CN1038728C - 在传代细胞系中生产传染性囊病病毒 - Google Patents
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Abstract
本发明涉及含IBDV抗原的传染性囊炎病毒(IBDV)疫苗,IBDV抗原物来自用IBDV感染的哺乳动物细胞系。
Description
本发明与利用永久性哺乳动物细胞系增殖感染鸟类的传染性囊病病毒(IBDV)、及IBDV抗原有关。
鸟类病毒通常在鸡胚中,靠来自鸡胚的细胞底物,如原代、次代鸡胚成纤维细胞(CEF),原代鸡肝细胞或鸡肾细胞产生。也可在活体动物器官中产生,如在法氏囊中。此种来源得到的病毒,在世界上被广泛用于制备灭活疫苗和活疫苗。
用动物和鸡胚来制备疫苗的主要缺点在于它们的质量不能保证。甚至无特殊病原体的小鸡,也会出忽意料地受到感染,使之不适合于疫苗的生产。偶尔,这类感染在一段时间内检测不到。
使用永久性细胞系能为这一问题提供一个理想的解决办法。但是至今尚未得到适合于疫苗生产的小鸡细胞系。大多数鸟类细胞系由淋巴母细胞样细胞组成,它们取自患淋巴样白血病即Marek′s疾病的动物。
从正常鸡胚成纤维细胞形成永久性细胞系的尝试,尚未成功。偶尔,由正常胚胎获得了细胞系,但在所有例子中,随后即发现它们都含有了反转录病毒基因组,有些甚至可排出病毒颗粒。
现已发现,能在鸡胚成纤维细胞培养物上生长的IBDV株(如D78株和SP株)也可有效地培养于哺乳动物细胞系中。已发现,无需使病毒适应哺乳动物细胞底物。而且发现,在哺乳动物细胞中,产量往往远高于CEF系统。病毒的产量通常用“单位体积中的感染性病毒颗粒”(EID50/ml;TCID50/ml)表示。另一确定病毒产量的方法是确定抗原量。用免疫化学技术,如ELISA法,可将病毒制备物的抗原含量与一标准制备物相比较(此标准物抗原量的单位数已定)。用此两类测定方法进行测定哺乳动物细胞系系统的产量都远高于CEF系统。
而且,令人惊呀的是,这一可观的产量是在细胞浓度比常规所用生产抗原的CEF系统为低的情况下得到的。哺乳动物细胞系的最适细胞浓度比用CEF的传统生产方法中的细胞浓度低3~6倍。这一结果表明,对于IBDV,一般说来哺乳动物细胞作底物要比CEF为好。同时发现,以此制备的抗原,作为疫苗至少与由CEF制备的抗原同样有效。
根据本发明,适合于生产IBDV的哺乳动物细胞系是,例如,Vero细胞、黑猩猩肝细胞、水牛Vervet细胞,以及鼠3T3细胞。
可在细胞培养瓶中静置培养和在旋转瓶中培养哺乳动物细胞。其它,通常规模更大的培养系统有,培养非贴附性细胞的搅拌器(发酵器),培养贴附性细胞的微载体系统,以及可用于培养这两类细胞的中空纤维系统。此外,还有许多其它培养贴附性细胞的培养系统。后一系统的一个共同特点是它们有一很大的可供细胞附着的表面。
哺乳动物细胞培养需用复杂的培养液。它们通常由基础液(培养基)和一种或多种添加物组成,基础液在化学上业经明确,而添加物则尚无明确的化学定义。添加物通常为富含蛋白质的溶液,如血清和蛋白水解产物。血清对于细胞生长和分裂确是必不可少的。许多培养系统都加入浓度为1~10%(v/v)的胎牛血清(FoCs)或禁食小牛的血清(FaCS)。只有在特别的情况下,有可能在适应一段时间后,将哺乳动物细胞培养在不含血清,甚至不含蛋白的培养基中。
根据本发明,IBD病毒如果需要,可于事先减毒后作为活病毒组成疫苗,或者作为灭活病毒成为疫苗。
含活病毒的疫苗,可采取悬液或冷冻干燥的形式制备和出售。冷冻干燥的疫苗最好含一种或几种稳定剂。适合的稳定剂有,例如,SPGA(Bovarnik(1950):J.Bacteriology 59;509),碳水化合物(如山梨糖、甘露糖、淀粉、蔗糖、葡聚糖、葡萄糖),蛋白质(如清蛋白、酪蛋白)和它的降解物、含蛋白质的材料(如牛血清、脱脂奶)及缓冲液(如碱金属磷酸盐)。如果需要,还可加入一种或多种有佐剂作用的化合物。适于这一目的化合物有,例如,氢氧化铝、磷酸盐或氧化物、矿物油(如Bayol F,Marcol 52)和皂角苷。
灭活IBD病毒的目的在于消除病毒的复制和毒性。总的来说,这可通过化学或物理方法实现。可用酶、甲醛、β-丙酸内酯、氮杂环丙烷或其衍生物、有机溶剂(如卤化烃)、和/或去垢剂(如Tween、TritonX、脱氧胆酸钠、硫酸甜菜碱或乙酰三甲基胺盐处理病毒,进行化学灭活。如果必要,灭活剂随后可被中和,例如,用甲醛灭活的材料可用硫代磷酸中和。使病毒受高能辐射,如紫外光、X射线、γ-射线,可很好地实现物理灭活。如果需要,可在处理后将PH值调回至7左右。
在灭活的病毒材料中,通常要加入佐剂(如上面提到的例子),如果需要,还要加入一种或几种乳化剂如Tween和Span。
根据本发明,疫苗适合于使家禽(如鸡和火鸡)抵抗IBD(Gumboro′s疾病)的感染。
根据本发明,可通过肌肉注射、皮下注射、或卵内注射、眼滴、鼻滴、饮水或喷雾方式给予疫苗。
根据本发明(其它疫苗)与IBDV材料一起组成联合疫苗也包括在本发明中。对于灭活疫苗,这一IBDV材料可与新城鸡瘟病毒、感染性支气管炎病毒、卵片综合征病毒、呼肠病毒、细菌(如大肠杆菌)、寄生虫(如艾氏球虫)结合。与新城鸡瘟病毒和/或Marek病毒联合很适合于组成活的联合疫苗。
实施例
在哺乳动物细胞系上制备IBDV
细胞培养
细胞材料存于玻璃安瓿里,置液氮中保存。开始细胞培养时,将一安瓿中的材料迅速化冻,并用细胞培养液慢慢稀释。将细胞悬液低速离心,除去冷冻剂中的二甲基亚砜。将沉下的细胞再悬浮于完全细胞培养基中,并将细胞接种于合适的培养瓶。通常,将细胞培养在1∶1的M199/F10混合培养基中,或培养在补充了胰蛋白磷酸肉汤的MEM培养基中。培养基含2~10%Focs,如果需要,加入抗生素和杀真菌剂。细胞在37℃下静置培养(组织培养瓶)或在旋转瓶(490cm2)中培养。当细胞密度达到形成了一致密的单层时,用胰蛋白酶处理细胞,为继续培养作准备。
病毒的生产、收获和灭活
溶解冷冻干燥的毒种,或者融化低温冷冻的毒种。用细胞培养液稀释,使其浓度尽可能达到合适的比例。以100~10-4TCID50/细胞的感染复数(M.O.I.)感染细胞。
如果需要,可在接种细胞后直接加入毒种。感染细胞可孵育至10天。通常在病毒感染后的2~10天收获病毒。收集培养瓶中的上清液,用福尔马林灭活。为此,向细胞悬液中加入甲醛直至浓度为0.05~0.2%。混合物在20~22℃下孵育1~3天。
生产疫苗
为作疫苗用,将灭活的IBDV抗原引入油包水乳化剂中(Marcol 52)。
在原代鸡胚成纤维细胞(CEF)上生产IBDV
CEF的制备:
从10~11日龄的、无特殊病原体的孵鸡蛋中,按技术熟练者所熟知的方法制备CEFs。
CEFs的培养
用与培养传代细胞系相同的培养基培养CEFs。在培养基中加入Focs或Facs,使浓度达5%。用细胞培养液稀释经胰蛋白酶处理后所得的浓缩细胞悬液,使之浓度为0.5-10×10细胞/ml。将细胞悬液转入组织培养瓶或旋转瓶中。细胞在37~39.5℃下培养约24小时后,便形成一致密集单层。
生产、收获、灭活病毒
用细胞培养液溶解并稀释冷冻干燥的毒种,使体积大到足以分成合适的量以接种细胞。病毒按100~10-5TCID50/细胞的浓度加入。如果需要,可在接种细胞后直接加入毒种。
感染细胞孵育48~96小时后从培养瓶中收集上清液,并用0.2%甲醛在室温下灭活24小时。确定IBDV抗原量。
借助定量夹心ELISA法确定IBDV抗原。
1.用IBDV特异性抗体包被微量滴定板;
2.使各孔充满系列稀释的抗原样品,保温;
3.小孔然后与酶联IBDV特异性抗体一起保温;
4.接着向孔中加入酶底物;
5.一段时间后,用稀硫酸终止酶促反应。用分光光度法测定孔内物颜色程度。将测得的吸收值与已知浓度系列稀释标准抗原制备物相比较,以ELISA单位/ml(EU/ml)表示抗原量的值。
病毒滴定
用原代鸡胚成纤维细胞在微量滴定板上测定感染性病毒的滴度。首先,每ml含5×105细胞的原代CEF悬液配成以10倍比稀释的样品稀释系列,然后在微滴定板小孔中加入200μl样品稀释物,每份样测6~10次。将盘置37°~39℃的CO2温箱中,保温4~7天。随后,镜检出现的细胞致病效应。以TCID50/ml计算滴度。通常,病毒滴度用10log TCID50/ml表示。测定中和病毒的抗体。
通过微滴定板中的微中和实验,确定中和病毒的抗体。为此,将双份稀释的血清样品稀释系列与1000TCID50的IBDV在不含血清的细胞培养基上于37℃的CO2培养箱中培养1~2小时。然后,悬于完全细胞培养基中的105个原代鸡胚细胞加入每一孔。将板置37℃CO2培养箱中保温4~7天,随后,镜检出现的细胞致病效应。病毒中和(VN)滴度现被定义为细胞致病效应完全消失时的最高稀释度的倒数。通常,VN滴度用2logVN表示。
例1
在原代鸡胚成纤维细胞上大规模生产D78抗原
从11日龄,无特殊病原体鸡胚中获得CEFs。这些细胞接种到1.585m2的旋转培养瓶中的补充了5%FaCS的M199/F10细胞培养基中,接种量为1-3×106细胞。每瓶装300ml细胞悬液。
将瓶在38.5~39.5℃下孵育18~24小时,然后向瓶中加入毒种以及过量的细胞。每一旋转瓶加含3~9×106细胞/ml和104~106TCID50D78毒种的悬液100ml。然后,旋转瓶在38.5~39.5℃下再保温48~120小时,随后,收集病毒悬液,并用甲醛灭活。八批产品的抗原量测定结果概括在表1中。
表1
抗原量(EU/ml)最低值 最高值 平均
397 1996 946
例2
在最适实验室条件下的原代鸡胚成纤维细胞上制备D78抗原
从11日龄无特殊病原体鸡胚中得到CEFs。将细胞以1.5,3.0和6.0×106细胞/ml的浓度接种在490cm2的塑料转瓶中,细胞悬于补充了5%Focs的M199/F10细胞培养液中,每瓶用100ml悬液。
同时,每瓶加入107.3TCID50毒种。使得感染量分别为0.12,0.06,0.03TCID50/细胞。转瓶于37℃下保温。保温2天后取样,第三天收获。用ELISA法测定样品及收获物中的抗原量。结果如表2所示。
表2起始细胞 抗原量(EU/ml) 抗原量/106细胞浓 度 2天 3天 (EU/106细胞)
p.i. p.i. 2天 3天
p.i. p.i.1.5×106 2779 3218 1852 21453.0×106 4353 5361 1451 17876.0×106 6467 6652 1078 1109
结论:在最适实验室条件下,非常有可能提高CEF上抗原的产量。但当细胞浓度增加时,每一细胞的产量大大下降。
例3
在Vero细胞上制备IBDV抗原
A.在长成完全单层细胞上的感染
将Vero细胞以0.25×106胞/ml的浓度接种于两个490cm2的转瓶中。每瓶中加入悬浮在补充了胰蛋白
磷酸肉汤和5%Focs的Eagle′s MEM中的细胞100ml。在37℃下,一瓶培养4天,另一瓶培养5天。然后,弃去细胞培养液,每一转瓶加入106TCID50毒种。将细胞一毒种混合物在37℃下培养30分钟后加入100ml细胞培养基。再分别培养8或7天后,收集病毒悬液,测抗原量,发现,培养7天后,为19413 EU/ml,8天后,为19706EU/ml。
B.在悬浮状态下细胞感染IBDV
培养物保温7天,感染后第45,96,144小时取样。借助ELISA法确定这些样品中抗原的含量(表3)。
表3
M.0.1 不同时间取样的抗原EU/ml 144小时后抗原毒株 TCID50细胞 45小时 96小时 144小时 量/106接种细胞
(EU/106细胞)D78 10-1 9044 20737 22937 22937D78 10-2 1197 20016 17759 17739D78 10-3 161 13324 18233 18233SP 10-1 3543 28439 26883 26883SP 10-2 509 21799 23165 23155SP 10-3 64 15121 13934 13934
结论:在与例2相应条件下,在哺乳动物细胞系上生产IBDV其抗原产量约为从CEF生产而得的抗原高10倍。单层细胞和细胞悬液上的产量大致相等。
例4
Vero细胞上产生的IBDV感染性病毒滴度
将vero细胞以1×106细胞/ml接种在490cm2的转瓶中,每一转瓶加入100ml细胞悬液,细胞悬浮于补充了胰蛋白
磷酸肉汤和5%Focs的Eagle′s MEM中。同时以0.01 TCID50/细胞的量,加入D78或SP毒种。将瓶于37℃下保温。在培养过程中取样,测定其抗原量和其中所含的感染性病毒。用ELISA法确定抗原量(EU/ml)。用原代CEFs(10 log TCID50/ml)借助微量滴定实验确定感染性病毒滴度。结果如表4所示。
表4毒株 产 率
4.5小时P.I. 96小时P.I. 144小时P.I.
EU/ml 10log EU/ml 10log EU/ml 10log
TCID50/ml TCID50/ml TCID50/mlSP 1197 8.9 20016 10.2 17739 9.9D78 509 8.2 21799 9.7 23165 9.5
例5
活疫苗
SPF(无特殊病原体鸡)(3周龄)四只一组,共两组,在第0天用产自Vero细胞的活IBD病毒D78系接种。每只鸡眼滴0.1ml病毒。一组中每只接受106 TCID50,另一组每只接受104.5TCID50。此外,一组四只以同样废水用产自原代CEFs的D78疫苗接种。接种16天后采血,检测IBDV中和抗体的存在。
结果见表5
表5疫 苗 剂 量 血清中和滴度
(10log TCID50/鸡) (2log VN)D78/Vero 6.0 9.1±1.51)D78/Vero 4.5 10.6±1.4D78/CEF 5.7 10.2±1.81)2log VN±标准差
许多小鸡亦进行了同样的实验,它们在接种了病毒3,6,16天后摘除囊并在组织学上对囊进行了急性和亚急性病变的检查。用产自CEF的病毒免疫的小鸡的囊与用产自Vero细胞的病毒免疫的小鸡的囊未见有任何差异。
结论:在Vero细胞上生产的IBD活疫苗具有与在CEF上生产的活疫苗完全一样的免疫原性和无害性。
例6
在黑猩猩肝细胞系中制备IBDV D78株
将100ml黑猩猩细胞悬液(浓度为0.6×106细胞/ml)接种于490cm2的转瓶中。按10-4TCID50/细胞的浓度加入IBDV毒种。孵育7天后,抗原含量为27042 EU/ml,大约相应于45×103EU/106接种细胞。
例7
在鼠细胞系中制备IBDV D78株
将100ml NTH3 T3鼠细胞悬液(浓度为0.3×106细胞/ml)接种于490cm2转瓶中,随后,按10-4TCID50/细胞的浓度加入毒种(D78株)。孵育7天后,病毒的抗原含量为3514EU,约相当于12×103EU/106接种细胞。
例8
培养在CEF或Vero细胞中IBDV抗原的免疫原性的比较
将从原代CEFs或Vero细胞制备而得的D78和SP抗原用福尔马林灭活,并在矿物油中乳化。每一只四周龄,无特殊病原体小鸡(white Leghorns),用0.5ml上述乳化液肌肉注射接种疫苗。免疫六周后,从小鸡身上采血。血清在56℃下灭活30分钟。其所含中和病毒的抗体在原代CEFs上测定。结果概括如表6。
表6病毒抗原 生产细胞 抗原量 中和抗体滴度1)(2log VN)D78 CEF 2924 14.4±1.3D78 CEF 1072 13.7±1.7D78 Vero 2750 14.6±1.5D78 Vero 690 13.1±1.9SP Vero 4000 14.6±1.1SP Vero 1000 13.5±1.31)10只鸡平均值±标准差例9培养于CEF或Vero细胞的IBDV抗原免疫效果的比较八周龄的小鸡,10只一组分成几组,接种灭活的D78-油乳化疫苗。每只鸡肌肉注入0.5ml疫苗。一组鸡用产自Vero细胞的抗原免疫,两组用来自CEF的抗原免疫,抗原皆按例2和例3所述方法,产自转瓶。接种疫苗六周后,采集小鸡血清,测定中和病毒的抗体。结果概括于表7。
表7疫 苗 2log VN±标准误差D78/CEF1 11.2±1.4D78/CEF2 13.1±0.9D78/Vero 14.5±1.7
统计学分析表明,D78/Vero诱导的滴度明显高于由D78/CEF诱导的滴度(学生T检验;P<0.05 CEF2)。
Claims (4)
1.一种制备灭活的传染性囊病病毒(IBDV)疫苗的方法,该方法包括:
(a)在哺乳动物细胞系中培养传染性囊病病毒,
(b)从培养物中收集传染性囊病病毒抗原物质,
(c)灭活所收集的传染性囊病病毒抗原物质,
(d)将灭活的抗原物质与可作药用的载体或辅助剂混合。
2.按照权利要求1所述的方法,其特征在于抗原物质是在用传染性囊病病毒感染细胞2-10天后收集。
3.按照权利要求1所述的方法,其特征在于哺乳动物细胞系是一猿类细胞系。
4.按照权利要求3所述的方法,其特征在于猿类细胞系为Vero细胞系。
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JP (1) | JP2749887B2 (zh) |
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EP0352835A1 (en) | 1990-01-31 |
DE68919449D1 (de) | 1995-01-05 |
HUT51904A (en) | 1990-06-28 |
JP2749887B2 (ja) | 1998-05-13 |
US6129920A (en) | 2000-10-10 |
CA1331444C (en) | 1994-08-16 |
KR0156732B1 (ko) | 1998-11-16 |
AU628063B2 (en) | 1992-09-10 |
EP0352835B1 (en) | 1994-11-23 |
ES2067528T3 (es) | 1995-04-01 |
HU218738B (hu) | 2000-11-28 |
HU211246A9 (en) | 1995-11-28 |
KR900001382A (ko) | 1990-02-27 |
US5602022A (en) | 1997-02-11 |
AU3825289A (en) | 1990-01-25 |
ZA895232B (en) | 1990-04-25 |
NZ229997A (en) | 1991-11-26 |
US5192539A (en) | 1993-03-09 |
CN1039359A (zh) | 1990-02-07 |
DE68919449T2 (de) | 1995-05-04 |
JPH0278634A (ja) | 1990-03-19 |
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