摘要
目的建立三色混合荧光HPV假病毒中和检测系统，并评价其在检测HPV九价疫苗免疫血清免疫原性的应用。方法分别选取红色、绿色和蓝色荧光蛋白的质粒N31-MCHREEY、N31-EGFP和N31-mTagBFP构建HPV6/11/16/18/31/33/45/52/58假病毒，利用双抗夹心法和病毒半数组织培养感染剂量法检测HPV假病毒浓度和感染滴度；利用单色荧光与三色混合荧光HPV16/33/45假病毒，检测HPV九价疫苗免疫小鼠血清的中和滴度，以验证三色混合荧光HPV假病毒系统的准确性；采用单色荧光和三色混合荧光HPV6/11/16/18/31/33/45/52/58假病毒系统检测HPV九价疫苗免疫食蟹猴血清的中和滴度，判断其是否可应用于HPV九价疫苗免疫血清的免疫原性检测。结果含有绿色、红色和蓝色荧光质粒的HPV16型假病毒的浓度为5~6 μg/ml，HPV6/11/18/31/33/45/52/58的三色混合荧光假病毒浓度为1~3 μg/ml。获得了分别含有EGFP、Mcherry和mTagBFP报告基因的9个型别的HPV假病毒，且满足中和检测需要；9个型别单色荧光HPV假病毒的滴度值均在1×104~1×105之间，即它们具有相似的感染滴度。检测HPV九价疫苗免疫的小鼠血清时，单色荧光与三色混合荧光HPV16/33/45假病毒系统所得中和滴度值差异均无统计学意义（P值均〉0.05）；检测HPV九价疫苗免疫的食蟹猴血清时，单色荧光与三色混合荧光A、B和C组HPV6/11/16/18/31/33/45/52/58假病毒所得中和滴度值差异均无统计学意义（P值均〉0.05）。结论成功地建立了三色混合荧光HPV假病毒中和检测系统；验证其与单色荧光HPV假病毒系统具有一致性；证实其可应用于检测HPV九价疫苗免疫血清的免疫原性检测中。
ObjectiveTo establish a triple-color pseudovirion-based neutralization assay （PBNA） and evaluate its capability of detecting immunogenicity of the sera generated by the immunization of HPV 9-valent vaccine.MethodsHPV pseudovirus （PsVs） 6/11/16/18/31/33/45/52/58 with the encapsidated fluorescence expressing red fluorescent plasmid N31-MCHREEY, green fluorescent N31-EGFP or blue fluorescent N31-mTagBFP were generated. The concentration of HPV PsVs and the infection titers of HPV PsVs were detected by double-antibody sandwich ELISA and TCID50, respectively. The single- and triple color HPV 16/33/45 PsVs were used to detect the neutralization titers of mice sera immunized with HPV 9-valent vaccine and confirmed the accuracy and specificity of the triple-color PBNAs. Then, the single- and triple color HPV 6/11/18/31/33/45/52/58 PsVs were employed to detect the neutralization titers of cynomolgus macaques sera immunized with HPV 9-valent vaccine and determined whether the triple-color PBNAs could be applied to evaluate the immunogenicity of the sera generated by the immunization of HPV9-valent vaccine.ResultsThe concentration of HPV16 PsVs encapsulating green, red or blue fluorescent plasmid was 5.0 to 6.0 μg/ml and HPV6/11/18/31/33/45/52/59 triple-color HPV PsVs was about 1.0 to 3.0 μg/ml. 9 types HPV PsVs containing EGFP, Mcherry or mTagBFP reporter plasmid were obtained and the concentration can meet the need of neutralization detection. 9 types single-color fluorescent HPV PsVs had similar infectivity against 293FT cells with the infection titer values between 1×104 and 1×105. The results of PBNAs showed that there was no significant difference in the anti-HPV neutralization titers of mice sera induced by HPV 9-valent vaccine between single-color and triple-color HPV16/33/45 PsVs （P〉0.05）. Similarly, there was also no significant difference in the anti-HPV neutralization titers of cynomolgus macaques sera induced by HPV 9-valent vaccine between single-color and triple-color HPV6/11/18/31/33/45/52/58 PsVs （P〉0.05）.ConclusionWe successfully established the triple-color PBNAs and verified the accuracy and specificity of triple-color PBNAs consistent with single-color PBNAs. The triple-color PBNAs can be applied to evaluate the immunogenicity of HPV 9-valent vaccine＇s immune serum.
作者
魏双萍
范飞
陈洁
柳欣林
杨与柔
王致萍
宋硕
李智海
魏旻希
王大宁
李少伟
夏宁邵
Wei Shuangping;Fan Fei;Chen Jie;Liu Xinlin;Yang Yurou;Wang Zhiping;Song Shuo;Li Zhihai;Wei Minxi;Wang Darting;Li Shaowei;Xia Ningshao(National Institute of Diagnostics and Vaccine Development of Infectious Diseases,State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics,School of Life Sciences,Xiamen University,Xiamen 361102,China)
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2018年第10期1039-1044,共6页
Chinese Journal of Preventive Medicine
基金
国家自然科学基金（31670935）
关键词
疫苗
中和试验
免疫血清
人乳头瘤病毒
三色荧光假病毒
Vaccine
Neutralization tests
Immune sera
Human papillomavirus
HPV triple-color pseudovirus
