HIV-1 diversity considerations in the application of the Intact Proviral DNA Assay (IPDA)

Abstract

The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials.

Fig. 1. Interindividual HIV diversity can lead to detection failure by the IPDA.

a IPDA 2D ddPCR plots. Ψ-single-positive (Q1, blue), Ψ- and env- double-positive (Q2, orange), double-negative (Q3, gray) and env single-positive (Q4, green) events for an IPDA true-positive individual, a presumed IPDA true-negative individual and a presumed case of Ψ detection failure. Plots show merge of four replicate wells. b IPDA detection failure due to interindividual HIV diversity. In silico analysis of 337 linked Ψ and env probe sequences from proviral DNA of participant BC-004, who originally failed detection in env, revealed single nucleotide mismatches (red, inset) to the IPDA env probe. These sequences yielded in silico Ψ+(blue), env+(green) and intact (Ψ+env+; orange) provirus frequencies as shown on left. Experimental results using the published IPDA and autologous env probe are shown on right. Source data, including the 337 Ψ and env probe sequences and their classifications, are provided in the Source Data File. c HIV polymorphisms in IPDA primer and probe region sequences for participants with Ψ or env detection failure. Hyphens (-) indicate matches to the IPDA primer or probe; red letters indicate mismatches; asterisk indicates insertion location. d Correlation between reservoir size as measured by IPDA (Intact Proviruses/Million CD4 + T-cells) and QVOA (Infectious Units/Million CD4 + T-cells). When data from all 37 virally-suppressed participants are included, Spearman’s ρ = 0.03 with a two-tailed p = 0.83. After excluding 11 presumed IPDA detection failures (red datapoints), Spearman’s ρ = 0.35 with a two-tailed p = 0.07. Two individuals for whom no replication-competent viruses were detected (IUPM = 0) are plotted on the X-axis. QVOA replicates are detailed the methods; IPDA data represent the point estimate from four merged technical replicates per sample. Source data are provided in the Source Data file. e Correlation between QVOA and HIV gag copies/million CD4 + T-cells. Analysis comprises 31 unique participants, yielding Spearman’s ρ = 0.42, two-tailed p = 0.02. Two individuals for whom no replication-competent viruses were detected (IUPM = 0) are plotted on the X-axis. HIV gag copies report the point estimate from at least four merged technical replicates for each sample. Source data are provided in the Source Data File.

Fig. 2. Intraindividual HIV diversity can lead to reservoir underestimation by IPDA.

a Env gp120 amino acid and IPDA env probe nucleotide sequences from participant 91C33 identified by GenBank accession numbers. 91C33 harbored one HIV subpopulation that matched the IPDA env probe and was sensitive to bNAbs 3BNC117 and 10-1074, and another that harbored a mismatch at position 4 of the IPDA env probe and was resistant to both bNAbs. Hyphens (-) indicate matches to the reference; red letters denote mismatches (IPDA env probe) or amino acid substitutions (bNAb). Sequence is abbreviated seq. b ddPCR env plots from one of 2 independent experiments assessing the ability of the IPDA env reaction to detect representative within-host sequences MH632945 (env probe match) and MH632950 (env probe mismatch), present as purified env PCR products of equal length and comparable quantities. Positive droplets are green and negative droplets are grey. See Supplementary Fig. 3 for additional experiments. c Virus 3, isolated from participant OM5346’s reservoir, was sensitive to 10-1074-mediated ADCC but resistant to 3BNC117; vice-versa for virus 4. Box and error bars indicate mean and standard deviation of three technical replicates, from one of two independent experiments. Open circles denote individual datapoints. Source data are provided in the Source Data File. d 1D IPDA env ddPCR plots from one of 2 independent experiments for cells infected in vitro with virus 3 (IPDA env probe match) or virus 4 (G13A mismatch). Both viruses were detectable using an alternative primer/probe set (Supplementary Fig. 6). e Possible impacts of intraindividual HIV diversity on IPDA-measured reservoir changes following a hypothetical intervention. The solid line indicates the observed effect by IPDA; dashed line indicates the true effect. An intervention’s impact on the reservoir could be overestimated when a subset of within-host HIV sequences are undetectable by IPDA and resistant to treatment (e.g., 91C33 during treatment with 3BNC117 or 10-1074, or OM5346 during treatment with 10-1074). Conversely, an intervention’s impact on the reservoir could be underestimated when a subset of within-host HIV sequences are undetectable by IPDA and sensitive to treatment (e.g., OM5346 during treatment with 3BNC117). In a worst-case scenario, one could erroneously conclude that the treatment had no effect.

Fig. 3. HIV sequence-specific optical bleed-through prohibits tight thresholding on negative populations.

a 2D and env 1D plots of OM5346 virus 3 Ψ region sequence when tested as a synthetic DNA gene fragment (Virus 3 Ψ gBlock) without corresponding env template. Minimal Ψ- to env- channel spillover occurs and the positive droplet threshold can be drawn tightly to the double-negative population without consequence (note the presence of one false double-positive droplet). b 2D and env 1D plots of OM5346 virus 4 Ψ region sequence, tested as a synthetic DNA gene fragment (Virus 4 Ψ gBlock), without corresponding env template. Drawing a tight threshold causes optical bleed-through of Ψ (FAM) fluorescence into the env (VIC) channel to yield a false-positive env (and by extension, false-positive intact) signal. c 2D and env 1D plots of OM5346 Virus 4 Ψ region sequence, tested as a synthetic DNA gene fragment (Virus 4 Ψ gBlock) without corresponding env template, with a threshold drawn at an appropriate distance from the double-negative population. This threshold accommodates the fluorescence shift and thus avoids the creation of false-positive intact or env-positive droplet population (note the presence of a single false double-positive droplet). Representative plots from one of three technical replicates from one experiment are shown.

Fig. 4. Performance of the secondary env primer/probe set.

a A secondary env primer/probe set rescued detection in all 9 cases of IPDA env detection failure (p = 0.004, two-sided Wilcoxon signed-rank test). b A secondary env primer/probe set performs comparably to the IPDA in samples detectable by the latter assay. Black datapoints denote 33 participants whose reservoir was detectable by both IPDA and secondary env primer/probe sets; red datapoints denote three participants whose reservoir was detectable by IPDA but not using the secondary env primer/probe set. Excluding the latter three points yielded a Spearman’s ρ = 0.82, p < 0.0001 (shown on figure). Including these three red datapoints yielded a Spearman’s ρ = 0.68, p < 0.0001. Datapoints show point estimate from four merged technical replicates for each participant sample. Source data are provided in the Source Data File.