EV71灭活疫苗(人二倍体细胞)灭活工艺验证

Verification of inactivation process for inactivated enterovirus 71 vaccine(human diploid cells)

目的对肠道病毒71型（enterovirus 71,EV71）灭活疫苗（人二倍体细胞）的灭活工艺进行验证。方法取3批次经甲醛灭活的EV71灭活病毒收获液样品,分别连续盲传4代,并设病毒对照盲传4代。观察每代次样品的细胞病变情况,采用微量滴定法检测病毒感染性滴度,ELISA法检测EV71特异抗原含量,实时定量PCR法检测病毒核酸水平及病毒负链产生情况。结果 3批灭活的疫苗病毒收获液样品经4次盲传后,各代次均未出现明显的细胞病变,经微量滴定法均未检测到EV71感染性滴度。灭活的病毒收获液盲传至第2代时,病毒负链检测已为阴性;至第3代时,EV71抗原检测为阴性;至第4代时,病毒核酸检测为阴性。病毒对照经盲传4代后,各代次均出现明显细胞病变;自第2代开始,感染性滴度维持在106~107 CCID50/ml,抗原含量为200~370 U/ml,且均可检测出明显的病毒核酸及病毒负链增殖。结论通过经典的＂细胞上盲传3代＂的检测方法,并结合实时定量RT-PCR对病毒核酸和负链进行检测,证实经甲醛灭活的EV71灭活疫苗样品已完全灭活。

Objective To verify the inactivation procedure for inactivated enterovirus 71（EV71）vaccine（human diploid cells）. Methods Samples were taken from three batches of harvests of EV71 inactivated with formaldehyde for four blind passages, using the harvests before inactivation as control. Each passage was observed for cytopathic effect（CPE）, and determined for infectious titer by microtitration, for EV71-specific antigen content by ELISA, and for viral DNA level and production of virus negative strand by real-time quantitative PCR（q RT-PCR）. Results After four blind passages, no obvious CPEs were observed in three batches of inactivated vaccine of various passages, and no infectious EV71 titers were detected by microtitration. After two blind passage, no negative strand of virus was detected. The test result for EV71 antigen was negative after three blind passages, while that for viral DNA was negative after four blind passages.However, obvious CPEs were observed in virus control of various passages after four blind passages, while the infectious titers were 106~ 107CCID50/ ml, the antigen content was 200 ~ 370 U / ml, and obvious propagation of viral DNA and negative strand were observed, after two blind passages. Conclusion The inactivated EV71 samples were inactivated with formaldehyde completely by typical three blind passages in cells combined with q RT-PCR.