Identification of mechanisms underlying the attenuation of viruses by codon pair deoptimization

使用传统方法开发疫苗费时、费力且不可预测，在最需要应对新出现的传染病时会失败。密码子对去优化减毒（CPD）是一种新的减毒策略，可以高效快速地减毒各种病毒，包括脊髓灰质炎病毒和登革病毒。CPD减毒是基于病毒基因组的大规模重新编码。它涉及病毒基因中密码子的重组，而不改变编码蛋白质的密码子偏好或氨基酸组成。重组的目的是使宿主基因组中代表性不足的密码子对的数量最大化，因为密码子对去优化的病毒复制效率远低于其野生型亲本。CPD具有巨大的潜力，因为它适用于许多动物或人类病毒和其他病原体的减毒。然而，CPD方法的主要缺点是导致衰减的分子机制仍然难以捉摸。CPD将数百个核苷酸突变引入到重新编码的基因中，但尚不知道哪些引入的遗传变化是减毒的原因。在这个问题得到解决之前，将不可能在临床环境中利用重新编码的病毒疫苗。此外，不可能通过监测减毒病毒的遗传变化来检查减毒的逆转。一旦我们了解CPD减毒的分子基础，就有可能改进减毒设计并生产更好和更安全的病毒疫苗。本项目的主要目标是确定CPD减毒的确切分子机制。为了实现本项目的目标，我们将使用H1N1流感病毒，一种重要的人类病原体，作为模型病毒。我们已经制备了一系列重新编码的H1N1突变体，其中我们独立地改变了两个主要序列特征，这两个主要序列特征被怀疑是减毒的基础-代表性不足的密码子对的数量和类型，以及CpG二核苷酸的数量。我们的初步研究是这一提议的关键，因为它们明确地证实了将代表性不足的密码子对而不是CpG二核苷酸引入重新编码的基因中会导致体外重新编码病毒的减毒。在这个项目中，我们将解决CPD减毒的四个主要未知数。我们将i）准确识别哪些密码子对主要负责重新编码的病毒的减毒; ii）建立代表性不足的密码子对如何干扰导致减毒的分子机制; iii）量化减毒病毒在体外广泛传代后恢复毒力的能力; iv）比较密码子去优化、密码子对去优化或CpG最大化的病毒的减毒水平和疫苗效力，并确定三种重编码方法中的哪一种对于体内H1N1病毒的减毒是上级的。

Vaccine development using traditional approaches is time-consuming, laborious and unpredictable and fails when response to newly emerging infectious diseases is needed the most. Attenuation by codon pair deoptimization (CPD) is a new strategy that has enabled highly efficient and rapid attenuation of a wide variety of viruses, including poliovirus and Dengue virus. The attenuation by CPD is based on large-scale recoding of viral genomes. It involves reshuffling of codons in viral genes without changing the codon bias or amino acid composition of the encoded proteins. The goal of reshuffling is to maximize the number of codon pairs that are underrepresented in the host genome, because codon pair deoptimized viruses replicate much less efficiently as their wild type parents. CPD has an immense potential, because it is applicable for attenuation of many animal or human viruses and other pathogens. The major drawback of the CPD approach is, however, that the molecular mechanisms that result in attenuation have remained elusive. CPD introduces several hundred nucleotide mutations into recoded genes, but it is not known which of the introduced genetic changes are responsible for attenuation. Until this problem is solved it will not be possible to utilize recoded viral vaccines in the clinical settings. Also, it will not be possible examine the reversion of attenuation through monitoring of genetic changes in attenuated viruses. Once we understand the molecular basis of attenuation by CPD it will be possible to refine the attenuation design and produce better and safer viral vaccines. The main goal of this project is to identify the exact molecular mechanisms that underlie the attenuation by CPD. To attain the objective of this project we will use H1N1 influenza virus, an important human pathogen, as a model virus. We have prepared a series of recoded H1N1 mutants in which we independently varied two main sequence features that are suspected to underlie the attenuation – the number and type of underrepresented codon pairs, and the number of CpG dinucleotides. Our preliminary studies are key for this proposal, because they unequivocally confirmed that introduction of underrepresented codon pairs, but not CpG dinucleotides into recoded genes leads to attenuation of recoded viruses in vitro. During this project we will solve four major unknowns of the attenuation by CPD. We will i) identify exactly which codon pairs are principally responsible for attenuation of recoded viruses; ii) establish how underrepresented codon pairs perturb molecular mechanisms that lead to attenuation; iii) quantify the capacity of attenuated viruses to revert to virulence upon extensive passage in vitro; iv) compare the level of attenuation and vaccine efficacy of codon-deoptimized, codon pair-deoptimized, or CpG-maximized viruses and determine which of the three recoding methods is superior for attenuation of H1N1 virus in vivo.