Background: Extracellular vesicles (EVs) are cell-derived vesicles with diverse functions in intercellular communication including disease and infection, and EVs seem to influence HIV-1 pathogenesis. EVs isolated from HIV-1–uninfected semen (SE), but not blood (BE), contain factors that interfere with HIV-1 infection and replication in target cells. The reason for this dichotomy is unknown. Furthermore, the effect of HIV-1 infection and antiretroviral (ARV) drugs on the anti–HIV-1 effects of SE and BE is unknown. Here, we characterize EVs and EV-free plasma isolated from HIV-infected donor semen and blood and their effects on HIV infection. Methods: EVs and EV-free plasma were purified from autologous blood and semen of HIV-negative, HIV-infected antiretroviral therapy (ART)-naïve, and HIV-infected ART-treated participants. HIV infection was assessed in a TZM-bl cell reporter system. ARV concentrations were analyzed using liquid chromatography-mass spectrometry. Results: SE isolated from both HIV-negative and HIV-infected, ART-naïve donors inhibited HIV-1 infection, but BE and semen and blood EV-free plasma did not. By contrast, BE, SE, and EV-free plasma from HIV-infected, ART-treated donors inhibited HIV-1. Importantly, exosomes isolated from ART-treated donors contained concentrations of ARV drugs (ART-EVs) at biologically relevant inhibitory levels. Conclusions: The HIV-1–inhibitory phenotype of SE is independent of donor HIV-1 or ART status, and ARV drugs and their metabolites are SE- and BE-associated in vivo.

Quantification of antiretroviral (ARV) drug concentrations in peripheral blood mononuclear cells (PBMCs) and tissue isolated mononuclear cells (TIMCs) from lymph node (LNMC) and rectum (RMC) is an important measure of bio-distribution. Normalization of drug concentrations is critical to represent tissue drug concentrations and to analyze both intra-individual and inter-individual variability in drug distribution. However, a molecular method to normalize intracellular drug concentrations in PBMCs and TIMCs methanol extracts is currently unavailable. In this study, a novel droplet digital PCR (ddPCR) assay was designed to amplify RPP30 gene sequence conserved in human and non-human primates (NHP). Genomic DNA (gDNA) isolated from 70 percent methanol embedded PBMCs and TIMCs was used as ddPCR template to quantitate precise RPP30 copies to derive cell counts. The novel molecular method quantitated RPP30 copies in human and rhesus macaque gDNA templates with greater accuracy and precision than qPCR. RPP30 ddPCR derived cell counts are strongly correlated with automated cytometer based cell counts in PBMC (R = 0.90, p = 0.001 and n = 20); LNMC (R = 0.85 p = 0.0001 and n = 22) and RMC (R = 0.92, p = 0.0001 and n = 20) and achieved comparable normalized drug concentrations. Therefore, the RPP30 ddPCR assay is an important normalization method in drug bio-distribution and pharmacokinetic studies in humans and NHPs.

Purpose Macrophages are an important cellular reservoir in HIV, and exist in two phenotypically dissimilar subsets, the pro-inflammatory M1 phenotype, and the anti-inflammatory M2 phenotype. The role of these two subsets is uncertain. We hypothesized that differences in drug efflux transporters exist between the subsets, which would result in altered intracellular drug concentrations between these cells. Methods U937 monocytic cells were polarized to the M1 or M2 phenotype via treatment with interferon-gamma and LPS, or interleukins 4, 13, and LPS, respectively. PGP function was assessed with Hoechst 33342, and expression via western blotting. Intracellular lopinavir was assessed via LC-MS/MS. Data was confirmed with primary monocyte derived macrophages. Results We observed significant differences in intracellular concentrations of lopinavir, a PGP substrate, with higher concentrations in M1 cells. PGP function and expression was higher in the M2 macrophages. These results were confirmed with primary monocyte derived macrophages. Conclusions This data shows that there are previously unreported differences in P-glycoprotein expression between macrophage subsets, and suggests that there may be differences for other transporters. These differences can play a role in intracellular drug concentrations in these cells, and may allow for low-level HIV replication.

Adequate distribution of antiretroviral drugs to infected cells in HIV patients is critical for viral suppression. In humans and primates, HIV- and SIV-infected CD4 T cells in adipose tissues have recently been identified as reservoirs for infectious virus. To better characterize adipose tissue as a pharmacological sanctuary for HIV-infected cells, in vitro experiments were conducted to assess antiretroviral drug efficacy in the presence of adipocytes, and drug penetration in adipose tissue cells (stromal-vascular-fraction cells and mature adipocytes) was examined in treated humans and monkeys. Co-culture experiments between HIV-1-infected CD4 T cells and primary human adipocytes showed that adipocytes consistently reduced the antiviral efficacy of the nucleotide reverse transcriptase inhibitor tenofovir and its prodrug forms tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF). In HIV-infected persons, LC-MS/MS analysis of intracellular lysates derived from adipose tissue stromal-vascular-fraction cells or mature adipocytes suggested that integrase inhibitors penetrate adipose tissue, whereas penetration of nucleoside/nucleotide reverse transcriptase inhibitors such as TDF, emtricitabine, abacavir, and lamivudine is restricted. The limited distribution and functions of key antiretroviral drugs within fat depots may contribute to viral persistence in adipose tissue.

Background The secondary lymphoid tissues (LTs), lymph nodes (LNs) and gut-associated lymphoid tissue (GALT) are considered reservoirs for HIV. Antiretrovirals (ARVs) have lower penetration into LT. In vitro models predictive of ARV LT penetration have not been established. Objectives To develop an in vitro model of LT bioavailability using human lymphoid endothelial cells (HLECs) and investigate its predictability with in vivo pharmacokinetic (PK) studies in mice. Methods ARV bioavailability in HLECs was evaluated at the maximum plasma concentration (Cmax) observed in HIV-infected patients. ARVs were: abacavir, atazanavir, darunavir, dolutegravir, efavirenz, elvitegravir, emtricitabine, maraviroc, raltegravir, rilpivirine, ritonavir, tenofovir disoproxil fumarate and the PK booster cobicistat. The LT PK of representative drugs showing high (efavirenz), intermediate (dolutegravir) and low (emtricitabine) HLEC bioavailability was investigated in BALB/c mice given 50/10/30 mg/kg efavirenz/dolutegravir/emtricitabine orally, daily for 3 days. The concordance of in vitro and in vivo ARV bioavailability was examined. Results ARVs showed high (>67th percentile; rilpivirine, efavirenz, elvitegravir and cobicistat), intermediate (67th–33rd percentile; ritonavir, tenofovir disoproxil fumarate, dolutegravir and maraviroc) and low (<33rd percentile; atazanavir, darunavir, raltegravir, emtricitabine and abacavir) HLEC bioavailability. The hierarchy of efavirenz, dolutegravir and emtricitabine bioavailability in LN, gut and brain tissues of mice was: efavirenz>dolutegravir>emtricitabine. Conclusions ARVs displayed distinct HLEC penetration patterns. PK studies of representative ARVs in LT of mice were concordant with HLEC bioavailability. These findings support further development of this approach and its translational predictability in humans.

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The secondary lymphoid tissues (LT), lymph nodes (LN) and gut-associated lymphoid tissue are the primary sites of HIV replication and where the latent pool of virus is maintained. We compared the pharmacokinetics of tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) in LT of 13 HIV-infected persons receiving a TDF-containing antiretroviral regimen who subsequently switched to a TAF-containing regimen. Study participants were on stable antiretroviral therapy for ≥12 months with plasma HIV-RNA < 48 copies/mL for 6 months before enrollment and entry CD4 cell counts > 300 cells/µL. Intracellular concentrations of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) were quantified in PBMCs and in mononuclear cells obtained from LN, ileum and rectal tissues. With TAF, the TFV-DP concentrations in PBMCs and LN were 7.3-fold and 6.4-fold higher (ratios of geometric means of TAF to TDF), respectively, compared with TDF; ileal and rectal concentrations, however, were lower with geometric mean ratios of 0.14 and 0.18, respectively. A statistically significant relationship was observed between PBMC and LN concentrations of TFV-DP. During TDF-containing therapy, the expected effect of cobicistat to increase TFV plasma concentrations was observed, as were higher TFV-DP concentrations in PBMCs and mononuclear cells from LN, ileum and rectal tissues. The higher TFV-DP concentrations achieved with TAF in the LN provides the first human correlate of the observation in animals that TAF produced higher tenofovir LN concentrations. The ability to increase LN concentrations allows investigations of whether antiretroviral regimens with improved LN pharmacokinetics elicit a more complete virologic response in that compartment.