CN1604961A - 用于生产病毒抗原的禽胚胎颗粒生物质 - Google Patents
用于生产病毒抗原的禽胚胎颗粒生物质 Download PDFInfo
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Abstract
本发明提供了一种包含具有约0.5mm至10.0mm颗粒大小的禽胚胎颗粒的生物质及其在病毒抗原生产方法中的用途。还提供了一种利于病毒疾病的改善和预防的疫苗的制备方法。
Description
发明背景
用于生产病毒抗原的传统方法包括卵内生产，即在感染的含胚卵中的生产；或细胞培养生产，即原代细胞或已被感染的细胞系。一般用于生产抗原的卵内方法涉及许多难于自动化、劳动集约、费时且易受污染的步骤。通常细胞培养生产需要使用尽可能小的单个细胞和细胞团块，这就需要将组织用机械力或酶最大程度地分解或分离。这种处理可能导致许多细胞的裂解，从而可能引起细胞蛋白质的高度污染，而后者很难从所需产物中分离。生产蜱传脑炎病毒抗原和流感病毒疫苗的方法在US 5,391,491和US 5,698,433中分别有描述。这些方法使用直径＞100μm至＜1000μm的禽胚胎细胞团块并且需要酶促步骤。
因此，本发明的目的是提供一种用于病毒抗原生产的生物质，其克服了卵内抗原生产以及利用单个细胞、细胞系或微米量级细胞团块生产抗原的缺陷。
本发明的另一个目的是提供一种病毒抗原的生产方法，该方法获得的病毒抗原产量较高且可能在商业范围内使用。
本发明的优点是生物质易于处理，且这种抗原生产方法提供了经济的步骤并显著减少感染的机会。
本发明的特征是有效抵抗病毒感染和疾病的疫苗可更容易且更经济地生产。
本发明进一步的目的和特征将在下面的详细说明中更加明显。
发明概述
一种用于生产病毒抗原的生物质，该生物质包含颗粒大小约为0.5-10.0mm的禽胚胎颗粒，其中所述颗粒被病毒感染。
本发明还提供了一种用于生产病毒抗原的方法，其包括：
a)在培养基中用病毒感染约0.5mm至10.0mm大小的禽胚胎颗粒以形成生物质；
b)在升高的温度下对所述生物质充氧(oxygenate)以形成充氧混合物；以及
c)过滤所述混合物以获得含有所需病毒抗原产物的滤过物。
本发明进一步提供一种用于制备疫苗的方法，其包括：
a)在培养基中用病毒感染约0.5mm至10.0mm大小的禽胚胎颗粒以形成生物质；
b)在升高的温度下对所述生物质充氧以形成充氧混合物；
c)过滤所述混合物以获得含有所需病毒抗原产物的滤过物。
d)将所述滤过物与药理学上可接受的液体载体混合；以及
e)任选加入免疫原性促进佐剂。
发明详述
用于生产病毒抗原的传统方法包括在感染的含胚卵如母鸡卵中的生产，在已被感染的原代细胞培养中或在感染的细胞系中的生产或使用微米量级的禽胚胎细胞团块的生产。所有这些方法都涉及多个步骤和/或酶促断裂和例如离心，沉降等分离技术。
令人惊奇的是，现已发现一种包含禽类，优选鸡的被病毒感染的胚胎颗粒的生物质可用于有效且经济地生产病毒抗原，其中颗粒大小约0.5-10.0mm，优选约1.0-3.0mm。根据本发明生物质的禽胚胎颗粒可通过例如高剪切混合、快速多挡板搅拌以及匀浆等传统的机械粉碎方法获得，其中优选匀浆。例如，取自孵育9-12天，优选11天的母鸡卵的鸡胚胎可用无菌缓冲溶液洗涤，并用例如胰蛋白
磷酸盐肉汤的培养基稀释至每升约50至250，优选约80-120，更优选约100胚胎的浓度，并且经机械粉碎提供具有约0.5mm至10.0mm，优选约1.0mm至3.0mm颗粒大小的禽胚胎颗粒悬浮液。该悬浮液随后可用病毒或病毒原种(master seed)感染以提供合乎本发明的生物质。适合的病毒包括能够在禽胚胎细胞中复制的任何病毒或其它抗原，例如呼肠孤病毒、传染性法氏囊病病毒(IBDV)、马立克病病毒(MDV)、新城疫病毒(NDV)、传染性支气管炎病毒(IBV)、痘病毒、鸡贫血病毒(CAV)、产卵下降综合症(Egg Drop Syndrome，EDS)、火鸡鼻气管炎(TRT)病毒、肺病毒、传染性喉气管炎(ILT)病毒、脑脊髓炎病毒、流感病毒、狂犬病病毒、瘟热病毒、出血性肠炎病毒、肝炎病毒、鹦鹉热衣原体、副鸡嗜血杆菌等，优选禽类病毒，更优选传染性法氏囊病病毒。
相应的，本发明提供了一种用于病毒抗原生产的方法，其包括在培养基中用病毒，优选禽病毒，更优选传染性法氏囊病病毒感染具有约0.5mm至10.0mm，优选约1.0mm至3.0mm颗粒大小的禽胚胎颗粒以形成生物质；在升高的温度下对所述生物质充氧以形成充氧混合物；以及过滤所述混合物以获得含有所需病毒抗原产物的滤过物。
适于本发明方法中使用的培养基包括胰蛋白
磷酸盐肉汤，EMEM，DMEM，(Anhui Chemicals生产)或任何适于生物培养的传统培养基，优选胰蛋白
磷酸盐肉汤。
适于本发明方法中使用的升高的温度是约90°F至110°F，优选约95°F至105°F，更优选98°F至102°F的温度。
本发明方法中获得的充氧混合物可含有约40％至60％，优选45％至55％，更优选50％的溶解氧。
在实际操作中，在具有每升培养基约50-250，优选约80-120，更优选约100胚胎浓度的培养基，例如胰蛋白
磷酸盐肉汤中，具有约0.5mm至10.0mm，优选约1.0mm至3.0mm颗粒大小的禽胚胎颗粒的悬浮液用病毒，优选禽病毒，更优选传染性法氏囊病病毒感染以获得生物质：所述生物质在约90°F至110°F，优选约95°F至105°F，更优选约98°F至102°F的升高的温度下充氧，以获得含有约40％至60％，优选约45％至55％，更优选约50％溶解氧的充氧混合物；该充氧混合物通过约50微米至100微米，优选约70微米至80微米，更优选约75微米的筛子过滤，从而获得含有所需抗原产物的滤过物。这样得到的抗原原液可以通过例如稳定剂、抗氧化剂、消泡剂等传统赋形剂加以处理并通过冷冻或冷冻干燥保藏用于日后的疫苗制备，或者可能被用于疫苗的制剂中。
相应的，本发明还提供了一种用于制备疫苗的方法，其包括将通过上述方法生产的抗原原液与药理学上可接受的载体混合，并和任选加入免疫原性促进佐剂。
适于本发明的疫苗制备方法中使用的药理学上可接受的载体包括任何适于兽用药物制剂的传统液体载体，优选适于在组织培养基中使用的平衡盐溶液。
适于本发明的疫苗制备方法中使用的免疫原性促进佐剂包括任何当与抗原联合施用时能够增强或促进动物免疫反应的化合物，例如表面活性剂，即十六胺、十八胺、溶血卵磷脂、二甲基二(十八烷基)溴化铵、N，N-双十八烷基-N’-N-双(2-羟乙基-丙二胺)、甲氧基十六烷基甘油、Pluronic多元醇、皂角苷、QuilA等；聚阴离子，例如吡喃、硫酸葡聚糖、聚肌苷酸多聚胞苷酸的多核苷酸复合物、聚丙烯酸、聚羧乙烯、氢氧化铝、磷酸铝等；肽，例如胞壁酰二肽、二甲基甘氨酸、吞噬作用激素等；油乳状液；免疫调节剂，例如白介素-1、白介素-2、白介素-12、GM-CSF等；或其组合。
在实际操作中，根据上述本发明的抗原生产方法得到的、优选禽病毒抗原、更优选传染性法氏囊病病毒抗原的病毒抗原原液与例如磷酸缓冲盐溶液等药理学上可接受的载体和任选的免疫原性促进佐剂混合，以获得具有高于最小保护剂量约1.2log抗原浓度的疫苗产物。
这样制得的疫苗可进一步用例如稳定剂、抗氧化剂、消泡剂等兽用疫苗中通常使用的传统赋形剂处理。
在本发明的一个实施方案中，病毒抗原原液可在疫苗制备前灭活。根据本发明的抗原生产方法制备的病毒抗原原液可通过传统的灭活方式失活，例如使用如吖丙啶、β-丙醇酸内酯、福尔马林、硫柳汞、戊二醛、十二烷基硫酸钠等化学灭活剂或其混合物，优选福尔马林进行的化学灭活。所述抗原也可以通过加热或通过在紫外光下的补骨脂素被灭活。
为了更清楚地理解本发明，列出下述的实施例。这些实施例仅仅是说明性的而并不应以任何方式理解为限制本发明的范围或基本要素。实际上，除了在此显示和描述的之外，根据下述实施例和前文的描述，本发明的多种改进对于本领域技术人员来说是显而易见的。这些改进也落在本发明的保护范围内。
除非另外提到，所有的份数都是重量份。
实施例1
传染性法氏囊病病毒(IBDV)抗原的制备
从在99°F下孵育11天的母鸡卵中获取的胚胎用含庆大霉素(30mg/mL)的磷酸盐缓冲液在室温下洗涤三次。经洗涤的胚胎在胰蛋白
磷酸盐肉汤中稀释至每升大约100个胚胎。利用Chemineer(Greerco)生产的W250V(Gifford-Wood)型匀浆器，缝隙预设为3.0mm，将稀释的胚胎匀浆至1.0至3.0μm的颗粒大小。所得悬浮液与每胚胎0.2mL X+4 Lukert株工作种(USDA-APHIS认定的IBDV)(5.5 TCID50/mL)机械混合20-30分钟。所得混合物在pH7.1，3.0psi，100.4°F和20胚胎/升的浓度下充氧至最终溶解氧(DO)含量为50％DO。48小时后，充氧混合物通过75微米的筛子过滤，在室温下与稳定剂H1以1∶1的比例混合一小时并于≤40°F保藏。
通过使用上述步骤，获得具有9.2 TCID50/mL效价的IBDV抗原原液。TCID代表组织培养感染剂量。
1稳定剂H配方：以下列出的成分按照所列出的顺序混合。每一成分在下一成分加入前充分溶解。必要时进行加热至最高温度为60℃。
成分 重量/体积
纯化水 0.5kg/L
Pharmatone，American Labs生产 5.0g/L
Peptone Bacto，Becton Dickinson生产 45.0g/L
蔗糖 50.0g/L
Yt型N-Z-胺，Quest International生产 25.0g/L
谷氨酸一钠 5.0g/L
纯化水 Q.S.
实施例2
病毒抗原的制备
用与实施例1基本相同的步骤，用适合的病毒，获得表1中所示的抗原原液。
表1
抗原
效价
呼肠孤病毒 8.26 TCID50/mL
传染性法氏囊病病毒 9.20 TCID50/mL
新城疫病毒 8.60 EID1 50/Ml
1EID＝胚胎感染剂量
实施例3
传染性法氏囊病病毒疫苗的制备
根据实施例1中描述的步骤制备并冷冻的传染性法氏囊病抗原原液在室温下解冻，并且用以1∶1比例混合的稳定剂H和D-mem1稀释至高于最小保护剂量1.2log的最终抗原浓度。经稀释的原液经机械搅拌至少15分钟并置于单或多剂量小瓶中。
1D-mem是JRH Bioscience生产的极限必需培养基。
Claims (20)
1、一种用于生产病毒抗原的生物质，该生物质包含颗粒大小约为0.5-10.0mm的禽胚胎颗粒，其中所述颗粒被病毒感染。
2、根据权利要求1的生物质，其中所述颗粒大小为约1.0mm至3.0mm。
3、根据权利要求1的生物质，其中所述禽胚胎是鸡胚胎。
4、根据权利要求1的生物质，其中所述病毒选自呼肠孤病毒、传染性法氏囊病病毒、马立克病病毒、新城疫病毒、传染性支气管炎病毒、痘病毒、鸡贫血病毒、产蛋下降综合症、火鸡鼻气管炎病毒、肺病毒、传染性喉气管炎病毒、脑脊髓炎病毒、流感病毒、狂犬病病毒、瘟热病毒、出血性肠炎病毒、肝炎病毒、副鸡嗜血杆菌和鹦鹉热衣原体。
5、根据权利要求1的生物质，所述病毒是禽病毒。
6、根据权利要求5的生物质，其中所述病毒是传染性法氏囊病病毒。
7、—种用于生产病毒抗原的方法，其包括：
a)在培养基中用病毒感染具有约0.5mm至10.0mm颗粒大小的禽胚胎颗粒以形成生物质；
b)在升高的温度下对所述生物质充氧以形成充氧混合物；以及
c)过滤所述混合物以获得含有所需病毒抗原产物的滤过物。
8、根据权利要求7的方法，其中所述胚胎颗粒大小为约1.0mm至3.0mm。
9、根据权利要求7的方法，其中所述培养基是胰蛋白
磷酸盐肉汤。
10、根据权利要求7的方法，其中所述充氧混合物含有约40％至60％的溶解氧。
11、根据权利要求7的方法，其中所述升高的温度是约95°F至105°F。
12、根据权利要求7的方法，其中所述病毒选自呼肠孤病毒、传染性法氏囊病病毒、马立克病病毒、新城疫病毒、传染性支气管炎病毒、痘病毒、鸡贫血病毒、产蛋下降综合症、火鸡鼻气管炎病毒、传染性喉气管炎病毒、脑脊髓炎病毒、流感病毒、狂犬病病毒、瘟热病毒、肠炎病毒、肝炎病毒和衣原体病毒。
13、根据权利要求7的方法，其中所述病毒是禽病毒。
14、根据权利要求13的方法，其中的病毒是传染性法氏囊病病毒。
15、一种用于制备病毒疫苗的方法，其包括：
a)在培养基中用病毒感染具有约0.5mm至10.0mm颗粒大小的禽胚胎颗粒以形成生物质；
b)在升高的温度下对所述生物质充氧以形成充氧混合物；
c)过滤所述混合物以获得含有所需病毒抗原产物的滤过物；
d)将所述滤过物与药理学上可接受的液体载体混合；以及
e)任选加入免疫原性促进佐剂。
16、根据权利要求15的方法，其中所述病毒选自呼肠孤病毒、传染性法氏囊病病毒、马立克病病毒、新城疫病毒、传染性支气管炎病毒、痘病毒、鸡贫血病毒、产蛋下降综合症、火鸡鼻气管炎病毒、传染性喉气管炎病毒、脑脊髓炎病毒、流感病毒、狂犬病病毒、瘟热病毒、肠炎病毒、肝炎病毒和衣原体病毒。
17、根据权利要求15的方法，其中所述病毒是禽病毒。
18、根据权利要求17的方法，其中所述病毒是传染性法氏囊病病毒。
19、根据权利要求18的方法，其中所述禽胚胎颗粒具有约1.0mm至3.0mm的颗粒大小。
20、根据权利要求19的方法，其中所述颗粒是鸡胚胎颗粒。
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| Publication number | Publication date |
|---|---|
| US20090226487A1 (en) | 2009-09-10 |
| CO5590972A2 (es) | 2005-12-30 |
| BR0215013A (pt) | 2004-11-09 |
| RS55404A (en) | 2006-10-27 |
| HU225995B1 (en) | 2008-02-28 |
| NZ534070A (en) | 2006-01-27 |
| AR037908A1 (es) | 2004-12-22 |
| EP2157173A1 (en) | 2010-02-24 |
| HRP20040641A2 (en) | 2004-10-31 |
| EP1458851A1 (en) | 2004-09-22 |
| US20080102507A1 (en) | 2008-05-01 |
| HUP0402548A2 (hu) | 2005-03-29 |
| AU2002360659B2 (en) | 2009-01-08 |
| CN100408675C (zh) | 2008-08-06 |
| MXPA04005888A (es) | 2004-09-13 |
| CA2471131A1 (en) | 2003-07-10 |
| TWI267553B (en) | 2006-12-01 |
| AU2002360659A1 (en) | 2003-07-15 |
| PL371381A1 (en) | 2005-06-13 |
| TW200301305A (en) | 2003-07-01 |
| WO2003055988A1 (en) | 2003-07-10 |
| KR20040070250A (ko) | 2004-08-06 |
| JP2005514018A (ja) | 2005-05-19 |
| US20040023358A1 (en) | 2004-02-05 |
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