Abstract Body

HIV-associated neurocognitive disorders (HAND) remain prevalent despite antiretroviral therapy (ART). The relationship of CSF extracellular vesicles (EVs) to HAND is unclear. We performed a cross-sectional and longitudinal study to investigate the association of CSF EVs with HAND and CNS injury-related biomarkers (NFL, S100B, neopterin) in HIV+ subjects on ART.

CSF NFL, S100B, and neopterin were measured by ELISA in 112 subjects (67 HIV+ virally suppressed on ART with nadir CD4 ∠300 cells/μl, age range 36-78 years, 73% male, 57% white and 45 HIV- controls matched for age, gender, race). CSF EVs were isolated from 49 HIV+ (24 cognitively impaired (NCI) and 25 unimpaired) and 16 HIV- controls. EVs were characterized by electron microscopy, nanoparticle tracking analysis, and immunoblotting for exosome markers (CD9, CD63, CD81, FLOT-1). CSF EV protein cargo was analyzed by untargeted LC-MS/MS in 12 subjects. GO analysis used geneXplain TRANSFAC.

CSF NFL, S100B, and neopterin levels were higher in subjects with HIV, detectable plasma VL, low CD4 count, and NCI compared to corresponding controls (p<0.05), and increasing NFL and S100B levels were associated with cognitive decline over 2 years. CSF EVs were more abundant in HIV+ vs. HIV- subjects (p<0.001), and NCI vs. unimpaired subjects (p=0.01). CSF EV concentrations correlated with NFL (r=0.567, p<0.001) and S100B (r=0.389, p=0.0015). Proteomics analysis identified >800 proteins enriched or uniquely detected in CSF EVs compared to EV-depleted CSF, and suggested CSF EVs originate from myeloid cells (CD14, CHI3L1, CSF1R, MARCO, MRC1), astrocytes (S100B, GFAP, PEA15, SLC1A3), and neurons (NFL, NFASC, NPTN, ENO2) and carry proteins related to exosomes (CD9, CD81, FLOT-1, ALIX), inflammatory/immune responses (GAS6, LBP, HLAs), stress responses (HSPs, SOD, PARK7, PRDXs, TXN), and blood-brain-barrier (BBB) (AGRN, AQP4, DAG1, VCAM1). HLA-DR levels were higher in CSF EVs from NCI but not unimpaired subjects vs. HIV- controls (p=0.03), suggesting activated macrophages/microglia are a potential source of CSF EVs in HAND.

CSF EVs are more abundant in HIV+ compared to HIV- individuals, and neurocognitively impaired compared to unimpaired individuals. CSF EVs correlate with known biomarkers of CNS injury and carry protein cargo related to neuronal injury, inflammation, stress responses, and BBB function, suggesting applications as novel biomarkers of CNS injury in HIV patients on ART.