Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Department of Microbiology, Chiang Mai University, Faculty of Medicine, Chiang Mai, Thailand.
J Virol. 2020 Dec 22;95(2). doi: 10.1128/JVI.01374-20.
Species A rotaviruses (RVs) are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years. Currently available RV vaccines were adapted from wild-type RV strains by serial passage of cultured cells or by reassortment between human and animal RV strains. These traditional methods require large-scale screening and genotyping to obtain vaccine candidates. Reverse genetics is a tractable, rapid, and reproducible approach to generating recombinant RV vaccine candidates carrying any VP4 and VP7 genes that provide selected antigenicity. Here, we developed a vaccine platform by generating recombinant RVs carrying VP4 (P[4] and P[8]), VP7 (G1, G2, G3, G8, and G9), and/or VP6 genes cloned from human RV clinical samples using the simian RV SA11 strain (G3P[2]) as a backbone. Neutralization assays using monoclonal antibodies and murine antisera revealed that recombinant VP4 and VP7 monoreassortant viruses exhibited altered antigenicity. However, replication of VP4 monoreassortant viruses was severely impaired. Generation of recombinant RVs harboring a chimeric VP4 protein for SA11 and human RV gene components revealed that the VP8* fragment was responsible for efficient infectivity of recombinant RVs. Although this system must be improved because the yield of vaccine viruses directly affects vaccine manufacturing costs, reverse genetics requires less time than traditional methods and enables rapid production of safe and effective vaccine candidates. Although vaccines have reduced global RV-associated hospitalization and mortality over the past decade, the multisegmented genome of RVs allows reassortment of VP4 and VP7 genes from different RV species and strains. The evolutionary dynamics of novel RV genotypes and their constellations have led to great genomic and antigenic diversity. The reverse genetics system is a powerful tool for manipulating RV genes, thereby controlling viral antigenicity, growth capacity, and pathogenicity. Here, we generated recombinant simian RVs (strain SA11) carrying heterologous VP4 and VP7 genes cloned from clinical isolates and showed that VP4- or VP7-substituted chimeric viruses can be used for antigenic characterization of RV outer capsid proteins and as improved seed viruses for vaccine production.
A 型轮状病毒(RV)是导致 5 岁以下婴幼儿严重急性胃肠炎的主要原因。目前可用的 RV 疫苗是通过对培养细胞进行连续传代或在人源和动物 RV 株之间进行重配,从野生型 RV 株中开发而来。这些传统方法需要进行大规模的筛选和基因分型,以获得疫苗候选物。反向遗传学是一种可行、快速且可重复的方法,可以生成携带任何 VP4 和 VP7 基因的重组 RV 疫苗候选物,这些基因提供了选择的抗原性。在这里,我们使用猴轮状病毒 SA11 株(G3P[2])作为骨架,从人类 RV 临床样本中克隆 VP4(P[4]和 P[8])、VP7(G1、G2、G3、G8 和 G9)和/或 VP6 基因,生成了一种携带 VP4、VP7 和/或 VP6 基因的重组 RV 疫苗平台。使用单克隆抗体和鼠抗血清进行的中和测定表明,重组 VP4 和 VP7 单重重组病毒表现出改变的抗原性。然而,VP4 单重重组病毒的复制受到严重损害。携带 SA11 和人 RV 基因成分嵌合 VP4 蛋白的重组 RV 的生成表明,VP8* 片段是重组 RV 有效感染性的原因。尽管该系统需要改进,因为疫苗病毒的产量直接影响疫苗的制造成本,但与传统方法相比,反向遗传学需要的时间更少,并且能够快速生产安全有效的疫苗候选物。尽管过去十年疫苗已降低了全球与 RV 相关的住院和死亡率,但 RV 的多节段基因组允许不同 RV 种和株的 VP4 和 VP7 基因重组。新型 RV 基因型及其组合的进化动态导致了极大的基因组和抗原多样性。反向遗传学系统是操纵 RV 基因的有力工具,从而控制病毒的抗原性、生长能力和致病性。在这里,我们生成了携带从临床分离株克隆的异源 VP4 和 VP7 基因的重组猴轮状病毒(SA11 株),并表明 VP4 或 VP7 替代的嵌合病毒可用于 RV 外壳蛋白的抗原性特征分析,以及作为改进的疫苗生产种子病毒。