Blanchard Emmeline L, Loomis Kristin H, Bhosle Sushma M, Vanover Daryll, Baumhof Patrick, Pitard Bruno, Zurla Chiara, Santangelo Philip J
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA.
CureVac, Paul-Ehrlich-Straße 15, 72076 Tübingen, Germany.
Mol Ther Nucleic Acids. 2019 Mar 1;14:52-66. doi: 10.1016/j.omtn.2018.11.002. Epub 2018 Nov 20.
The characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile, and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening.
先天免疫激活的特征描述对于疫苗和治疗方法的开发至关重要，包括基于RNA的疫苗，这是一种很有前景的方法。目前的测量方法可量化I型干扰素和炎性细胞因子的产生，但它们无法分离单个信号通路，无法提供组织内的动力学激活或空间信息，也无法转化为临床研究。在这里，我们展示了使用邻近连接分析（PLA）来检测细胞和组织样本中的模式识别受体（PRR）激活。首先，我们使用特征明确的可溶性激动剂验证了PLA的敏感性和特异性。接下来，我们对体外转录（IVT）mRNA引起的PRR激活以及体外序列和碱基修饰的影响进行了表征。最后，我们通过PLA在小鼠体内肌内（i.m.）注射IVT mRNA后，建立了组织切片中PRR激活的测量方法。总体而言，我们的结果表明，PLA是一种用于监测PRR激活以进行疫苗、佐剂和治疗筛选的有价值、通用且灵敏的工具。
