CROI 2019 Abstract eBook
(Spearman r=0.3; p=0.03). A non-significant decrease in sPDL-1 values was observed in HIV-1-infected subjects with controlled viraemia with vs. without STI (0.57ng/ml vs. 0.88 ng/ml, p= 0.29). Conclusion: sPD-L1 levels are significantly elevated during HIV-1 infection, despite control of viraemia. This fact opens new avenues for this biomarker as a predictor factor of virological failure or VL during the clinical follow up of PLWH. 248 IMPACT OF HIV INFECTION AND ANTIRETROVIRAL THERAPY ON IMMUNE CELLULAR FUNCTIONS Marek Korencak 1 , Enrico Richter 1 , Bruce T. Schultz 1 , Patrick Juszczak 1 , Buena D. Reyes 2 , Sandra Winning 2 , Joachim Fandrey 2 , Stefan Esser 2 , Hendrik Streeck 1 1 Institute for HIV Research, Essen, Germany, 2 University Hospital Essen, Essen, Germany Background: Despite viral suppression by ART and restoration of CD4 T cells, immune activation and exhaustion persist in many of HIV infected individuals which might result in overall decreased cellular activity. In this study we analyze cellular metabolism, function and proliferation in context of HIV infection and immunological parameters. Methods: Glycolysis and oxidative phosphorylation of lymphocytes from HIV infected treatment-naïve (n=12), ART-treated (n=12) and HIV negative (n=12) individuals were analyzed using extracellular flux analyzer and expression of key metabolic genes was measured by qPCR. Changes in HIF1α transcription factor were analyzed by western blot. We assessed the impact of ART regimens on proliferation capacity by CSFE staining ex vivo. We used ICS staining to determine changes in cellular function and phenotype using multicolor flow cytometry. Comparison of mitochondrial mass was done by qPCR, mitochondrial membrane potential and production of mitochondrial ROS by flow cytometry. Results: Respiration of CD4, CD8 T cells and NK cells from HIV infected treatment-naïve individuals was significantly reduced compared to HIV negative subjects (p<0.001, p<0.0001, p<0.05). Both respiration and glycolysis of CD8 T cells were in strong correlation with expression of inhibitory receptor PD-1 (p<0.0001) and immune activation (HLA-DR+, CD38+; p<0.0001). While we expected ART to restore metabolic profiles, we observed that the respiration of CD4 T cells was significantly decreased (p<0.001). This was in particular the case for INSTI containing regimens. We observed that cells from these individuals showed significantly lower ex vivo proliferation compared to CD4 T cells from individuals receiving either PI (p<0.05) or NNRTI (p<0.001). We next assessed the impact of individual ART on CD4 T cells. Both INSTI, EVG and DLG, but not RAL, dramatically reduced respiration (EVG p<0.05; DLG p<0.0001) without having an impact on glycolysis, Glut1 and PGK1 expression or HIF1α. We also observed decreased secretion of IL-2 (p<0.001), MIP-1β (p<0.001), CD107a (p<0.05) and INFγ (p<0.05) indicating impaired function of the cells. Both INSTI increased mitochondrial mass (EVG p<0.01; DLG p<0.05) and mitochondrial reactive oxidative species (EVG p<0.0001; DLG p<0.05), but had no impact on mitochondrial membrane potential. Conclusion: We identified significant interference of INSTI with CD4 T cell respiration, proliferation and immune responses resulting in decreased immune cellular function. 249 HIV-1 DIVERSITY IN GUT IS ASSOCIATED WITH RESIDUAL MUCOSAL VIRUS PRODUCTION ON ART Manon Nayrac 1 , Mary Requena 2 , Nicolas Jeanne 2 , Maud Mavigner 3 , Claire Loiseau 4 , Romain Carcenac 2 , Michelle Cazabat 2 , Jacques Izopet 2 , Pierre Delobel 2 1 INSERM, Toulouse, France, 2 CHU de Toulouse, Toulouse, France, 3 Emory Vaccine Center, Atlanta, GA, USA, 4 Australian Institute of Tropical Health and Medicine, Cairns, Australia Background: HIV-1 persists in cellular reservoirs and some anatomical compartments despite antiretroviral therapy (ART). We compared HIV-1 in gut and blood compartments on ART, regarding differences in target cells, residual HIV-1 DNA and RNA, coreceptor usage, and virus diversity. Methods: Peripheral blood and duodenum samples were obtained from 17 HIV-1-infected subjects with sustained plasma VL <50 c/mL for 5 years. Blood and gut CD4+ T cells were phenotyped by flow cytometry (BD LSRII). HIV-1 DNA was quantified in sorted blood and gut CD4+ T cells by qPCR. HIV-1 RNA was quantified in duodenum tissue by qRT-PCR. Virus quasispecies were characterized by next-generation sequencing of C2V3C3 env (454 GS Junior), with data cleaning and coreceptor usage prediction by Pyrovir software. Viral diversity in blood and gut compartments was assessed by haplotype numbers, adjusted-Shannon entropy, and Hill numbers. Phylogenetic analyses were
performed using CLUSTAL W. A non-parametric test for panmixia was used to look for compartmentalization. Results: CD4+ T cells in the gut were mainly CD45RO+CCR7- effector memory cells (88.2% vs 13.1% in blood, P<0.01). CD4+ T cells were more frequently activated (HLA-DR+, 15% vs 8.2%, P<0.05) and proliferating (Ki67+, 2.7% vs 2%, P<0.01), and expressed much more CCR5 (83% vs 5.7%, P<0.01) in gut than in blood. HIV-1 DNA was 6.7-fold higher in gut than in blood CD4+ T cells (P<0.01). Low-level HIV-1 RNA was detected in duodenum tissue of 13/14 subjects (1-7 c/mg). HIV-1 quasispecies displayed compartmentalization between the gut and blood (test for panmixia, P<0.01). In the blood, 9 subjects harbored only R5 viruses vs 8 R5/X4 dual-mixed (DM) viruses, while in the gut 13 harbored only R5 viruses vs 4 DM viruses. Virus diversity in V3 env region was reduced in gut vs blood compartment: median haplotype numbers (7 vs 10, P<0.01), median adjusted-Shannon entropy (0.14 vs 0.18, P<0.05). Virus diversity in the gut, assessed by adjusted-Shannon entropy of C2V3C3 env, correlated with mucosal CCR5+CD4+ T cell frequency (ρ=0.71, P<0.05), and residual mucosal HIV-1 RNA level (ρ=0.57, P<0.05). Conclusion: HIV-1 persists in the gut mucosa on ART with increased levels of infected cells compared to blood CD4+ T cells, and low-level mucosal HIV-1 RNA production. Virus diversity was reduced with enrichment in R5 viruses and compartmentalization in gut compared to blood. Virus diversity in the gut was associated with residual mucosal virus production. 250 ASSOCIATION BETWEEN HIV ANTIBODIES AND D-DIMER: ROLE OF “DEFECTIVE” PROVIRUSES Hiromi Imamichi 1 , Tracey D. Zhai 1 , Nicole E. Winchester 1 , Francesca Scrimieri 2 , Mindy Smith 1 , Thomas Buerkert 1 , Ivery Davis 2 , Adam Rupert 2 , Robin L. Dewar 2 , Joseph A. Kovacs 3 , H. Clifford Lane 1 1 National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA, 2 Leidos Biomedical Research, Inc, Frederick, MD, USA, 3 NIH, Bethesda, MD, USA Background: Persistent inflammation and immune activation can be seen in HIV-infected individuals who have achieved prolonged suppression of plasma viremia. The potential contributions from transcription/translation- competent yet “defective” HIV-1 proviruses (“Zombies”) to sustained aberrant immune activation and inflammation has recently received attention. The purpose of the study was to identify pathway(s) that contribute to persistent immune activation by looking at associations among proviral genome burden, transcription of “defective” proviruses, levels of biomarkers for inflammation and immune activation, and magnitude of anti-HIV Ab responses. Methods: 15 HIV-infected pts on suppressive cART with pVL<40 copies/mL for >2 yrs (range 2.1-10.8) and 5 pts with pVL≥40 copies/mL (range 2,044-26,100) were studied. HIV genomes were assessed by 5’LTR-to-3’LTR PCR single-genome amplification and direct amplicon sequencing. Levels of proviral DNA and cell-associated HIV-RNA were determined by a semiquantitative PCR. Levels of D-dimer, IL-6 and hsCRP were measured on plasma by ELISA assays. Western blots (WB) were performed using the Cambridge Biotech HIV-1 Western Blot kit. A WB score, reflecting the magnitude of anti-HIV Ab responses, was assigned to each patient. Results: Persistence of HIV antibodies was seen in all pts, irrespective of virological status or duration of viral suppression. A positive correlation was observed between WB scores and D-dimer levels (p=0.0083), indicating that the magnitude of the anti-HIV Ab responses could serve as a biomarker for identifying a state of immune activation. WB scores were also associated with proviral copy numbers (p=0.0087). Incomplete WB banding profiles, involving the absence of p31 and p17 bands, were found exclusively in pts with pVL<40 and were associated with the presence of “defective” proviruses (1.5-8 kb in length). Conclusion: Identifying the source and the mechanism(s) that contribute to persistent immune activation and inflammation in individuals with HIV infection is of critical importance in advancing our understanding of HIV-1 pathogenesis. The close relationship found between the magnitudes of anti-HIV Ab responses and D-dimer indicates a novel interplay between antigen production and immune activation/coagulation. “Defective” proviruses that overwhelmingly persist during suppressive cART can serve as a persistent source of viral antigen productions and stimulate the adaptive and innate immune systems.
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