Molecular Characterization of In Vivo Adjuvant Activity in Ferrets Vaccinated against Influenza Virus. Mustela putorius furo

The 2009 H1N1 influenza pandemic has prompted a significant need for the development of efficient, single-dose, adjuvanted vaccines. Here we investigated the adjuvant potential of CpG oligodeoxynucleotide (ODN) when used with a human seasonal influenza virus vaccine in ferrets. We found that the CpG ODNadjuvanted vaccine effectively increased antibody production and activated type I interferon (IFN) responses compared to vaccine alone. Based on these findings, pegylated IFN- 2b (PEG-IFN) was also evaluated as an adjuvant in comparison to CpG ODN and complete Freund’s adjuvant (CFA). Our results showed that all three vaccines with adjuvant added prevented seasonal human A/Brisbane/59/2007 (H1N1) virus replication more effectively than did vaccine alone. Gene expression profiles indicated that, as well as upregulating IFN-stimulated genes (ISGs), CpG ODN enhanced B-cell activation and increased Toll-like receptor 4 (TLR4) and IFN regulatory factor 4 (IRF4) expression, whereas PEG-IFN augmented adaptive immunity by inducing major histocompatibility complex (MHC) transcription and Ras signaling. In contrast, the use of CFA as an adjuvant induced limited ISG expression but increased the transcription of MHC, cell adhesion molecules, and B-cell activation markers. Taken together, our results better characterize the specific molecular pathways leading to adjuvant activity in different adjuvant-mediated influenza virus vaccinations. Overall design: In this experiment, 3 ferrets in each group immunizied with different adjuvanted human seasonal vaccines of CFA plus vaccine, CpG plus vaccine, pegylated IFN-alpha plus vaccine and vaccine alone (PBS plus vaccine) and 4 ferrets from control group (PBS only) were anesthetized at day 1 post vaccination. The whole blodd was collected for RNA extraction and purification on the scheduled date. The subsequent gene expression analysis was performed with Affymetrix GeneChip Canine Genome 2.0 Array.

2009年甲型H1N1流感大流行催生了对高效单剂量佐剂疫苗的迫切需求。本研究针对CpG寡脱氧核苷酸（CpG oligodeoxynucleotide, ODN）与季节性人源流感病毒疫苗联合使用时的佐剂潜力，在雪貂模型中展开探究。研究发现，相较于单纯疫苗，CpG ODN佐剂疫苗可有效提升抗体生成水平，并激活I型干扰素（type I interferon, IFN）应答。基于上述发现，本研究同时将聚乙二醇化IFN-α2b（PEG-IFN）作为佐剂，与CpG ODN及完全弗氏佐剂（complete Freund’s adjuvant, CFA）开展对比评估。
结果显示，三款添加佐剂的疫苗均较单纯疫苗更有效地抑制了季节性人源A/Brisbane/59/2007（H1N1）病毒的复制。基因表达谱分析表明，CpG ODN除上调干扰素刺激基因（IFN-stimulated genes, ISGs）的表达外，还可增强B细胞活化，并上调Toll样受体4（Toll-like receptor 4, TLR4）及干扰素调节因子4（IFN regulatory factor 4, IRF4）的表达；而PEG-IFN则通过诱导主要组织相容性复合体（major histocompatibility complex, MHC）转录及Ras信号通路活化，增强适应性免疫应答。与之形成对比的是，使用完全弗氏佐剂作为佐剂仅能诱导有限的ISG表达，但可上调MHC、细胞黏附分子及B细胞活化标志物的转录。综上，本研究结果进一步明确了不同佐剂介导的流感病毒疫苗接种中，驱动佐剂活性的特异性分子通路。
总体实验设计：本实验中，各实验组分别为完全弗氏佐剂联合疫苗组、CpG ODN联合疫苗组、聚乙二醇化IFN-α2b联合疫苗组，以及单纯疫苗组（磷酸盐缓冲液PBS联合疫苗），每组各纳入3只雪貂；另设仅注射PBS的空白对照组，含4只雪貂。所有雪貂均于接种后第1天实施麻醉，并按预定时间采集全血用于RNA提取与纯化。后续基因表达分析采用Affymetrix GeneChip犬基因组2.0阵列完成。