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

doi:10.1016/j.xcrm.2021.100243

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

Cell Rep Med. 2021 Apr 12;2(4):100243. doi: 10.1016/j.xcrm.2021.100243. eCollection 2021 Apr 20.

Authors

Claire N Levy¹, Sean M Hughes¹, Pavitra Roychoudhury², Daniel B Reeves², Chelsea Amstuz², Haiying Zhu³, Meei-Li Huang³, Yulun Wei³, Marta E Bull^{4

5}, Noah A J Cassidy⁶, Jan McClure³, Lisa M Frenkel^{4

5

7

8}, Mars Stone^{9

10

11}, Sonia Bakkour^{9

10

11}, Elizabeth R Wonderlich¹², Michael P Busch^{9

10}, Steven G Deeks^{11

13}, Joshua T Schiffer^{2

6

14}, Robert W Coombs^{3

7}, Dara A Lehman^{6

8}, Keith R Jerome^{2

3

8}, Florian Hladik^{1

2

7}

Affiliations

¹ Department of Obstetrics & Gynecology, University of Washington, Seattle, WA, USA.
² Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
³ Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA.
⁴ Seattle Children's Research Institute, Seattle, WA, USA.
⁵ Department of Pediatrics, University of Washington, Seattle, WA, USA.
⁶ Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
⁷ Department of Medicine, University of Washington, Seattle, WA, USA.
⁸ Department of Global Health, University of Washington, Seattle, WA, USA.
⁹ Vitalent Research Institute, San Francisco, CA, USA.
¹⁰ Department of Laboratory Medicine, University of San Francisco, San Francisco, CA, USA.
¹¹ School of Medicine, University of San Francisco, San Francisco, CA, USA.
¹² Department of Infectious Disease Research, Southern Research, 431 Aviation Way, Frederick, MD, USA.
¹³ Division of HIV, Infectious Diseases and Global Medicine, Zuckerberg San Francisco General Hospital, San Francisco, CA, USA.
¹⁴ Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Abstract

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.

Keywords: HIV cure; HIV reservoir; IPDA; digital PCR; genital mucosa; intact proviral DNA assay; intestinal mucosa; multiplexing; rectal; viral outgrowth assay.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

DNA, Viral / analysis
HIV Infections / genetics*
HIV Seropositivity / genetics
HIV-1 / genetics*
Humans
Polymerase Chain Reaction* / methods
Proviruses / genetics*
Viral Load / methods
Virus Latency / genetics

Substances

DNA, Viral

Abstract

Publication types

MeSH terms

Substances

Grants and funding