Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
PLoS One. 2020 Aug 6;15(8):e0237218. doi: 10.1371/journal.pone.0237218. eCollection 2020.
Influenza is an infectious respiratory illness caused by influenza viruses. Despite yearly updates, the efficacy of influenza vaccines is significantly curtailed by the virus antigenic drift and antigenic shift. These constant changes to the influenza virus make-up also challenge the development of a universal flu vaccine, which requires conserved antigenic regions shared by influenza viruses of different subtypes. We propose that it is possible to bypass these challenges by the development of an influenza vaccine based on conserved proteins delivered in an adjuvanted nanoparticle system. In this study, we generated influenza nanoparticle constructs using trimethyl chitosan nanoparticles (TMC nPs) as the carrier of recombinant influenza hemagglutinin subunit 2 (HA2) and nucleoprotein (NP). The purified HA2 and NP recombinant proteins were encapsulated into TMC nPs to form HA2-TMC nPs and NP-TMC nPs, respectively. Primary human intranasal epithelium cells (HNEpCs) were used as an in vitro model to measure immunity responses. HA2-TMC nPs, NP-TMC nPs, and HA2-NP-TMC nPs (influenza nanoparticle constructs) showed no toxicity in HNEpCs. The loading efficiency of HA2 and NP into the TMC nPs was 97.9% and 98.5%, respectively. HA2-TMC nPs and NP-TMC nPs more efficiently delivered HA2 and NP proteins to HNEpCs than soluble HA2 and NP proteins alone. The induction of various cytokines and chemokines was more evident in influenza nanoparticle construct-treated HNEpCs than in soluble protein-treated HNEpCs. In addition, soluble factors secreted by influenza nanoparticle construct-treated HNEpCs significantly induced MoDCs maturation markers (CD80, CD83, CD86 and HLA-DR), as compared to soluble factors secreted by protein-treated HNEpCs. HNEpCs treated with the influenza nanoparticle constructs significantly reduced influenza virus replication in an in vitro challenge assay. The results indicate that TMC nPs can be used as influenza vaccine adjuvants and carriers capable of delivering HA2 and NP proteins to HNEpCs.
流感是由流感病毒引起的传染性呼吸道疾病。尽管每年都进行更新，但流感疫苗的功效还是会因病毒抗原漂移和抗原转变而显著降低。流感病毒的这些不断变化也给通用流感疫苗的开发带来了挑战，通用流感疫苗需要不同亚型流感病毒共有的保守抗原区域。我们提出，通过开发基于保守蛋白的流感疫苗，并将其递送至佐剂纳米颗粒系统中，可以绕过这些挑战。在这项研究中，我们使用三甲基壳聚糖纳米颗粒（TMC nPs）作为重组流感血凝素亚单位 2（HA2）和核蛋白（NP）的载体，生成了流感纳米颗粒构建体。纯化的 HA2 和 NP 重组蛋白被包裹到 TMC nPs 中，分别形成 HA2-TMC nPs 和 NP-TMC nPs。原代人鼻上皮细胞（HNEpCs）被用作体外模型来测量免疫反应。HA2-TMC nPs、NP-TMC nPs 和 HA2-NP-TMC nPs（流感纳米颗粒构建体）在 HNEpCs 中没有显示出毒性。HA2 和 NP 加载到 TMC nPs 中的效率分别为 97.9%和 98.5%。HA2-TMC nPs 和 NP-TMC nPs 比单独的可溶性 HA2 和 NP 蛋白更有效地将 HA2 和 NP 蛋白递送至 HNEpCs。在流感纳米颗粒构建体处理的 HNEpCs 中诱导的各种细胞因子和趋化因子比在可溶性蛋白处理的 HNEpCs 中更明显。此外，与可溶性蛋白处理的 HNEpCs 分泌的可溶性因子相比，流感纳米颗粒构建体处理的 HNEpCs 分泌的可溶性因子显著诱导 MoDCs 成熟标志物（CD80、CD83、CD86 和 HLA-DR）。在体外挑战试验中，用流感纳米颗粒构建体处理的 HNEpCs 显著降低了流感病毒的复制。结果表明，TMC nPs 可用作流感疫苗佐剂和载体，能够将 HA2 和 NP 蛋白递送至 HNEpCs。
