CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

475 MASS SPEC IMAGING REVEALS ASSOCIATIONS BETWEEN ARVS, VIRUS, AND CELLS IN LYMPH NODES Elias Rosen 1 , Claire Deleage 2 , Nicole White 1 , Craig Sykes 1 , Lourdes Adamson 3 , Yuri Fedoriw 1 , Jacob D. Estes 2 , Paul Luciw 3 , Angela Kashuba 1 1 University of North Carolina Chapel Hill, Chapel Hill, NC, USA, 2 Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 3 University of California Davis, Davis, CA, USA Background: Recent data suggest that lymph nodes (LN) may harbor HIV infection despite HAART, possibly due to reduced ARV penetration. Here, we combine mass spectrometry imaging (MSI) and in situ hybridization (ISH) and immunohistochemistry (IHC) to evaluate the biodistribution of ARVs relative to viral and target cell expression in LN of rhesus macaques (RM). Methods: Axillary LN were collected and snap frozen at necropsy from RT-SHIV infected RM after 10 days of dosing with emtricitabine (FTC) + tenofovir (TFV) (N=6) in combination with either efavirenz (EFV) + raltegravir (RAL) (N=3), cohort 1, or maraviroc (MVC) + atazanavir (ATZ) (N=3), cohort 2. Tissue accumulation of ARVs and metabolites was measured by LC-MS/MS (analyte-specific limits of detection [LOD]: 1-8 ng/ml homogenate) and infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI (LOD: 0.05-0.37 ng/mg tissue) from 10 μm thick cryosections. Tissue and plasma measurements were compared based on an assumed tissue density of 1.06 g/ ml. Serial sections of tissue were analyzed for viral RNA (vRNA) by RNAscope ISH and for CD4+ cells by IHC. Results: Concentrations of ARVs in LN [LC-MS/MS (μg/g): 0.35-2 TFV, EFV, MVC; 0.05 – 0.2 FTC, ATZ; 0.02-0.03 RAL] exceeded concentrations in plasma (6 to 10-fold increase) for all ARVs except RAL (8-fold decrease). MSI revealed that ARVs accumulated in LN heterogeneously, with total ARV LN exposure (total coverage: cohort 1= 33-75%; 2 = 67-100%) and co-localization with target CD4+ cells (CD4+ coverage: cohort 1 = 65-75%; 2 > 90%) varying between dosing regimens. Combination of MSI and ISH showed localization of vRNA in secondary LN follicles where ARV concentration was poor or undetectable. Up to 50% of vRNA was not co-localized with any ARV. Concentrations of ARVs exceeding in vitro IC 50 values were limited where co-localization was determined, with EFV and MVC providing the greatest exposure ([ARV] > IC 50 : 3-16% and 41-47%, respectively). Conclusion: The spatial distributions of drug and viral RNA expression observed provide contextual information underscoring the influence of location and microenvironment within a tissue compartment, otherwise lost by tissue homogenization with LC-MS/MS analysis and single cell analytical methods. Fusing MSI and ISH images can inform drug distribution in the context of viral replication, and provides a unique means of spatially resolving pharmacokinetic- pharmacodynamic relationships.

474 DIFFERENTIAL BRAIN TISSUE PENETRATION OF ANTIRETROVIRALS AND FLUCONAZOLE Melanie Nicol 1 , Jonee Taylor 1 , Katelyn Pastick 1 , James Fisher 1 , Carol Karuganda 2 , Joshua Rhein 1 , Darlisha A. Williams 1 , David Meya 2 , David R. Boulware 1 , Robert Lukande 3 1 University of Minnesota, Minneapolis, MN, USA, 2 Infectious Disease Institute, Kampala, Uganda, 3 Makerere University, Kampala, Uganda Background: The central nervous system (CNS) is believed to be a significant reservoir for pathogens such as HIV and Cryptococcus; however, current understanding of drug penetration into the CNS is limited and largely based on cerebrospinal fluid (CSF) concentrations. However, CSF is not brain tissue. Herein we used tissues collected post-mortem from HIV-infected Ugandan subjects co-infected with Cryptococcus to characterize the relative distribution of antiretrovirals and antifungal agents across plasma and CNS compartments. Methods: Following obtainment of written, informed consent from next of kin, post-mortems were performed on five subjects co-infected with HIV and cryptococcal meningitis. Tissues from cerebellum, pons, and CSF were snap frozen in liquid nitrogen. Whole blood was collected from femoral vein into EDTA tubes and stored on ice for 1 hour before separating plasma from cell pellets. All samples were transferred to -80 ºC for storage. Following tissue homogenization, drug quantification was performed using high performance (efavirenz) or ultrahigh performance (tenofovir, lamivudine, fluconazole) liquid chromatography coupled with triple quadrupole mass spectrometer. Calibrator standards and quality control (QC) samples were prepared in the matrix to match the sample tested; bovine brain homogenate was used for brain tissue, a solution of salt and proteins for CSF, and lithium heparin for plasma. Data are reported as median (range). Results: Post-mortems were performed 5.2 (2.2-13.7) hours following death. Three individuals receiving daily tenofovir/lamivudine/efavirenz (300/300/600 mg) had detectable drug in all tissue compartments. Likewise, four individuals receiving fluconazole (400-1200 mg daily) had detectable drug in all compartments. CSF: plasma ratios were similar to values reported in the literature. Drug exposure in brain tissues was consistently lower than CSF for tenofovir, lamivudine, and fluconazole and higher than CSF for efavirenz (see Table). Conclusion: Tenofovir, lamivudine, and fluconazole exposure in CSF over- predicted brain tissue penetration. In contrast, CSF exposure of efavirenz under-predicted brain exposure. These findings highlight the limitations of CSF as a surrogate for overall CNS drug exposure. Validation in larger cohorts and in additional CNS tissue compartments is warranted. These data support the use of post-mortem tissues to assess drug distribution to and within the CNS, relevant for HIV reservoir eradication.

Poster Abstracts

476 EFFECT OF SEX, SHIV, AND DRUG TRANSPORTERS ON LYMPH NODE ANTIRETROVIRAL PENETRATION Erin M. Burgunder 1 , John K. Fallon 1 , Nicole White 1 , Amanda Schauer 1 , Craig Sykes 1 , Lourdes Adamson 2 , Jason R. Pirone 1 , Paul Luciw 2 , Philip C. Smith 1 , Angela Kashuba 1 1 University of North Carolina Chapel Hill, Chapel Hill, NC, USA, 2 University of California Davis, Davis, CA, USA Background: Despite antiretroviral (ARV) therapy, HIV persists in tissue reservoirs throughout the body. Lymph node ARV penetration is crucial for HIV

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