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

Antiretroviral therapy (ART) can attain prolonged undetectable HIV-1 in plasma and cerebrospinal fluid (CSF), but brain injury remains prevalent in people living with HIV-1 infection (PLHIV). We investigated cell-associated (CA)-HIV-1 RNA transcripts in cells in CSF and blood, using the highly sensitive Double-R assay, together with proton Magnetic Resonance Spectroscopy (1H MRS) of major brain metabolites, in sixteen PLHIV. 14/16 CSF cell samples had quantifiable CA-HIV-1 RNA, at levels significantly higher than in their PBMCs (median 9,266 vs 185 copies /106 CD4+ T-cells; p<0.0001). In individual PLHIV, higher levels of HIV-1 transcripts in CSF cells were associated with greater brain injury in the frontal white matter (Std ß=-0.73; p=0.007) and posterior cingulate (Std ß=-0.61; p=0.03). 18-colour flow cytometry revealed that the CSF cells were 91% memory T-cells, equally CD4+ and CD8+ T-cells, but fewer B cells (0.4 %), and monocytes (3.1%). CXCR3+CD49d+integrin ß7-, CCR5+CD4+ T-cells were highly enriched in CSF, compared with PBMC (p <0.001). However, CA-HIV-1 RNA could not be detected in 10/16 preparations of highly purified monocytes from PBMC, and was extremely low in the other six. Our data show that elevated HIV-1 transcripts in CSF cells were associated with brain injury, despite suppressive ART. The cellular source is most likely memory CD4+ T cells from blood, rather than trafficking monocytes. Future research should focus on inhibitors of this transcription to reduce local production of potentially neurotoxic and inflammatory viral products.

HIV-1 viral blips are associated with repeated and increasingly high levels of cell-associated HIV-1 RNA transcriptional activity.

OBJECTIVE: Some HIV+ patients, virally suppressed on ART, show occasional 'blips' of detectable HIV-1 plasma RNA. We used a new highly sensitive assay of cell-associated HIV-1 RNA to measure transcriptional activity in PBMCs and production of infectious virus from the viral reservoir, in patients with and without 'blips'. DESIGN/METHODS: RNA and DNA extracted from cells in 6 ml of peripheral blood, from suppressed patients with one to two 'blip' episodes over the past 2 years of ART (n = 55), or no 'blips' (n = 52), were assayed for HIV-1 RNA transcripts and proviral DNA targeting the highly conserved 'R' region of the LTR. Follow-up samples were also collected. Purified CD4+ T cells were cultured with anti-CD3/CD28/CD2 T-cell activator to amplify transcription and measure replication competent virus. RESULTS: HIV-1 RNA transcripts ranged from 1.3 to 5415 copies/106 white blood cells. 'Blip' patients had significantly higher levels vs. without blips (median 192 vs. 49; P = 0.0007), which correlated with: higher levels of inducible transcripts after activation in vitro, sustained higher HIV-1 transcription levels in follow-up samples along with increasing HIV-1 DNA in some, and production of replication-competent HIV-1. CONCLUSION: Viral 'blips' are significant reflecting higher transcriptional activity from the reservoir and contribute to the reservoir over time. This sensitive assay can be used in monitoring the size and activity of the HIV-1 reservoir and will be useful in HIV-1 cure strategies.