Development of a Genetically Stable Live Attenuated Influenza Vaccine Strain using an Engineered High-Fidelity Viral Polymerase
Development of a Genetically Stable Live Attenuated Influenza Vaccine Strain using an Engineered High-Fidelity Viral Polymerase
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使用工程化高保真病毒聚合酶开发遗传稳定的减毒活流感疫苗株
DOI：
10.1128/jvi.00493-21
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发表时间：
2021
影响因子：
5.4
通讯作者：
Saito Mineki
中科院分区：
文献类型：
--
作者：
Mori Kotaro;Ohniwa Ryosuke L.;Takizawa Naoki;Naito Tadasuke;Saito Mineki
RNA病毒表现出广泛的变异，称为准物种，由于病毒RNA依赖的RNA聚合酶容易出错的复制。虽然减毒活疫苗在预防RNA病毒感染方面是有效的，但在接种后存在毒力逆转的风险。为了验证高保真病毒聚合酶降低流感病毒准种多样性，从而抑制减毒表型逆转的假设，我们首先使用鸟苷类似物利巴韦林选择的连续病毒传代筛选高保真病毒聚合酶。因此，我们在聚合酶碱性蛋白1 （PB1）中发现了一个Leu66-to-Val单氨基酸突变。利用新一代测序分析和纯化流感病毒聚合酶的生化分析证实了PB1-L66V的高保真表型。正如预期的那样，与野生型（WT）相比，PB1-L66V表现出至少低两倍的突变率和低的错误掺入率。因此，接下来，我们基于流感减毒活疫苗FluMist（一种可以通过其突变限制病毒传播的流感减毒活疫苗LAIV），产生了具有温度敏感（ts）表型的PB1-L66V减毒病毒，并使用系列病毒传代检测了PB1-L66V减毒病毒的遗传稳定性。PB1-L66V突变阻止了ts表型向WT表型的逆转，这表明高保真病毒聚合酶可能有助于产生具有高遗传稳定性的LAIV，而不会还原为致病性病毒。重要性：目前使用的LAIV是为2至49岁的个人主动免疫而规定的。然而，它不被批准用于婴儿和老年人，他们实际上最需要它，因为它可能会延长病毒的传播，并在这些人身上引起明显的感染，因为他们的免疫系统薄弱。最近，在培养细胞中证实了目前使用的LAIV株的ts表型向致病性病毒的逆转。因此，应考虑与LAIV毒力增强相关的突变的产生。在这项研究中，我们分离了一种新的流感病毒株，该株在PB1中具有Leu66-to-Val单氨基酸突变，其保真度明显高于WT。我们产生了一种新的具有该突变的LAIV候选株。该菌株具有较高的遗传稳定性，无ts表型逆转。因此，我们的高保真菌株可能有助于开发更安全的LAIV。
RNA viruses demonstrate a vast range of variants, called quasispecies, due to error-prone replication by viral RNA-dependent RNA polymerase. Although live attenuated vaccines are effective in preventing RNA virus infection, there is a risk of reversal to virulence after their administration. To test the hypothesis that high-fidelity viral polymerase reduces the diversity of influenza virus quasispecies, resulting in inhibition of reversal of the attenuated phenotype, we first screened for a high-fidelity viral polymerase using serial virus passages under selection with a guanosine analog ribavirin. Consequently, we identified a Leu66-to-Val single amino acid mutation in polymerase basic protein 1 (PB1). The high-fidelity phenotype of PB1-L66V was confirmed using next-generation sequencing analysis and biochemical assays with the purified influenza viral polymerase. As expected, PB1-L66V showed at least two-times-lower mutation rates and decreased misincorporation rates, compared to the wild type (WT). Therefore, we next generated an attenuated PB1-L66V virus with a temperature-sensitive (ts) phenotype based on FluMist, a live attenuated influenza vaccine (LAIV) that can restrict virus propagation by ts mutations, and examined the genetic stability of the attenuated PB1-L66V virus using serial virus passages. The PB1-L66V mutation prevented reversion of the ts phenotype to the WT phenotype, suggesting that the high-fidelity viral polymerase could contribute to generating an LAIV with high genetic stability, which would not revert to the pathogenic virus.IMPORTANCEThe LAIV currently in use is prescribed for actively immunizing individuals aged 2 to 49 years. However, it is not approved for infants and elderly individuals, who actually need it the most, because it might prolong virus propagation and cause an apparent infection in these individuals, due to their weak immune systems. Recently, reversion of the ts phenotype of the LAIV strain currently in use to a pathogenic virus was demonstrated in cultured cells. Thus, the generation of mutations associated with enhanced virulence in LAIV should be considered. In this study, we isolated a novel influenza virus strain with a Leu66-to-Val single amino acid mutation in PB1 that displayed a significantly higher fidelity than the WT. We generated a novel LAIV candidate strain harboring this mutation. This strain showed higher genetic stability and no ts phenotype reversion. Thus, our high-fidelity strain might be useful for the development of a safer LAIV.