Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.
Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.
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DOI：
10.1371/journal.pone.0034834
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Chong P
中科院分区：
文献类型：
--
作者：
Chou AH;Liu CC;Chang CP;Guo MS;Hsieh SY;Yang WH;Chao HJ;Wu CL;Huang JL;Lee MS;Hu AY;Lin SC;Huang YY;Hu MH;Chow YH;Chiang JR;Chang JY;Chong P
肠病毒71型（EV71）在亚洲引起了几次手足口病的流行，现在被认为是一种重要的嗜神经病毒。目前迫切需要针对EV71感染的有效药物和预防性疫苗。基于灭活脊髓灰质炎病毒疫苗的成功，一种化学灭活EV71候选疫苗原型已经开发出来，目前正在进行人体1期临床试验。在本报告中，我们介绍了一种基于无血清细胞的EV71疫苗的开发。对制造过程中每一步的优化进行了研究、表征和量化。在上游工艺开发中，使用滚瓶技术筛选不同的市售含血清或无血清的细胞培养基，以检测细胞生长和病毒产量。根据Vero细胞生长情况和EV71病毒产生情况选择VP-SFM无血清培养基。在上游工艺（病毒收获、滤除和浓缩）之后，以每种工艺40升的中试规模研究了凝胶过滤液相色谱和/或蔗糖梯度超离心的组合下游纯化工艺。虽然用色谱法和蔗糖梯度超离心法分离得到了极纯的EV71感染性病毒颗粒，但通过基于vp2的定量ELISA测定，疫苗的总产率为7-10%。采用层析法进行下游纯化，病毒产率为30-43%。为了保持病毒中和表位的完整性和疫苗产品的稳定性，发现0.025%福尔马林在37°C下处理3至6天的病毒灭活效果最好。此外，发现福尔马林灭活病毒粒子候选疫苗在4°C下可稳定18个月，并且与明矾佐剂配制的微克病毒蛋白可以在小鼠，大鼠，兔子和非人灵长类动物中诱导强烈的病毒中和抗体反应。这些结果提供了有价值的信息，支持目前基于细胞的无血清EV71候选疫苗进入人体I期临床试验。
Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7–10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30–43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37°C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4°C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.
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影响因子：
5.5
作者：
Chung, Yao-Chi;Ho, Mei-Shang;Hu, Yu-Chen
通讯作者：
Hu, Yu-Chen
影响因子：
5.5
作者：
Liu, Chia-Chyi;Lian, Wei-Cheng;Wu, Suh-Chin
通讯作者：
Wu, Suh-Chin
DOI：
10.1186/1479-0556-5-6
发表时间：
2007-04-19
期刊：
Genetic vaccines and therapy
影响因子：
--
作者：
Tung WS;Bakar SA;Sekawi Z;Rosli R
通讯作者：
Rosli R
影响因子：
3.7
作者：
Liu CC;Guo MS;Lin FH;Hsiao KN;Chang KH;Chou AH;Wang YC;Chen YC;Yang CS;Chong PC
通讯作者：
Chong PC
影响因子：
5.5
作者：
Liu, Chia-Chyi;Chou, Ai-Hsiang;Chong, Pele
通讯作者：
Chong, Pele