Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure
Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure
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DOI：
10.1073/pnas.1619242114
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发表时间：
2017-01-17
影响因子：
11.1
通讯作者：
Buchholz, Ursula J.
中科院分区：
文献类型：
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作者：
Le Nouen, Cyril;McCarty, Thomas;Buchholz, Ursula J.
通过大量同义但次优替换重新编码病毒基因组可提供候选减毒活疫苗。由于涉及许多变化，这些候选疫苗的减毒风险应该较低。然而，它们在选择压力下的遗传稳定性很大程度上未知。我们在强选择压力的背景下评估了去优化的人呼吸道合胞病毒（RSV）候选疫苗的表型逆转。 RSV 的密码子对去优化 (CPD) 版本是减毒的且对温度敏感。在逐渐升高的温度下连续传代期间，11个ORF中的9个含有2,692个同义突变的CPD RSV没有失去温度敏感性，保持遗传稳定，并且在34℃/35℃及以上的温度下受到限制。然而，仅在聚合酶 L ORF 中含有 1,378 个同义突变的 CPD RSV 很快就失去了实质性的减毒作用。对病毒群体的综合序列分析发现了 L ORF 中许多不同的潜在减毒突变，以及令人惊讶的许多出现在其他 ORF 中的突变。表型分析显示，病毒转录抗终止因子 M2-1 中 CPD 区域之外的两个竞争突变中的任何一个，都显着逆转了 CPD L 基因的缺陷转录，并在体外显着恢复了病毒的适应性，并且在这两个突变之一的情况下，也在体内显着恢复了病毒的适应性。矛盾的是，在 Min L 中引入 M2-1、N、P 和 L 蛋白各一个突变，导致病毒体内减毒作用增强，但免疫原性增强。因此，除了提供关于基因组规模去优化RNA病毒适应性的见解外，稳定性研究还可以产生改进的合成RNA病毒候选疫苗。
Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 degrees C/35 degrees C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these twomutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.