Abstract
Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection; HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1–2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10–15 years on therapy, there were as many as 3.5–5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.
Highlights
Successful combination antiretroviral therapy completely blocks ongoing viral replication [1,2,3] and improves infection-associated morbidity and mortality, but does not cure the infection [4,5,6,7]
We focused on two abundant proviruses, one present in the HORMAD2 gene, which accounts for approximately 20% of all proviruses in one patient, and an infectious provirus, AMBI-1, which accounted for 3.1% of proviruses in another [16,30]
Because the kinetics of clonal expansion of individual HIV-infected cells during a participant who had clones of HIV-infected cells for which we had previously determined the Combination antiretroviral therapy (cART) have not been described, we developed Droplet Digital PCR (ddPCR) assays to quantify the proviruses in specific integration sites [16]
Summary
Successful combination antiretroviral therapy (cART) completely blocks ongoing viral replication [1,2,3] and improves infection-associated morbidity and mortality, but does not cure the infection [4,5,6,7]. Siliciano and coworkers reported that the fraction of defective proviruses increases during cART [14,15], and Pinzone et al observed that, on average, the fraction of the proviruses that are intact declines slowly during cART [28], despite a relatively constant total proviral load These shifts in HIV populations take place in the setting of ongoing immune recovery and increases in CD4+ T cells after cART is initiated. These findings shed new light on the dynamics of the population of HIV-infected cells during cART and have implications for the understanding of persistence of viral DNA and ongoing pathogenesis. These findings have relevance to efforts to control or cure HIV, because the populations of infected cells targeted by eradication strategies will be fundamentally different depending on timing of these interventions
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