JPH07561B2 - Bovine adenovirus type 7 vaccine - Google Patents
Bovine adenovirus type 7 vaccineInfo
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- JPH07561B2 JPH07561B2 JP59190099A JP19009984A JPH07561B2 JP H07561 B2 JPH07561 B2 JP H07561B2 JP 59190099 A JP59190099 A JP 59190099A JP 19009984 A JP19009984 A JP 19009984A JP H07561 B2 JPH07561 B2 JP H07561B2
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Description
【発明の詳細な説明】 〔発明の利用分野〕 本発明は、牛アデノウイルス7型ウイルスを山羊由来の
組織培養細胞で増殖させ、得られたウイルス浮遊液を用
いて調製したワクチンに関し、さらに詳しくは、牛アデ
ノウイルス7型ウイルスを山羊由来の肺,肝,脾,腎,
精巣,胸腺,甲状腺,その他の組織由来の培養細胞に接
種し、30〜37℃で培養又は継代順化させることにより、
牛由来の組織培養細胞と同程度にウイルスを増殖させ、
得られたウイルス浮遊液を原材料として調製された生ワ
クチン又は不活化ワクチンに関するものである。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.
牛アデノウイルス7型ウイルスは牛アデノウイルスの多
くの型(9型以上)の中で牛に対する病原性がもっとも
強く、牛に感染すると2〜3日の潜伏期で41〜42℃の発
熱がみられ、5〜7日間持続し、鼻汁,流涙,咳等の呼
吸器症状、又は発熱末期にかけて激しい下痢をともなっ
た消化器症状を発症させる。幼牛はこれらの呼吸器症状
及び消化器症状を同時に発現する場合が多く、このよう
な場合の死亡率は高い。近年、乳用ホルスタイン種雄子
牛の集団的な肉用肥育が行われるようになり、大きな被
害がみられている。本ウイルスは妊娠中に感染すると胎
児感染を起し、流産又は虚弱子症候群の原因ともなって
いる。さらに、本ウイルスはしばしば他のウイルス、例
えば、牛のパラインフルエンザウイルス3型,アールエ
スウイルス,レオウイルス,ライノウイルス,牛伝染性
鼻気管炎ウイルス,牛ウイルス性下痢,粘膜病ウイル
ス,牛パルボウイルス,牛コロナウイルス,牛ロタウイ
ルス等との2種又は3種以上のウイルスとの混合感染を
起すことがあり、このような場合の被害はさらに大きく
なる。このような状況のなかで本ウイルス感染症に対す
る有効なワクチンの開発が急務とされている。言うまで
もなく、ワクチンの製造については、宿主動物に対する
外来の病原ウイルスの混入のない動物材料又は培養細胞
を用い、ウイルスを高度に増殖させる必要があるが、本
ウイルスを牛以外の動物又は培養細胞で高度に増殖させ
る培養細胞等の検索は容易でなく、実際にも未だ見出さ
れていないためこれまでに提案されていない。したがっ
て、野外からのウイルス分離や分離ウイルスの弱毒化に
は、もっぱら子牛精巣培養細胞が用いられており、ワク
チンの製造に適する動物材料又は培養細胞の開発が望ま
れている。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.
本発明はかかる観点から、牛以外の動物由来の培養細胞
を用いて高度に牛アデノウイルスを増殖させることので
きる培養法の提供を目的としてなされたものである。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.
また本発明の他の目的は、かかる培養法によって増殖さ
れたウイルスより製造されたワクチンを提供するところ
にある。Another object of the present invention is to provide a vaccine produced from the virus propagated by such a culture method.
前記した目的に従って、本発明者等は牛以外の動物、す
なわち、モルモット,ハムスター,豚,サル及び山羊由
来の培養細胞を用いて、本ウイルスの増殖性を検討し
た。これによると、モルモット,ハムスター及びサル由
来の培養細胞では、いずれも良好な成績は得られず、豚
の精巣初代培養細胞では少量のウイルス増殖がみられた
が、継代順化によってもウイルスの増殖性を高めること
には成功していない。これに対し、山羊由来の培養細胞
では非常に良好な結果が得られることを知見した。かか
る観点から、本発明者は牛以外の動物として山羊を選
び、得られた組織から培養細胞を調製し、同培養細胞で
の本ウイルスの増殖性を検討したところ、本ウイルスが
牛由来の培養細胞と同程度に増殖すること、さらには本
培養細胞から得られたウイルス浮遊液から調製した生ワ
クチン及び不活化ワクチンが牛由来の培養細胞から得ら
れたワクチンと同程度の有効性を保持していることを見
い出し本発明をなすに至ったものである。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.
以下本発明を実施例に基づいて更に詳細に説明するが、
本発明は実施例のみに限定されるものではない。Hereinafter, the present invention will be described in more detail with reference to Examples.
The invention is not limited to the examples.
(1)山羊由来組織細胞の培養 各種動物の組織由来の細胞培養法は、一般的には細胞を
消化剤で単離させ、その単離細胞を細胞培養液に浮遊さ
せ、宿主動物の体温に近い温度(36〜37℃)で培養し、
細胞層を形成させる単層培養法が用いられ、他に、組織
を可能な限り細切し、その組織由来の性状を保持させた
状態で培養液に浮遊させ、宿主動物の体温に近い温度で
培養する器官培養法が用いられる。単層培養法には静置
培養法,低速回転培養法,マイクロキャリアー培養法,
多重層培養法等の応用が可能である。本発明ではこれら
の各培養法が適用可能であるが、本例では単層培養法の
静置培養法を行なった。(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.
細胞培養用の山羊組織は胎児又は生後30日以内の子山羊
から得たものが、より良好な結果が得られる。各組織を
無菌的に採取し、細切した後、約200倍量の0.01Mリン酸
緩衝食塩液(以下PBSという)に浮遊させ、軽く撹拌し
ながら組織を洗浄し、適当な遠心沈殿管に入れ、500〜8
00rpmで2分間遠心分離し、沈殿に集められた組織を約2
00倍量の0.25%トリプシンPBS溶液、又は0.01%ディス
パーゼPBS溶液中に浮遊させ、7〜10℃又は30〜32℃で
スターラー撹拌しながら細胞をばらばらに消化単離させ
る。単離細胞浮遊液を適当な沈殿管に入れ、800〜1000r
pmで2分間遠心分離し、沈殿に集められた単離細胞を、
約100倍量の牛アデノウイルス7型ウイルス抗体陰性の
山羊血清を5%の割合に含む平衡リン酸緩衝食塩液に浮
遊させ、撹拌洗浄後、800〜1000rpmで2分間遠心分離す
る。沈殿に集められた単離細胞を上記同様にして洗浄す
る。この操作は軽遠心した上清がほとんど透明になるま
でくりかえす。ついで、沈殿に集められた単離細胞を細
胞増殖用培養液(牛アデノウイルス7型ウイルス抗体陰
性の山羊血清10%、TPB(Tryptose Phosphate Broth)
0.295%及び7%NaHCO3溶液1%を含むイーグルMEM(Mi
nimum Essential Medium)溶液)に1ml当たり5〜8×1
05個になるように浮遊し、細胞培養びんの容量にしたが
って規定量ずつ分注し、37℃で4〜5日間静置培養して
細胞層を形成させる。細胞の継代及び凍結保存は常法に
よって行う。通常これらの培養細胞は5代以上の継代が
可能である。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)ウイルスの山羊由来培養細胞での培養 実施例1 弱毒ウイルスの培養 生後7日の子山羊から肺,肝,脾,腎,胸腺及び精巣を
採取し、前記の細胞培養法で各培養細胞を調製し、試験
には200ml容量の培養びんに10mlずつ分注し、37℃で4
日間培養した継代2代目の細胞を用いた。又、ウイルス
増殖用の対照培養細胞として子牛精巣培養細胞を用い
た。ウイルス株は子牛精巣培養細胞に30℃の低温培養法
で継代順化された弱毒ウイルスBT−3株(農林水産省家
畜試験場より分与)を用いた。ウイルス接種は各培養細
胞の培養液を除去し、PBSで細胞表面を1回洗浄後、10
5.0TCID50/ml(1ml中の50%組織培養感染ウイルス量)
のウイルス浮遊液を1mlずつ接種し、37℃で60分間感作
してウイルスを吸着させた後、接種材料を除去し、細胞
維持用培養液(牛アデノウイルス7型ウイルス抗体陰性
の山羊血清2%、TPB0.295%及び7%NaHCO3溶液2%を
含むイーグルMEM溶液)を10mlずつ注加後、37℃と30℃
で静置培養しながらCPE(Cyto Pathogenic Effect:細胞
変性効果)出現状況を観察した。又、37℃では培養7日
目に、30℃では培養14日目に培養液を採取し、子牛精巣
培養細胞を用いてウイルス含有量を測定した。対照培養
細胞として用いた子牛精巣培養細胞は37℃では7日目、
30℃では12日目に培養液を採取した。結果は下記表1に
みられるように、37℃培養ではウイルス接種後7日目に
はすべての培養細胞に20〜50%以上のCPEが出現し、そ
の培養液1ml中のウイルス含有量は、20%程度のCPEを示
した肝,脾,腎ではそれぞれ105.8,106.0,105.7TCID
50、50%以上のCPEを示した肺,胸腺,精巣ではそれぞ
れ107.8,107.8,107.6TCID50であった。対照培養細胞
のCPEは80%以上で、その培養液1ml中のウイルス含有量
は107.8TCID50であった。30℃培養では培養14日目では
肺,胸腺,精巣に20〜50%のCPEが出現したが、その他
の培養細胞ではCPEが出現しなかった。これらの培養液1
ml当たりのウイルス含有量は、CPEの出現した肺,胸
腺,精巣ではそれぞれ106.2,106.3,106.0TCID50、CPE
の出現しなかった肝,脾,腎ではそれぞれ103.5,1
04.0,103.8TCID50であった。30℃対照培養細胞のCPEは
12日目に80%以上となり、その培養液1ml当たりのウイ
ルス含有量は106.4TCID50であった。以上の成績は本ウ
イルスが山羊由来の培養細胞で牛由来の培養細胞と同程
度に増殖することを示している。(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.
実施例2 強毒ウイルスの培養 生後3日の子山羊から肺,腎,甲状腺,胸腺及び精巣を
採取し、前記の細胞培養法で各培養細胞を調製し、試験
には500ml容量の培養びんに30mlずつ分注し、37℃で4
日間培養した継代2代目の細胞を用いた。又、ウイルス
増殖用の対照培養細胞として子牛精巣培養細胞を用い
た。ウイルス株は牛に病原性を示す強毒な袋井株(農林
水産省家畜試験場より分与)を用いた。ウイルス接種は
各培養細胞の培養液を除去し、PBSで細胞表面を1回洗
浄後、105.0TCID50/mlのウイルス浮遊液を5mlずつ接種
し、37℃で60分間感作してウイルスを吸着させた後、接
種材料を除去し、細胞維持用培養液を50mlずつ注加後、
37℃で静置培養しながらCPE出現状況を観察した。又、
培養液の一部をウイルス接種後5日,8日及び10日目に抜
き取り、そのウイルス含有量を子牛精巣培養を用いて測
定した。結果は下記表2にみられるように、CPEの出現
は胸腺細胞で4日目から、肺及び甲状腺細胞で7日目か
ら認められたが、腎及び精巣細胞では培養10日目まで認
められなかった。ウイルス増殖のピークは大部分の培養
細胞で5日目前後とみられ、7日目以降CPEの認められ
た肺,甲状腺,胸腺由来の培養液1ml当たりのウイルス
含有量は5日目でそれぞれ106.5,105.0,106.8TCID50
で、10日目ではそれぞれ106.8,105.3,106.8TCID50で
あった。CPEの出現しなかった腎及び精巣細胞の培養液
では10日目で105.0,106.0TCID50/mlの値を示した。対
照として用いた子牛精巣培養細胞ではウイルス接種後3
日目からCPEが出現し、8日目には80%以上となり、そ
の培養液1ml当たりのウイルス含有量は108.0TCID50であ
った。以上の成績は、牛アデノウイルス7型強毒ウイル
ス袋井株が山羊由来の培養細胞で増殖することを示して
いるが、その程度は牛由来の培養細胞に若干劣ることが
示されている。 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.
実施例3 強毒ウイルスの継代順化 実施例2において、山羊由来の培養細胞では強毒ウイル
ス袋井株の増殖が牛由来の培養細胞の場合よりも劣る傾
向が示されたので、本ウイルスの山羊由来培養細胞への
継代順化を試みた。試験に用いた培養細胞は実施例2に
用いた子山羊の肺,胸腺及び精巣細胞の継代4〜8代の
ものを用いた。ウイルスは実施例2の培養10日目に採取
した各培養細胞系のものを用いた。ウイルスの継代は10
4.7〜105.3TCID50/mlのウイルス浮遊液を、実施例2と
同様の方法で接種し、吸着させた後培養液を加え、37℃
で7日間培養する方法で7〜10代行った。その結果、下
記表3にみられるようにいずれの培養細胞でも培養7日
目までのCPEは、ウイルスの継代が進むにつれてその程
度が強く現れるようになり、肺培養細胞では継代3代目
から50%,8代目から80%以上,胸腺培養細胞では2代目
から50%,6代目から80%以上,精巣培養細胞では2代目
まではCPEが出現しなかったが、3代目で20%,6代目で5
0%,8代目で80%以上となった。培養液中のウイルス含
有量はCPEの程度が強く現れるのに平行して増加し、肺
培養細胞では継代7代以降、胸腺培養細胞では継代4代
以降、精巣培養細胞では継代7代以降いずれも107.5TCI
D50/ml以上となり、その最高値は胸腺培養細胞継代5代
目の108.0TCID50/mlであった。以上の成績は強毒ウイル
ス袋井株が山羊由来の培養細胞に順化し、牛由来の培養
細胞の場合と同程度に増殖することを示している。 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)ワクチンの調製 ワクチンには、対象動物に病原性を示さないように弱毒
化された生きたウイルスを用いた生ワクチンと、弱毒ウ
イルス又は強毒ウイルスを薬剤又はその他の方法で殺
し、免疫原性のみを保持させた不活化ワクチンとがあ
り、以下これらの各実施例について示す。 (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.
実施例4 生ワクチンの調製及び牛への応用生ワクチン
の調製 胎令137日の山羊胎児から肺,肝,脾,腎,胸腺,筋
肉,皮膚の各組織を採取し、前記(1)の細胞培養法に
よって各培養細胞を調製した。試験には500ml容量の培
養びんに30mlずつ分注し、37℃で4日間培養した継代3
代目の各細胞を用いた。ウイルス株は弱毒ウイルスBT−
3株を山羊胸腺培養細胞で3代限界希釈継代したクロー
ンを同培養細胞でさらに2代継代増殖させたBT−3−GT
h5株を用いた。ウイルス接種は各培養細胞の培養液を除
去し、細胞表面をPBSで1回洗浄後、105.3TCID50/mlの
ウイルス浮遊液を5mlずつ接種し、37℃で60分間感作し
てウイルスを吸着させた後、接種材料を除去し、細胞維
持用培養液50mlずつを注加し、32〜34℃で静置培養し
た。培養7日目には20〜80%のCPEが出現したので、各
培養細胞ごとに培養液を採取混合した。各培養液1ml当
たりのウイルス含有量は、CPEが20〜50%であった肝,
脾,腎の各培養細胞由来では、それぞれ105.0,105.8,
105.5TCID50であったが、CPEが80%以上を示したその他
4種の培養細胞由来では107.5〜107.7の高い値が得られ
た。高い値の得られた前記4種の細胞培養液を混合し、
軽遠心した上液をワクチン調製用のウイルス浮遊液と
し、これを2分し、その一方にはA安定剤(乳糖10%及
びポリビニールピロリドン0.3%溶液)を、他方にはB
安定剤(ペプトン10%及びポリビニールピロリドン0.3
%溶液)を等量加え、10ml容量のバイアルに1mlずつ分
注し、凍結乾燥後、減圧下で封じ、前者をLotNo.1、後
者をLotNo.2とし、2ロットの生ワクチンを調整した。
本ワクチンを10mlずつの溶解用液(0.01M平衡リン酸緩
衝食塩液)で溶解し、子牛精巣培養細胞を用いてウイル
ス含有量を測定した結果、LotNo.1では105.5TCID50/m
l、LotNo.2では105.6TCID50/mlであった。この成績は、
弱毒ウイルスの約103.0TCID50を牛に注射すると十分な
免疫が付与されることから、山羊由来培養細胞で得られ
たウイルス浮遊液から高力価の生ワクチン製造が可能な
ことを示している。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. .
生ワクチンの安全性 前記により調整した生ワクチンLotNo.1及び2の各2本
ずつを2mlの溶解用液で溶解(含有ウイルス量106.8及び
106.9TCID50)し、これを体重178〜193kgのアデノウイ
ルス7型ウイルス抗体陰性の子牛1頭ずつに皮下注射
し、1頭を非注射対照牛として同居させた。これらの子
牛は毎日元気・食欲の異常、鼻汁及び下痢の有無等の臨
床症状の観察、体温及び白血球数の計測を14日間、血
液,糞便及び鼻汁からのワクチンウイルスの回収を10日
間実施した。又、ワクチン注射後7日、14日、21日目に
血清中の牛赤血球凝集抑制(HI)抗体価を測定した。そ
の結果、試験牛は観察期間中まったく異常を示さず経過
し、体温及び白血球数にも異常が認められなかった。
又、血液,糞便及び鼻汁からのウイルス回収もすべて陰
性であった。HI抗体はワクチン注射牛では7日目から検
出され、その値は20倍と40倍、14日目には40倍と80倍、
21日目にはいずれも160倍となった。一方、同居牛は同
居21日後においてもHI抗体は検出されなかった。以上の
結果は下記表4に示した通りであり、本ワクチンウイル
スが有効量(103.0TCID50)の約5000倍量を子牛に注射
しても安全であることを示している。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 ).
生ワクチンの免疫原性 前記生ワクチンLotNo.1及び2を10mlの溶解用液で溶解
し、さらに溶解用液を用いて200倍に希釈して1ml当たり
のウイルス含有量がLotNo.1では103.2TCID50,LotNo.2で
は103.3TCID50になるように調整したものを注射材料と
した。供試牛は体重167〜235kgの牛アデノウイルス7型
ウイルス抗体陰性の子牛5頭を選出した。2頭にはLotN
o.1希釈ワクチンの1mlずつを、2頭にはLotNo.2希釈ワ
クチンの1mlずつを、それぞれ皮下注射し、1頭は強毒
ウイルス攻撃対照として、ワクチン注射牛に同居させ
た。これらの子牛は毎日元気・食欲の異常、鼻汁及び下
痢の有無等の臨床症状の観察、体温及び白血球数の計測
を14日間行うとともに、7日ごとに28日目までHI及び中
和抗体価を測定した。さらに、これらの試験牛はワクチ
ン注射後28日目に強毒ウイルス袋井株の107.0TCID50を
皮下注射する方法で攻撃し、臨床症状の観察、体温及び
白血球数の計測、血中からの攻撃ウイルスの回収を14日
間行うとともに攻撃後7日目及び14日目のHI及び中和抗
体価を測定した。結果は表5にみられるように、ワクチ
ン注射による臨床症状,体温及び白血球数の異常はいず
れの牛にも認められなかった。HI及び中和抗体応答はワ
クチン注射後7日目には4頭中3頭(75%)に、14日目
には全例に認められ、強毒ウイルス攻撃時(ワクチン注
射後28日目)にはHI抗体で40〜80倍、中和抗体で16〜64
倍の値が検出された。これらのワクチン注射牛は強毒ウ
イルス攻撃後も臨床症状,体温及び白血球数の異常をま
ったく示さず経過し、血中ウイルスの検出も陰性であ
り、さらに攻撃後の抗体上昇がまったく認められず、10
7.0TCID50の攻撃ウイルスに対し完全な防御を示した。
一方、ワクチン非注射対照牛は典型的なアデノウイルス
7型感染症の症状を示して耐過し、血清中には高いHI及
び中和抗体が産生された。以上の成績は、山羊由来の培
養細胞を宿主として作出された生ワクチンは103.0TCID
50前後の注射ウイルス量で約1カ月後には野外の強毒ウ
イルスの感染を防御することを示している。 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.
実施例5 不活化ワクチンの調製及び牛への応用不活化
ワクチンの調製 30日令の子山羊から肺,胸腺及び精巣の各組織を採取
し、前記(1)の細胞培養法によって各培養細胞を調製
した。試験には500ml容量の培養びんに30mlずつ分注
し、37℃で5日間培養した継代3代目の細胞を用いた。
ウイルス株は強毒ウイルス袋井株の山羊精巣培養細胞継
代7代目のウイルスを用いた。ウイルス接種は各培養細
胞の培養液を除去し、細胞表面をPBSで1回洗浄後、10
5.5TCID50/mlのウイルス浮遊液を5mlずつ接種し、37℃
で60分間感作してウイルスを吸着させた後、接種材料を
除去し、細胞維持用培養液50mlずつを加え、37℃で静置
培養し、ウイルス接種後7日目に各培養細胞ごとに培養
液を採取混合した。ついで軽遠心後、その上液の一部は
ウイルス含有量測定用とし、残部にはホルマリンを0.2
%の割合に添加し4℃で7日間不活化した。不活化ウイ
ルス浮遊液に1/10量のリン酸アルミニウムゲルを添加
し、2日後30ml容量のバイアルに30mlずつ分注後封栓
し、肺,胸腺及び精巣培養細胞由来の順にLotNo.1,2,3
とし、3ロットの不活化ワクチンを調製した。これらの
不活化ワクチン調製用ウイルス浮遊液の不活化前のウイ
ルス含有量は、それぞれ107.5,108.0,108.0TCID50で
あった。以上の成績は本不活化ワクチン中に十分な抗原
量が含有されていることを示している。また、この他に
山羊由来の肝,脾,筋肉,皮膚の各組織を用いて調整し
た不活化ワクチンについても、十分な抗原量の含有され
ていることが確認された。 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.
不活化ワクチンの安全性 前項で試作した不活化ワクチン3ロットを、体重210〜2
30kgの牛アデノウイルス7型ウイルス抗体陰性の子牛1
頭ずつに、それぞれ10mlずつ頸部筋肉内に注射し、元気
・食欲の異常、鼻汁及び下痢の有無等の臨床症状の観察
と体温測定を14日間行うとともに、ワクチン注射後7
日,14日及び21日目にHI抗体価を測定した。その結果は
下記表6に示す通りであり、試験牛は観察期間中まった
く異常を示さず経過した。血清中のHI抗体はワクチン注
射後14日目までは検出されなかったが、21日目に全例に
20〜80倍の値が検出された。この成績は、本不活化ワク
チン中の強毒ウイルスが完全に不活化され、子牛に対し
て安全なことを示している。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.
不活化ワクチンの免疫原性 前項2−(1)で試作した不活化ワクチン3ロットを、
体重178〜213kgの牛アデノウイルス7型ウイルス抗体陰
性の子牛2頭ずつに、それぞれ3mlずつ28日間隔で2回
頸部筋肉内に注射し、元気・食欲の異常、鼻汁及び下痢
の有無等の臨床症状の観察と体温測定をワクチン注射後
14日間実施するとともに、ワクチン第1回注射直前、第
2回注射直前及び第2回注射後14日目に血清中のHI及び
中和抗体を測定した。結果は表7にみられるように、試
験牛は観察期間中まったく異常を示さず経過し、ワクチ
ン第2回注射直前にはいずれも10〜40倍のHI抗体及び2
〜16倍の中和抗体が検出された。これらのHI及び中和抗
体価はワクチンの第2回注射後14日目にはさらに上昇
し、HI抗体は40〜320倍、中和抗体は32〜256倍の値を示
した。以上の成績は、本不活化ワクチンが有効な免疫原
性を保持していることを示している。 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.
〔発明の効果〕 本発明によって初めて、牛アデノウイルス7型ウイルス
を牛以外の動物(山羊由来)の組織培養を用いて培養増
殖でき、しかも牛由来の培養細胞から得られたワクチン
と同程度の有効性を有するワクチンが、この山羊由来の
培養細胞から得られたウイルス浮遊液より調製できるた
め、これによって宿主動物に由来する病原ウイルスの混
入のない、本ウイルス感染症に対する有効なワクチンを
初めて提供することができるという効果が得られる。 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.
フロントページの続き (56)参考文献 特開 昭50−18619(JP,A) 特開 昭48−13519(JP,A) 特開 昭57−56431(JP,A) 特公 昭48−2767(JP,B1) 特公 昭49−40924(JP,B1)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)
来の組織培養細胞を用いて増殖させ、得られたウイルス
浮遊液を継代培養することにより、あるいは不活化する
ことにより調製されたことを特徴とする生もしくは不活
化牛アデノウイルス7型ワクチン。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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP59190099A JPH07561B2 (en) | 1984-09-11 | 1984-09-11 | Bovine adenovirus type 7 vaccine |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP59190099A JPH07561B2 (en) | 1984-09-11 | 1984-09-11 | Bovine adenovirus type 7 vaccine |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS6168428A JPS6168428A (en) | 1986-04-08 |
JPH07561B2 true JPH07561B2 (en) | 1995-01-11 |
Family
ID=16252353
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP59190099A Expired - Lifetime JPH07561B2 (en) | 1984-09-11 | 1984-09-11 | Bovine adenovirus type 7 vaccine |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH07561B2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0650734B1 (en) * | 1993-10-28 | 1999-03-10 | Division Of Microbiology, Kyoto Biken Laboratories, Inc. | Pentavalent live viral vaccine |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5250530B2 (en) * | 1972-08-24 | 1977-12-24 | ||
JPS5018619A (en) * | 1973-03-28 | 1975-02-27 |
-
1984
- 1984-09-11 JP JP59190099A patent/JPH07561B2/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
JPS6168428A (en) | 1986-04-08 |
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