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. 2021 Dec 11;25(1):103615.
doi: 10.1016/j.isci.2021.103615. eCollection 2022 Jan 21.

HIV reservoir quantification using cross-subtype multiplex ddPCR

Noah A J Cassidy  1 , Carolyn S Fish  1 , Claire N Levy  2 , Pavitra Roychoudhury  3   4 , Daniel B Reeves  3 , Sean M Hughes  2 , Joshua T Schiffer  3   5 , Sarah Benki-Nugent  6 , Grace John-Stewart  6   5   7   8 , Dalton Wamalwa  9 , Keith R Jerome  3   4 , Julie Overbaugh  1 , Florian Hladik  2   3   5 , Dara A Lehman  1   6
Affiliations

HIV reservoir quantification using cross-subtype multiplex ddPCR

Noah A J Cassidy et al. iScience. .

Abstract

A major barrier to conducting HIV cure research in populations with the highest HIV burden is the lack of an accurate assay to quantify the replication-competent reservoir across the dominant global HIV-1 subtypes. Here, we modify a subtype B HIV-1 assay that quantifies both intact and defective proviral DNA, adapting it to accommodate cross-subtype HIV-1 sequence diversity. We show that the cross-subtype assay works on subtypes A, B, C, D, and CRF01_AE and can detect a single copy of intact provirus. In longitudinal blood samples from Kenyan infants infected with subtypes A and D, patterns of intact and total HIV DNA follow the decay of plasma viral load over time during antiretroviral therapy, with intact HIV DNA comprising 7% (range 1%-33%) of the total HIV DNA during HIV RNA suppression. This high-throughput cross-subtype reservoir assay will be useful in HIV cure research in Africa and Asia, where HIV prevalence is highest.

Keywords: Genotyping; Virology.

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Figures

None
Graphical abstract
Figure 1
Figure 1
Quantification of a panel of HIV DNA subtype samples (A–D) Quantification across the 5T-IPDA target regions (colors as in key) relative to the number of copies quantified by the env primer/probe set. The 5T-IPDA subtype-B primers and probes were used to quantify HIV DNA samples shown on the x axis derived from (A) patients infected with HIV subtype B viruses and (B) patients infected with HIV subtypes (A, C, and D) and CRF01_AE. The cross-subtype primers and probes were used to quantify HIV DNA samples derived from (C) patients infected with HIV subtype B viruses and (D) patients infected with HIV subtypes (A, C, and D) and CRF01_AE.
Figure 2
Figure 2
Impact of subtype diversity on detection of intact sequences by CS-IPDA (A) Droplet clusters on mixtures of all possible single-, dual-, and triple-combination plasmids of CS-IPDA targets with subtype B sequences (top), and on 3-target subtype A and D plasmids (middle and bottom) using 5T-IPDA (left) and CS-IPDA (right) primers/probes. (B) CS-IPDA on single-target plasmids with a sequence from LANL that had the most mismatches relative to CS-IPDA and prevalent at >1% of sequences in the LANL HIV database. (C) CS-IPDA on 3-target plasmids with mismatches in the 5′pol probe (top) and env probe (bottom). (D) CS-IPDA results from 2 Kenyan infants at 2 timepoints each. (E) Sequences used in the plasmid controls shown above with degenerate bases in the CS-IPDA primers and probes shown in green, bases that mismatch only the 5T-IPDA in blue, bases that mismatch only the CS-IPDA in orange, and bases that mismatch both 5T-IPDA and CS-IPDA in red.
Figure 3
Figure 3
Longitudinal patterns of total and intact HIV DNA and HIV RNA during ART in Kenyan infants CS-IPDA quantification of HIV in PBMC samples from 5 to 8 timepoints over 42–96 months of ART treatment in 6 HIV-infected Kenyan infants infected with subtypes A or D. Patterns of total HIV DNA (blue circles) and intact HIV DNA (red squares) in copies per million T cells (left Y axis). HIV RNA (green triangles) copies per mL (right Y axis, limit of detection 150 copies/mL).
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