CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

Conclusion: We detected a different profile of RASs in the compartment explored concerning the NS3 and NS5a target region. So, in particular for GT1a the liver compartment could be responsible for the emergence of resistant variant not detected in the corresponding plasma sample by viral population analysis. This finding may have implication especially for GT1a patients with virological failure and absence of RASs in plasma sample at re-treatment. 612 SECOND HITS PROMOTE HEPATOCYTE DEATH AND LIVER INJURY IN HIV INFECTION Murali Ganesan 1 , Raghubendra S. Dagur 1 , Edward Makarov 1 , Srivatsan Kidambi 2 , Larisa Y. Poluektova 1 , Natalia Osna 1 1 University of Nebraska Medical Center, Omaha, NE, USA, 2 University of Nebraska– Lincoln, Lincoln, NE, USA Background: Liver disease became second cause of non-AIDS-related death in HIV-infected patients. Although immune and central nervous systems are well- characterized targets of HIV-infection, hepatocytes (Hep) were never considered as permissive cells for HIV. We hypothesize that “second hits” as HCV-co- infection or alcohol exposure make Hep HIV-permissive, facilitate apoptotic Hep death and promote liver inflammation and fibrosis development by activating non-parenchymal liver cells by apoptotic Hep engulfment. Methods: Primary human hepatocytes or their experimental prototype, Huh7.5-CYP (RLW) cells were infected with HIV-1 ADA and then either exposed to HCV (co-infection model) or to ethanol (HIV+ ethanol model). HIVgag RNA was measured in these cells by RT-PCR and reverse transcriptase (RT) activity as evidence of HIV replication was determined in cell supernatants. As apoptotic cell death indication, we used cleaved caspase 3 (Western Blot) and M30 (ELISA). After engulfment of hepatic apoptotic bodies (AB) by monocyte- derived macrophages (MDM) and hepatic stellate cells (HSC, LX2 cell line) inflammasome activation and pro-fibrotic markers were quantified by RT-PCR. Results: We observed that both HCV co-infection and co-treatment with ethanol substantially increased HIV gag RNA in hepatocytes and RT in cell supernatants. This increase was associated with enhanced HIV replication inside of cells since the removal of surface structures by low acid wash did not decrease HIV RNA levels triggered by HCV or ethanol exposure. Both insults push HIV-infected Hep to apoptosis prevented by co-treatment with pan-caspase inhibitor. Furthermore, apoptosis was attenuated by AZT, suggesting that it is initiated by HIV replication. AB generated from HIV-infected Hep spread the virus to intact MDM. Engulfment of HIV+ AB Hep activated inflammasome (based on NLRP3, caspase 1, IL-1 β and IL-18 expression) in MDM and pro-fibrotic markers (Col1A1, TGFβ and prostaglandin D receptor 2) in HSC. Activation of fibrotic changes in HSC was AB Hep -specific since engulfment of AB from HIV+ lymphocytes induced pro-inflammatory, but not pro-fibrotic events Conclusion: We conclude that second hits, like co-infection with HCV and co-treatment with ethanol increase permissiveness of hepatocytes to HIV- infection and trigger their apoptosis, thereby initiating the cross-talk between hepatocytes, macrophages and stellate cells to promote liver inflammation and fibrosis progression. 613 INFLAMMATORY CHEMOKINES LINKED TO HIV GENETIC DIVERSITY DURING HIV/HBV COINFECTION Xiao Qian Wang 1 , Bethany A. Horsburgh 1 , Katie Fisher 1 , Jennifer M. Zerbato 2 , Jennifer Audsley 2 , Anchalee Avihingsanon 3 , Ajantha Solomon 2 , Julia Stout 2 , Sharon R. Lewin 2 , Sarah Palmer 1 1 The Westmead Institute for Medical Research, Westmead, NSW, Australia, 2 Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia, 3 HIV–NAT, Thai Red Cross AIDS Research Centre, Bangkok, Thailand Background: HIV-hepatitis B virus (HBV) co-infected individuals experience higher rates of liver disease than mono-infected individuals. Previous studies have found that HIV co-infection can impact the natural course of HBV infection, but the reverse has not been confirmed. We aimed to determine the frequency of intact provirus in HIV-HBV co-infected individuals prior to ART initiation and whether this frequency was associated with any clinical parameters. Methods: HIV-HBV co-infected individuals and HIV mono-infected individuals naïve to ART were recruited in Bangkok Thailand as part of a prospective observational cohort study. HIV proviruses were sequenced from peripheral blood CD4+ T-cells using full-length individual proviral sequencing (FLIPS). Primers were adapted for specificity to HIV subtype AE and single near full-length HIV proviruses (92% of the genome) were sequenced using Next Generation Sequencing. Genetically intact HIV proviruses were identified as

those lacking inversions, stop codons/hypermutation, insertions, deletions or frameshifts. Results: A total of 522 HIV proviruses were sequenced and analysed from 17 HIV-HBV co-infected individuals, and 165 proviruses from 4 HIV mono-infected individuals; both cohorts being naïve to ART. Both the co-infected and mono- infected individuals had a similar and high proportion of genetically intact provirus (range = 7-66% and 23-59% respectively). Intact sequences from these cohorts had genetic diversity ranging 0.2-2% and 0.3-1.6% for the co-infected and mono-infected cohorts respectively. The mean diversity of genetically- intact provirus was lower in the mono-infected (0.7%) than the co-infected cohort (1.0%), but this did not reach significance (p=0.28). No correlation was found between HBV infection parameters (HBV DNA, HBsAg, and ALT levels, HBeAg status) and the proportion of genetically intact HIV proviruses or their genetic diversity in the co-infected individuals. However, higher levels of the inflammatory chemokines CCL2 in the blood and CXCL10 in the liver were associated with increases in overall genetic diversity of HIV (p=0.028 and p=0.0016). Conclusion: Genetically unique and intact HIV proviral sequences were commonly identified in untreated HIV-HBV co-infected and HIV mono-infected participants. The frequency of intact virus was far higher than previous studies of individuals on suppressive ART. Inflammatory chemokines were associated with the genetic diversity of HIV proviruses in HIV-HBV co-infected participants. 614 INFLUENCE OF HCV INFECTION ON HIV-1 SPLICING IN CHRONICALLY COINFECTED PATIENTS Paula Martínez-Román 1 , Mª Rosa López-Huertas 2 , Claudia Palladino 3 , Marta Sánchez-Carrillo 1 , Luz Martin-Carbonero 4 , Lourdes Domínguez-Domínguez 5 , Pablo Ryan 6 , Ignacio Santos 7 , Sara de la Fuente Moral 8 , José Alcamí Pertejo 1 , Salvador Resino 1 , Amanda Fernández-Rodríguez 1 , Mayte Coiras 1 , Verónica Briz 1 , for the COVIHEP 1 Institute de Salud Carlos III, Majadahonda, Spain, 2 Instituto Ramón y Cajal de Investigación Sanitaria, Madrid, Spain, 3 Universidade de Lisboa, Lisbon, Portugal, 4 Hospital La Paz Institute for Health Research, Madrid, Spain, 5 Hospital Universitario 12 de Octubre, Madrid, Spain, 6 Hospital Universitario Infanta Leonor, Madrid, Spain, 7 Hospital Universitario de La Princesa, Madrid, Spain, 8 Hospital Puerta de Hierro, Madrid, Spain Background: HIV/HCV coinfection influences HIV-1 reservoir size. We previously observed a higher quantity of HIV-1 proviral DNA in coinfected patients regarding to HIV-monoinfected individuals. However, it is unknown whether this coinfection may also induce a higher proviral transcription, thereby increasing the viral load and influencing in the reservoir size. We assess if HCV coinfection influences HIV-1 proviral transcription and splicing forms in isolated, resting CD4+ (rCD4) T cells and the remaining non-resting PBMCs. Methods: Cross-sectional study: 29 (49.1%) HIV-1/HCV coinfected subjects and 28 (50.9%) HIV-1 patients. PBMCs were obtained from 50 ml of peripheral blood and rCD4 T-cells were isolated (CD4+CD25-HLADR-CD69-). Total RNA was extracted from rCD4+ cells and the remaining non-resting PBMCs, and then analyzed by qPCR to measure the unspliced (~9kb), single spliced (~4kb) and multiple spliced (~2kb) transcripts. Linear correlations between viral reservoir size and viral splicing were also determined. Results: An increase in HIV-1 reservoir size was observed in HIV+/HCV+ patients regarding to the HIV+ group [84.9 (48.3-154.2) vs. 28.5 (8.5-97.7) proviral DNA copies/10^6 rCD4 cells, respectively (p=0,003)]. Analysis of HIV-1 alternative slicing showed 3.2-fold increase of multiple spliced transcripts in HIV/HCV patients (ΔRQ=13.6 vs 4.3; p>0.05). Not significant increase in unspliced and single spliced forms (19.9- and 5.8-fold, respectively) was also observed in the remaining non-resting from HIV+/HCV+ subjects (Fig1). A significant positive correlation in HIV+/HCV+ individuals was identified between HIV reservoir size and some viral spliced forms. Conclusion: Splicing of HIV transcripts is necessary for viral transcription. We previously observed that coinfection of HCV and HIV influences HIV reservoir size. Now we found that rCD4 cells isolated from HIV/HCV patients showed an increase of multiple spliced transcripts, suggesting that HIV-1 regulator Tat could be more active in these cells, yielding a higher number of viral particles and increasing the reservoir size. Moreover, coinfection with HCV could enhance HIV proviral transcription and splicing due to an interaction between HCV proteins and the cellular splicing machinery. The positive correlation between reservoir size and some viral splicing forms may support this hypothesis. This

Poster Abstracts

CROI 2019 232