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).
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.
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 IC50 values were limited where co-localization was determined, with EFV and MVC providing the greatest exposure ([ARV] > IC50 : 3-16% and 41-47%, respectively).
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.