Le Nouën Cyril, McCarty Thomas, Brown Michael, Smith Melissa Laird, Lleras Roberto, Dolan Michael A, Mehedi Masfique, Yang Lijuan, Luongo Cindy, Liang Bo, Munir Shirin, DiNapoli Joshua M, Mueller Steffen, Wimmer Eckard, Collins Peter L, Buchholz Ursula J
RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892;
RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 2017 Jan 17;114(3):E386-E395. doi: 10.1073/pnas.1619242114. Epub 2017 Jan 3.
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 °C/35 °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 two mutations, 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.
通过大量同义但次优的替换对病毒基因组进行重新编码可提供减毒活疫苗候选株。由于涉及众多变化，这些疫苗候选株的毒力恢复风险应较低。然而，它们在选择性压力下的遗传稳定性在很大程度上尚不清楚。我们在强选择性压力背景下评估了去优化的人呼吸道合胞病毒（RSV）疫苗候选株的表型回复。RSV的密码子对去优化（CPD）版本具有减毒和温度敏感性。在逐渐升高的温度下连续传代时，在11个开放阅读框中的9个中含有2692个同义突变的CPD RSV并未丧失温度敏感性，保持遗传稳定，并且在34℃/35℃及以上温度受到限制。然而，仅在聚合酶L开放阅读框中含有1378个同义突变的CPD RSV很快就失去了显著的减毒效果。对病毒群体的全面序列分析在L开放阅读框中鉴定出许多不同的潜在减毒突变，令人惊讶的是，在其他开放阅读框中也出现了许多此类突变。表型分析表明，在CPD区域之外的病毒转录抗终止因子M2-1中的两个竞争性突变中的任何一个，都能显著逆转CPD L基因的缺陷转录，并在体外显著恢复病毒适应性，并且这两个突变之一在体内也能恢复病毒适应性。矛盾的是，在M2-1、N、P和L蛋白中各引入一个突变到Min L中，会产生一种在体内减毒增加但免疫原性增强的病毒。因此，除了提供关于基因组规模去优化的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病毒疫苗候选株。
