CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Activity against HIVIII B 1.9nM, p<0.0001; 1:40, IC 50

in MT4 cells was higher for SDNs compared to aqueous solutions (1:4, IC 50

= 10.1 ± 0.5nM versus 15.7 ± 0.3nM, p<0.0001; 1:8, IC 50

= 14.9 ± 0.6 versus 24.1 ±

= 17.4 ± 0.7 versus 31.7 ± 1.2nM, p<0.0001, respectively) consistent with higher cellular accumulation. Conclusions: Combination SDNs have increased cellular accumulation and enhanced antiviral activity in cell-based assays. Notably, equivalent protease inhibition in vitro with LPV doses requiring 10-fold less RTV was observed. Due to RTV intolerance and long-term toxicities associated with chronic exposure to RTV, strategies to reduce RTV dose are worthy of further investigation. 528 HIV reservoir targeted antiretroviral nanofabrication facilitates viral clearance Pavan Puligujja 1 ; JoEllyn McMillan 1 ; Shantanu Balkundi 2 ; Prasanta K. Dash 1 ; James Hilaire 1 ; Santhi Gorantla 1 ; Larisa Poluektova 1 ; Xin-Ming Liu 1 ; Howard Gendelman 1 1 University of Nebraska Medical Center, Omaha, NE, US; 2 Kansas University Innovation and Collaboration, Lawrence, KS, US Background: Limitations in adherence, toxicity and reservoir targeting underlie the needs for improvements in antiretroviral therapy (ART). We posit that long-acting nanoformulated antiretroviral therapy (nanoART) can address this need by improving current drug pharmacokinetics (PK) and biodistribution. The development of monocyte- macrophage targeting ligands could affect drug depots and target sanctuaries of viral infections. To this end, we developed a folic acid (FA) crystalline nanofabrication for ritonavir-boosted atazanavir (FA-nanoATV/r). The PK and pharmacodynamics properties were, until now, not known. Methods: The size, shape, zeta potential, and polydispersity of FA poloxamer 407 ATV/r nanoparticles were determined following high-pressure homogenization. NOD/ scid - IL-2R γ c null (NSG) mice were reconstituted with human peripheral blood lymphocytes (hu-PBL) and treated with 10, 50 and 100 mg/kg of native drug, nanoATV/r, FA-nanoATV/r or left untreated. The mice were challenged with HIV-1 ADA (10 4 tissue culture infective dose50, TCID 50 ) 24 hrs later and sacrificed 14 days after treatment. In further experiments human CD34+ hematopoietic stem cells (HSC) reconstituted NSG mice at 6-8 weeks age were infected for four weeks with HIV-1 ADA. The mice were then treated every two weeks at week 4, 6 and 8 and sacrificed on week ten post-infection. Bioavailability and tissue biodistribution were determined by ultra performance liquid chromatography-tandemmass spectrometry. Pharmacodynamics was scored by measures of HIV-1p24 levels or viral load determined by immunohistochemistry, reverse transcriptase polymerase chain reaction or by viral RNA copies (Cobas Aplicor V1.5) in spleen, liver, lung or plasma. Results: FA-nanoATV/r led to an increase in plasma and spleen, liver and lung tissue drug levels up to > 5-fold over uncoated nanoATV/r with protection of CD4+ to CD8+ T cell ratios in Hu-PBL mice. Number of infected HIV-1p24 cells and viral RNA levels in liver, spleen and or lung with FA-nanoATV/r were reduced up to > 2 logs compared to untreated and > 1 log over nanoATV/r treated hu-PBL animals. Two of four HSC NSGmice challenged with HIV-1 ADA showed no detectable virus with the other two demonstrating viral reductions of up to 2 logs. Conclusions: FA targeted ART nanofabrications are a significant means to improve PK and PD. Reservoir targeting could lead to platforms for chemical viral eradication measures. 529 The Macrophage Proteome Defines the Long Acting Antiretroviral Therapy Cell Depot Dongwei Guo ; Mariluz Araínga; Jayme Horning; Pawel Ciborowski; Xin-Ming Liu; JoEllyn McMillan; Howard Gendelman University of Nebraska Medical Center, Omaha, NE, US Background: Long-acting antiretroviral therapy (nanoART) nanoformulations can improve drug adherence, reduce systemic toxicities, and facilitate clearance of human immunodeficiency virus (HIV). While, monocyte-macrophage depots of nanoART are contained in recycling and late endosomes whether these organelles provide strategic advantages for viral elimination is not known. Methods: We applied quantitative SWATH-MS proteomic and cell profiling to nanoformulated atazanavir (nanoATV) treated HIV-1 ADA infected human monocyte-derived macrophages (MDM). DAVID and KEGG bioinformatics tools were used to define the mechanistic pathways that facilitate cell-nanoATV carriage while limiting viral growth. Results: Both HIV-1 and nanoATV engaged phagolysosomal trafficking pathways. However, MDM phagolysosomal proteins were dysregulated in opposing directions. Paired- samples Z-test showed that an upregulation of phagolysosomal proteins by HIV-1 was reversed by nanoATV. DAVID and KEGG bioinformatics tools defined the signaling pathways for cell-nanoATV carriage that limiting viral growth. These phagolysosomal protein sets were linked to macrophage functions that included phagocytosis, antigen presentation, secretory activities, phagocytic and cell migration responses. KEGG pathway analyses indicated phagosome signalling as one of the main pathways related to HIV infection and nanoATV treatment. KEGG revealed the up-regulation of Rab5 and -7 proteins in HIV-1 infected cells; in contrast these same proteins were down-regulated in nanoATV-treated HIV- infected cells. Western blot results showed a clear down regulation in the expression of Rab5, 7, 11 and LAMP1 proteins after nanoATV treatment. Measures of reverse transcriptase activity in infected cell treated with nanoATV showed attenuation of viral infection. Cytokine assay detected IL12 and TNF were increased in HIV-1 infected macrophages but reduced with nanoATV treatment. Conclusions: Late and recycling macrophage endosomes ensure viral replication. These same pathways are nanoART depots. The modulation of phagolysosomal pathways by nanoART in the setting of HIV-1 infection provides a strategic therapeutic advantage enabling viral elimination. 530 Primary CD4 Subsets Are Similarly Loaded by Tenofovir Alafenamide (TAF) Christian R. Frey;Yang Liu; Darius Babusis; Adam Palazzo; Adrian S. Ray; Michael D. Miller; Kathryn M. Kitrinos; Christian Callebaut Gilead Sciences, Inc., Foster City, CA, US Background: Tenofovir alafenamide (TAF), a new prodrug of the HIV-1 NRTI tenofovir (TFV), shows improved antiviral activity in monotherapy clinical studies, at lower doses than tenofovir disoproxil fumarate (TDF). TAF delivers TFV more efficiently than TDF to lymphoid cells with a 5-fold increase in intracellular TFV-diphosphate (TFV-DP) level and a 90% reduction in plasma TFV. It had not previously been established that TFV-DP levels are evenly distributed among primary human CD4+ T-lymphocytes (CD4) subsets following TAF treatment. Therefore, intracellular TFV-DP levels were evaluated in CD4 subsets at clinically relevant TAF concentrations. Methods: In vitro TAF loading studies were conducted using primary cells from healthy human donors. PBMCs, including total CD4, as well as, naïve, effector, central memory and effector memory CD4 T-cell subsets were evaluated. Total CD4 were enriched using magnetic beads and CD4 subsets were isolated by cell sorting based on CD3, CD4, CD45RA, and CCR7 expression. TAF loading was evaluated using a 2h pulse incubation followed by washout and then incubation in drug free media to mimic in vivo TAF exposure. Cell extracts were prepared and TFV, TFV-MP, and TFV-DP levels were measured by LC/MS/MS. Results: Cell loading studies in PBMCs demonstrated that a 2h pulse and 22h washout of 200-400 nM TAF achieved TFV-DP levels comparable to those observed in vivo following clinical dosing of TAF. There was minimal variation in the levels of intracellular TAF metabolites between donors. Additionally, comparable TFV-DP levels were achieved after a 2h pulse with either 200 or 400 nM TAF in total CD4, as well as in the CD4 subsets, with a trend for higher TFV-DP levels in memory cells compared to other CD4s subsets. For each of the CD4 subsets evaluated, there was no significant decrease in TFV-DP levels at 24h, indicating a uniformly long intracellular half-life in all cell populations, consistent with observations of TFV-DP levels in PBMCs following TAF dosing in clinical studies. Conclusions: CD4 subsets were similarly loaded by TAF in vitro, achieving high and persistent levels of TFV-DP. The sustained levels of TFV-DP 24h post-treatment suggest that high levels of TFV-DP will be maintained across most relevant CD4 cell subsets in patients receiving TAF. The higher levels in memory subsets may have implications for maintaining viral suppression in latent viral reservoirs and for future cure efforts.

Poster Abstracts

345

CROI 2015