JPH07561B2 - Bovine adenovirus type 7 vaccine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Use of the Invention] The present invention relates to a vaccine prepared by growing bovine adenovirus type 7 virus in tissue culture cells derived from goat and using the resulting virus suspension. Uses bovine adenovirus type 7 virus in goat-derived lung, liver, spleen, kidney,
By inoculating cultured cells derived from testis, thymus, thyroid, and other tissues and culturing or subculturing at 30 to 37 ° C,
Proliferate the virus to the same degree as tissue-derived cells derived from cows,
The present invention relates to a live vaccine or inactivated vaccine prepared by using the obtained virus suspension as a raw material.

[Background of the Invention]

Bovine adenovirus type 7 virus has the strongest pathogenicity to cattle among many types of bovine adenoviruses (type 9 and above), and when infected with cattle, a fever of 41 to 42 ° C is observed in the incubation period of 2 to 3 days. It lasts 5 to 7 days, and causes respiratory symptoms such as nasal discharge, tearing, and cough, or digestive symptoms accompanied by severe diarrhea toward the end of fever. Calves often develop these respiratory and digestive symptoms simultaneously and the mortality in such cases is high. In recent years, collective fattening of dairy Holstein male calves has been carried out, and serious damage has been observed. When the virus is infected during pregnancy, it causes fetal infection, which causes miscarriage or frail child syndrome. In addition, this virus is often another virus, such as bovine parainfluenza virus type 3, R-S virus, reovirus, rhinovirus, bovine infectious rhinotracheitis virus, bovine viral diarrhea, mucosal disease virus, bovine parvovirus, A mixed infection with two or three or more viruses such as bovine coronavirus and bovine rotavirus may occur, and the damage in such a case is further increased. Under such circumstances, there is an urgent need to develop an effective vaccine against this viral infection. Needless to say, for the production of the vaccine, it is necessary to highly propagate the virus by using animal material or cultured cells free from the contamination of the host animal with foreign pathogenic virus. It has not been proposed so far because the search for highly proliferated cultured cells and the like is not easy and has not been found yet. Therefore, calf testis cultured cells are mainly used for virus isolation from the field and attenuation of the isolated virus, and development of animal material or cultured cells suitable for vaccine production is desired.

[Object of the Invention]

From this point of view, the present invention has been made for the purpose of providing a culture method capable of highly proliferating bovine adenovirus using cultured cells derived from animals other than bovine.

Another object of the present invention is to provide a vaccine produced from the virus propagated by such a culture method.

[Outline of Invention]

According to the above-mentioned object, the present inventors examined the proliferative property of this virus using cultured cells derived from animals other than cattle, that is, guinea pig, hamster, pig, monkey and goat. According to this, good results were not obtained in cultured cells derived from guinea pigs, hamsters, and monkeys, and a small amount of virus was observed in primary testis cultured cells of pigs. It has not been successful in increasing proliferation. On the other hand, it was found that very good results were obtained with goat-derived cultured cells. From this point of view, the present inventor selected goat as an animal other than bovine, prepared cultured cells from the obtained tissue, and examined the proliferative ability of the virus in the cultured cells. Proliferates to the same extent as cells, and live vaccines and inactivated vaccines prepared from virus suspension obtained from main culture cells retain the same efficacy as vaccines obtained from cultured cells of bovine origin. That is, the present invention has been completed.

Hereinafter, the present invention will be described in more detail with reference to Examples.
The invention is not limited to the examples.

(1) Culturing of goat-derived tissue cells Cell culture methods derived from tissues of various animals are generally carried out by isolating cells with a digestive agent, suspending the isolated cells in a cell culture medium, and adjusting the body temperature of the host animal. Incubate at close temperature (36-37 ℃),
A monolayer culture method for forming a cell layer is used. In addition, the tissue is cut into pieces as much as possible, suspended in a culture medium while maintaining the properties derived from the tissue, and the temperature is close to the body temperature of the host animal. An organ culture method of culturing is used. The monolayer culture method includes static culture method, low speed rotation culture method, microcarrier culture method,
Applications such as the multi-layer culture method are possible. In the present invention, each of these culturing methods can be applied, but in this example, the stationary culture method of the monolayer culturing method was performed.

Better results are obtained when goat tissue for cell culture is obtained from a fetus or a goat less than 30 days old. Aseptically collect each tissue, cut into small pieces, suspend in approximately 200 volumes of 0.01M phosphate buffered saline (hereinafter referred to as PBS), wash the tissue with gentle agitation, and place in an appropriate centrifuge tube. Put, 500-8
Centrifuge at 00 rpm for 2 minutes, and collect the collected tissue for about 2 minutes.
The cells are suspended in 00 volumes of 0.25% trypsin PBS solution or 0.01% dispase PBS solution, and the cells are digested and isolated while stirring with a stirrer at 7 to 10 ° C or 30 to 32 ° C. Place the isolated cell suspension in a suitable sedimentation tube, 800-1000r
The isolated cells collected in the precipitate by centrifugation at pm for 2 minutes
About 100 times the amount of bovine adenovirus type 7 virus antibody-negative goat serum is suspended in an equilibrium phosphate buffered saline containing 5%, washed with stirring, and then centrifuged at 800 to 1000 rpm for 2 minutes. The isolated cells collected in the precipitate are washed as above. This procedure is repeated until the supernatant obtained by light centrifugation is almost transparent. Then, the isolated cells collected in the precipitate were added to a cell growth medium (goat serum 10% bovine adenovirus 7 virus antibody-negative, TPB (Tryptose Phosphate Broth)).
Eagle MEM (Mi) containing 0.295% and 1% 7% NaHCO ₃ solution
nimum Essential Medium) solution) 5-8 x 1 per 1 ml
0 ⁵ to float so that, aliquoted specified amount according to the volume of cell culture bottle min, allowed to stand for 4-5 days of incubation at 37 ° C. to form a cell layer. Cell subculture and cryopreservation are performed by a conventional method. Usually, these cultured cells can be passaged for 5 generations or more.

(2) Culturing of virus in goat-derived cultured cells Example 1 Culture of attenuated virus Lung, liver, spleen, kidney, thymus, and testis were collected from a postnatal day 7 goat and each cultured cell was subjected to the above cell culture method. For each test, dispense 10 ml into a 200 ml culture bottle and test at 37 ℃.
Second-passage cells cultured for a day were used. In addition, calf testis culture cells were used as control culture cells for virus propagation. As the virus strain, an attenuated virus BT-3 strain (distributed by the Ministry of Agriculture, Forestry and Fisheries Livestock Research Institute) was used which was subcultured to calf testis culture cells by a low temperature culture method at 30 ° C. For virus inoculation, remove the culture solution of each cultured cell, wash the cell surface once with PBS, and
^5.0 TCID ₅₀ / ml (50% tissue culture infectious virus amount in 1 ml)
1 ml each of the virus suspension was inoculated, and after sensitizing at 37 ° C for 60 minutes to adsorb the virus, the inoculum was removed, and the cell maintenance medium (bovine adenovirus 7 virus antibody-negative goat serum 2 %, TPB 0.295% and 7% NaHCO ₃ solution 2% in Eagle MEM solution) 10 ml each, and then 37 ℃ and 30 ℃
The appearance of CPE (Cyto Pathogenic Effect) was observed while statically culturing. The culture solution was collected on day 7 of culture at 37 ° C and on day 14 of culture at 30 ° C, and the virus content was measured using calf testis culture cells. Calf testis cultures used as control cultures were at 37 ° C on day 7,
The culture solution was collected on day 12 at 30 ° C. As can be seen from the results shown in Table 1 below, in 37 ° C. culture, 20 to 50% or more of CPE appeared in all cultured cells 7 days after virus inoculation, and the virus content in 1 ml of the culture solution was 10 ^5.8 , 10 ^6.0 , and 10 ^5.7 TCID in liver, spleen, and kidney showing 20% CPE, respectively.
_The lung, thymus, and testis with CPE of ₅₀ and 50% or more were 10 ^7.8 , 10 ^7.8 , and 10 ^7.6 TCID ₅₀ , respectively. The CPE of the control cultured cells was 80% or more, and the virus content in 1 ml of the culture was 10 ^7.8 TCID ₅₀ . In culture at 30 ℃, 20 to 50% of CPE appeared in lung, thymus, and testis on day 14 of culture, but CPE did not appear in other cultured cells. These cultures 1
The virus content per ml was 10 ^6.2 , 10 ^6.3 , 10 ^6.0 TCID ₅₀ , and CPE in the lung, thymus, and testis where CPE appeared, respectively.
10 ^3.5 and 1 in liver, spleen, and kidney, respectively
The values were 0 ^4.0 and 10 ^3.8 TCID ₅₀ . CPE of 30 ° C control culture cells
On day 12, it was 80% or more, and the virus content per ml of the culture was 10 ^6.4 TCID ₅₀ . The above results indicate that this virus proliferates in goat-derived cultured cells to the same extent as in bovine-derived cultured cells.

Example 2 Cultivation of virulent virus Lung, kidney, thyroid, thymus and testis were collected from a postnatal day 3 goat and each cultured cell was prepared by the above-mentioned cell culture method, and a 500 ml culture bottle was used for the test. Dispense 30 ml aliquots, 4 at 37 ℃
Second-passage cells cultured for a day were used. In addition, calf testis culture cells were used as control culture cells for virus propagation. The virus strain used was a virulent Fukuroi strain that is pathogenic to cattle (distributed by the Ministry of Agriculture, Forestry and Fisheries Livestock Experiment Station). For virus inoculation, remove the culture solution of each cultured cell, wash the cell surface once with PBS, inoculate 5 ml each of 10 ^5.0 TCID ₅₀ / ml virus suspension, and sensitize at 37 ° C for 60 minutes to infect the virus. After adsorbing, remove the inoculum and add 50 ml of cell maintenance medium,
The appearance of CPE was observed while statically culturing at 37 ° C. or,
A part of the culture solution was sampled on the 5th, 8th and 10th days after the virus inoculation, and the virus content was measured using a calf testis culture. As shown in Table 2 below, the appearance of CPE was observed in thymocytes from day 4 and in lung and thyroid cells from day 7, but not in kidney and testis cells until day 10 of culture. It was The peak of viral growth was observed around the 5th day in most of the cultured cells, and after 7th day, the virus content per ml of the culture solution derived from lung, thyroid, and thymus where CPE was observed was 10 ^{6.5 on the} 5th day. , 10 ^5.0 , 10 ^6.8 TCID ₅₀
On the 10th day, the ^values were 10 ^6.8 , 10 ^5.3 , and 10 ^6.8 TCID ₅₀ , respectively. In the culture medium of kidney and testis cells in which CPE did not appear, values of 10 ^5.0 and 10 ^6.0 TCID ₅₀ / ml were shown on the 10th day. In the calf testis culture cells used as a control, 3 after virus inoculation
CPE appeared from the day, and became 80% or more on the 8th day, and the virus content per 1 ml of the culture was 10 ^8.0 TCID ₅₀ . The above results show that the bovine adenovirus type 7 virulent virus Fukuroi strain grows in goat-derived cultured cells, but the extent thereof is shown to be slightly inferior to that of bovine-derived cultured cells.

Example 3 Passive acclimation of virulent virus In Example 2, since the proliferation of goat-derived cultured cells was inferior to that of bovine-derived cultured cells, it was shown that An attempt was made to subculture the goat-derived cultured cells. As the cultured cells used in the test, the lung, thymus and testis cells of the goat used in Example 2 were used at passages 4 to 8. The viruses used were those of each cultured cell line collected on the 10th day of culture in Example 2. 10 passages of virus
A virus suspension of ^4.7 to 10 ^5.3 TCID ₅₀ / ml was inoculated in the same manner as in Example 2, and after adsorbing, a culture solution was added and the mixture was incubated at 37 ° C.
The cells were cultured for 7 days at 7 to 10 generations. As a result, as shown in Table 3 below, in any of the cultured cells, the degree of CPE up to the 7th day of culture became stronger as the passage of the virus progressed, and in the lung cultured cells, from the 3rd passage. 50%, 80% or more from the 8th generation, 2% to 50% from the 2nd generation in thymic culture cells, 80% or more from the 6th generation, CPE did not appear in the 2nd generation in testis culture cells, but 20%, 6 5th generation
0%, 80% or more in the 8th generation. The virus content in the culture medium increased in parallel with the strong appearance of CPE, and was found in passage 7 and above in lung culture cells, passage 4 and above in thymocytes, and passage 7 in testis culture cells. All later 10 ^7.5 TCI
It was D ₅₀ / ml or more, and the maximum value was 10 ^8.0 TCID ₅₀ / ml at the 5th passage of thymocyte culture. The above results indicate that the virulent virus Fukuroi strain adapts to goat-derived cultured cells and proliferates to the same extent as in the case of bovine-derived cultured cells.

(3) Preparation of vaccine As a vaccine, a live vaccine using a live virus that has been attenuated so as not to show pathogenicity in a target animal, and an attenuated virus or a virulent virus that is killed by a drug or other method to immunize There is an inactivated vaccine that retains only the originality, and each of these examples will be described below.

Example 4 Preparation of Live Vaccine and Application to Cattle Preparation of Live Vaccine Each tissue of lung, liver, spleen, kidney, thymus, muscle, and skin was collected from a goat fetus of 137 days old, and the cells of (1) above were collected. Each cultured cell was prepared by the culture method. For the test, 30 ml each was dispensed into a 500 ml culture bottle and cultured at 37 ° C for 4 days. Passage 3
Each cell of the passage was used. The virus strain is the attenuated virus BT-
BT-3-GT obtained by further subculturing clones obtained by subculturing 3 strains of goat thymus cultured cells for 3 generations and further subcultured for 2 passages of the same cultured cells.
The h5 strain was used. For virus inoculation, remove the culture solution of each cultured cell, wash the cell surface once with PBS, inoculate 5 ml each of 10 ^5.3 TCID ₅₀ / ml virus suspension, and sensitize at 37 ° C for 60 minutes to obtain virus. After adsorbing, the inoculum was removed, 50 ml of each cell maintenance medium was added, and static culture was performed at 32-34 ° C. Since 20 to 80% of CPE appeared on the 7th day of culture, the culture solution was collected and mixed for each cultured cell. The virus content per 1 ml of each culture was 20-50% of CPE in the liver,
From cultured cells of spleen and kidney, 10 ^5.0 , 10 ^5.8 ,
Although it was 10 ^5.5 TCID ₅₀ , a high value of 10 ^7.5 to 10 ^7.7 was obtained from the other 4 types of cultured cells showing CPE of 80% or more. Mixing the above-obtained four types of cell culture medium,
The lightly centrifuged upper solution was used as a virus suspension for vaccine preparation, and this was divided into two parts. One was A stabilizer (10% lactose and 0.3% polyvinylpyrrolidone solution) and the other was B
Stabilizer (peptone 10% and polyvinylpyrrolidone 0.3
% Solution), added in 1 ml aliquots to 10 ml capacity vials, lyophilized and sealed under reduced pressure, the former was Lot No. 1 and the latter was Lot No. 2 to prepare 2 lots of live vaccine.
Dissolving the vaccine solution for dissolution of 10ml each with (0.01 M equilibrated phosphate buffered saline), a result of measuring the virus content with calves testicular cell cultures, LotNo.1 the 10 ^5.5 TCID ₅₀ / m
It was 10 ^5.6 TCID ₅₀ / ml for L and Lot No. 2. This grade is
Injecting approximately 10 ^3.0 TCID ₅₀ of the attenuated virus into cattle provides sufficient immunity, indicating that it is possible to produce high-titer live vaccine from virus suspension obtained from goat-derived cultured cells. .

Safety of Live Vaccine Two of each of the live vaccines Lot Nos. 1 and 2 prepared above were dissolved in 2 ml of the lysis solution (containing virus amount 10 ^6.8 and
10 ^6.9 TCID ₅₀ ) of the adenovirus type 7 virus antibody-negative calves weighing 178 to 193 kg were subcutaneously injected into each calf, and one calf cohabited as a non-injection control calf. These calves were observed daily for abnormalities in energy and appetite, observation of clinical symptoms such as presence or absence of nasal discharge and diarrhea, measurement of body temperature and white blood cell count for 14 days, and recovery of vaccine virus from blood, feces and nasal discharge for 10 days. . Further, the bovine hemagglutination inhibitory (HI) antibody titer in serum was measured on the 7th, 14th and 21st days after the vaccination. As a result, the test cows did not show any abnormality during the observation period, and neither the body temperature nor the white blood cell count was observed.
In addition, virus recovery from blood, feces and nasal discharge were all negative. HI antibody was detected in vaccinated cows from the 7th day, the values were 20 times and 40 times, and on the 14th day, 40 times and 80 times,
On the 21st day, all increased 160 times. On the other hand, no HI antibody was detected in the same cow 21 days after living together. The above results are shown in Table 4 below, and it is shown that the vaccine virus is safe to be injected into calves in an amount of about 5000 times the effective amount (10 ^3.0 TCID ₅₀ ).

Immunogenicity of live vaccine Lot No. 1 and 2 of the above-mentioned live vaccine were dissolved in 10 ml of the lysis solution, and further diluted 200-fold with the lysis solution to obtain a virus content of 10 ^3.2 per 1 ml in Lot No. 1. TCID ₅₀ and Lot No. 2 were adjusted to 10 ^3.3 TCID ₅₀ and used as injection materials. Five calves having a body weight of 167 to 235 kg and being negative for bovine adenovirus type 7 virus antibody were selected as test cows. LotN for two
1 ml each of o.1 diluted vaccine and 2 ml each of 1 ml of Lot No. 2 diluted vaccine were subcutaneously injected into 2 animals, and 1 animal was allowed to cohabit with vaccinated cows as a control for virulent virus challenge. These calves were observed daily for clinical symptoms such as abnormalities of appetite, presence of nasal discharge and diarrhea, measurement of body temperature and white blood cell count for 14 days, and HI and neutralizing antibody titers every 7 days until the 28th day. Was measured. In addition, these test cattle were challenged by subcutaneous injection of 10 ^7.0 TCID ₅₀ of the virulent virus Fukuroi strain on the 28th day after vaccination, observation of clinical symptoms, measurement of body temperature and white blood cell count, and attack from blood. The virus was recovered for 14 days, and the HI and neutralizing antibody titers were measured 7 and 14 days after the challenge. As shown in Table 5, no clinical signs, abnormalities in body temperature and white blood cell count due to vaccination were observed in any of the cattle. HI and neutralizing antibody responses were observed in 3 out of 4 (75%) 4 days after vaccination and in all 14 days after vaccination, during virulent virus challenge (28 days after vaccination) HI antibody 40 to 80 times, neutralizing antibody 16 to 64
Doubled values were detected. These vaccinated cows did not show any clinical symptoms, abnormalities in body temperature and white blood cell count after the virulent virus attack, the blood virus was also negatively detected, and no increase in antibody after the attack was observed. Ten
^It showed complete protection against an attacking virus of ^7.0 TCID ₅₀ .
On the other hand, the non-vaccinated control cattle showed typical adenovirus type 7 infection symptoms and survived, and high HI and neutralizing antibody were produced in the serum. The above results show that 10 ^3.0 TCID of the live vaccine produced using goat-derived cultured cells as a host.
_It has been shown that an injection virus amount of about ₅₀ protects against the infection of field virulent virus after about 1 month.

Example 5 Preparation of inactivated vaccine and application to cattle Preparation of inactivated vaccine Each tissue of lung, thymus and testis was collected from a 30 day old goat and each cultured cell was prepared by the cell culture method of the above (1). Prepared. For the test, 30 ml of each was dispensed into a 500 ml culture bottle and cultured at 37 ° C. for 5 days, and the cells at the third passage were used.
As the virus strain, the virulent virus Fukuroi strain of the 7th generation of goat testis culture cell passage was used. For virus inoculation, remove the culture solution of each cultured cell, wash the cell surface once with PBS, and then
^5.5 TCID ₅₀ / ml virus suspension was inoculated in 5ml aliquots at 37 ℃
After sensitizing the cells for 60 minutes to adsorb the virus, remove the inoculum, add 50 ml each of the cell culture medium, incubate at 37 ° C, and incubate the cells 7 days after virus inoculation. The culture solution was collected and mixed. Then, after a light centrifugation, a part of the supernatant was used for virus content measurement, and the balance was 0.2% formalin.
% And inactivated at 4 ° C. for 7 days. 1/10 volume of aluminum phosphate gel was added to the inactivated virus suspension, and after 2 days, 30 ml each was dispensed into 30 ml vials and sealed. Lot No. 1 and 2 were derived from lung, thymus and testis culture cells in this order. , 3
And 3 lots of inactivated vaccine were prepared. The virus contents of these virus suspensions for inactivated vaccine preparation before inactivation were 10 ^7.5 , 10 ^8.0 , and 10 ^8.0 TCID ₅₀ , respectively. The above results indicate that the inactivated vaccine contains a sufficient amount of antigen. In addition, it was also confirmed that the inactivated vaccine prepared using goat-derived liver, spleen, muscle, and skin tissues contained a sufficient amount of antigen.

Safety of killed vaccine 3 lots of killed vaccine prototyped in the previous section were weighed between 210 and 2
30 kg of bovine adenovirus type 7 virus antibody-negative calf 1
Inject 10 ml each into the cervical muscle of each head, observe clinical symptoms such as abnormal health / appetite, presence of nasal discharge and diarrhea, and measure body temperature for 14 days.
HI antibody titers were measured on days 14, 14 and 21. The results are shown in Table 6 below, and the test cattle passed without any abnormality during the observation period. HI antibody in serum was not detected until 14 days after vaccination, but in all cases on day 21
20 to 80 times higher values were detected. This result indicates that the virulent virus in this inactivated vaccine is completely inactivated and safe for calves.

Immunogenicity of inactivated vaccine 3 lots of inactivated vaccine prepared as a trial in the previous section 2- (1)
2 calves of 178 to 213 kg bovine adenovirus type 7 antibody-negative calves were each intramuscularly injected twice with a volume of 3 ml each at 28-day intervals, and the presence / absence of health / appetite abnormalities, nasal discharge and diarrhea, etc. Observation of clinical symptoms and temperature measurement after vaccination
The test was performed for 14 days, and HI and neutralizing antibody in serum were measured immediately before the first injection of the vaccine, immediately before the second injection, and 14 days after the second injection. As shown in Table 7, the test cows showed no abnormality during the observation period, and 10-40 times higher HI antibody and 2 were observed immediately before the second vaccine injection.
~ 16-fold neutralizing antibody was detected. The HI and neutralizing antibody titers were further increased 14 days after the second injection of the vaccine, and the HI antibody showed a value of 40 to 320 times and the neutralizing antibody showed a value of 32 to 256 times. The above results indicate that this inactivated vaccine retains effective immunogenicity.

EFFECTS OF THE INVENTION According to the present invention, for the first time, bovine adenovirus type 7 virus can be cultivated and propagated using tissue culture of animals other than bovine (goat-derived), and at the same level as a vaccine obtained from cultured cells derived from bovine. Since an effective vaccine can be prepared from the virus suspension obtained from this goat-derived cultured cell, this provides for the first time an effective vaccine against this viral infectious disease, which is free of host virus-derived pathogenic viruses. The effect of being able to do is obtained.

Continuation of the front page (56) Reference JP-A-50-18619 (JP, A) JP-A-48-13519 (JP, A) JP-A-57-56431 (JP, A) JP-B-48-2767 (JP , B1) Japanese Patent Publication Sho 49-40924 (JP, B1)

Claims (1)

[Claims] 1. A bovine adenovirus type 7 virus is grown using tissue culture cells derived from goat, and the virus suspension obtained is subcultured or inactivated. Characterized live or inactivated bovine adenovirus type 7 vaccine.