Abstract Antiretroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-virus producing cells as a standard to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: 1) EDITS (envelope detection by induced transcription-based sequencing) assay; 2) HIV-Flow; and 3) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24 respectively; and 4) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivity of these assays is sufficient to detect a rare HIV-producing/env mRNA+/p24+ cell in a million uninfected cells. These high-throughput technologies thus provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. Importance Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV virus or the virus’ major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV Envelope mRNA (EDITS assay) and gag mRNA and p24 (Flow-FISH, HIV-Flow and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thus providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

Detecting sources of immune activation and viral rebound in HIV infection