KR100622399B1 - Vaccine Comprising the Mixture of Inactivated TGEV and PEDV, and Preparation Method thereof - Google Patents

Abstract

The present invention provides a mixed inactivating vaccine having antigens of inactivated TGEV and PEDV. In addition, the present invention (1) culturing the TGEV in ST cells, culturing the PEDV in Vero cells; (2) separating the supernatant containing the high titer TGEV and PEDV (10 ^7.0 TCID ₅₀ / ml or more) by centrifuging the culture solution of step 1; (3) inactivating each virus by adding a virus inactivator to the supernatant obtained in step 2; (4) inoculating the inactivated culture solution into ST and Vero cells to confirm the presence of cell degeneration; And (5) provides a method of producing a mixed inactivating vaccine comprising the step of mixing the fluorinated virus culture solution. In addition, the present invention is characterized in that the inactivated vaccine according to the present invention is inoculated twice twice each 5 to 7 weeks before and 2 to 3 weeks before the delivery of pigs to deliver TGEV and PEDV antibodies from colostrum of sows to piglets. Provided is a method for preventing swine viral diarrhea.

According to the above configuration, it is possible to sufficiently prevent TGEV and PEDV, which are the main causes of major viral viral diarrheal disease, and to minimize the damage caused by diarrheal disease in piglets, while having excellent safety.

Description

Vaccine Comprising the Mixture of Inactivated TGEV and PEDV, and Preparation Method Etc.             

1 is a manufacturing process diagram of a mixed inactivating vaccine presented in a preferred embodiment according to the present invention

Figure 2 shows the results of safety investigation (temperature change) in sows after vaccination according to the present invention. [One. Temperature unit: ℃, 2. 39, 40, 77, 79: sow number]

Figure 3 is a diagram showing a neutralizing antibody titer of the change in the antibody of the outdoor pregnant sows inoculated with the vaccine according to the present invention.

The present invention relates to a vaccine for the prevention of swine viral diarrheal diseases and a method for producing the same, and more particularly to a mixed inactivating vaccine for the prevention of disease caused by TGEV and PEDV, the main cause of the disease It is about.

TGE and PED are diarrheal diseases caused by the transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). It is characterized by high mortality and morbidity due to dehydration.

TGEV is estimated to have spread domestically from imported bovine pigs in Gyeonggi-do in 1954. Since then, it has been consistently occurring in winter. Since PEDV was first reported in Korea in 1993, TGE and PED have been occurring regardless of season. In recent years, PEDs have been much higher than TGE, causing economic damage to hog farmers. According to data released by the National Veterinary Research and Quarantine Service during December 2003, 79 PED cases were reported and TGE was 2 cases.

In order to prevent such diarrheal disease, so far, based on passive immunization, which delivers the antibody formed by inoculating live sows before delivery to the sows through colostrum. Domestic vaccine producers produce and sell live PED vaccines. Japanese foreign PED vaccines and PED / TGE mixed vaccines are imported. However, in the case of live vaccines, in particular, the incidence of diarrhea in piglets is increasing every year due to the lack of vaccination due to the high distrust of preventive efficacy in outdoor farms for PEDV.

Accordingly, in order to effectively prevent the diseases, it is necessary to deliver sufficient protective antibodies to colostrum in mammals through colostrum. To do this, effective sows can be given to sows, and an effective vaccine that can be used safely is urgently needed.

The present invention has been made to solve the problems of the prior art as described above, its purpose is to sufficiently prevent TGEV and PEDV, the main cause of major viral viral diarrheal disease, while excellent safety and damage caused by diarrheal disease in piglets It is to provide a combination inactivating vaccine that is possible to minimize the.

Another object of the present invention is to provide a method for preparing a mixed inactivating vaccine for the prevention of TGEV and PEDV, the causative agent of porcine viral diarrhea.

Still another object of the present invention is to provide a method for preventing swine viral diarrhea in which protective antibodies against TGEV and PEDV, which are the causes of swine viral diarrhea, are effectively delivered to piglets.

As a means for achieving the above object, the present invention provides a mixed inactivating vaccine comprising the inactivated TGEV and PEDV as an antigen.

The present invention preferably provides a mixed inactivating vaccine in which an immunopotentiator is added to the vaccine.

The present invention preferably provides a mixed inactivating vaccine in which the antigen is used to inactivate each virus in TGEV culture cultured in ST cells and PEDV culture cultured in Vero cells.

Preferably, the culture is inoculated with the virus by inoculating each cell, and then cultured by adding serum-free α-MEM containing 5-10 μg / ml trypsin to the cell maintenance medium. Provided is a mixed inactivating vaccine comprising a supernatant obtained by freeze-thawing and centrifugation when the cytopathic effect of.

In addition, the present invention

(1) culturing TGEV in ST cells and culturing PEDV in Vero cells;

(2) separating the supernatant containing TGEV and PEDV by centrifuging the culture solution of step 1;

(3) inactivating each virus by adding a virus inactivator to the supernatant obtained in step 2;

(4) inoculating the inactivated culture solution into ST and Vero cells to confirm the presence of cell degeneration; And

(5) It provides a method of producing a mixed inactivating vaccine comprising the step of mixing the fluorinated virus culture solution.

The present invention preferably provides a method of adding an immunopotentiator in step 5.

Preferably, the supernatant of step 2 is inoculated with each cell to inoculate a virus and then cultured by adding serum-free α-MEM containing 5-10 μg / ml trypsin as a cell maintenance medium. A supernatant obtained by lyolysis and centrifugation when ˜90% of the cytopathic effect appears (viral titer is 10 ^7.0 TCID ₅₀ / ml or more in both TGEV and PEDV) is provided.

In addition, the present invention was inoculated twice (preferably about 3 ml each) of the mixed inactivation vaccine according to the present invention 5 to 7 weeks before and 2 to 3 weeks after delivery of the pigs so that TGEV and PEDV antibodies from colostrum of sows. It provides a method for preventing swine viral diarrhea, characterized in that it is delivered to the piglets.

Hereinafter, the content of the present invention in more detail as follows.

1 is a manufacturing process / application of the inactivated vaccine shown in a preferred embodiment according to the present invention. Referring to this, in the preparation of the mixed vaccine according to the present invention, first, to isolate the TGE and PED virus in the feces and intestine of pigs with outdoor diarrhea in order to obtain the virus to be used as the antigen of the inactivated vaccine, through passage And propagating the virus, and then inactivating the virus contained in the culture medium, mixing the two inactivated viruses with an immunostimulant, and sharing the disease.

Inactivated TGEV and PEDV do not use live viruses and thus can dispel the distrust of preventive efficacy in outdoor farms.

TGEV and PEDV can be isolated from pig feces or intestines of farmers with swine onset of diarrhea. For these cultures, it is preferable to use swine testicle cell lines (hereinafter, abbreviated as ST) in the case of TGEV, and monkey kidney cells (hereafter Vero) in the case of PEDV. Preference is given to using. Each virus cultivated using the cells can be usefully used as an antigen presentation excellent in antibody type performance.

The culturing process preferably includes the step of subcultured while adding trypsin to the medium while maintaining proliferation and immunogenicity. The concentration of trypsin is not particularly limited, but is preferably about 5 to 10 µg / ml.

In order to obtain a virus from the culture medium, the obtained culture solution is freeze-thawed when a cytopathic effect of 80-90% is obtained, and the supernatant obtained after centrifugation is separated. The separated supernatant contains the respective viruses. At this time, the virus titer is more than 10 ^7.0 TCID ₅₀ / ml in both TGEV and PEDV.

In order to inactivate the virus contained in the supernatant, a predetermined inactivating agent is added. High titers of the virus can be effectively inactivated by binary ethyleneamine (BEI). The concentration of the inactivating agent may vary depending on the titer of the virus contained in the supernatant, but it is preferable to adjust the final concentration to 0.001M by adding BEI, for example, 0.1M.

Thereafter, the inactivation reaction includes agitation and stirring at 37 ° C. for about 10 hours by adjusting the pH of the supernatant to pH 8.0, which is a weakly alkaline region.

After inactivation is performed, 2M sodium thiosulfate is added to neutralize the inactivating agent used and stirred at 37 ° C. for about 1 hour. Next, the pH of the supernatant is adjusted to about 7.0 to 7.5, and the inactivated TGEV and PEDV cultures are inoculated into ST and Vero cells, respectively, to confirm whether the virus is inactivated or not.

The mixed vaccine according to the present invention includes a process of mixing the culture medium of the two inactivated viruses. The composition ratio of the two cultures at the time of mixing does not require a special limitation, but preferably in the same amount, it is good to add a predetermined immune enhancer for the immune boosting. Examples of such adjuvants are various, for example, IMS1313 from Seppic, France.

The mixed inactivated vaccine obtained through the above process may be administered by oral or intramuscular inoculation. The dose of vaccine is sufficient to inoculate twice (preferably about 3 ml each) 5-7 weeks before delivery and 2-3 weeks before delivery. In the above case it can be confirmed through the embodiment of the present invention that the TGEV and PEDV antibodies can be effectively delivered to piglets from colostrum of sows. In addition, the mixed inactivation vaccine according to the present invention has been proved to be safe in the field test, it can be confirmed that it can maintain a sufficient protective antibody can completely resolve the high confidence in the live vaccine administration of the farm.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Preparation of Mixed Inactivation Vaccine

1. Virus isolation and propagation

TGEV isolated from field diarrheal disease-producing farms were passaged in 34 generations from ST cells, and a strain (named 175L strain) having excellent proliferation and antigenicity was selected and disclosed as a vaccine strain. PEDV was a vaccine strain that selected a strain (named SM98P strain) with excellent proliferative and antigenic properties by culturing 110 generations of vero cells from three-day-old mammal pigs with severe diarrhea.

TGEV cultures used ST cells, and PED virus cultures used vero cells. Cell proliferation medium contained 5% fetal bovine serum (FBS, Gibco BRL), penicillin (10,000 units / ml) and streptomycin sulfate in α-MEM (α-minimum essential medium, Gibco-BRL, USA). (10,000mg / ml) and amphotericin B (25ug / ml) were added and used, and the cell maintenance medium was prepared using the same composition as the growth medium except for FBS. Monolayers of ST and vero cells were washed three times with PBS, and each cell was inoculated with TGEV and PEDV. After inoculation for 1 hour at 37 ° C, serum-free α-MEM containing 5-10ug / ml trypsin (Difco, USA) was added to the cell maintenance medium and cultured at 37 ° C for 1-2 days, followed by 80-90% When the cytopathic effect of the cells were lyophilized three times and centrifuged at 3,000 g for 20 minutes, the supernatant was used for vaccine production. At this time, the viral titers of TGEV and PEDV are all 10 ^7.0 TCID ₅₀ / ml or more.

2. Virus inactivation

TGEV and PEDV cultures were inactivated with BEI according to the method of Girard et al. (1977, Bull. Off. Int. Epizoot. Inactivation of O1 FMD virus by the binary ethylene imine (BEI)). That is, 2% 2-bromoethylamine hydrobromide (2-BEA) was dissolved in 0.2N NaOH solution and treated for 1 hour in a 37 ° C constant temperature water bath to prepare 0.1M of BEI. 0.1M BEI was added to the virus culture to adjust the final concentration to 0.001M, and then the virus culture was adjusted to pH8.0 with NaOH solution to inactivate the virus while shaking and stirring at 37 ° C for 10 hours. After inactivation, sterilized 2M sodium thiosulfate was added to a final concentration of 2 mM, stirred at 37 ° C. for 1 hour to neutralize BEI, and then the pH was adjusted to 7.0 to 7.5. The inactivated TGE and PED cultures were added to ST and vero cells. Each inoculation was confirmed for virus inactivation with or without cytopathic effect and then used for vaccine production.

3. Preparation of the vaccine

The same amount of adjuvant was mixed with the inactivated TGEV and PEDV cultures, stirred for 30 minutes or more, and then subdivided and stored in a cold dark place at 4 ° C to investigate the safety, immunogenicity and shelf life of the test vaccine. The immunopotentiator used in the manufacture of the test bag is IMS1313 from Seppic, France.

Experimental Example 1 Vaccine Safety and Immunogenicity

The safety of the vaccine was determined in mice, guinea pigs and 90-day-old pigs, and the changes in body temperature and feed intake were examined in pregnant sows.

Mice were inoculated with 0.5 ml of 10 mice each in the abdominal cavity and guinea pigs were inoculated with 2 ml of 4 vaccines. Six pigs were inoculated into the muscles of the muscles of the muscles, twice the amount of outdoor use, and 3ml of the first sows were inoculated into pregnant sows. After inoculation, mice and guinea pigs observed clinical symptoms for 14 days, and raised pigs for 21 days, but no mortality or other side effects were observed (see Table 1). Changes were measured to measure the thermal response following vaccination, but normal body temperature and diarrhea and mortality of 38-39 ° C. were not observed, confirming safety (see FIG. 2).

Table 1 Safety investigation of TGE, PED inactivated combination vaccine

animal Inoculation head Inoculation amount / inoculation site Clinical observation (14-21 days) mouse 10 0.5ml / IP clear Guinea pig 4 2ml / IP clear Cultivation money 3 6ml / IM clear

The immunogenicity of the vaccine against guinea pigs was immunized twice with 3 ml intervals of 1 ml each of 8 guinea pigs, and blood samples were collected at 2 and 4 weeks after inoculation to measure serum neutralizing antibody titers. Immunogenicity against gestational sows was inoculated with 3 ml of muscle muscles at 5-7 weeks before delivery and 2 to 3 weeks before delivery. In guinea pigs it showed a high antibody rise as shown in Table 2 below. In pregnant sows, TGEV serum neutralized antibody titers were 256-512 times in colostrum, 512-8,192 times in colostrum, and in the case of PEDV, they were 64-256 times in serum and 256-512 in colostrum (Tables 3 and 4).

 Table 2: Investigation of immunogenicity in guinea pigs of TGE, PED inactivated combination vaccine

virus Inoculation head Neutralizing antibodies after vaccination (log ₂ ) 3 weeks after the first vaccination 2 weeks after the 2nd vaccination TGEV 8 2.13 ± 1.73 9.13 ± 1.13 PEDV 8 0.40 ± 0.70 4.50 ± 1.60 Control 6 0 0

<Table 3> Immunogenicity of TGEV in Pregnant Pigs with TGE and PED Mixed Inactivation Vaccine

division Sows TGEV SN Titer number Before vaccination After the first After the second Delivery time 10 days after attack beestings 10 days Public money ^* 413 <2 8 64 512 2048 1024 8 4195 <2 2 8 256 2048 1024 4 Outdoor sows 50 128 128 512 256 512 8192 64 424 256 256 512 256 256 1024 NT 4193 32 32 512 512 256 512 8 Contrast money 10 <2 <2 <2 <2 512 <8 <8

 *: Inoculated with 3ml each for 5-7 weeks and 2-3 weeks before delivery.

<Table 4> Immunogenicity of PEDV in Pregnant Pigs with TGE and PED Mixed Inactivation Vaccine

division Sows PEDV SN Titer number Before vaccination After the first After the second Delivery time After attack beestings 10 days Public money ^* 451 <2 4 32 128 64 256 16 470 <2 8 64 128 128 512 16 Outdoor sows 517 64 NT NT 256 256 512 32 614 32 NT NT 64 128 512 16 656 64 NT NT 256 256 512 16 Control 19 <2 <2 <2 <2 128 <8 <8

 *: Inoculated with 3ml each for 5-7 weeks and 2-3 weeks before delivery.

Experimental Example 2 Efficacy Test of Vaccine

Maternal pigs vaccinated 5-7 weeks before delivery and 2-3 weeks before delivery were given oral inoculation with 1 ml of outdoor poultry TGEV (all 3 weeks) and PEDV (CJ98P) in control sows. The clinical symptoms and mortality were examined to determine the protective effect of the vaccine against mammalian pigs. It was found that the piglets had a 100% survival rate and had a protective effect against TGEV in the mammal pigs of the vaccine. When challenged with PEDV, all piglets survived, while the control pigs showed a 37.5% survival rate, indicating a protective effect against PEDV (Tables 5 and 6).

Table 5 Investigation of TGEV Protective Effects of Piglets in Vaccine Sows

division Sows Attack ^* Attack After challenge Weight change number Head Age Snowy exchange rate Survival Rate (%) ^** Attack After attack (10 days) Public announcement 413 14 4 14/14 (100) 10/14 (71.4) 2.10 2.67 4195  11 6 11/11 (100) 9/11 (81.8) 2.17 2.75 Outdoor sow 50 9 4 3/9 (33.3) 9/9 (100) 1.76 3.41 424 4 2 0/4 (0) NT 1.20 NT 4193 7 3 2/7 (28.6) 7/7 (100) 1.65 2.73 Counter money 10 9 4 9/9 (100) 2/9 (22.2) 1.31 1.40

* Oral inoculation of 1 ml (10 ^4.0 PID ₅₀ / ml) of TGEV (all 3) Poisoned Poultry

  ** Survival head up to 10 days after challenge

<Table 6> Investigation of PEDV Protective Effect of Piglets in Sows Containing TGE and PED Mixed Inactivation Vaccination

division Sows Attack ^* Attack After challenge Weight change (kg) number Head Age Snowy exchange rate Survival Rate (%) ^** Attack After attack (10 days) Public announcement 451 10 4 0/10 (0) 10/10 (100) 1.37 2.64 451  8 3 0/8 (0) 8/8 (100) 1.39 2.64 Outdoor sow 547 8 4 0/8 (0) 8/8 (100) 1.85 3.0 614 12 3 0/12 (0) 12/12 (100) 1.79 2.94 656 9 5 5/9 (55.5) 9/9 (100) 1.78 2.88 Counter money 19 8 3 8/8 (100) 3/8 (37.5) 1.45 1.39

* Oral inoculation of 1 ml (10 ^4.0 PID ₅₀ / ml) of PEDV (CJ98P) Poisoned Poultry

  ** Survival head up to 10 days after challenge

Experimental Example 3 Preservation Test of Vaccine

In order to investigate the preservation of the vaccine, the vaccine was stored at 4 ° C. in a dark place for 12 months, and 5 ml of guinea pigs were inoculated twice every 3 weeks at 3 ml intervals for 5 guinea pigs. Examination of the preservation of the shelf life of 12 months or more confirmed (Table 7).

<Table 7> Conservation test of vaccine

Guinea pig inoculation Retention period and neutralizing antibody value (log ₂ ) January June December TGEV PEDV TGEV PEDV TGEV PEDV 5 7.25 ± 2.10 5.25 ± 0.96 7.67 ± 0.58 5.16 ± 0.40 7.56 ± 1.51 5.44 ± 1.13

Experimental Example 4 Safety and Immunogenicity Test in the Field Test

Referring to FIG. 3, the antibody value was 16-fold before vaccination, but the antibody was elevated and maintained at 512-fold antibody titer to TGE and 64-fold to PED after inoculation of the vaccine according to the present invention. You can check it. This shows that the vaccine according to the present invention maintains sufficient protective antibodies even in field tests. In addition, 6500 pregnant sows were tested for safety, but a slight fever after inoculation resulted in a slight inoculation reaction with a longer feed intake interval.

Table 8 Field safety survey of vaccines

area Farm water  Sows Safety Investigation on Pregnancy Pigs after Vaccination Game 3 1,100  -Delayed feed intake time after inoculation (difference between farms)-no side effects such as similar acids                                                                                                                                                                                    Chungnam One 500 Gyeongbuk 3 1,490 Gyeongnam 10 1,930 Former north 4 1,370 Jeju 3 650 system 23 6,500

The vaccine according to the present invention can sufficiently prevent TGEV and PEDV, which are the main causes of major viral viral diarrheal diseases, while minimizing the damage caused by diarrheal disease in piglets with excellent safety. Experimental results also show that the protective antibodies are effectively delivered to the piglets in the inoculation of pregnant sows twice each, about 3ml 5-7 weeks before delivery and 2-3 weeks before delivery, confirming that the vaccine is a useful vaccine.

As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the present invention described in the claims below. It will be appreciated that it can be changed.

Claims (8)

Mixed inactivation vaccines with antigens of high titer TGEV and PEDV greater than 10 ^7.0 TCID ₅₀ / mL inactivated The vaccine of claim 1, further comprising an immunopotentiator. 2. The vaccine according to claim 1, wherein the antigen is one in which each virus is inactivated in a TGEV culture cultured in ST cells and a PEDV culture cultured in Vero cells. According to claim 3, wherein the culture was inoculated and adsorbed to each of the cells, and then cultured by adding a serum-free α-MEM containing 5-10μg / ml trypsin as a cell maintenance medium 80-90% Vaccine, characterized in that the supernatant obtained by lyolysis and centrifugation when the cytopathic effect of (1) inoculating TGEV into ST cells, inoculating PEDV into Vero cells, and then adsorbing serum-free α-MEM containing 5-10 μg / mL trypsin as a cell maintenance medium; (2) freezing and thawing when cultured in step 1 showed a cytopathic effect of 80-90%, and centrifuging to obtain a supernatant containing TGEV and PEDV having a titer of at least 10 ^7.0 TCID ₅₀ / mL; (3) inactivating each virus by adding a virus inactivator to the supernatant obtained in step 2; (4) inoculating the inactivated culture solution into ST and Vero cells to confirm the presence of cell degeneration; And (5) a method of producing a mixed inactivating vaccine, comprising mixing the inactivated virus culture solution 6. A process according to claim 5, wherein an immunopotentiator is added in step 5. delete delete

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