Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications

Abstract

HIV-1 DNA persists in infected cells despite combined antiretroviral therapy (cART), forming viral reservoirs. Recent trials of strategies targeting latent HIV reservoirs have rekindled hopes of curing HIV infection, and reliable markers are thus needed to evaluate viral reservoirs. Total HIV DNA quantification is simple, standardized, sensitive, and reproducible. Total HIV DNA load influences the course of the infection and is therefore clinically relevant. In particular, it is predictive of progression to AIDS and death, independently of HIV RNA load and the CD4 cell count. Baseline total HIV DNA load is predictive of the response to cART. It declines during cART but remains quantifiable, at a level that reflects both the history of infection (HIV RNA zenith, CD4 cell count nadir) and treatment efficacy (residual viremia, cumulative viremia, immune restoration, immune cell activation). Total HIV DNA load in blood is also predictive of the presence and severity of some HIV-1-associated end-organ disorders. It can be useful to guide individual treatment, notably, therapeutic de-escalation. Although it does not distinguish between replication-competent and -defective latent viruses, the total HIV DNA load in blood, tissues, and cells provides insights into HIV pathogenesis, probably because all viral forms participate in host cell activation and HIV pathogenesis. Total HIV DNA is thus a biomarker of HIV reservoirs, which can be defined as all infected cells and tissues containing all forms of HIV persistence that participate in pathogenesis. This participation may occur through the production of new virions, creating new cycles of infection and disseminating infected cells; maintenance or amplification of reservoirs by homeostatic cell proliferation; and viral transcription and synthesis of viral proteins without new virion production. These proteins can induce immune activation, thus participating in the vicious circle of HIV pathogenesis.

FIG 1

Several forms of HIV DNA compose the total HIV DNA and participate in HIV pathogenesis. The integrated form, the provirus, is the more persistent form and permits production of virions when quiescent infected cells are reactivated. Virions can infect new cells and propagate infection and the HIV reservoir. The provirus form persists in all cells during cell proliferation. Episomal forms with 1-LTR or 2-LTR persist and are diluted in some daughter cells during cell proliferation. Linear unintegrated HIV DNA is the more labile form and is essentially present when the virus is replicating. Defective provirus, with a deletion, nonsense mutation, or hypermutation, cannot produce new virions but can produce transcripts and viral proteins which could activate the immune system and participate in HIV pathogenesis.

FIG 2

Reproducibility of total HIV DNA quantification in two positive controls by real-time PCR (59). For this experiment, two different pools of blood cells (in EDTA) from HIV-infected patients were prepared. Aliquots were frozen at −80°C. One aliquot of each control (high-level positive control [A] and moderate-level positive control [B]) was tested in all assays performed during a >9-month period to verify the assays reproducibility over time. Results presented here are for 69 quantifications performed by different technicians and with different thermocyclers and with four lots of Biocentric reagents, for the high-level positive control (A) and moderate-level positive control (B). Means (red lines), standard deviations, coefficients of variation, and 95% confidence intervals were, respectively, 3.30 log copies/10⁶ leukocytes, 0.11 log, 0.03, and ±0.22 log for the high-level positive-control sample and 2.40 log copies/10⁶ leukocytes, 0.18 log, 0.07, and ±0.35 log for the moderate-level positive-control sample.

FIG 3

Spectrum of total HIV-1 DNA levels in PBMC during HIV infection. The natural history data are from HIV DNA quantified for 552 adults at the time of the primary infection (PRIMO cohort, ANRS) (81), for 271 patients who had seroconverted 6 to 24 months previously (SEROCO cohort, ANRS) (84), and for 121 perinatally infected children (median age, 6 years), of whom 46.6% and 20.3% were at CDC stage B and C, respectively (ANRS 1244/1278) (94). The data set for “during antiretroviral therapy” is for HIV DNA quantified during antiretroviral therapy initiated during the primary infection and continued for 2 years (90 patients; OPTIPRIM trial, ANRS) (109) or for a median of 3.6 years, with HIV RNA levels of <50 copies/ml for a median of 3.1 years (n = 35) (123). HIV DNA was quantified during antiretroviral therapy initiated early during the chronic phase in 116 adults (CD4 cell count, >350/mm³; plasma HIV RNA, <4.7 log copies/ml), with antiretroviral therapy for a median of 5.3 years (SALTO trial, ANRS) (220) and in 272 adults treated later (median CD4 cell nadir, 228/mm³; plasma HIV RNA, 5.3 log copies/ml) for a median of 7.3 years, with HIV RNA levels at <50 copies/ml for a median of 3.9 years (123). HIV DNA was quantified in 44 adults with advanced therapeutic failure and AIDS (CD4 count, ≤200/mm³; HIV RNA level, >4 log; genotypic score showing two or fewer active drugs) (ETOILE trial, ANRS) (197). For the HIV control data set, HIV DNA were quantified for patients who controlled the infection naturally (plasma HIV RNA undetectable for >10 years in the absence of antiretroviral treatment [15 patients]) (HIV controllers cohort, ANRS) (89) or after treatment interruption (14 patients) (VISCONTI study, ANRS) (92). The same standardized assay was used in the same laboratory. A broad range of HIV DNA levels was found. Medians and interquartile ranges are indicated.

FIG 4

Anatomical HIV reservoirs. Reservoir cells are highly disseminated.