Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA
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...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2019-03-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253118302907 |
| _version_ | 1811305850190430208 |
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| author | Emmeline L. Blanchard Kristin H. Loomis Sushma M. Bhosle Daryll Vanover Patrick Baumhof Bruno Pitard Chiara Zurla Philip J. Santangelo |
| author_facet | Emmeline L. Blanchard Kristin H. Loomis Sushma M. Bhosle Daryll Vanover Patrick Baumhof Bruno Pitard Chiara Zurla Philip J. Santangelo |
| author_sort | Emmeline L. Blanchard |
| collection | DOAJ |
| description | 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. Keywords: in vitro-transcribed mRNA, innate immune responses, proximity ligation assay, protein-protein interactions |
| first_indexed | 2024-04-13T08:33:51Z |
| format | Article |
| id | doaj.art-ce9a7b51eb514c22a442138d2bda3bc5 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-13T08:33:51Z |
| publishDate | 2019-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-ce9a7b51eb514c22a442138d2bda3bc52022-12-22T02:54:10ZengElsevierMolecular Therapy: Nucleic Acids2162-25312019-03-01145266Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNAEmmeline L. Blanchard0Kristin H. Loomis1Sushma M. Bhosle2Daryll Vanover3Patrick Baumhof4Bruno Pitard5Chiara Zurla6Philip J. Santangelo7Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USAWallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USAWallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USAWallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USACureVac, Paul-Ehrlich-Straße 15, 72076 Tübingen, GermanyIn-Cell-Art, 21 rue de la Noue Bras de Fer, 44200 Nantes, FranceWallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USAWallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA; Corresponding author: Philip J. Santangelo, Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA.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. Keywords: in vitro-transcribed mRNA, innate immune responses, proximity ligation assay, protein-protein interactionshttp://www.sciencedirect.com/science/article/pii/S2162253118302907 |
| spellingShingle | Emmeline L. Blanchard Kristin H. Loomis Sushma M. Bhosle Daryll Vanover Patrick Baumhof Bruno Pitard Chiara Zurla Philip J. Santangelo Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA Molecular Therapy: Nucleic Acids |
| title | Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA |
| title_full | Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA |
| title_fullStr | Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA |
| title_full_unstemmed | Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA |
| title_short | Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA |
| title_sort | proximity ligation assays for in situ detection of innate immune activation focus on in vitro transcribed mrna |
| url | http://www.sciencedirect.com/science/article/pii/S2162253118302907 |
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