Abstract
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.
Highlights
Here we demonstrated the use of proximity ligation assays (PLAs) as a sensitive, quantitative, and specific tool to measure pattern recognition receptor (PRR) activation in cells and in tissue upon in vitro-transcribed (IVT) mRNA delivery
We compared PRR activation with modulated protein levels in vitro, as well as over time in response to known agonists. This validated the specificity of PLA to detect individual PRR pathways, as interactions unique to each pathway were identified and quantified, indicating both the antibodies’ specificity for the target complex molecules and the assay’s specificity for complex formation
This demonstrated the versatility of PLA, as it was successfully optimized for several PRR pathways whose activation occurred in distinct cellular compartments: TLR4 on the cell surface ported modulatory effect of modified nucleosides in IVT mRNA
Summary
The primary controllers of the innate immune system are pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are transmembrane receptors on the cell surface, such as TLR4, which recognizes bacterial wall lipids, or in endosomes, such as TLR7, which detects single-stranded RNA (ssRNA). Cytoplasmic PRRs include retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), which recognize short double-stranded RNA (dsRNA) with 50 triphosphorylated (50ppp) ends or longer dsRNA, respectively. PRRs form complexes with adaptor molecules to begin signaling cascades that lead to type I interferon (IFN) and cytokine induction (Figure 1A).[1,13,14,15,16,17,18]
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