We have previously shown that ARV distribution within lymphoid tissue can be highly heterogeneous. Understanding potential consequences of diverse ARV accumulation requires quantitative methods to characterize ARV proximity to virus and target cells. Here, we developed a novel analytical approach based on a combination of mass spectrometry imaging (MSI), in situ hybridization (ISH) and immunohistochemistry (IHC) to understand the consequences of ARV distribution in lymph nodes (LN).
Axillary LN were collected and snap frozen at necropsy from RT-SHIV infected rhesus macaques dosed 10 days with emtricitabine (FTC) + tenofovir (TFV) (N=6) in combination with either efavirenz (EFV) + raltegravir (RAL) (N=3), cohort FTER, or maraviroc (MVC) + atazanavir (ATZ) (N=3), cohort FTMA. Tissue accumulation of ARVs and metabolites was measured by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI from 10 m thick cryosections at 0.1 mm spatial resolution. Serial sections of tissue were analyzed for viral RNA (vRNA) by RNAscope ISH and for CD4+ cells by IHC. Spatial relationships were evaluated by nearest neighbor search (NNS) on co-registered images using MATLAB (Fig A).
MSI simultaneously measured all detectable ARVs (FTC, RAL < limits of detection: 0.05-0.37 ng/mg tissue) and the blood biomarker, HEME. Based on NNS analysis between ARVs and HEME (reflecting ARV in the vasculature), 57% of all ARVs in LN were ≤0.1 mm from HEME. The greatest tissue penetration was found for TFV and EFV (up to 1.7 mm from HEME). The degree of colocalization between ARVs and vRNA varied (Fig B. TFV: 1-9%; ATZ: 4-16%; MVC: 54-68%; EFV: 89-99%). Yet NNS analysis indicated that >95% of all vRNA was ≤0.1 mm of a detectable ARV response in each cohort (Fig C). However, proximity of vRNA to ARV concentrations >in vitro IC50 values was farther (vRNA≤0.3 mm: FTER=0-60%; FTMA=88-97%). Similar results were observed for NNS analysis of CD4+ T cells (Fig D. CD4≤0.3 mm: FTER=0-94%; FTMA=96-99%).
A quantitative approach has been developed for analysis of spatial relationships between drug and targets such as blood, virus, and T cells. ARV coverage extends to >73% of the LN, better for CD4 (>64%) than vRNA (>58%), but may not be adequate everywhere relative to known inhibitory concentrations. The flexibility of this framework allows ARVs to be evaluated individually or in aggregate, and offers a tool help optimize pharmacokinetics and pharmacodynamics to ARV treatment.