Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia; Institute of Vector Borne Disease, Monash University, Clayton, Victoria, Australia.
Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia.
Vaccine. 2019 May 9;37(21):2857-2863. doi: 10.1016/j.vaccine.2019.03.062. Epub 2019 Apr 15.
The overwhelming increase of dengue virus (DENV) infections in recent years shows that current strategies to combat dengue do not work. The lack of a highly effective dengue vaccine and the limited effectivity of vector controls exacerbate this situation. To point the way to a novel method of creating DENV vaccine candidates, here we disrupted the codon usage in a DENV-2 reporter replicon to generate variants with different replication characteristics. Six different mutated constructs containing stretches of altered codon usage in the non-structural genes were generated. The mutated sequences were deoptimized to the least favorable codons for human cells. We studied the replication efficiency of these constructs by measuring luciferase reporter activity, relative RNA fold change, and NS1 secretion. Our findings showed that the level of virus attenuation is closely associated with the amount of codon deoptimization. Indeed, replication was completely abolished in extensively-deoptimized constructs D2Rep-6 and D2Rep-5, intermediate with constructs D2Rep-4 (771 bp silent mutations) and D2Rep-3 (756 bp silent mutations) and restored almost to wildtype levels with constructs D2Rep-2 (394 silent mutations) and D2Rep-1 (48 silent mutations). We also determined that the position of codon deoptimization within the genome is crucial to the degree of attenuation observed. Based on our analysis, we propose that the design for an ideal DENV vaccine candidate could include 700-1500 silent mutations within the NS2A and NS3 genes. Our results suggest that codon deoptimization is an ideal strategy that can readily be used to develop a DENV vaccine candidate with "fine-tuned" attenuation.
近年来，登革热病毒（DENV）感染的大量增加表明，目前对抗登革热的策略并不奏效。缺乏高效的登革热疫苗和媒介控制的有限效果加剧了这种情况。为了为开发登革热疫苗候选物指明新的方法，我们在这里打乱了 DENV-2 报告子复制子中的密码子使用，以产生具有不同复制特征的变体。生成了包含非结构基因中改变的密码子使用的不同长度的六个不同突变构建体。突变序列被去优化为最不利于人类细胞的密码子。我们通过测量荧光素酶报告基因活性、相对 RNA 倍数变化和 NS1 分泌来研究这些构建体的复制效率。我们的研究结果表明，病毒衰减的程度与密码子去优化的程度密切相关。实际上，在广泛去优化的构建体 D2Rep-6 和 D2Rep-5 中，复制完全被消除，在中度去优化的构建体 D2Rep-4（771 个沉默突变）和 D2Rep-3（756 个沉默突变）中复制几乎恢复到野生型水平，在构建体 D2Rep-2（394 个沉默突变）和 D2Rep-1（48 个沉默突变）中复制完全恢复到野生型水平。我们还确定了基因组中密码子去优化的位置对观察到的衰减程度至关重要。基于我们的分析，我们提出理想的登革热疫苗候选物的设计可以包括 NS2A 和 NS3 基因内的 700-1500 个沉默突变。我们的结果表明，密码子去优化是一种理想的策略，可以很容易地用于开发具有“微调”衰减的登革热疫苗候选物。
