Elevation of cell-associated HIV-1 RNA transcripts in CSF CD4+ T cells, despite suppressive antiretroviral therapy, is linked to in vivo brain injury

Objective Despite effective antiretroviral therapy (ART), brain injury remains prevalent in people living with HIV-1 infection (PLHIV) possibly due to ART lacking direct inhibition of transcription with continued local production of viral transcripts and neurotoxic proteins, such as Tat, rather than cell-free whole virion toxicity. We quantified cell-associated (CA) HIV-1 RNA-transcripts in CSF and blood, in relation to proton Magnetic Resonance Spectroscopy (1H MRS) of major brain metabolites, in well characterised PLHIV. Methods RNA was extracted from cells in 16 paired samples of CSF and blood, from PLHIV on fully suppressive ART. HIV-1 CA-RNA copies were measured using the highly sensitive Double-R assay and normalized /million CD4+ T cells. 18-colour flow cytometry was used to count and analyse CD4+ T cells and monocytes in CSF and blood. The concentrations of major brain metabolites from 1H MRS in frontal white matter (FWM), posterior cingulate cortex (PCC), and caudate areas were measured. Brain injury in each voxel was defined using a composite score derived by principal component analysis. Results 14/16 CSF cell samples had quantifiable HIV-1 CA-RNA transcripts, at levels significantly higher than in their PBMCs (median 9,266 vs 185 copies /106 CD4+ T cells; p<0.0001). Higher levels of CSF transcripts were associated with greater brain injury in the FWM (Std beta=-0.73; p=0.007) and PCC (Std beta=-0.61; p=0.03). CSF cells were 91% memory T cells, equally CD4+ (median 3,605) cells and CD8+ T cells (3,632), but contained much fewer B cells (0.4 %), NK cells (2.0%) and monocytes (3.1%; 378 cells; >90% CD14+CD16+ phenotype). CXCR3+CD49d+integrin beta7-negative, CCR5+ CD4+ T cells were significantly enriched in CSF, compared with PBMC (p <0.001). Transcriptional activity in CSF cells was highly correlated with levels of transcriptional activity in CD4+ T cells in PBMC (r=0.76; p=0.002). In contrast, HIV-1 RNA in highly purified monocytes from PBMC was detected in only 6/16 samples. Conclusions Elevated HIV-1 transcripts in CSF cells were associated with in vivo brain injury, despite suppressive ART. The cellular source is most likely the predominant CXCR3+ CD49d+ integrin beta7- CCR5+ memory CD4+ T cells, not monocytes. Inhibitors of transcription to reduce local production of potentially neurotoxic proteins, should be developed.