Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Vaccine. 2021 Mar 19;39(12):1708-1720. doi: 10.1016/j.vaccine.2021.02.024. Epub 2021 Feb 25.
Enterovirus A71 (EV-A71) causes hand, foot and mouth disease (HFMD) in young children. It is associated with severe neurological complications and death. This study aims to develop a live-attenuated vaccine by codon deoptimization (CD) and codon-pair deoptimization (CPD) of EV-A71. CD is generated by introducing the least preferred codons for amino acids while CPD increases the presence of underrepresented codon pairs in the specific genes. CD and CPD chimeras were generated by synonymous mutations at the VP2, VP3, VP1 and 2A gene regions, designated as XYZ. All twelve deoptimized viruses were viable with similar replication kinetics, but the plaque sizes were inversely proportional to the level of deoptimization. All the deoptimized viruses showed attenuated growth in vitro with reduced viral protein expression at 48 h and lower viral RNA at 39 °C. Six-week-old ICR mice were immunized intraperitoneally with selected CD and CPD X and XY vaccine candidates covering the VP2-VP3 and VP2-VP3-VP1 genes, respectively. All vaccine candidates elicited high anti-EV-A71 IgG levels similar to wild-type (WT) EV-A71. The CD X and CPD X vaccines produced robust neutralizing antibodies but not the CD XY and CPD XY. On lethal challenge, offspring of mice immunized with WT, CD X and CPD X were fully protected, but the CD XY- and CPD XY-vaccinated mice had delayed symptoms and eventually died. Similarly, active immunization of 1-day-old suckling mice with CD X, CPD X and CD XY vaccine candidates provided complete immune protection but CPD XY only protected 40% of the challenged mice. Histology of the muscles from CD X- and CPD X-vaccinated mice showed minimal pathology compared to extensive inflammation in the post-challenged mock-vaccinated mice. Overall, we demonstrated that the CD X and CPD X elicited good neutralizing antibodies, conferred immune protection and are promising live-attenuated vaccine candidates for EV-A71.
肠道病毒 A71（EV-A71）可引起婴幼儿手足口病（HFMD）。它与严重的神经并发症和死亡有关。本研究旨在通过密码子去优化（CD）和密码子对去优化（CPD）开发 EV-A71 的活减毒疫苗。CD 通过引入对氨基酸最不优选的密码子产生，而 CPD 增加特定基因中代表性不足的密码子对的存在。通过 VP2、VP3、VP1 和 2A 基因区域的同义突变生成 CD 和 CPD 嵌合体，分别命名为 XYZ。所有 12 种去优化病毒均具有活力，复制动力学相似，但蚀斑大小与去优化水平成反比。所有去优化病毒在体外生长均减弱，在 48 小时时病毒蛋白表达降低，在 39°C 时病毒 RNA 降低。6 周龄 ICR 小鼠经腹腔免疫接种选择的 CD 和 CPD X 和 XY 疫苗候选物，分别覆盖 VP2-VP3 和 VP2-VP3-VP1 基因。所有疫苗候选物均诱导高抗 EV-A71 IgG 水平，与野生型（WT）EV-A71 相似。CD X 和 CPD X 疫苗产生了强大的中和抗体，但 CD XY 和 CPD XY 疫苗没有。在致死性挑战中，WT、CD X 和 CPD X 免疫小鼠的后代完全受到保护，但 CD XY 和 CPD XY 疫苗接种的小鼠出现症状延迟，最终死亡。同样，1 日龄乳鼠主动免疫接种 CD X、CPD X 和 CD XY 疫苗候选物可提供完全免疫保护，但 CPD XY 仅保护 40%的挑战小鼠。与未挑战的假疫苗接种小鼠的广泛炎症相比，CD X 和 CPD X 疫苗接种小鼠的肌肉组织病理学显示最小的病理。总的来说，我们证明 CD X 和 CPD X 诱导了良好的中和抗体，提供了免疫保护，是有前途的 EV-A71 活减毒疫苗候选物。
