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. 2020 Aug 4;117(31):18692-18700.
doi: 10.1073/pnas.2006816117. Epub 2020 Jul 20.

Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA

Francesco R Simonetti  1 , Jennifer A White  1 , Camille Tumiotto  2 , Kristen D Ritter  3 , Mian Cai  3 , Rajesh T Gandhi  4 , Steven G Deeks  5 , Bonnie J Howell  6 , Luis J Montaner  7 , Joel N Blankson  1 , Albine Martin  3 , Gregory M Laird  3 , Robert F Siliciano  8   9 , John W Mellors  2 , Janet D Siliciano  1
Affiliations

Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA

Francesco R Simonetti et al. Proc Natl Acad Sci U S A. .

Abstract

A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1+ adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/106 CD4+ T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/106 CD4+ T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.

Keywords: HIV; IPDA; cure; latency; reservoir.

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Conflict of interest statement

Competing interest statement: Aspects of the IPDA are the subject of patent application PCT/US16/28822 filed by Johns Hopkins University with R.F.S. as an inventor and licensed to AccelevirDx. R.F.S. holds no equity interest in AccelevirDx. R.F.S. consults for Merck and AbbVie on HIV cure-related issues. K.D.R. and M.C. are employees of AccelevirDx. A.M. and G.M.L. are employees of and equity holders in AccelevirDx. B.J.H. is an employee of Merck.

Figures

Fig. 1.
Fig. 1.
Overview of the IPDA (A) Map of the HIV-1 genome indicating positions of the Ψ (blue arrow) and env (green arrow) amplicons. (B) Illustration of the common types of fatal defects detected by the IPDA. Precision maps of sequenced proviruses are available elsewhere (5, 8, 9). (C) Overview of the IPDA. In addition to the HIV-1 proviral discrimination reaction illustrated here, the IPDA includes a parallel ddPCR reaction targeting a host gene to allow determination of input cell number and correction for shearing between Ψ and env amplicons (9, 19). (D) Representative 2D ddPCR plot from the HIV-1 proviral discrimination component reaction of the IPDA. The typical droplet counts follow the pattern Q3 >>> Q1 ∼ Q4 > Q2.
Fig. 2.
Fig. 2.
IPDA analysis of peripheral blood CD4+ T cells from 400 HIV-1–infected individuals on ART provides absolute frequencies and relative composition of the population of persistent proviruses. (A) Number of CD4+ T cell genomes analyzed per assay, as measured by the copy reference/DNA shearing component reaction of the IPDA. The horizontal line indicates median value. n = 400 (B) Absolute frequencies of intact and defective proviruses per 106 total CD4+ T cells as measured by the IPDA. Total proviruses were determined as the sum of intact, 5′ defective, and 3′ defective proviruses from each donor. Horizontal line indicated median values. n = 400 (C) Average relative composition of persistent proviruses as measured by the IPDA. (D) Landscape of persistent proviruses in ART-treated individuals with HIV-1. The QVOA detects an average of 1/106 resting CD4+ T cells, based on previous studies (, , –22). The value for the frequency of cells with intact proviruses (38/106 resting CD4+ T cells) is based on the mean frequency measured in B, corrected for the fraction of cells with small defects not detected by the IPDA (∼30% of Q2 droplets) (9). The value for total infected cells is based on data from B, corrected for the fraction of proviruses (4%) not detected by either of the IPDA amplicons. (E) Frequency of intact and total proviruses per million CD4+ T cells, as measured by the IPDA. The dashed lines reference relative ratios of intact:total proviruses. Correlation was performed on log-transformed data (Spearman’s ρ = 0.76; P < 0.0001).
Fig. 3.
Fig. 3.
IPDA amplicon signal failure due to polymorphisms in primer or probe binding sites is infrequent. (A) Frequency of amplicon signal failure for the Ψ and env amplicons. Of the 400 individuals analyzed in this study, we observed Ψ amplicon signal failure in samples from 3.5% and env amplicon signal failure in samples from 2.8%. (B) Representative 2D ddPCR plot from an HIV-1 proviral discrimination reaction illustrating Ψ amplicon signal failure. Ψ amplicon signal failure is easily identified by the absence of droplets in Q1 and Q2. (C) Representative 2D ddPCR plot from an HIV-1 proviral discrimination reaction illustrating env amplicon signal failure, which is readily identified by the absence of droplets in Q2 and in Q4. (D–G) Targeted sequencing (Methods) in individuals with signal failure revealing polymorphisms that account for a lack of signal. Polymorphisms at the 3′ end of the Ψ forward primer account for the Ψ signal failure observed in donor 04036 (B) and another donor, 04022. Donor 04052 (C) and another donor, 04122, showed normal Ψ amplification but failed env amplification. In these cases, polymorphisms in the env primers and probe were responsible. Donor 04036 showed no polymorphisms in this region and a normal droplet distribution.
Fig. 4.
Fig. 4.
Custom IPDA oligos can resolve problematic mismatches, salvage amplification in the affected amplicon, and allow detection of intact proviruses. (A) IPDA dot plot showing ψ amplicon failure. (B) Restoration of the normal droplet pattern using custom primers based on single genome sequencing (SGS) of the ψ region. (C) SGS sequences of the ψ forward and reverse primer regions with custom primer design. A single nucleotide length polymorphism at the 3′ end of the ψ forward primer may have precluded amplification. There was also a two-nucleotide insertion in the ψ reverse primer. The use of alternate primers allowed successful ψ amplification shown in B. *Sequences are shown as sense strand nucleotides. The ψ reverse primers used in the IPDA are the reverse complements of the sequences shown.
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