Reverse Genetics Approach for Developing Rotavirus Vaccine Candidates Carrying VP4 and VP7 Genes Cloned from Clinical Isolates of Human Rotavirus
Reverse Genetics Approach for Developing Rotavirus Vaccine Candidates Carrying VP4 and VP7 Genes Cloned from Clinical Isolates of Human Rotavirus
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DOI：
10.1128/jvi.01374-20
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发表时间：
2021-01-01
影响因子：
5.4
通讯作者：
Kobayashi, Takeshi
中科院分区：
文献类型：
--
作者：
Kanai, Yuta;Onishi, Misa;Kobayashi, Takeshi
A类轮状病毒（RV）是婴儿和5岁以下儿童严重急性胃肠炎的主要原因。目前可用的RV疫苗是通过培养细胞的连续传代或通过人和动物RV毒株之间的重配而从野生型RV毒株改编的。这些传统方法需要大规模筛选和基因分型以获得候选疫苗。反向遗传学是一种易于处理、快速和可重复的方法，用于产生携带任何VP4和VP7基因的重组RV疫苗候选物，这些基因提供选定的抗原性。在这里，我们通过使用猿猴RV SA 11株（G3P [2]）作为骨架，生成携带从人RV临床样本克隆的VP4（P [4]和P [8]）、VP7（G1、G2、G3、G8和G9）和/或VP6基因的重组RV，开发了一种疫苗平台。使用单克隆抗体和鼠抗血清的中和试验表明，重组VP4和VP7单抗病毒表现出改变的抗原性。然而，VP4单克隆抗体病毒的复制严重受损。携带SA11和人RV基因组分的嵌合VP4蛋白的重组RV的产生揭示了VP8 * 片段负责重组RV的有效感染性。尽管由于疫苗病毒的产量直接影响疫苗制造成本，因此必须改进这一系统，但反向遗传学所需时间比传统方法更少，并能够快速生产安全有效的候选疫苗。重要性尽管疫苗在过去十年中减少了全球RV相关的住院和死亡率，RV的多节段基因组允许来自不同RV物种和毒株的VP4和VP7基因的重配。新型RV基因型及其星座的进化动力学导致了巨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，我们产生了重组猴RV（SA 11株）携带异源的VP4和VP7基因克隆从临床分离株，并表明VP4或VP7取代的嵌合病毒可用于RV外壳蛋白的抗原性表征，并作为疫苗生产的改进种子病毒。
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.IMPORTANCE 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 VP4or VP7-substituted chimeric viruses can be used for antigenic characterization of RV outer capsid proteins and as improved seed viruses for vaccine production.