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

Autor:	Blanchard EL; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA., Loomis KH; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA., Bhosle SM; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA., Vanover D; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA., Baumhof P; CureVac, Paul-Ehrlich-Straße 15, 72076 Tübingen, Germany., Pitard B; In-Cell-Art, 21 rue de la Noue Bras de Fer, 44200 Nantes, France., Zurla C; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA., Santangelo PJ; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA. Electronic address: philip.santangelo@bme.gatech.edu.
Jazyk:	angličtina
Zdroj:	Molecular therapy. Nucleic acids [Mol Ther Nucleic Acids] 2019 Mar 01; Vol. 14, pp. 52-66. Date of Electronic Publication: 2018 Nov 20.
DOI:	10.1016/j.omtn.2018.11.002
Abstrakt:	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. (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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