Meng Jia, Lee Sujin, Hotard Anne L, Moore Martin L
mBio. 2014 Sep 23;5(5):e01704-14. doi: 10.1128/mBio.01704-14.
Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness in children for which there is no licensed vaccine. Live-attenuated RSV vaccines are the most clinically advanced in children, but achieving an optimal balance of attenuation and immunogenicity is challenging. One way to potentially retain or enhance immunogenicity of attenuated virus is to mutate virulence genes that suppress host immune responses. The NS1 and NS2 virulence genes of the RSV A2 strain were codon deoptimized according to either human or virus codon usage bias, and the resulting recombinant viruses (dNSh and dNSv, respectively) were rescued by reverse genetics. RSV dNSh exhibited the desired phenotype of reduced NS1 and NS2 expression. RSV dNSh was attenuated in BEAS-2B and primary differentiated airway epithelial cells but not in HEp-2 or Vero cells. In BALB/c mice, RSV dNSh exhibited a lower viral load than did A2, and yet it induced slightly higher levels of RSV-neutralizing antibodies than did A2. RSV A2 and RSV dNSh induced equivalent protection against challenge strains A/1997/12-35 and A2-line19F. RSV dNSh caused less STAT2 degradation and less NF-κB activation than did A2 in vitro. Serial passage of RSV dNSh in BEAS-2B cells did not result in mutations in the deoptimized sequences. Taken together, RSV dNSh was moderately attenuated, more immunogenic, and equally protective compared to wild-type RSV and genetically stable.
Respiratory syncytial virus (RSV) is the leading cause of infant viral death in the United States and worldwide, and no vaccine is available. Live-attenuated RSV vaccines are the most studied in children but have suffered from genetic instability and low immunogenicity. In order to address both obstacles, we selectively changed the codon usage of the RSV nonstructural (NS) virulence genes NS1 and NS2 to the least-used codons in the human genome (deoptimization). Compared to parental RSV, the codon-deoptimized NS1/NS2 RSV was attenuated in vitro and in mice but induced higher levels of neutralizing antibodies and equivalent protection against challenge. We identified a new attenuating module that retains immunogenicity and is genetically stable, achieved through specific targeting of nonessential virulence genes by codon usage deoptimization.
呼吸道合胞病毒（RSV）是导致儿童下呼吸道疾病的最重要病原体，目前尚无获批的疫苗。减毒活RSV疫苗在儿童中是临床进展最为显著的，但要在减毒和免疫原性之间实现最佳平衡具有挑战性。一种可能保留或增强减毒病毒免疫原性的方法是突变抑制宿主免疫反应的毒力基因。根据人类或病毒的密码子使用偏好，对RSV A2株的NS1和NS2毒力基因进行密码子去优化，通过反向遗传学拯救得到重组病毒（分别为dNSh和dNSv）。RSV dNSh表现出NS1和NS2表达降低的预期表型。RSV dNSh在BEAS - 2B和原代分化气道上皮细胞中减毒，但在HEp - 2或Vero细胞中不减毒。在BALB/c小鼠中，RSV dNSh的病毒载量低于A2株，但其诱导的RSV中和抗体水平略高于A2株。RSV A2和RSV dNSh对攻击株A/1997/12 - 35和A2 - line19F诱导的保护作用相当。在体外，RSV dNSh导致的STAT2降解和NF - κB激活比A2株少。RSV dNSh在BEAS - 2B细胞中的连续传代未导致去优化序列发生突变。综上所述，与野生型RSV相比，RSV dNSh适度减毒、免疫原性更强且保护作用相当，并且遗传稳定。
呼吸道合胞病毒（RSV）是美国乃至全球婴儿病毒性死亡的主要原因，且尚无可用疫苗。减毒活RSV疫苗在儿童中研究最多，但存在遗传不稳定和免疫原性低的问题。为了解决这两个障碍，我们将RSV非结构（NS）毒力基因NS1和NS2的密码子使用情况选择性地改变为人类基因组中使用最少的密码子（去优化）。与亲本RSV相比，密码子去优化的NS1/NS2 RSV在体外和小鼠中均减毒，但诱导产生更高水平的中和抗体以及对攻击的等效保护作用。我们通过密码子使用去优化特异性靶向非必需毒力基因，鉴定出一种新的减毒模块，该模块保留免疫原性且遗传稳定。
