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

Significance Despite clinically effective antiretroviral therapy (ART), replication-competent HIV-1 persists as latent proviral DNA capable of rekindling viral replication when ART is stopped. To cure the infection, therapies are being developed to eliminate latent HIV-1. Accurate assays for intact or rebound-competent HIV-1 are important to this effort. We previously developed the intact proviral DNA assay (IPDA) as a more accurate and scalable assay for intact HIV-1 proviruses. Here we present IPDA analysis in a diverse cohort of 400 ART-treated individuals. We confirm that intact proviruses, although vastly outnumbered by defective proviruses, are present at significantly higher frequencies than previously detected by viral outgrowth assays. We also show that IPDA amplicon signal issues, observed in 6.3% of samples, result from sequence polymorphisms. 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.