Sabin株脊髓灰质炎灭活疫苗在动物体内的免疫原性及安全性评价

Immunogenicity and safety of inactivated poliovirus vaccine derived from Sabin strain in animals

目的评价Sabin株脊髓灰质炎灭活疫苗（inactivated poliovirus vaccine derived from Sabin strain,s IPV）在动物体内的免疫原性和安全性。方法利用篮式生物反应器在Vero细胞上培养Sabin株脊髓灰质炎病毒,收获病毒液,经超滤浓缩、柱层析纯化、病毒灭活后,制备三价s IPV,参考《中国药典》三部（2010版）相关附录方法检测宿主细胞蛋白（host cell protein,HCP）、宿主细胞DNA及牛血清白蛋白残留量,HPLC法检测纯度;采用SDS-PAGE、Western blot法及透射电子显微镜对病毒的特异性进行鉴定;将s IPV于第0和21天免疫Wister大鼠,微量中和法检测免疫后大鼠血清中和抗体水平,并与野毒株IPV（IPV derived from wild strain,w IPV）组进行比较;通过观察ICR小鼠单次肌肉和静脉注射给予s IPV后产生的毒性反应及豚鼠反复给予s IPV后出现的速发型全身过敏反应,评价疫苗的安全性。结果 s IPV残余牛血清白蛋白、宿主细胞DNA、HCP检测结果分别低于2.5 ng/ml、100 pg/ml和100 ng/ml,疫苗纯度达95%以上;病毒形态及直径大小与脊髓灰质炎病毒一致,兔抗脊髓灰质炎病毒多克隆抗体可与对应型别的脊灰病毒的VP1、VP2、VP3发生特异性抗原抗体结合反应,相对分子质量分别在31 000-38 000、27 000-32 000和26 000-28 000之间;二免后,s IPV大鼠血清中和抗体阳转率及GMT均高于w IPV组;小鼠的急性毒性和豚鼠全身主动过敏反应均为阴性。结论制备的s IPV具有良好的免疫原性和安全性。

Objective To evaluate the immunogenicity and safety of inactivated poliovirus vaccine derived from Sabin strain（s IPV） in animals. Methods Sabin strain was cultured in Vero cells by using basket bioreactor, and the virus liquid was harvested, concentrated by ultrafiltration, purified by column chromatography and inactivated. The prepared trivalent s IPV was determined for host cell protein（HCP）, host cell DNA and residual bovine serum albumin by the methods in Chinese Pharmacopoeia（Volume Ⅲ, 2010 edition）, and for purity by HPLC. The specificity of virus was identified by SDS-PAGE, Western blot and transmission electron microscopy. Wister rats were immunized with s IPV on days 0 and 21, of which the serum neutralizing antibody levels were determined by micro-neutralization test and compared with those induced by IPV derived from wild strain（w IPV）. The safety of s IPV was evaluated by observation of toxic reaction in ICR mice after a single immunization by intramuscular and intravenous routes and immediate hypersensitivity after repeat immunization. Results The residual bovine serum albumin, host cell DNA and HCP contents in s IPV were less than 2. 5 ng / ml, less than 100 pg / ml and less than 100 ng / ml respectively, while the vaccine purity was more than95%. The morphology and size of virus were consistent with those of poliovirus. Rabbit anti-poliovirus polyclonal antibody showed specific binding to the VP1, VP2 and VP3 of poliovirus, with relative molecular masses of 31 000 - 38 000,27 000 - 32 000 and 26 000 - 28 000, respectively. Both the positive rate and GMT of neutralizing antibody in sera of rats after the second immunization with s IPV were higher than those with w IPV. However, both the results of acute toxicity test in mice and systemic active anaphylactic reaction test in guinea pigs were negative. Conclusion The prepared s IPV showed good immunogenicity and safety.