High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay
Robert W. Yucha,
Kristen S. Hobbs,
Emily Hanhauser,
Louise E. Hogan,
Wildaliz Nieves,
Mehmet O. Ozen,
Fatih Inci,
Vanessa York,
Erica A. Gibson,
Cassandra Thanh,
Hadi Shafiee,
Rami El Assal,
Maja Kiselinova,
Yvonne P. Robles,
Helen Bae,
Kaitlyn S. Leadabrand,
ShuQi Wang,
Steven G. Deeks,
Daniel R. Kuritzkes,
Utkan Demirci,
Timothy J. Henrich
Affiliations
Robert W. Yucha
Division of Infectious Diseases, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, United States
Kristen S. Hobbs
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
Emily Hanhauser
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
Louise E. Hogan
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
Wildaliz Nieves
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
Mehmet O. Ozen
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States
Fatih Inci
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States
Vanessa York
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
Erica A. Gibson
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
Cassandra Thanh
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
Hadi Shafiee
Division of Renal Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, United States
Rami El Assal
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States
Maja Kiselinova
HIV Translational Research Unit, Department of Internal Medicine, Ghent University and Ghent University Hospital, Ghent, Belgium
Yvonne P. Robles
Division of Infectious Diseases, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, United States
Helen Bae
Harvard University, Faculty of Arts & Sciences, Cambridge, MA 02138, United States
Kaitlyn S. Leadabrand
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
ShuQi Wang
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affliliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
Steven G. Deeks
Positive Health Program, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
Daniel R. Kuritzkes
Division of Infectious Diseases, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, United States
Utkan Demirci
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA 94304, United States
Timothy J. Henrich
Division of Experimental Medicine, University of California, San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, United States
Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous—increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.