The Cerebrospinal Fluid Virome in People Living with HIV

Study objective: A relevant proportion of central nervous system (CNS) inflammation and disorders in people living with HIV (PWH) remains unexplained despite effective HIV virological suppression. To evaluate the presence and composition of cerebrospinal fluid (CSF) virome in PWH on suppressive antiretroviral therapy and its relationship with mental health and CNS inflammation. We hypothesized that CSF virome and immune system have intertwined effects upon neuronal and astrocytic damage, blood-brain barrier (BBB) functioning, and CNS inflammation underlying mood and cognitive disorders. Materials and methods: This pilot observational cross-sectional study evaluated the CSF virome of 81 PWH that underwent lumbar puncture between 2010 and 2019 at Amedeo di Savoia Hospital, Turin. Inclusion criteria were age>18 years, being on NRTI-based ART, and plasma HIV-RNA<200 cp/mL. Substance abuse and CNS disorders were exclusion criteria. Prokaryotic and eukaryotic viruses (DNA+RNA) were enriched and sequenced (QIAseq® Single Cell RNA Library). After host decontamination and background removal, taxonomy and species abundance were estimated by Kraken2 v2.1.2 and Bracken v2.7. The α (Observed, Shannon, Simpson) and β (Bray-Curtis) diversities and principal component analysis were performed. Virome composition was compared to the following variables: demographics, viro-immunological status, mood/neurocognition (BDI-II and 13 neurocognitive tests) and CSF biomarkers (tau, p-tau, neopterin, S100β, 1-42 β Amyloid, intrathecal synthesis, CSAR, cells, proteins, glucose). Unsupervised twostep cluster analysis on α diversity indexes was performed for further exploratory analysis. Results: Participants were mainly middle-age (50±11.2 years), white (93.8%) male (71.6%). Depressive mood and cognitive impairment were detected in 17.3% and 42% of them. Median CD4+ T cell count was 455. CSF virome was not detected in 23 samples, while a median of 305 (122-1035) viral reads was observed in 58 (71.6%) samples. Among the latter, all presented bacteriophages (mostly Siphoviridae, Myoviridae, Podoviridae; 231 [110-1,057] reads), and 7.4% also eukaryotic viruses (EBV, HCV, HHV-6, TTV, HPV-96/-201; 122 [79-303] reads). Higher α diversity was associated with higher CSF HIV RNA, lower CSF glucose, and lower CD4+ T-cell count. All these associations were confirmed by bivariate correlations (ρ=+0.264; -0.444; -0.223; p<0.05 for all). No other differences in α- and in β-diversity were observed by clinical groups and biomarker concentrations. Cluster analysis identified two groups: 22.6% participants at the lowest extreme of richness and evenness vs. 77.4% with higher α diversity; the latter cluster was characterized by higher intrathecal synthesis (e.g., Tibbling p=0.004, Tourtelotte p=0.015), higher CSF HIV RNA (p=0.012), lower CSF glucose (p=0.010), and worse depression scores (p=0.037). Conclusion: Both prokaryotic and eukaryotic viruses were found in the CSF of antiretroviral-treated PWH. Our results suggest that worse viro-immunological profiles (lower CD4+ T cell and higher CSF HIV RNA) may correlate with higher diversity of CSF virome. Such differences in richness and evenness were also associated with differences in CSF humoral immune responses (intrathecal synthesis), and in depressive mood but not in cognitive impairment rates. Further insights are needed to confirm these findings and address the interplay among HIV, CSF virome, and neuroinflammation.