The SD12-F120 is a live-attenuated genotype I strain of Japanese encephalitis virus (JEV) and was obtained by serial passage of wild-type strain SD12 on BHK-21 cells combined with multiple plaque purification and virulence selection in mice. The large scale production and vast clinical trials always demand ideal safety and efficacy profile of live-attenuated vaccines. In the present study, SD12-F120VC has undergone serial passaging of P1-P30 in WHO qualified Vero cells to assess the potential effect of adaptation to growth on Vero cells. The series of experiments showed that vaccine SD12-F120VC (Vero cell adapted) variants have consistently increased in peak virus titer compared to early passages and have good adaptation to growth in Vero cells. The animal experiments showed that Vero cell adapted SD12-F120VC variants have attenuation phenotype in suckling mice and the plaque morphology for all SD12-F120VC variants was small. Vaccination of mice with SD12-F120VC vaccine produced complete protection for homologous SD12 genotype I strain, but failed to give the complete protection of vaccinated mice against the challenge of heterologous N28 genotype III strain. In response to immunization of SD12-F120VC in mice, the neutralizing antibodies titer against homologous SD12-F120VC and SD12 (GI) was higher than heterologous N28 (GIII) strain. The prM protein has 6 amino acid substitutions, of which 5 amino acid changes were confined at the start of the pr domain in the ∼40 amino acids, and some mutations in the pr domain of prM might contribute to Vero cell adaptation. Our findings in this study are important for validation, evaluation and quality control study of live attenuated flaviviruses vaccines and show that Vero cells are a suitable substrate for the production of a safe and stable live-attenuated JEV vaccine.

SD12-F120 是日本脑炎病毒 (JEV) 的减毒基因 I 型活毒株，通过在 BHK-21 细胞上连续传代野生型毒株 SD12 并结合多斑纯化和小鼠毒力选择获得。大规模生产和大量临床试验总是要求减毒活疫苗具有理想的安全性和有效性。在本研究中，SD12-F120VC 在 WHO 合格的 Vero 细胞中经历了 P1-P30 的连续传代，以评估适应生长对 Vero 细胞的潜在影响。这一系列实验表明，与早期传代相比，疫苗 SD12-F120VC（Vero 细胞适应型）变体的峰值病毒滴度持续增加，并且对 Vero 细胞中的生长具有良好的适应性。动物实验表明，Vero 细胞适应的 SD12-F120VC 变体在哺乳小鼠中具有减毒表型，并且所有 SD12-F120VC 变体的斑块形态都很小。用 SD12-F120VC 疫苗接种小鼠对同源 SD12 基因型 I 株产生完全保护，但未能完全保护接种的小鼠免受异源 N28 基因型 III 株的攻击。响应于小鼠中 SD12-F120VC 的免疫接种，针对同源 SD12-F120VC 和 SD12 (GI) 的中和抗体效价高于异源 N28 (GIII) 菌株。prM 蛋白有 6 个氨基酸取代，其中 5 个氨基酸变化被限制在 pr 结构域的起始处，约 40 个氨基酸，prM pr 结构域中的一些突变可能有助于 Vero 细胞适应。黄病毒疫苗，并表明 Vero 细胞是生产安全稳定的 JEV 减毒活疫苗的合适底物。