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

• Type of Material: Article
• Authors: Emmeline L. Blanchard, Georgia Institute of Technology; Kristin H. Loomis, Georgia Institute of Technology; Sushma M. Bhosle, Georgia Institute of Technology; Daryll Vanover, Georgia Institute of Technology; Patrick Baumhof, CureVac; Bruno Pitard, In-Cell-Art; Chiara Zurla, Georgia Institute of Technology; Philip Santangelo, Emory University
• Language: English
• Date: 2019-03-01
• Publisher: Elsevier (Cell Press): OAJ
• Publication Version: Final Published Version
• Copyright Statement: © 2018 The Author(s)
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.omtn.2018.11.002
• Title of Journal or Parent Work: Molecular Therapy - Nucleic Acids
• ISSN: 2162-2531
• Volume: 14
• Start Page: 52
• End Page: 66
• Grant/Funding Information: The views, opinions, and/or findings expressed are those of the author(s) and should not be interpreted as representing the official views or policies of the Department of Defense, the U.S. Government, or the National Science Foundation.; This work was supported by Defense Advanced Research Projects Agency (DARPA), Sanofi Pasteur, and the RNArmorVax Consortium to P.J.S. and by the National Science Foundation Graduate Research Fellowship Program (grant DGE-1650044) to E.L.B.
• Keywords: innate immune responses; proximity ligation assay; protein-protein interactions; in vitro-transcribed mRNA
• Research Categories: Health Sciences, Immunology; Engineering, Biomedical

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.