CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

38LB ART WithWeekends Off Is Noninferior to Continuous ART in Young People on EFV+2NRTI Karina M. Butler On behalf of the BREATHERTrialTeam Our Lady’s Children’s Hospital, Dublin, Ireland Background: For HIV-1 infected young people (YP) facing lifelong ART, short cycle therapy (SCT) with long-acting agents offers the potential for drug-free weekends, less toxicity and better adherence, as well as cost savings. Methods: BREATHER is a randomised, non-inferiority trial in Europe, Thailand, Uganda, Argentina & US. YP (>8, ≤ 24 yrs) on efavirenz (EFV)+2NRTIs and HIV-RNA (VL)<50c/ml for >12 months were randomised to continue daily ART (CT) or change to SCT (5 days on; 2 days off ART). Followup was for minimum 48 weeks, with 0, 4, 12, then 12 weekly visits. The primary outcome was the difference between arms in proportion with VL>50c/ml (confirmed) by 48 weeks, estimated using Kaplan-Meier method (12% non-inferiority margin) adjusted for region and age. Results: 199 YP (11 countries) were randomised; 99 SCT, 100 CT and followed for median 86 weeks: 53%male, median age 14yrs (21% ≥ 18yrs); 35% Uganda site; 56% black, 19% Asian; 21% Caucasian; median CD4% 34%; CD4 count 793 cells/mm 3 . By week 48, only one YP on CT was lost to followup. The SCT arm had 27% decreased drug exposure as measured by adherence questionnaire and a MEMs substudy showing median cap openings/week of 5 SCT vs 7 CT. By 48 weeks, 6 SCT vs 7 CT had confirmed VL>50c/ml; difference (90% CI) -1.2% (-7.3, 4.9); 2 SCT vs 4 CT had confirmed VL>400c/ml; difference 2% (-6, 2.0). All 6 SCT with VL>50c/ml resumed daily ART; 5/6 resuppressed, 3 on same regimen, 2 with switch; 2 others on SCT resumed daily ART for other reasons. Overall, 4 SCT vs 11 CT (p=0.1) changed ART regimen: 8 for toxicity, 4 simplification, 2 compliance and 1 VL failure. 7 YP (2 SCT, 5 CT) had major NNRTI mutations at VL failure; 2 (1 SCT, 1 CT) had M184V. 2 YP had new CDC B events (1 SCT, 1 CT). There were no significant differences in toxicity between SCT and CT: grade 3/4 AEs (8 vs 12), ART-related AEs (2 vs 8), SAEs (7 vs 6). At week 48, there was no evidence that SCT led to increased inflammation using an extensive panel of markers. YP expressed preference for SCT in a qualitative substudy and in pre- and post-trial questionnaires, particularly as it enabled weekend time with friends. 98% YP are in a 2-year followup extension of the trial. Conclusions: Non-inferiority of VL suppression in YP on EFV-based firstline ART with VL<50c/ml was demonstrated for SCT vs CT, with similar resistance, safety and inflammatory marker profiles. Detailed evaluation of the immunological and virological impact of SCT is planned. Background: The individual steps of the HIV entry process have been defined in detail. However, uncertainty prevails on the stoichiometric requirements of the entry process, namely the number of envelope trimers that need to be engaged to trigger membrane fusion. Here, we provide estimates of the HIV-1 entry stoichiometry utilizing a combined approach that incorporates experimental analyses and mathematical modeling. Methods: To estimate the stoichiometry of entry (T) we analyzed pseudotyped virus stocks carrying mixed envelope trimers consisting of functional (wt) and dominant-negative mutant env, where a single dominant-negative env subunit incorporated into a trimer renders the trimer non-functional. Pseudovirus stocks expressing mixed trimers with different ratios of wt and dominant-negative env were produced in 293-T cells and virus infectivity determined by titration on TZM-bl reporter cells. Analysis of the data to derive estimates of T was performed using mathematical models we developed for this purpose. Virus entry kinetics were determined by a time-of-inhibitor addition experiment employing T-20 to stop virus infection of target cells at defined timepoints post-infection. Results: Analysis of 11 divergent HIV-1 strains revealed that isolates differ in their requirements for trimer numbers during entry, with estimates ranging from 1 to 7 trimers and most isolates depending on 2 to 3 trimers to complete infection. Highlighting the biological relevance of our finding, a high entry stoichiometry proved to correlate with low virus strain infectivity and slow entry kinetics. This was confirmed studying the naturally occurring point mutation N160K in gp120 and by extensive analysis of mutant viruses lacking the V1V2 domain. In both cases changes in infectivity were reflected in different estimates of the entry stoichiometry compared to the matching wt envelopes. Conclusions: While HIV can overcome partial envelope protein deficiencies by increasing the number of trimers involved in the entry process, this restricts infectivity to those virions that carry higher numbers of trimers, thereby reducing the overall infectivity of a virus population. Our data thus add an important concept to the growing body of information on molecular aspects of HIV entry and membrane fusion. 40 HIV-1 Accessory Protein Function: Evaluating VPU-Dependent Host Factor Degradation Prashant Jain ; Kevin Olivieri; Quy Nguyen; Paul De Jesus; Sumit Chanda Sanford-BurnhamMedical Research Institute, La Jolla, CA, US Background: Cellular proteins called “restriction factors” are important in counteracting HIV-1 infection through cell intrinsic immune mechanisms. BST2 (Tetherin) is a cellular factor that prevents virion release by trapping HIV-1 on the plasma membrane. The HIV-1 accessory protein VPU is known to down-regulate BST2, thereby aiding in virion release. We hypothesize that VPU may target additional host factors to facilitate immune evasion. Here, we describe a ‘High Content Imaging’ based proteomic screen to identify host factors degraded by the HIV-1 VPU. Methods: To identify novel host targets of VPU, 950 V5 tagged ISGs (Interferon Stimulated Genes), as well as VPU-Flag or a control LacZ-Flag were co-expressed in 293T cells. The cells were immunostained for V5 or Flag epitopes and imaged with Opera QEHS high content imaging microscope. Mean fluorescence intensity (MFI) for ISG-V5 was deduced in cells expressing either VPU-Flag or the LacZ-Flag. ISGs showing decreased MFI following VPU-Flag versus LacZ-Flag co-expression were considered as degraded by VPU. Results: Following plate median normalization of fold change in V5 staining intensities, 10 host ISGs were identified as true hits for VPU dependent degradation. Confirmatory studies involving a secondary screen and immunoblot analysis of cell lysates confirmed the degradation of 6 ISGs identified in the primary screen. UBE2L6, an E2 ligase for ISG15 conjugation, was a primary hit and selected for downstream verification and functional analysis. Cross profiling ISG degradation following co-expression of HIV1 accessory proteins VPU, VIF and NEF, indicated a VPU specific degradation of UBE2L6. Importantly, we observed a significant and VPU dependent reduction in endogenous UBE2L6 as well as ISG15 conjugation in THP1 cells infected with HIV-1 VSVg wt or a VPU deficient HIV-1 VSVg. Our data indicates that HIV1 VPU modulates cellular ISG15 conjugation machinery by targeting the E2 ligase UBE2L6. Session O-3 Oral Abstracts Room 615 10:00 am– 12:00 pm Cellular Dynamics, Sensing, and Viral Restriction 39 Envelope Trimer Numbers Required for Entry Steer HIV-1 Infectivity and Entry Kinetics Oliver Brandenberg 2 ; Carsten Magnus 2 ; Peter Rusert 2 ; Roland Regoes 1 ; Alexandra Trkola 2 1 ETH Zurich, Zurich, Switzerland; 2 University of Zurich, Zurich, Switzerland

Oral Abstracts

97

CROI 2015