Improved methods of measuring the latent HIV reservoir with DNA size selection and droplet digital PCR
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Abstract
For HIV-infected patients undergoing antiretroviral treatment (ART), the latent reservoir is a major barrier to cure. Formed in the acute stages of infection, the latent reservoir consists of cells in the host’s body where HIV can hide from the immune system and remain in a quiescent state. When a patient stops ART, the virus quickly rebounds, proliferates, and begins infecting more cells. The majority of assays available to measure the latent HIV reservoir measure HIV DNA and RNA using polymerase chain reaction (PCR). However, these methods can overestimate the reservoir size, because unintegrated and replication-incompetent DNA comprise a larger proportion of HIV nucleic acids than does replication competent HIV provirus.
To measure integrated HIV DNA from infected donor samples provided by the HIV Reservoir Assay Validation and Evaluation Network (RAVEN) and The University of Texas Health Sciences Center at Houston (UTHealth), I applied pulsed-field gel electrophoresis (PFGE) to reduce the amount of unintegrated HIV DNA such as episomal 2-long-terminal-repeat (2-LTR) circles before quantitation with droplet digital PCR (ddPCR). This assay could prove useful to measure changes in the latent HIV reservoir, especially in clinical trials that aim to reduce its size through a variety of compounds.
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Attribution 4.0 International