Novel approaches towards characterising the HIV reservoir: A combined microfluidic and target enrichment approach

The latent reservoir in human immunodeficiency virus (HIV) infection is a small population of long-lived infected cells that remain quiescent during suppressive antiretroviral therapy (ART). This reservoir is widely considered to be the major barrier to HIV cure, as in the absence of effective therapy the cells can become reactivated and cause recrudescent viraemia. A number of assays have been developed in an attempt to both quantify and improve our understanding of the reservoir. However, despite early promise the key factors that contribute to its establishment and persistence remain elusive. The work undertaken in this thesis was directed primarily at improving the suite of tools available to clinicians and researchers targeting the latent reservoir in HIV infection. To date, the assays that have been designed to quantify and characterise the reservoir have limitations leading to either imprecise measurements or potentially biased analysis of viral sequences. By developing a modified target enrichment protocol, it was shown that diverse full-length proviral sequences can be captured whilst avoiding the bias commonly introduced by PCR-based sequencing methods. In addition, this pipeline allows for the identification of integration sites from the same sample. The major hurdle facing reservoir assays is the inability to distinguish a latently infected cell from an uninfected cell by cell surface markers. To bypass this requirement, this thesis presents a microfluidics pipeline aimed at using the resident provirus as a means of identifying a latently infected cell. By encapsulating single cells in water-in-oil droplets, it is possible to lyse the cells and perform a PCR reaction targeting the HIV genome. In the presence of HIV, droplets fluoresce and are labelled for dielectrophoretic sorting. Post sorting, nucleic acids can be reclaimed from the droplets for downstream analysis on a purified subset of latently infected cells. The two assays proposed within this thesis have the potential to profoundly change our understanding of HIV latency. In addition to this, as clinicians enter a new era of treatment interruption studies, changes in reservoir size and composition are becoming a desirable metric. Overall, the foundation laid down in this thesis provides a solid base from which to begin applying both assays to clinical cohorts.