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

点击翻译按钮获取中文摘要

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.