人乳头瘤病毒16型假病毒中和实验的建立和初步应用

探讨了应用多质粒磷酸钙共转染方法在293FT细胞中生产HPV16(human papillomavirus type 16)假病毒。蛋白印迹检测显示在转染后细胞的裂解上清中具有很好的L1蛋白活性,通过透射电镜可观察到形态与天然病毒粒子相似的假病毒颗粒。对293FT细胞的感染实验显示,该假病毒可有效将EGFP报告质粒导入靶细胞中进行表达,经测定其滴度约为2×107TU/mL。通过与4株HPV16对照单抗的中和实验证明该假病毒可有效应用于中和实验。应用该方法从18株抗HPV16L1的单克隆抗体中鉴定获得了2株中和单抗3D10、PD1。所建立的HPV16假病毒生产和中和实验方法具有快速高效、低成本和易于检测的优点,适于进行较大规模应用,为快速准确鉴定HPV16中和单抗和候选疫苗的免疫保护效果提供了有效手段。

Establishment and Application of Human Papillomavirus Type 16 Pseudovirions Neutralization Assay

Human papillomaviruses (HPV) are causally associated with cervical cancer and genital warts. Lack of permissive and productive cell cultures for HPV has hindered the study of HPV and evaluation of virus-neutralizing antibodies. So generation of infectious virions in vitro is highly desirable. In this report,we got high titer infectious HPV16 pseudovirions by calcium phosphate co-transfection of codon optimized HPV16 capsid genes L1 and L2 and reporter plasmids into 293FT cell line. Electron micrograph indicated that the pseudovirions were morphologically similar with the intact HPV16 virions. To evaluate the feasibility of using the pseudovirions to identify neutralizing monoclonal antibodies (mAbs),pseudovirions were incubated with 2-fold gradient dilution of the well identified mAbs V5,E70,U4 and D9 and then used to infect 293FT cells preplated in 96-well tissue culture plate. The infection of pseudovirions could be inhibited by neutralizing mAbs V5,E70 and U4 that recognize surface conformational epitopes on L1 VLP,but not by mAb D9 that is reactive to a linear epitope buried in VLP,which indicated that the pseudovirions could be used to evaluate the neutralization efficiency of mono-and polyclonal antibodies. The pseudovirions were then employed to identify neutralizing mAbs from 18 mAbs generated previously in our lab,8 of which were conformational and 10 were linear. PD1 and 3D10,both of which recognized conformational epitopes on L1 VLP,had obviously strong neutralizing efficiency,with the neutralizing titer reached 81,920 and 20,480 respectively,while none of the linear mAbs were neutralizing,which reflected that rare linear mAbs have neutralization activity. The mechanism of PD1 and 3D10 block the infection of HPV16 pseudovirions need to be further studied. The technologies about generation of HPV16 pseudovirions and screening neutralizing mAb in our report are economical and efficient,can be easily used in large scale. They pave the way for rapid and precise evaluation of the protection efficiency of our prophylactic HPV vaccine being developed now.