Development of an Assay Evaluating the Inducible HIV‐1 Latent Reservoir Based on Reverse Transcription Droplet Digital PCR for Unspliced/Intact Viral RNA

T lymphocytes, which cannot be recognized by the immune system. Accurately assessing the active latent reservoir by HIV-1 RNA is crucial for the clinical diagnoses and treatment. Reverse transcription (RT)-polymerase chain reaction (PCR)-based assays are commonly employed to detect HIV-1 reservoirs in clinical settings, but single-site probe designs limit their ability to distinguish between intact and defective HIV-1 proviral transcripts. In this study, we present and optimize a RT-droplet digital PCR-based assay (RT-ddPCR) that accurately quantifies unspliced/intact intracellular HIV-1 RNA, which sensitively detects the activity of HIV-1 latent reservoirs. By testing with various latency-reversing agents (LRAs) in multiple HIV-1 latent cell line models, we demonstrated that our method is more accurate than traditional RT-PCR-based assays for HIV-1 RNA. Moreover, the unspliced/intact HIV-1 RNA assay was used to monitor HIV-1 latent reservoir activity in individuals undergoing analytical treatment interruption (ATI) after antiviral therapeutic intervention. The level of unspliced/intact HIV-1 RNA in peripheral blood mononuclear cells (PBMCs), with an increase in unspliced/intact viral RNA levels detectable before viral rebound in plasma, positively correlated with the initial viral load at rebound. Compared to culture-based methods for detecting inducible reservoirs, this approach significantly reduces the required cell quantity, operational complexity, and detection time. The highly sensitive RT-ddPCR detection of unspliced/intact HIV-1 RNA shows good correlation with the viral rebound following ATI, which will also be valuable for predicting inducible viral reservoir size. This finding supports the assay's utility for faster and more accurate prediction of viral rebound and timely initiation of intervention therapy.