Development of novel molecular and microfluidics tools for identification and characterization of latent HIV-1 reservoir

Abstract

The existence of latent HIV-1 reservoir (LR) in all HIV-1 infected patients serves as a major obstacle to completely cure HIV-1 infection. However, up to now there is still no available assay that provides an accurate measurement of the reservoir size. This thesis aims to address this challenge from different aspects with several novel technologies, using both molecular and microfluidics-based tools. To find a proper tool to identify the latent HIV-1 reservoir, in Paper I and II, LIPS assay, RNAflow, and RNAscope assay were optimized and evaluated for indirect and direct detection of latent HIV-1 reservoir. The results indicated the LIPS method might not be sufficient for latent HIV-1 reservoir detection, although it has been proposed to quantify the latent HIV-1 reservoir indirectly. Furthermore, the optimized RNAscope technique performed better than RNAflow for transcription and translation competent latent HIV-1 reservoir identification. The RNAscope was also found to be independent of the HIV-1 subtype and can be applied to patient samples at single cell level. As there are currently no available surface biomarkers for latent HIV-1 reservoir, in Paper III, transcriptomics and proteomics-based analysis method for high-throughput selection of potential biomarker were established and applied to different patient groups. Twelve membrane protein-coding genes were identified as downregulated in the patient group who were hypothesized to have lower latent reservoir. These proteins might have the potential to be used as surface biomarkers for latent HIV-1 reservoir. CD4+ T cells, monocyte/macrophages, and natural killer cells are believed to be the primary source for HIV-1 reservoirs in peripheral blood. In paper IV, a microfluidic chip was developed to simultaneously isolate these three mononuclear leukocyte cell types directly from whole blood. The microfluidic method reduces the sample volume requirement and is a promising tool for latent HIV-1 reservoir study. Together, though further improvement and clinical verification are necessary, the work in this thesis has contributed to the advancement of latent HIV-1 reservoir characterization and may facilitate future development of the latent HIV-1 reservoir targeting and clearance methods with the ultimate goal – to cure HIV-1 infection.