HIV Transcriptional Regulation in the Brain of Virally Suppressed PWH

Approximately 40 million people live with HIV (PWH) globally. Although antiretroviral therapy effectively suppresses plasma viral loads, treatment interruption leads to rapid viral rebound due to the persistence of HIV reservoirs. These reservoirs are distributed across anatomical compartments including the blood, gastrointestinal tract, lymphoid tissue, and the central nervous system. This thesis focuses on the HIV reservoir within the brain, which remains poorly defined during viral suppression but may contribute to neurocognitive disorders that affect >20% of virally suppressed people with HIV. The aim of this thesis was to quantify and characterise the persistence and impact of HIV DNA and RNA within the brain of ART-suppressed PWH. HIV DNA was quantified in post-mortem frontal cortex brain tissue of virally suppressed (n=16) and non-virally suppressed (n=17) PWH at similar levels using droplet digital PCR. Both intact and defective proviruses were identified using the intact proviral DNA assay, with intact HIV proviruses detected in 12/16 virally suppressed PWH and 14/17 non-virally suppressed PWH. Defective proviruses were identified in all individuals, comprised ~87% of total genomes identified and were associated with increased Mx1 expression in myeloid cells. Together, these findings confirm that ART does not eliminate brain reservoirs, which may contribute to ongoing neuroinflammation. To evaluate whether the HIV reservoir within the brain is capable of initiating viral transcription, HIV long terminal repeats (LTRs) were isolated from frontal cortex brain and matched peripheral tissue of virally suppressed PWH (n=5). HIV LTR sequences were phylogenetically similar between brain and peripheral compartments, and identical sequences were identified in both compartments in 2/5 PWH. Common mutations were observed in key transcription factor binding sites, including Sp1, NF-кB and C/EBP, with most variants either maintaining or reducing predictive motif binding. LTRs from both brain and peripheral compartments remained functional and capable of driving basal and Tat activated transcription, indicating that the HIV reservoir within the brain is capable of initiating transcription. Next, to determine if viral transcripts were actively being produced during viral suppression, HIV TAR, Long-LTR, Pol, PolyA and Tat/Rev RNA transcripts were measured in frontal cortex brain tissue and peripheral blood mononuclear cells (PBMCs) of PWH. HIV RNA transcripts were detected in frontal cortex tissue of all PWH (n=24) regardless of viral suppression with ART, although at lower frequencies than observed in PBMCs. Levels of HIV TAR transcripts in the frontal cortex were similar between virally suppressed and non-virally suppressed PWH. All assessed HIV transcripts were detected in the frontal cortex of 2/11 virally suppressed and 7/13 non-virally suppressed PWH, demonstrating transcriptional completion and splicing in the brain of a subset of PWH regardless of viral suppression. HIV p24 protein was detected using immunofluorescence in all individuals expressing Tat/Rev transcripts, confirming active viral protein production. HIV RNA was associated with an increased expression of Mx1 within myeloid cells, suggesting that persistent viral transcription may contribute to neuroinflammation. Together, these findings demonstrate that the brain is a persistent HIV reservoir, containing both intact and defective HIV proviruses that are transcriptionally active during suppressive ART. This activity may drive local neuroinflammation through viral RNA and protein production and contribute to neurological dysfunction. These findings highlight the importance of the brain reservoir and its need for consideration in the development of therapeutic strategies, including the production of transcriptional inhibitors which may decrease neuroinflammation and alleviate neurocognitive disorders.