Measuring the latent reservoir in vivo

Abstract

Current efforts toward achieving a cure for HIV are focused on developing strategies to eliminate latently infected CD4+ T cells, which represent the major barrier to virus eradication. Sensitive, precise, and practical assays that can reliably characterize and measure this HIV reservoir and can reliably measure the impact of a candidate treatment strategy are essential. PCR-based procedures for detecting integrated HIV DNA will overestimate the size of the reservoir by detecting replication-incompetent proviruses; however, viral outgrowth assays underestimate the size of the reservoir. Here, we describe the attributes and limitations of current procedures for measuring the HIV reservoir. Characterizing their relative merits will require rigorous evaluation of their performance characteristics (sensitivity, specificity, reproducibility, etc.) and their relationship to the results of clinical studies.

Figure 1. Assays to measure the latent HIV reservoir.

(A) Blood plasma (or seminal plasma or cerebrospinal fluid) can be assayed for residual virion production with highly sensitive assays of HIV RNA that use large volumes of specimen, as described in the text. Latently infected cells from blood or from tissues that are more difficult to obtain can be examined for several different analytes. Extracted DNA can be assayed for total, integrated, or 2-LTR forms either by qPCR or ddPCR. (B) HIV RNA transcripts can be measured in resting cells either directly or after induction in the presence of antiretroviral drug to prevent cell-to-cell transmission. caRNA can be assayed for specific transcripts, and cfRNA can be measured for the production of virions. The induction of replication-competent provirus is assayed in the absence of antiretroviral drug and the addition of cells that permit the propagation HIV infection, as measured by the generation of p24 antigen or HIV RNA.