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.

HIV proviral burden, genetic diversity, and dynamics in viremic controllers who subsequently initiated suppressive antiretroviral therapy

“…Intact HIV-1 copies (Ψ and env double-positive droplets) were corrected for DNA shearing based on the frequency of RPP30 and RPP30-Shear double positive droplets. The median DNA shearing index, which measures the proportion of sheared DNA in a sample, was 0.40 (IQR 0.39-0.43), which is in line with acceptable levels in this assay (29, 81).…”

“…For this reason, targeted HIV sequencing of the IPDA Ψ and env regions was performed for all participants prior to IPDA measurement, and autologous primers/probes substituted where necessary. The substituted primer and probe sequences (5' -> 3') were: acceptable levels in this assay (29,81).…”

“…The published HIV primers/probes however can sometimes fail to detect autologous HIV sequences due to naturally-occurring polymorphism (81, 82), thus requiring autologous primer/probes. For this reason, targeted HIV sequencing of the IPDA Ψ and env regions was performed for all participants prior to IPDA measurement, and autologous primers/probes substituted where necessary.…”

Longitudinal clonal dynamics of HIV-1 latent reservoirs measured by combination quadruplex polymerase chain reaction and sequencing

“…For example, VOA is labor intensive, variable from assay to assay (10), and ultimately underestimates the latent reservoir (22,23). IPDA is a sensitive, highthroughput assay (24), but is limited to detecting proviruses that are sequence compatible with its 2 Packaging Signal (PS) and env oligonucleotide probe sets (18,39). In addition, IPDA lacks a sequence verification step to determine if a provirus is truly intact or defective (20).…”

An Improved Tat/Rev Induced Limiting Dilution Assay With Enhanced Sensitivity and Breadth of Detection

“…Recently, Bruner et al developed the intact proviral DNA assay (IPDA), which utilizes ddPCR to simultaneously detect two regions of the HIV-1 genome (Y sequence and env) often deleted in replication-deficient proviruses (16). Although IPDA is superior to the total HIV-1 DNA amplification, the assay still overestimates the proportion of the intact virus, as it overlooks defects in other regions of the viral genome (17,18). Importantly, IPDA also is restricted by an additional technical limitation that the inducibility of intact proviral genomes identified by the assay remains to be demonstrated.…”