Application of Next Generation Sequencing (RNA-seq and miRNA-seq) to study the molecular signature of Aluminium hydroxide adjuvant in ovine peripheral blood mononuclear cells (PBMCs) [RNA-Seq]. Application of Next Generation Sequencing (RNA-seq and miRNA-seq) to study the molecular signature of Aluminium hydroxide adjuvant in ovine peripheral blood mononuclear cells (PBMCs) [RNA-Seq]

We report the application of RNA sequencing technology for high-throughput profiling of the aluminium hydroxide adjuvant mechanism of action in an in vivo experiment on sheep. We mapped about 52 million sequence reads per sample to the sheep genome (build Oar3.1) and identified 21,274 genes in the PBMCs of sheep treated with commercial vaccines or with the adjuvant alone. A total of 2473, 2980 and 429 differentialy expressed genes were identified in the vaccine tf VS vaccine t0, adjuvant tf VS adjuvant t0 and adjuvant tf VS vaccine tf comparisons, respectively. Previously reported genes related to alterations caused by aluminium adjuvant were found differentially expressed, namely: NLRP3, IL1B, IL8, TNF, NFKB2, RELA and RELB. In addition, most of the genes related to apoptosis were up-regulated in both group after treatment. In contrast, other genes related to immune response, inflammatory response and cell-cell signalling were up-regulated in Vac-injected sheep and whereas down-regulated in Adj-injected animals. Taken together, our analysis provides characterization of alterations in the transcriptome caused by the aluminium adjuvant and identifies increased expression of inflammatory cytokines, NF-KB family genes and apoptotic genes. Overall design: PBMCs Total RNA profiles of six age-matched Rasa sheep before and after inoculation (t0 and tf) of nine different commercial vaccines based of aluminium hydroxide (the vaccine group composed of 3 animals) or with the aluminium adjuvant alone in an equivalent dose (the adjuvant group composed of 3 animals) were generated by deep sequencing on an Illumina HiSeq2000 sequencer. In total, the study is composed of three biological replicates per group and thirteen libraries were sequenced (there was a tecnical replicate of a sample at the end of the experiment and two other samples where sequenced in two different sequencing runs).

本研究报道了RNA测序（RNA sequencing）技术在绵羊体内实验中用于氢氧化铝佐剂（aluminium hydroxide adjuvant）作用机制高通量谱分析的应用。我们将每个样本约5200万条测序读段（sequence reads）比对至绵羊基因组（版本Oar3.1），并在经商用疫苗或单独佐剂处理的绵羊外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs）中鉴定出21274个基因。分别在疫苗组tf vs t0、佐剂组tf vs t0以及佐剂组tf vs疫苗组tf的比较中，鉴定出2473、2980和429个差异表达基因（differentially expressed genes）。此前已报道的与铝佐剂诱导的表达改变相关的基因均被检出差异表达，包括NLRP3、IL1B、IL8、TNF、NFKB2、RELA及RELB。此外，两组经处理后，绝大多数与细胞凋亡（apoptosis）相关的基因均呈现上调表达。与之相反，在接种疫苗的绵羊中，其余与免疫应答、炎症应答及细胞间信号传导相关的基因呈现上调表达，而在接种佐剂的动物中这些基因则呈下调表达。综上，本研究分析阐明了氢氧化铝佐剂诱导的绵羊转录组（transcriptome）表达改变，并鉴定出炎症细胞因子、NF-κB家族基因及凋亡相关基因的表达上调。

实验设计概述：本研究对6只年龄匹配的拉沙绵羊进行接种处理，分别接种9种基于氢氧化铝的商用疫苗（疫苗组，含3只动物）或等效剂量的单独氢氧化铝佐剂（佐剂组，含3只动物），并在接种前（t0）及接种后（tf）两个时间点采集其外周血单个核细胞，通过Illumina HiSeq2000测序仪进行深度测序，获取总RNA表达谱。本研究每组设置3个生物学重复，共构建13个文库并完成测序（实验结束后对1个样本进行了技术重复，另有2个样本在两次独立的测序运行中完成测序）。