Naito Tadasuke, Mori Kotaro, Ushirogawa Hiroshi, Takizawa Naoki, Nobusawa Eri, Odagiri Takato, Tashiro Masato, Ohniwa Ryosuke L, Nagata Kyosuke, Saito Mineki
Department of Microbiology, Kawasaki Medical School, Okayama, Japan
Laboratory of Virology, Institute of Microbial Chemistry, Tokyo, Japan.
J Virol. 2017 Feb 28;91(6). doi: 10.1128/JVI.01073-16. Print 2017 Mar 15.
Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production. Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.
接种疫苗被认为是控制流感最有效的预防手段。开发一种生长良好且遗传稳定的母病毒，可通过基因重配用于制备疫苗病毒，对于提高疫苗性能和生产效率至关重要。在此，我们描述了一种高保真、高生长的流感疫苗母病毒株的产生，该毒株是在高生长的A/波多黎各/8/1934（PR8）母病毒的PB1聚合酶中发生了单个V43I氨基酸变化。引入PB1-V43I突变以提高复制保真度，从而设计出错误率低的H1N1疫苗株。与亲本PR8病毒相比，PR8-PB1-V43I病毒表现出良好的复制能力。为了比较在鸡蛋中适应的效率以及导致抗原性改变的基因突变的发生情况，我们构建了A（H1N1）pdm09和PR8-PB1-V43I病毒之间的6:2基因重配病毒；血凝素（HA）和神经氨酸酶（NA）来自A（H1N1）pdm09病毒，其他基因来自PR8病毒。在连续的鸡蛋传代过程中，负责鸡蛋适应性突变的突变发生在PB1-V43I重配病毒的HA中；然而，相比之下，具有野生型PB1的6:2重配病毒的HA基因中引入了抗原性突变。这项研究表明，具有V43I替代的PB1聚合酶的突变PR8病毒可作为母病毒，用于产生具有高聚合酶保真度、低基因复制错误率以及在用于疫苗生产的鸡蛋中病毒增殖期间抗原多样性降低的高生长疫苗病毒。接种疫苗是预防流感最有效的选择。流感大流行出现的威胁使得必须具备快速生产疫苗病毒的能力。然而，由于流感病毒表现出高突变率，必须更新疫苗以确保疫苗与流行毒株之间的HA和NA抗原良好匹配。在此，我们产生了一种遗传稳定的A/波多黎各/8/1934（H1N1）骨架的母病毒，其编码一种工程化的高保真病毒聚合酶。重要的是，在应用高保真PR8骨架后，在A（H1N1）pdm09候选疫苗病毒的增殖过程中，HA基因未引入导致抗原性变化的突变。目前疫苗病毒的低错误率应会降低产生毒力增加的突变病毒的风险。因此，我们的研究结果有望有助于大流行前疫苗和减毒活疫苗的开发，其安全性高于目前的候选疫苗。
