Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed MRNA

Overview

Journal Mol Ther Nucleic Acids

Publisher Cell Press

Date 2018 Dec 23

PMID 30579042

Citations 13

Authors

Emmeline L Blanchard

Kristin H Loomis

Sushma M Bhosle

Daryll Vanover

Patrick Baumhof

Bruno Pitard

Chiara Zurla

Philip J Santangelo

Affiliations

Soon will be listed here.

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.

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