A perfusion host-microbe bioreactor (HMB) system that captures dynamic interactions of secreted metabolites between epithelial cells cocultured with a human gut anaerobe.

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes.

A Novel Rotavirus Reverse Genetics Platform Supports Flexible Insertion of Exogenous Genes and Enables Rapid Development of a High-Throughput Neutralization Assay.

Despite the success of rotavirus vaccines, rotaviruses remain one of the leading causes of diarrheal diseases, resulting in significant childhood morbidity and mortality, especially in low- and middle-income countries. The reverse genetics system enables the manipulation of the rotavirus genome and opens the possibility of using rotavirus as an expression vector for heterologous proteins, such as vaccine antigens and therapeutic payloads. Here, we demonstrate that three positions in rotavirus genome-the C terminus of NSP1, NSP3 and NSP5-can tolerate the insertion of reporter genes. By using rotavirus expressing GFP, we develop a high-throughput neutralization assay and reveal the pre-existing immunity against rotavirus in humans and other animal species. Our work shows the plasticity of the rotavirus genome and establishes a high-throughput assay for interrogating humoral immune responses, benefiting the design of next-generation rotavirus vaccines and the development of rotavirus-based expression platforms.

Development of a fully automated platform for agar-based measurement of viable bacterial growth.

Dynamic in vitro antibacterial studies provide valuable insight on effective dosing strategies prior to translating to in vivo models. Frequent sampling is required to monitor the pharmacodynamics (PD) of these studies, leading to significant work when quantifying the bacterial load of the samples. Spreading a bacterial suspension on agar to allow colony counting is a proven process for measuring very low levels of growth, but commercial automation equipment to handle agar plating and colony counting at scale is not readily available. We describe a process to greatly decrease the hands-on time required for PD assays by utilizing general-purpose liquid handling robots to plate bacteria and a custom-made plate imager to automate colony counting. The platform developed handles the biological assay from beginning to end as well as sample tracking at each step of the process. The process relies heavily on custom automation scheduling software to enable dynamic process decisions and coordinate data flow throughout. Using the described platform, we can efficiently quantify >100 PD samples per day while maintaining the necessary dynamic range of the assay. Alleviating the main bottleneck in the dynamic antibacterial studies has allowed us to accelerate the rate of experiments to provide antibacterial dosing data within shorter timelines.

In Vitro Pharmacokinetic/Pharmacodynamic Modeling of HIV Latency Reversal by Novel HDAC Inhibitors Using an Automated Platform.

Antiretroviral therapy is able to effectively control but not eradicate HIV infection, which can persist, leading to the need for lifelong therapy. The existence of latently HIV-infected cells is a major barrier to the eradication of chronic HIV infection. Histone deacetylase inhibitors (HDACis), small molecules licensed for oncology indications, have shown the ability to produce HIV transcripts in vitro and in vivo. The pharmacologic parameters that drive optimal HIV latency reversal in vivo are unknown and could be influenced by such factors as the HDACi binding kinetics, concentration of compound, and duration of exposure. This study evaluates how these parameters affect HIV latency reversal for a series of novel HDACis that differ in their enzymatic on and off rates. Varying cellular exposure, using automated washout methods of HDACi in a Jurkat cell model of HIV latency, led to the investigation of the relationship between pharmacokinetic (PK) properties, target engagement (TE), and pharmacodynamic (PD) responses. Using an automated robotic platform enabled miniaturization of a suspension cell-based washout assay that required multiple manipulations over the 48 h duration of the assay. Quantification of histone acetylation (TE) revealed that HDACis showed early peaks and differences in the durability of response between different investigated HDACis. By expanding the sample times, the shift between TE and PD, as measured by green fluorescent protein, could be fully characterized. The comprehensive data set generated by automating the assays described here was used to establish a PK/PD model for HDACi-induced HIV latency reversal.

Identification of proximal biomarkers of PKC agonism and evaluation of their role in HIV reactivation.

DESIGN: The HIV latent CD4+ T cell reservoir is broadly recognized as a barrier to HIV cure. Induction of HIV expression using protein kinase C (PKC) agonists is one approach under investigation for reactivation of latently infected CD4+ T cells (Beans et al., 2013; Abreu et al., 2014; Jiang et al., 2014; Jiang and Dandekar, 2015). We proposed that an increased understanding of the molecular mechanisms of action of PKC agonists was necessary to inform on biological signaling and pharmacodynamic biomarkers. RNA sequencing (RNA Seq) was applied to identify genes and pathways modulated by PKC agonists. METHODS: Human CD4+ T cells were treated ex vivo with Phorbol 12-myristate 13-acetate, prostatin or ingenol-3-angelate. At 3 h and 24 h post-treatment, cells were harvested and RNA-Seq was performed on RNA isolated from cell lysates. The genes differentially expressed across the PKC agonists were validated by quantitative RT-PCR (qPCR). A subset of genes was evaluated for their role in HIV reactivation using siRNA and CRISPR approaches in the Jurkat latency cell model. RESULTS: Treatment of primary human CD4+ T cells with PKC agonists resulted in alterations in gene expression. qPCR of RNA Seq data confirmed upregulation of 24 genes, including CD69, Egr1, Egr2, Egr3, CSF2, DUSP5, and NR4A1. Gene knockdown of Egr1 and Egr3 resulted in reduced expression and decreased HIV reactivation in response to PKC agonist treatment, indicating a potential role for Egr family members in latency reversal. CONCLUSION: Overall, our results offer new insights into the mechanism of action of PKC agonists, biomarkers of pathway engagement, and the potential role of EGR family in HIV reactivation.

Virologic resistance analysis from a phase 2 study of MK-5172 combined with pegylated interferon/ribavirin in treatment-naive patients with hepatitis C virus genotype 1 infection.

BACKGROUND: Virologic failure following treatment of hepatitis C virus (HCV) genotype 1 with direct-acting antiviral agents is often accompanied by the emergence of resistant variants. MK-5172 is an investigational once-daily protease inhibitor. We analyzed variants in treatment-naive noncirrhotic patients with virologic failure on MK-5172 (100-800 mg/day) plus pegylated interferon alfa/ribavirin (peg-IFN/RBV) during a phase 2 trial. METHODS: Population and selective clonal sequencing were performed at baseline and at virologic failure in the 4 MK-5172 dosing arms. MK-5172 activity was determined using a mutant replicon assay. RESULTS: Six of 266 (2.3%) MK-5172 recipients satisfied prespecified criteria for virologic failure, all with genotype 1a infection. Five patients with virologic failure were in the MK-5172 100-mg arm, including 4 patients with low plasma MK-5172 levels documented during triple therapy. Variants associated with >4-fold loss of potency were detected in 3 of the 4 patients with genotype 1a breakthrough while on MK-5172. The fifth patient had undetectable HCV-RNA levels at the end of triple therapy but subsequently broke through during the peg-IFN/RBV tail 16 weeks after completion of MK-5172. Three patients had D168 variants at virologic failure, including 2 with the D168A variant associated with a 95-fold loss of potency. The sole apparent relapse was actually a genotype 3a reinfection in the MK-5172 200-mg group. CONCLUSIONS: Virologic failure occurred uncommonly (6/266 [2.3%]) in MK-5172/peg-IFN/RBV recipients. The most prevalent treatment-emergent variants were detected at the D168 locus. D168A variants conferring approximately 2-log reduction in MK-5172 susceptibility emerged in 2 of the 4 evaluable patients with genotype 1a breakthrough. Clinical Trials Registration. NCT01353911.

Emergence of resistance-associated variants after failed triple therapy with vaniprevir in treatment-experienced non-cirrhotic patients with hepatitis C-genotype 1 infection: a population and clonal analysis.

BACKGROUND: Vaniprevir with P/R improved SVR rates over P/R alone in treatment-experienced patients with chronic HCV-genotype 1 infection, but treatment failure presents therapeutic challenges. We identified RAVs from non-cirrhotic patients failing to achieve SVR on vaniprevir-containing regimens from a dose/duration-ranging trial of triple-combination therapy. METHODS: Using population analysis, resistance sequencing was performed on all baseline samples and on samples at virologic failure in the vaniprevir arms. Longitudinal clonal analyses were performed on viral isolates from six vaniprevir recipients experiencing breakthrough viremia. RESULTS: Baseline RAVs were detected in two patients subsequently experiencing virologic failure. At virologic failure, the majority of RAVs had substitutions at R155, A156, or D168. Clonal analyses identified novel double/triple variants emerging with continuing vaniprevir dosing. CONCLUSIONS: RAVs were predominantly observed at R155, A156, and/or D168 during virologic failure on vaniprevir/P/R. Double/triple RAVs were identified in patients remaining viremic on triple therapy, suggesting evolution of resistance under selective pressure.