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。目前的测量方法可以量化 I 型干扰素和炎症细胞因子的产生，但它们不允许分离各个途径，不能提供组织内的动力学激活或空间信息，并且不能转化为临床研究。在这里，我们演示了使用邻近连接测定（PLAs）来检测细胞和组织样本中的模式识别受体（PRR）激活。首先，我们使用充分表征的可溶性激动剂验证了 PLA 的敏感性和特异性。接下来，我们表征了体外转录 (IVT) mRNA 的 PRR 激活，以及体外序列和碱基修饰的影响。最后，我们建立了在小鼠肌内 (im) 注射 IVT mRNA 后通过 PLA 测量组织切片中 PRR 激活的方法。总体而言，我们的结果表明，PLA 是一种有价值、多功能且灵敏的工具，可用于监测疫苗、佐剂和治疗筛选的 PRR 激活。