A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir

Cell Reports Medicine

Joshua T. Schiffer¹  Dara A. Lehman²  Steven G. Deeks³  Lisa M. Frenkel⁴  Michael P. Busch⁴  Sean M. Hughes⁵  Daniel B. Reeves⁵  Keith R. Jerome⁶  Sonia Bakkour⁷  Marta E. Bull⁷  Noah A.J. Cassidy⁷  Yulun Wei⁷  Claire N. Levy⁸  Pavitra Roychoudhury⁸  Mars Stone⁹  Elizabeth R. Wonderlich^1,10  Jan McClure^1,10  Meei-Li Huang^1,11  Haiying Zhu^1,11  Chelsea Amstuz^1,11  Robert W. Coombs^1,12  Florian Hladik^1,12 

[1] Department of Global Health, University of Washington, Seattle, WA, USA;Department of Laboratory Medicine, University of San Francisco, San Francisco, CA, USA;Department of Medicine, University of Washington, Seattle, WA, USA;Department of Pediatrics, University of Washington, Seattle, WA, USA;Gynecology, University of Washington, Seattle, WA, USA;School of Medicine, University of San Francisco, San Francisco, CA, USA;Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA;;Department of Obstetrics &Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA;Seattle Children’s Research Institute, Seattle, WA, USA;Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA;Vitalent Research Institute, San Francisco, CA, USA;

关键词: HIV cure;    HIV reservoir;    digital PCR;    multiplexing;    viral outgrowth assay;    intact proviral DNA assay;   

DOI  :  

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown