Cell Reports Medicine (Apr 2021)
A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir
- Claire N. Levy,
- Sean M. Hughes,
- Pavitra Roychoudhury,
- Daniel B. Reeves,
- Chelsea Amstuz,
- Haiying Zhu,
- Meei-Li Huang,
- Yulun Wei,
- Marta E. Bull,
- Noah A.J. Cassidy,
- Jan McClure,
- Lisa M. Frenkel,
- Mars Stone,
- Sonia Bakkour,
- Elizabeth R. Wonderlich,
- Michael P. Busch,
- Steven G. Deeks,
- Joshua T. Schiffer,
- Robert W. Coombs,
- Dara A. Lehman,
- Keith R. Jerome,
- Florian Hladik
Affiliations
- Claire N. Levy
- Department of Obstetrics & Gynecology, University of Washington, Seattle, WA, USA
- Sean M. Hughes
- Department of Obstetrics & Gynecology, University of Washington, Seattle, WA, USA
- Pavitra Roychoudhury
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
- Daniel B. Reeves
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
- Chelsea Amstuz
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
- Haiying Zhu
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA
- Meei-Li Huang
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA
- Yulun Wei
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA
- Marta E. Bull
- Seattle Children’s Research Institute, Seattle, WA, USA; Department of Pediatrics, University of Washington, Seattle, WA, USA
- Noah A.J. Cassidy
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
- Jan McClure
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA
- Lisa M. Frenkel
- Seattle Children’s Research Institute, Seattle, WA, USA; Department of Pediatrics, University of Washington, Seattle, WA, USA; Department of Medicine, University of Washington, Seattle, WA, USA; Department of Global Health, University of Washington, Seattle, WA, USA
- Mars Stone
- Vitalent Research Institute, San Francisco, CA, USA; Department of Laboratory Medicine, University of San Francisco, San Francisco, CA, USA; School of Medicine, University of San Francisco, San Francisco, CA, USA
- Sonia Bakkour
- Vitalent Research Institute, San Francisco, CA, USA; Department of Laboratory Medicine, University of San Francisco, San Francisco, CA, USA; School of Medicine, University of San Francisco, San Francisco, CA, USA
- Elizabeth R. Wonderlich
- Department of Infectious Disease Research, Southern Research, 431 Aviation Way, Frederick, MD, USA
- Michael P. Busch
- Vitalent Research Institute, San Francisco, CA, USA; Department of Laboratory Medicine, University of San Francisco, San Francisco, CA, USA
- Steven G. Deeks
- School of Medicine, University of San Francisco, San Francisco, CA, USA; Division of HIV, Infectious Diseases and Global Medicine, Zuckerberg San Francisco General Hospital, San Francisco, CA, USA
- Joshua T. Schiffer
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
- Robert W. Coombs
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA; Department of Medicine, University of Washington, Seattle, WA, USA
- Dara A. Lehman
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Global Health, University of Washington, Seattle, WA, USA
- Keith R. Jerome
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA; Department of Global Health, University of Washington, Seattle, WA, USA; Corresponding author
- Florian Hladik
- Department of Obstetrics & Gynecology, University of Washington, Seattle, WA, USA; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Medicine, University of Washington, Seattle, WA, USA; Corresponding author
- Journal volume & issue
-
Vol. 2,
no. 4
p. 100243
Abstract
Summary: Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4+ T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.
