呼吸道合胞病毒单克隆抗体逃逸株体外适合度变化

In vitro fitness change of respiratory syncytial virus Palivizumab escape mutants

目的Palivizumab（PZ）是针对呼吸道合胞病毒（respiratory syncytial virus，RSV）F蛋白的人源化单克隆抗体（McAb）。本研究观察PZ逃逸株体外适合度变化规律。方法采用竞争复制法测定两株Pz抵抗株（F212和MP4）的体外适合度。将等量F212和母株A2混合后在HEp-2细胞中连续转代，在不同时间点采用差异噬斑法、核酸序列图谱分析法和限制性片段多态性法分析混合病毒群体中F212和A2的比例。由于MP4在细胞培养和棉鼠肺内复制水平与A2相似，故将A2和MP4按照1：1、10：1、100：1和1000：1的比例混合并在HEp-2细胞中竞争复制，不同时点采用差异噬斑法和核酸序列图谱分析法分析混合病毒群体中MP4和A2比例。结果等比例混合的F212／A2混合群体在传代至10代时，F212已基本消失，传至35代时仍无F212信号，提示其适合度低于A2。A2／MP4捏合病毒群体中，除按1000：1比例预混外，其余所有比例预混的病毒中MP4均成为优势株，因而提示MP4的适合度高于A2。结论首次发现RSVPZ逃逸株适合度可超过原型株A2，了解适合度增加或降低的机制对减毒活疫苗的研究有理论指导意义。

Objective To assess the fitness of two Palivizumab（PZ）-resistant respiratory syncytial viruses （RSV） strains（F212 and MP4） in HEp-2 cells. Methods A mixture was made with equal amounts of F212 and A2 yirus and subcultured continuously in cell cultures. During cultivation the ratio of F212/A2 was measured by differential plaque assay, nucleotide sequence analysis and restriction fragment length poly-morphism at different time points. Because the replication levels of A2 and MP4 in cell cultures or in cotton rat lungs were similar, the mixtures of A2/MP4 virus at the ratios of 1： 1, 10： 1, 100：1 and 1000：1 were inoculated respectively in HEp-2 cell cuhures for the comparison in competitive replication. The relative amount of MP4 or A2 was tested by differential plaque assay and nucleotide sequence analysis at different time points. Results During continuous subcultures of F212/A2 at 1：1 ratio, F212 disappeared nearly at the 10th passage indicating its fitness was lower than that of A2 virus. Among all groups of virus mixtures with various ratios except for 1000：1 group, the MP4 virus became predominant, which showed that the fitness of MP4 was higher than that of A2. Conclusion The mutant virus strain MP4 is of more fitness than that of A2 virus in both in vitro and in vivo competitive replication. Understanding the increase and decrease mechanisms of mutant virus fitness could be useful for the research of attenuated vaccine.