Quantification of Total HIV1-DNA in Peripheral Blood Mononuclear Cells

Abstract

HIV reservoir measurement in patients is one of the challenges at the time of testing new treatment approaches aiming at eradicating HIV infection. HIV reservoirs are complex and disseminated in a large number of organs and lymphoid tissues. We chose to quantify total cell-associated HIV-DNA in PBMC as a marker of HIV reservoirs and described the method we developed. The marker was used in large cohort studies at different stages of HIV disease and in therapeutical trials. Our results show how informative is this marker, as well as that plasma HIV-RNA and CD4 T cell count are representative of each patient when measured in blood. Such a series of results might help to adapt simplification or structured interruption strategies, design new clinical trials targeting HIV reservoirs, and select populations that could benefit of such new treatments.

Towards an HIV-1 cure: measuring the latent reservoir

“…This renders the cells permissive for viral gene expression and induces release of HIV-1 from latently infected cells. Since the development of the VOA, other assays such as PCR-based ones that measure total HIV-1 proviral DNA, have been advanced as simpler measures of the LR [15–17]. Although both culture- and PCR-based methods are commonly used, there is little correlation between the two types of assays.…”

“…These methods provide a quicker and easier way to study viral persistence and can be applied to a variety of immune cell types. The most common PCR method for measuring the LR is a qPCR for proviral DNA in either unfractionated PBMCs [15,42–46], CD4 + T cells [47,48], or resting CD4 + T cells [49]. All methods involve isolating the desired cell populations from the peripheral blood of infected individuals and subsequently extracting the DNA.…”

Measuring the latent reservoir in vivo

“…The most common method for measuring HIV DNA is quantitative PCR (qPCR) for total DNA, which includes integrated and nonintegrated forms. Typically, qPCR is performed on DNA extracted from total peripheral blood mononuclear cells (PBMCs) or enriched CD4 + T cells using PCR primers and probes targeting conserved regions of the HIV genome (Pol or Gag) (10,11). A standard curve is constructed with known copy numbers of proviral DNA (for example, using a plasmid standard such as 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.…”

Relative efficacy of T cell stimuli as inducers of productive HIV-1 replication in latently infected CD4 lymphocytes from patients on suppressive cART

“…While this may result in a more accurate measurement, multiple activation rounds make this approach very time consuming. HIV DNA assays that measure integrated or total DNA (O’Doherty et al, 2002; Rouzioux et al, 2014; Strain et al, 2013) are relatively quick to perform. However, they tend to overestimate the true reservoir size by detecting mutated proviruses that can never be expressed, even upon cessation of cART.…”