Abstract
Antiretroviral therapy effectively suppresses, but does not eradicate HIV-1 infection. Persistent low-level HIV-1 can still be detected in plasma and cellular reservoirs even after years of effective therapy, and cessation of current treatments invariably results in resumption of viral replication. Efforts to eradicate persistent HIV-1 require a comprehensive examination of the quantity and genetic composition of HIV-1 within the plasma and infected cells located in the peripheral blood and tissues throughout the body. Single-molecule techniques, such as the single-copy assay and single-genome/proviral sequencing assays, have been employed to further our understanding of the source and viral dynamics of persistent HIV-1 during long-term effective therapy. The application of the single-copy assay, which quantifies plasma HIV-1 RNA down to a single copy, has revealed that viremia persists in the plasma and CSF after years of effective therapy. This low-level HIV-1 RNA also persists in the plasma following treatment intensification, treatment with latency reversing agents, cancer-related therapy, and bone marrow transplantation. Single-genome/proviral sequencing assays genetically characterise HIV-1 populations after passing through different selective pressures related to cell type, tissue type, compartment, or therapy. The application of these assays has revealed that the intracellular HIV-1 reservoir is stable and mainly located in CD4+ memory T cells. Moreover, this intracellular HIV-1 reservoir is primarily maintained by cellular proliferation due to homeostasis and antigenic stimulation, although cryptic replication may take place in anatomic sites where treatment is sub-optimal. The employment of single-genome/proviral sequencing showed that latency reversing agents broadly activate quiescent proviruses but do not clear the intracellular reservoir. Recently, full-length individual proviral sequencing assays have been developed and the application of these assays has revealed that the majority of intracellular HIV-1 DNA is genetically defective. In addition, the employment of these assays has shown that genetically intact proviruses are unequally distributed in memory T cell subsets during antiretroviral therapy. The application of single-molecule assays has enhanced the understanding of the source and dynamics of persistent HIV-1 in the plasma and cells of HIV-infected individuals. Future studies of the persistent HIV-1 reservoir and new treatment strategies to eradicate persistent virus will benefit from the utilization of these assays.
Highlights
The development of antiretroviral therapy (ART) for the treatment of human immunodeficiency virus (HIV-1) remains one of the great triumphs of modern medicine
Studies that measured HIV-1 RNA levels in paired plasma and cerebrospinal fluid (CSF) samples from ART-suppressed HIV-infected participants using single-copy assay (SCA) revealed that the proportion of samples with measurable HIV-1 RNA was lower in CSF samples (14–17%) compared to plasma samples (57–64%) and the median levels of HIV-1 RNA in the CSF were significantly lower (p ≤ 0.0001) [39]
One study estimated that the evolutionary rate was no greater than 0.0006 and 0.002 nucleotide substitutions/site during the 4–12 years of suppressive therapy for the participants treated during early and chronic infection, respectively [25]. These results suggest viral replication is not a major cause of persistence in the cellular populations analysed and that persistent intracellular HIV-1 DNA is most likely maintained by homeostatic and/or antigen-specific cellular proliferation [12, 25–27]
Summary
The development of antiretroviral therapy (ART) for the treatment of human immunodeficiency virus (HIV-1) remains one of the great triumphs of modern medicine. HIV-1 DNA persists as an integrated genome in longlived or slowly-dividing cellular reservoirs, current therapeutic approaches are unlikely to prove curative [2, 3]. In light of these challenges, treatments aimed at HIV-1 eradication stand out as a highly promising avenue to confront and defeat the HIV-1 epidemic [4, 5]. SCA has a broad dynamic range (1–106 copies/ml) and a limit of detection down to 1 copy of HIV RNA [7] Using this assay, it was found that approximately 80% of participants with plasma HIV-1 RNA levels below 50 copies/ml had quantifiable viremia [7, 8]. Monitoring the levels of persistent viremia is crucial for confirming the continued effectiveness of ART, and for determining the effectiveness of new curative treatment strategies for eliminating HIV-1
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