Assays that can verify full viral eradication are essential in the context of achieving a cure for HIV/AIDS. In vitro quantitative viral out growth assays (qVOA) are currently the gold standard for measuring latent HIV-1 but these assays often fail to detect very low levels of replication-competent virus. Here we investigated an alternative in vivo approach for sensitive viral detection using humanized mice (hmVOA). Peripheral blood CD4+ T cell samples from HIV subjects on stable ART with undetectable viral loads by RT-PCR were first assayed by in vitro qVOA. Corresponding patient samples in which no virus was detected by traditional qVOA were injected into humanized mice to allow viral outgrowth. Of the five in vitro qVOA virus negative samples, four gave positive viral outgrowth in the in vivo hmVOA assay suggesting that it is more sensitive in detecting latent HIV-1.

HIV replication within tissues may increase in response to a reduced exposure to antiretroviral drugs. Traditional approaches to measuring drug concentrations in tissues are unable to characterize a heterogeneous drug distribution. Here, we used mass spectrometry imaging (MSI) to visualize the distribution of six HIV antiretroviral drugs in gut tissue sections from three species (two strains of humanized mice, macaques, and humans). We measured drug concentrations in proximity to CD3+ T cells that are targeted by HIV, as well as expression of HIV or SHIV RNA and expression of the MDR1 drug efflux transporter in gut tissue from HIV-infected humanized mice, SHIV-infected macaques, and HIV-infected humans treated with combination antiretroviral drug therapy. Serial 10-μm sections of snap-frozen ileal and rectal tissue were analyzed by MSI for CD3+ T cells and MDR1 efflux transporter expression by immunofluorescence and immunohistochemistry, respectively. The tissue slices were analyzed for HIV/SHIV RNA expression by in situ hybridization and for antiretroviral drug concentrations by liquid chromatography–mass spectrometry. The gastrointestinal tissue distribution of the six drugs was heterogeneous. Fifty percent to 60% of CD3+ T cells did not colocalize with detectable drug concentrations in the gut tissue. In all three species, up to 90% of HIV/SHIV RNA was found to be expressed in gut tissue with no exposure to drug. These data suggest that there may be gut regions with little to no exposure to antiretroviral drugs, which may result in low-level HIV replication contributing to HIV persistence.

Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV-1, as they have the potential to maintain sustained viral inhibition with reduced treatment interventions. Such an option may represent a long-term treatment alternative to highly active antiretroviral therapy.Methods: We previously described a therapeutic approach, referred to as transcriptional gene silencing (TGS), whereby small noncoding RNAs directly inhibit the transcriptional activity of HIV-1 by targeting sites within the viral promoter, specifically the 5' long terminal repeat (LTR). TGS differs from traditional RNA interference (RNAi) in that it is characterized by concomitant silent-state epigenetic marks on histones and DNA. To deliver TGS-inducing RNAs, we developed functional RNA conjugates based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV-1 siRNA (dsiRNA), LTR-362.Results: We demonstrate here that high levels of processed guide RNAs localize to the nucleus in infected T lymphoblastoid CEM cell line and primary human CD4+ T-cells. Treatment of the aptamer-siRNA conjugates induced TGS with an ~10-fold suppression of viral p24 levels as measured at day 12 post infection. To explore the silencing efficacy of aptamer-siRNA conjugates in vivo, HIV-1-infected humanized NOD/SCID/IL2 rγnull mice (hu-NSG) were treated with the aptamer-siRNA conjugates. Systemic delivery of the A-1-stick-LTR-362 27-mer siRNA conjugates suppressed HIV-1 infection and protected CD4+ T cell levels in viremia hu-NSG mice.Principle conclusions: Collectively these data suggest that the gp120 aptamer-dsiRNA conjugate design is suitable for systemic delivery of small RNAs that can be used to suppress HIV-1.

Many murine and non-human primate animal models have been recently developed to understand Zika viral pathogenesis. However, a major limitation with these models is the inability to directly examine the human-specific immune response. Here, we utilized a BLT humanized mouse model endowed with a transplanted human immune system. Plasma viremia could be detected within 48h after viral challenge and viremia persisted for as long as 220 days in some mice. Neutralizing human antibody was detected in infected mice and mouse sera showed reactivity with the viral envelope and capsid proteins in a radio-immunoprecipitation assay. Human monocytes/macrophages, B cells and hematopoietic stem cells in the bone marrow were found to be virus infected. These data establish that BLT mice are permissive for Zika viral infection and are capable of generating viral-specific human immune responses thus providing a human surrogate model for future testing of vaccine and antiviral therapeutic candidates.

Objectives Drug transporters affect ART tissue disposition, but quantitative measures of drug transporter protein expression across pre-clinical species are not available. Our objective was to use proteomics to obtain absolute transporter concentrations and assess agreement with corresponding gene and immunometric protein data. Design In order to make interspecies comparisons, two humanized mouse (hu-HSC-Rag (n=41); BLT (n=13)) and one primate (rhesus macaque, (NHP, n=12)) models were dosed to steady-state with combination ART. Ileum and rectum were collected at necropsy and snap frozen for analysis. Methods Tissues were analyzed for gene (qPCR) and protein (LC-MS proteomics and Western blot) expression and localization (immunohistochemistry) of ART efflux and uptake transporters. Drug concentrations were measured by LC-MS/MS. Multivariable regression was used to determine the ability of transporter data to predict tissue ART penetration. Results Analytical methods did not agree, with different trends observed for gene and protein expression. For example, qPCR analysis showed a 2-fold increase in permeability glycoprotein (Pgp) expression in NHPs versus mice, however proteomics showed a 200-fold difference in the opposite direction. Proteomics results were supported by IHC staining showing extensive efflux transporter localization on the luminal surface of these tissues. ART tissue concentration was variable between species, and multivariable regression showed poor predictive power of transporter data. Conclusions Lack of agreement between analytical techniques suggests that resources should be focused on generating downstream measures of protein expression to predict drug exposure. Taken together, these data inform the use of pre-clinical models for studying ART distribution and the design of targeted therapies for HIV eradication.