【Nature Vaccines】MP-12疫苗在细胞培养连续传代时遗传的稳定性

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编者按：7月17日，美国德克萨斯大学在加尔维斯顿的医学分部的Nandadeva Lokugamage等人在Nature Vaccines 上发表了论文，成功地揭示了MP-12株在M-U795C,M-A3564G和L-G3104A上的变异造成病毒的衰减，及M-U795C,M-A3564G,L-U533C和L-G3750A的变异独立地造成温度敏感的表型，同时揭示了回复突变L-G3750A发生在MP-12在Vero或MRC-5细胞传代的过程中，也发生在重组株rMP12-∆NSs16/198在Vero细胞培养时的M-A3564G，L-U533C上，但MP-12及变异株rMP12-TOSVSs在有病毒性脑炎的老鼠大脑中没有发现回复突变。这项研究完善了对过多的病毒传代后MP-12疫苗株回复突变L-G3750A的潜在风险的认识，也为MP-12疫苗遗传稳定性的进一步研究提供了新的思路。
       里夫特裂谷热是撒哈拉以南非洲的地方病，但已传播到埃及，马达加斯加岛，沙特阿拉伯和也门。它由蚊子传播，引起怀孕反刍动物高流产率和胎儿畸形。通过暴露于感染动物的体液或被感染的蚊子叮咬传播给人。高的致死率见于有出血、黄疸、神经功能障碍或失明的住院病人。2013年，MP-12疫苗就已获准用于兽医，了解它在病毒传代时的遗传稳定性对人类大规模预防接种是必要的。


            
      首先，研究者将MP-12疫苗株lot-7-2-88及重组株rMP12-∆NSs16/198在Vero细胞上连续传代25次（图1a）,又将MP-12疫苗株lot-7-2-88在MRC-5细胞上连续穿代25次，做了三组实验（图1b）,对1,5,10,15,25代细胞培养物上清的病毒滴度进行了测定。

Fig.1 Virus titers during serial virus passages.

接着，研究者用微滴式数字PCR对Vero细胞培养的疫苗株的五个决定毒力衰减或温度敏感性表型的突变——M-795, M-3564, L-533, L-3104, 和L-3750进行分析（图2），结果只有一个突变在传代时回复突变超过0.2% 。
      

Fig.2 Detection of reversion mutants of the MP-12 vaccine during viral passages in Vero cells.
   在MRC-5细胞，MP-12也MP-795,M-3564,L-533和L-3104 也没有出现回复突变超过0.2%,除了L-3750（图3）。

Fig. 3 Detection of reversion mutants of the MP-12 vaccine during viral passages in MRC-5 cells.
      为评估MP-12疫苗株和变异株rMP12-TOSVSs在体内病毒复制时的回复突变的发生，研究者提取了两者引起脑炎的老鼠的脑组织的RNA，用微滴式数字PCR仪分析，没有一个脑标本在上述五个变异位点有回复突变（图4）。

Fig. 4  Detection of reversion mutations in MP-12 and rMP12-TOSNSs in brains of mice
      综上，这项研究显示了MP-12疫苗在Vero和MRC-5细胞上有限的复制周期中显著的稳定性，尽管L-V172A, L-M1244I和M-R1182G 的回复突变在病毒连续传代时被确证。对MP-12疫苗株进一步的安全性分析，着重点应在于体内和体外实验产生的突变基因型的相关毒力。
Title:Genetic stability of Rift Valley fever virus MP-12 vaccine during serial passages in culture cells
Abtract:Rift Valley fever is a mosquito-borne zoonotic disease endemic to Africa, which affects both ruminants and humans. Rift Valley fever causes serious damage to the livestock industry and is also a threat to public health. The Rift Valley fever virus has a segmented negative-stranded RNA genome consisting of Large (L)-segment, Medium (M)-segment, and Small (S)-segment. The live-attenuated MP-12 vaccine is immunogenic in livestock and humans, and is conditionally licensed for veterinary use in the US. The MP-12 strain encodes 23 mutations (nine amino acid substitutions) and is attenuated through a combination of mutations in the L-segment, Msegment, and S-segment. Among them, the M-U795C, M-A3564G, and L-G3104A mutations contribute to viral attenuation through the L-segment and M-segment. The M-U795C, M-A3564G, L-U533C, and L-G3750A mutations are also independently responsible for temperature-sensitive phenotype. We hypothesized that a serial passage of the MP-12 vaccine in culture cells causes reversions of the MP-12 genome. The MP-12 vaccine and recombinant rMP12-ΔNSs16/198 were serially passaged 25 times. Droplet digital polymerase chain reaction analysis revealed that the reversion occurred at L-G3750A during passages of MP-12 in Vero or MRC-5 cells. The reversion also occurred at M-A3564G and L-U533C of rMP12-ΔNSs16/198 in Vero cells. Reversion mutations were not found in MP-12 or the variant, rMP12-TOSNSs, in the brains of mice with encephalitis. This study characterizedgenetic stability of the MP-12 vaccine and the potential risk of reversion mutation at the L-G3750A temperature-sensitive mutation after excessive viral passages in culture cells.
doi:10.1038/s41541-017-0021-9.




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