimine into a 0.5-0.7% of sodium hydroxide solution to prepare a binary ethylenimine solution having a concentration of 0.1 mol/L; standing the solution at 37°C for 1-2 h to obtain an inactivator; and preserving the inactivator at 4°C for later use; (3) inactivation operation: adding the inactivator into the BTV suspension to inactivate the virus; then adding sodium thiosulfate to be uniformly mixed; preparing inactivated Bluetongue antigen suspension after the inactivation; and preserving the inactivated Bluetongue antigen suspension at 4°C for later use; (4) concentration and purification: adding PEG6000 and sodium chloride into the inactivated Bluetongue antigen suspension; stirring the suspension at 40 C until the added materials are fully dissolved; centrifuging the suspension; removing the supernatant; and resuspending the precipitate, wherein the obtained resuspended matter is a concentrated and purified virus suspension; (5) antigen emulsification: uniformly mixing the concentrated and purified virus suspension with a mixed solution of white oil and Span-80; ultrasonically emulsifying the solution to obtain emulsion; additionally taking the concentrated and purified virus suspension; adding Tween-80 and thimerosal into the suspension; fully and uniformly mixing the materials to prepare outer virus suspension; adding the outer virus suspension into the emulsion for ultrasonic emulsification again;

Claims

preparing a single Bluetongue inactivated vaccine after the emulsification; and preserving the single Bluetongue inactivated vaccine at 4°C for later use; (6) vaccine proportioning: respectively emulsifying the type 1 BTV and type 16 BTV inactivated antigens under the same condition according to the step (5) to obtain respective single serotype vaccines; and uniformly mixing the type 1 Bluetongue single inactivated vaccine and the type 16 Bluetongue single inactivated vaccine according to a ratio of 1:(2-3) (v/v), thereby obtaining the bivalent inactivated vaccine for BTV.

2. The preparation method of the bivalent inactivated vaccine for BTV according to claim 1, wherein the BTV in the step (1) comprises type 1, type 2, type 3, type 4, type 5, type 7, type 9, type 10, type 12, type 15, type 16, type 21 and type 24 of the BTV.

3. The preparation method of the bivalent inactivated vaccine for BTV according to claim 1, wherein in the step (1), a culture temperature is 37°C; culture time is 3-4 days; and after the virus culture fluid is dumped, an addition amount of the serum-free MEM cell culture fluid is 1/10 of the volume of the culture fluid.

4. The preparation method of the bivalent inactivated vaccine for BTV according to claim 1, wherein ultrasonic disruption time is 3-5 min; centrifuging time is 20 min; and a rotation speed is 3000 rpm.

5. The preparation method of the bivalent inactivated vaccine for BTV according to claim 1, wherein in the step (3), an addition amount of the inactivator is 6% (v/v) of the volume of the BTV suspension; a concentration of the inactivator is 0.1 mol/L; the inactivator is stirred in an incubator of 37°C for inactivating for 30 h; and a stirring rate is 100-200 rpm/min.

6. The preparation method of the bivalent inactivated vaccine for BTV according to claim 1, wherein in the step (3), an addition amount of the sodium thiosulfate is 0.6% (v/v) of the volume of the BTV suspension; and a molar concentration of the sodium thiosulfate is 1 mol/L.

Claims

7. The preparation method of the bivalent inactivated vaccine for BTV according to claim 1, wherein in the step (4), an addition amount of the PEG6000 is 5-9% of the weight of the inactivated BT antigen suspension; and the addition amount of the sodium chloride is 14% of the weight of the inactivated BT antigen suspension.

8. The preparation method of the bivalent inactivated vaccine for BTV according to claim 1, wherein in the step (4), the operation of resuspending the precipitate is as follows: 1/10 (v/v) of the total liquid of serum-free MEM cell culture fluid or 0.01 M of tris(hydroxymethyl)aminomethane hydrochloric acid with a pH of 7.2-7.4 is added into the precipitate.

9. The preparation method of the bivalent inactivated vaccine for BTV according to claim 1, wherein in the step (5), the mixed solution of white oil and Span-80 is prepared by uniformly mixing white oil and Span-80 according to a volume ratio of 47:3 and sterilizing the mixture at a high pressure for 15 min.

10. The preparation method of the bivalent inactivated vaccine for BTV according to claim 1 or 9, wherein the step (5) comprises: uniformly mixing 1 part of the concentrated and purified virus suspension and 2 parts of the mixed solution of white oil and Span-80; ultrasonically emulsifying the mixture to obtain emulsion; adding 6% of Tween-80 (v/v) and 0.01% of thimerosal (w/v) into 3 parts of the concentrated and purified virus suspension; fully and uniformly mixing the materials to prepare outer virus suspension; adding the outer virus suspension into the emulsion for ultrasonic emulsification again; preparing a single Bluetongue inactivated vaccine after the emulsification; and preserving the single Bluetongue inactivated vaccine at 4°C for later use.