In Situ Multiplexing to Identify, Quantify, and Phenotype the HIV-1/SIV Reservoir Within Lymphoid Tissue

Modern combination antiretroviral therapy (ART) regimens provide abiding viral suppression for most individuals infected with human immunodeficiency virus (HIV). However, the persistence of viral reservoirs ensures that eradication of HIV-1 (i.e., cure) or sustained ART-free remission (i.e., functional cure) remains elusive, necessitating continual, strict ART adherence and contributing to HIV-1-related comorbidities. Eradication of these viral reservoirs, which persist primarily within lymphoid tissue, will require a deeper understanding of the cellular neighborhoods in which latent and active HIV-1-infected cells reside. By pairing highly sensitive in situ hybridization (ISH) with an exceptionally flexible immunofluorescence (IF) approach, we describe a simple, yet highly adaptable multiplex protocol for investigating the quantity, distribution, and characteristics of HIV-1 viral reservoirs.