Refining the balance of attenuation and immunogenicity of respiratory syncytial virus by targeted codon deoptimization of virulence genes.
Refining the balance of attenuation and immunogenicity of respiratory syncytial virus by targeted codon deoptimization of virulence genes.
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DOI：
10.1128/mbio.01704-14
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发表时间：
2014-09-23
期刊：
影响因子：
6.4
通讯作者：
Moore ML
中科院分区：
文献类型：
--
作者：
Meng J;Lee S;Hotard AL;Moore ML
呼吸道合胞病毒（RSV）是儿童下呼吸道疾病的最重要病原体，目前尚无疫苗。RSV减毒活疫苗是临床上最先进的儿童疫苗，但实现减毒和免疫原性的最佳平衡具有挑战性。潜在地保留或增强减毒病毒的免疫原性的一种方式是突变抑制宿主免疫应答的毒力基因。根据人类或病毒密码子使用偏好对RSV A2毒株的NS 1和NS 2毒力基因进行密码子去优化，并通过反向遗传学拯救所得重组病毒（分别为dNSh和dNSv）。RSV dNSh表现出所需的NS 1和NS 2表达降低的表型。RSV dNSh在BEAS-2B和原代分化的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，但其诱导的RSV中和抗体水平略高于A2。RSV A2和RSV dNSh对攻毒毒株A/1997/12-35和A2-line 19 F诱导了等效的保护作用。RSV dNSh在体外引起的STAT 2降解和NF-κB活化比A2少。RSV dNSh在BEAS-2B细胞中的连续传代未导致去优化序列中的突变。总之，RSV dNSh是中度减毒的，更具免疫原性，与野生型RSV相比具有同等保护性，并且遗传稳定。呼吸道合胞病毒（RSV）是美国和世界范围内婴儿病毒性死亡的主要原因，并且没有可用的疫苗。减毒活RSV疫苗是在儿童中研究最多的，但具有遗传不稳定性和低免疫原性。为了解决这两个障碍，我们选择性地将RSV非结构（NS）毒力基因NS 1和NS 2的密码子使用改变为人类基因组中最少使用的密码子（去优化）。与亲本RSV相比，密码子去优化的NS 1/NS 2 RSV在体外和小鼠中减毒，但诱导更高水平的中和抗体和对攻击的等效保护。我们确定了一个新的减毒模块，保留免疫原性，是遗传稳定的，通过密码子使用去优化的非必需的毒力基因的特异性靶向。
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.