PDF(Pubmed)
Abstract：
Rabies virus (RABV) causes fatal neurological disease. Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) using inactivated-virus vaccines are the most effective measures to prevent rabies. In Japan, HEP-Flury, the viral strain, used as a human rabies vaccine, has historically been propagated in primary fibroblast cells derived from chicken embryos. In the present study, to reduce the cost and labor of vaccine production, we sought to adapt the original HEP-Flury (HEP) to Vero cells. HEP was repeatedly passaged in Vero cells to generate ten- (HEP-10V) and thirty-passaged (HEP-30V) strains. Both HEP-10V and HEP-30V grew significantly better than HEP in Vero cells, with virulence and antigenicity similar to HEP. Comparison of the complete genomes with HEP revealed three non-synonymous mutations in HEP-10V and four additional non-synonymous mutations in HEP-30V. Comparison among 18 recombinant HEP strains constructed by reverse genetics and vesicular stomatitis viruses pseudotyped with RABV glycoproteins indicated that the substitution P(L115H) in the phosphoprotein and G(S15R) in the glycoprotein improved viral propagation in HEP-10V, while in HEP-30V, G(V164E), G(L183P), and G(A286V) in the glycoprotein enhanced entry into Vero cells. The obtained recombinant RABV strain, rHEP-PG4 strain, with these five substitutions, is a strong candidate for production of human rabies vaccine.
摘要：
狂犬病病毒（RABV）导致致命的神经系统疾病。使用灭活病毒疫苗的暴露前预防（PrEP）和暴露后预防（PEP）是预防狂犬病的最有效措施。在日本,HEP-Flury,病毒株，用作人类狂犬病疫苗，历史上已在源自鸡胚的原代成纤维细胞中繁殖。在本研究中,为了降低疫苗生产的成本和劳动力，我们试图使原始的HEP-Flury（HEP）适应Vero细胞。HEP在Vero细胞中反复传代以产生十种(HEP-10V)和三十种传代(HEP-30V)菌株。HEP-10V和HEP-30V在Vero细胞中的生长明显优于HEP,具有类似于HEP的毒力和抗原性。完整基因组与HEP的比较揭示了HEP-10V中的三个非同义突变和HEP-30V中的另外四个非同义突变。通过反向遗传学构建的18个重组HEP菌株与用RABV糖蛋白假型的水泡性口炎病毒之间的比较表明，磷蛋白中的P（L115H）和糖蛋白中的G（S15R）的取代改善了HEP-10V中的病毒繁殖，在HEP-30V中,G(V164E),G(L183P),糖蛋白中的G(A286V)增强了进入Vero细胞的能力。获得的重组RABV菌株，rHEP-PG4菌株，有了这五个替换，是生产人类狂犬病疫苗的有力候选者。
