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Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR.

ABSTRACT: Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

SUBMITTER: Brandt LD

PROVIDER: S-EPMC8310066 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Little is known about the emergence and persistence of human immunodeficiency virus (HIV)-infected T-cell clones in perinatally infected children. We analyzed peripheral blood mononuclear cells (PBMCs) for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8 and 17.4 months of age and with viremia suppressed for 6 to 9 years. We obtained 8,662 HIV type 1 (HIV-1) integration sites from pre-ART samples and 1,861 sites from on-ART samples. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6 to 9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMCs infected ex vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B Our analyses indicate that, despite marked differences in T-cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.IMPORTANCE HIV-1 integrates its genome into the DNA of host cells. Consequently, HIV-1 genomes are copied with the host cell DNA during cellular division. Pediatric immune systems differ significantly from adults, consisting primarily of naive T cells, which have low expression of the HIV-1 coreceptor CCR5. This difference may result in variances in the number or size of infected cell clones that persist in children on ART. Here, we provide the most extensive analysis of the integration landscape of HIV-1 in children. We found that, despite the largely naive cell populations in neonatal immune systems, patterns of HIV-1 integration and the size of infected cell clones are as large and widespread as those in adults. Furthermore, selection for integration events in proto-oncogenes were observed in children despite early ART. If such cell clones persist for the life span of these individuals, there may be long-term consequences that have yet to be realized.

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Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR.

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