Department of Veterinary Science, National Institute of Infectious Diseases, Toyama, Shinjuku, Tokyo, Japan.
Antiviral Res. 2011 Jul;91(1):64-71. doi: 10.1016/j.antiviral.2011.04.016. Epub 2011 May 5.
Rabies virus (RABV) is highly neurotropic and causes acute infection of the central nervous system. Death can be averted by prompt post-exposure prophylaxis; however, after clinical symptoms appear, the mortality rate is almost 100% and no reliable treatment is available. In this study, we investigated whether intracellular immunization using single-chain variable fragments (scFvs) against RABV phosphoprotein (RABV-P) could inhibit RABV propagation in neuronal cells. Of four scFv clones derived from an scFv phage-displayed library, scFv-P19 showed extremely high transfection efficiency and stable expression in mouse neuroblastoma (MNA) cells. The intracellular affinity and inhibition of RABV propagation were investigated using RABV-infected MNA cells pretransfected with the scFv-P19 gene. The specific interaction between scFv and RABV-P was confirmed by an immunoprecipitation assay and an indirect immunofluorescence assay showing that these molecules colocalized in the cytoplasm. Measurements of the spread of RABV in a culture well and the virus titer in the supernatant showed that RABV inhibition peaked 3 days after infection, at 98.6% and 99.9% inhibition, respectively. Although the mechanism of RABV inhibition by scFv-P19 is not clear, this scFv-based intracellular immunization could be a candidate for future RABV therapeutic studies if combined with appropriate delivery and application systems.
狂犬病病毒（RABV）具有高度嗜神经性，可引起中枢神经系统的急性感染。及时进行暴露后预防即可避免死亡；然而，一旦出现临床症状，死亡率几乎为 100%，且尚无可靠的治疗方法。在本研究中，我们研究了针对 RABV 磷蛋白（RABV-P）的单链可变片段（scFv）的细胞内免疫接种是否可以抑制神经元细胞中的 RABV 增殖。从 scFv 噬菌体展示文库中获得的四个 scFv 克隆中，scFv-P19 在小鼠神经母细胞瘤（MNA）细胞中显示出极高的转染效率和稳定表达。使用预先转染 scFv-P19 基因的感染 RABV 的 MNA 细胞，研究了 scFv 对 RABV 增殖的细胞内亲和力和抑制作用。免疫沉淀测定和间接免疫荧光测定证实了 scFv 与 RABV-P 的特异性相互作用，表明这些分子在细胞质中共定位。测量培养孔中 RABV 的传播和上清液中的病毒滴度表明，RABV 抑制在感染后 3 天达到峰值，抑制率分别为 98.6%和 99.9%。尽管 scFv-P19 抑制 RABV 的机制尚不清楚，但如果与适当的递送和应用系统相结合，这种基于 scFv 的细胞内免疫接种可能成为未来 RABV 治疗研究的候选方法。
