CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

451 CEREBROSPINAL FLUID AND SERUM NEPRILYSIN IN PATIENTS INFECTED WITH HIV CLADES C AND B Sergio M. de Almeida 1 , Clea E. Ribeiro 1 , Indianara Rotta 1 , Mauro Piovesan 1 , Bin Tang 2 , Meire S. Batistela 1 , Scott L. Letendre 2 , Michael Potter 2 , Florin Vaida 2 , Ronald J. Ellis 2 1 Universidade Federal do Paranã, Curitiba, Brazil, 2 University of California San Diego, San Diego, CA, USA Background: The profile of neural injury biomarkers is not well defined in HIV infection; most previous studies are on HIV subtype B (HIV1-B). How HIV1 Tat induces the development of Aβ deposit in HIV1 patients is not fully understood. Neprilysin (NEP) is the dominant Aβ peptide-degrading enzyme. HIV1-B Tat protein is known to interfere with NEP function, but whether this is true of HIV1-C Tat, which has a defective chemokine dimotif, is not known. This study aimed to analyze the impact of HIV1 subtypes on NEP-mediated cleavage of Aβ by comparing CSF and serum levels of NEP between HIV+ (27 HIV1-B and 26 HIV1-C), healthy HIV- controls (n=13); and patients with Alzheimer’s disease (AD, n= 24). This was the first study to analyze NEP in patients with HIV1-C. Methods: Total NEP (both activated and unactivated forms), as well as Aβ oligomers 38,40 and 42 levels were measured in CSF and serum by immunoassays. Ratios of NEP to Aβ-38,40,42 and total Aβ were calculated for both CSF and serum. To estimate NEP intrathecal synthesis, we calculated CSF/ serum indexes of these ratios, and the NEP index=(CSF NEP X Serum Albumin)/ (Serum NEP X CSF Albumin). Comparisons between HIV(+) and HIV(-) were adjusted by linear regression for gender and age; HIV subtype comparisons were adjusted for nadir CD4 and plasma viral load suppression. The p-values were corrected for multiple testing with the Benjamini-Hochberg procedure. Results: Levels of NEP and CSF ratios were comparable for HIV1-C and B. Serum NEP was nonsignificantly lower for HIV1-C than HIV1-B (p= 0.060). The NEP/ Aβ-40 ratio in serumwas lower for HIV1-C than B (p=0.032). The CSF/serum index of NEP/Aβ-40, NEP/Aβ-42, and NEP/Aβ-total were lower for HIV1-B than C (p= 0.008, 0.005 and 0.017 respectively). The results of CSF/serum indexes corroborated the findings for serum. CSF NEP was comparable for HIV+, HIV-, and AD; although in serum NEP levels were higher for HIV than AD and CTRL. Conclusion: There was impact of HIV subtype on NEP. The ratio of NEP/Aβ-40 in serumwas lower for HIV1-C than HIV1-B. These results are consistent with previous reports of CSF Aß-42 levels decreased in HIV1-C compared with HIV1-B; suggesting higher amyloid ß deposition in HIV1-C than HIV1-B. 452 REGIONAL BRAIN HEME OXYGENASE CORRELATES WITH SIV LOAD IN ACUTE/CHRONIC INFECTION Rolando Garza 1 , Maria Diaz Ortiz 1 , Analise Gruenewald 1 , Fiorella Rossi 2 , Ferzin Sethna 3 , Guido Silvestri 4 , Michael R. Betts 1 , Dennis L. Kolson 1 1 University of Pennsylvania, Philadelphia, PA, USA, 2 Nova Southeastern University, Fort Lauderdale, FL, USA, 3 Princeton University, Princeton, NJ, USA, 4 Emory Vaccine Center, Atlanta, GA, USA Background: Persons with HIV-associated neurocognitive disorders (HAND) have impaired brain antioxidant responses, which is likely pathogenic. The pre- frontal cortex shows reduced levels of heme oxygenase-1 (HO-1), an inducible antioxidant enzyme that robustly suppresses oxidative stress and neurotoxin production. Within prefrontal cortex, HO-1 loss correlates with HIV load and macrophage activation. In an SIV macaque model, we determined brain region-specific relationships between HO-1 expression, parenchymal SIV load, and immune activation to validate the model for studies of host antioxidant responses to SIV/HIV infection. Methods: Rhesus macaques (2-3 yo, male, female) infected IV with SIVmac251 (500 TCID50) were sacrificed 5, 10, 13, 20, 41, and 90 days pi (n=3/day, n=18 total). Nine brain regions (frontal, deep frontal, pre-frontal, parietal, midbrain, basal ganglia, pons, medulla, and cerebellum) were analyzed for antioxidant gene expression (HO-1, HO-2, PRDX1, NQO1, GPX1, SOD1) by western blot and qPCR. Statistical testing was by two-way ANOVA with Tukey and multivariate linear regression. Results: HO-1 protein levels were lower in deep brain regions (midbrain, basal ganglia, pons, medulla) compared to cortical regions (frontal, pre-frontal cortex; p<.05). In several regions (midbrain, frontal cortex) HO-1 protein inversely correlated with HO-1 RNA (p<.020), suggesting feedback regulation. HO-1 protein correlated negatively with another antioxidant protein (PRDX1; p=0.0007), suggesting a compensatory response to HO-1 protein loss. Regional brain SIV load correlated positively with regional HO-1 RNA level (basal ganglia (p<.0001), pons (p=.026), pre-frontal cortex (p=.030), deep frontal lobe

(p=.048). Similarly, regional HO-1 RNA, correlated with plasma SIV load (p<.020 for all regions). These relationships were generally consistent over 90 days of infection. Conclusion: In the Rhesus macaque brain lower HO-1 protein expression in deep regions (midbrain, basal ganglia, pons, medulla) in comparison to cortical regions might contribute to the selective vulnerability of deep structures to SIV- induced injury. The correlation between regional SIV load and HO-1 RNA level (also seen in HIV-infected human brain prefrontal cortex) suggests that viral replication induces HO-1 transcription, as a host protective response. Similarities between macaque and human brain antioxidant responses to infection support the macaque model for testing HO-1 based neuroprotection strategies for HAND. 453 CSF EXTRACELLULAR VESICLES AS BIOMARKERS OF CNS INJURY IN HIV PATIENTS ON ART Debjani Guha , Sukrutha Chettimada, Vikas Misra, David Lorenz, Dana H. Gabuzda Dana–Farber Cancer Institute, Boston, MA, USA Background: 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. Methods: 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. Results: 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 frommyeloid 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. Conclusion: 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. 454 DELETERIOUS EFFECTS OF HIV-1 LATENCY-REVERSING AGENTS MEDIATED BY ASTROCYTES Alizé Proust , Corinne Barat, Mathieu Leboeuf, Jean Drouin, Michel J. Tremblay Laval University, Quebec City, QC, Canada Background: Despite the combined antiretroviral therapy potency, HIV-1 persists in long-lived latently infected cells. Thus, therapies capable of eliminating this latent viral reservoir are needed. A shock and kill strategy using latency-reversing agents (LRA) to reactivate HIV-1 has been proposed. However, the impact of these LRA on the central nervous system (CNS) remains elusive. Methods: In this study, we used primary fetal astrocytes and investigated the effects of LRA on their phagocytic, functional and secretory activities. Astrocytes were infected with VSV-G-pseudotyped HIV-1 before a 24h treatment with various blood-brain barrier (BBB)-permeable LRA at subcytotoxic doses, which allow HIV-1 reactivation based on previous in vitro and clinical studies. Cells and supernatants were then used to evaluate effects of infection and LRA on (i) yeast and amyloid beta (Aβ) phagocytosis by microscopy and flow cytometry, (ii) expression of complement component 3 (C3), a proxy

Poster Abstracts

CROI 2018 161