Identification of Influenza A/PR/8/34 Donor Viruses Imparting High Hemagglutinin Yields to Candidate Vaccine Viruses in Eggs

One of the important lessons learned from the 2009 H1N1 pandemic is that a high yield influenza vaccine virus is essential for efficient and timely production of pandemic vaccines in eggs. The current seasonal and pre-pandemic vaccine viruses are generated either by classical reassortment or reverse genetics. Both approaches utilize a high growth virus, generally A/Puerto Rico/8/1934 (PR8), as the donor of all or most of the internal genes, and the wild type virus recommended for inclusion in the vaccine to contribute the hemagglutinin (HA) and neuraminidase (NA) genes encoding the surface glycoproteins. As a result of extensive adaptation through sequential egg passaging, PR8 viruses with different gene sequences and high growth properties have been selected at different laboratories in past decades. The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously. Here, we use reverse genetics to analyze systematically the growth and HA antigen yield of reassortant viruses with 3 different PR8 backbones. A panel of 9 different HA/NA gene pairs in combination with each of the 3 different lineages of PR8 internal genes (27 reassortant viruses) was generated to evaluate their performance. Virus and HA yield assays showed that the PR8 internal genes influence HA yields in most subtypes. Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production. These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.

从2009年甲型H1N1流感大流行中汲取的重要经验之一是，高产流感疫苗病毒对于利用鸡胚高效且及时地生产大流行疫苗至关重要。当前的季节性及大流行前疫苗病毒，可通过经典基因重配或反向遗传学技术制备。两种技术路线均以高生长病毒——通常为A/波多黎各/8/1934（PR8）——作为全部或大部分内部基因的供体，同时以推荐纳入疫苗的野生型病毒作为供体，提供编码表面糖蛋白的血凝素（hemagglutinin，HA）与神经氨酸酶（neuraminidase，NA）基因。经过数十年鸡胚连续传代的适应性驯化，不同实验室在过去数十年间已筛选出多株基因序列各异且具备高生长特性的PR8毒株。这些亲缘关系相近但序列存在差异的PR8内部基因，对疫苗病毒在鸡胚中生长的影响此前尚未被系统研究。本研究采用反向遗传学技术，系统分析了携带3种不同PR8基因骨架的重配病毒的生长特性与HA抗原产量。我们构建了包含9组不同HA/NA基因对的毒株组合，并分别与3种不同谱系的PR8内部基因进行配对（共计27株重配病毒），以评估其生产性能。病毒产量与HA产量检测结果显示，PR8内部基因对多数亚型的HA产量均存在显著影响。尽管没有任何一种PR8内部基因组合在所有候选疫苗病毒中均表现最优，但特定PR8骨架与单个HA/NA基因对的组合可提升HA产量，进而加快疫苗生产速度。这些研究结果对于季节性疫苗的规模化生产，以及流感大流行期间的全球快速疫苗响应均具有重要参考价值。