Performance of the cobas® HBV RNA automated investigational assay for the detection and quantification of circulating HBV RNA in chronic HBV patients.

BACKGROUND: The amount of HBV RNA in peripheral blood may reflect HBV covalently closed circular DNA (cccDNA) transcriptional activity within infected hepatocytes. Quantification of circulating HBV RNA (cirB-RNA) is thus a promising biomarker for monitoring antiviral treatment. OBJECTIVES: We evaluated the performance of an automated, prototype quantitative HBV RNA assay for use on the Roche cobas® 6800/8800 systems. STUDY DESIGN: The sensitivity, specificity, linearity, and potential interference by HBV DNA of the cobas® HBV RNA assay were assessed using synthetic HBV armored RNA and clinical specimens. RESULTS: cobas® HBV RNA results were linear between 10 and 107 copies/mL in clinical samples of several HBV genotypes, and up to 109 copies/mL with synthetic RNA. Precision and reproducibility were excellent, with standard deviation below 0.15 log10 copies/mL and coefficients of variation below 5% throughout the linear range. The presence of HBV DNA had minimal (<0.3 log10 copies/mL) impact on HBV RNA quantification at DNA:RNA ratios of up to approximately one million. In a panel of 36 untreated patient samples, cirB-RNA concentrations were approximately 200-fold lower than HBV DNA. cirB-RNA was detected in all 13 HBeAg-positive patients (mean 6.0 log10 copies/mL), and in 20 of 23 HBeAg-negative patients (mean of quantifiable samples 2.2 log10 copies/mL). Finally, cirB-RNA was detected in 12 of 20 nucleoside analog-treated patients (mean of quantifiable samples 3.4 log10 copies/mL). CONCLUSIONS: The cobas® 6800/8800 investigational HBV RNA assay is a high throughput, sensitive and inclusive assay to evaluate the clinical relevance of cirB-RNA quantification in patients with chronic hepatitis B.

First identification of a recombinant form of hepatitis C virus in Austrian patients by full-genome next generation sequencing.

Hepatitis C virus (HCV) intergenotypic recombinant forms have been reported for various HCV genotypes/subtypes in several countries worldwide. In a recent study, four patients living in Austria had been identified to be possibly infected with a recombinant HCV strain. To clarify results and determine the point of recombination, full-genome next-generation sequencing using the Illumina MiSeq v2 300 cycle kit (Illumina, San Diego, CA, USA) was performed in the present study. Samples of all of the patients contained the recombinant HCV strain 2k/1b. The point of recombination was found to be within the HCV NS2 gene between nucleotide positions 3189-3200 based on H77 numbering. While three of four patients were male and had migration background from Chechnya (n = 2) and Azerbaijan (n = 1), the forth patient was a female born in Austria. Three of the four patients including the female had intravenous drug abuse as a risk factor for HCV transmission. While sequencing techniques are limited to a few specialized laboratories, a genotyping assay that uses both ends of the HCV genome should be employed to identify patients infected with a recombinant HCV strain. The correct identification of recombinant strains also has an impact considering the tailored choice of anti-HCV treatment.

An integrated chemical biology approach reveals the mechanism of action of HIV replication inhibitors.

Continuous flow (microfluidic) chemistry was employed to prepare a small focused library of dihydropyrimidinone (DHPM) derivatives. Compounds in this class have been reported to exhibit activity against the human immunodeficiency virus (HIV), but their molecular target had not been identified. We tested the initial set of DHPMs in phenotypic assays providing a hit (1i) that inhibited the replication of the human immunodeficiency virus HIV in cells. Flow chemistry-driven optimization of 1i led to the identification of HIV replication inhibitors such as 1l with cellular potency comparable with the clinical drug nevirapine (NVP). Mechanism of action (MOA) studies using cellular and biochemical assays coupled with 3D fingerprinting and in silico modeling demonstrated that these drug-like probe compounds exert their effects by inhibiting the viral reverse transcriptase polymerase (RT). This led to the design and synthesis of the novel DHPM 1at that inhibits the replication of drug resistant strains of HIV. Our work demonstrates that combining flow chemistry-driven analogue refinement with phenotypic assays, in silico modeling and MOA studies is a highly effective strategy for hit-to-lead optimization applicable to the discovery of future therapeutic agents.

Automated Multistep Continuous Flow Synthesis of 2-(1H-Indol-3-yl)thiazole Derivatives.

The multistep continuous flow assembly of 2-(1H-indol-3-yl)thiazoles using a Syrris AFRICA® synthesis station is reported. Sequential Hantzsch thiazole synthesis, deketalization and Fischer indole synthesis provides rapid and efficient access to highly functionalized, pharmacologically significant 2-(1H-indol-3-yl)thiazoles. These complex, drug-like small molecules are generated in reaction times of less than 15 min and in high yields (38%-82% over three chemical steps without isolation of intermediates).

A high-throughput drug screening system for HIV-1 transcription inhibitors.

Identification of HIV-1 transcription inhibitors was previously performed using infectivity assays. As de novo HIV-1 infection is highly sensitive to even minor compound toxicities, these assays are plagued by extremely high levels of false-positive hits. Hit identification is further complicated because infectivity assays lack target specificity. The authors demonstrate that it is possible to overcome these limitations by establishing a stable, chronically actively HIV-1-infected reporter cell line that exclusively identifies HIV-1 transcription inhibitors. In the reporter cell line, 2 spectrally separated fluorescence proteins serve as simultaneously accessible quantitative markers of HIV-1 expression and drug toxicity. The combined analysis of these markers drastically reduces the level of false-positive hits. As determination of fluorescence intensity in a plate-based format can be performed in a noninvasive manner, repeated measurements of fluorescence levels over several days after compound addition can be used to define the kinetic and dynamic characteristics of inhibitory drug effects. In addition, because of the stable nature of the reporter cell line, the assay requires no cell manipulation during assay preparation or assay analysis, rendering the system extremely cost-effective and reliable.

T20-insensitive HIV-1 from naive patients exhibits high viral fitness in a novel dual-color competition assay on primary cells.

The relationship between sensitivity to antiviral drugs and viral fitness is of paramount importance in understanding the long-term implications of clinical resistance. Here we report the development of a novel recombinant virus assay to study entry inhibitor-resistant HIV variants using a biologically relevant cell type, primary CD4 T-cells. We have modified the replication-competent molecular clone HIV(NL4-3) to express a reporter protein (Renilla luciferase), Green Fluorescent Protein (EGFP), or Red Fluorescent Protein (DsRed2) upon infection, thus allowing quantification of replication. Luciferase-expressing virus was used to evaluate drug sensitivity, while co-infection with viruses carrying the green and red fluorescent proteins was employed in the competitive fitness assay. Using envelope proteins from three T20 insensitive variants, lower levels of resistance were observed in primary CD4 T-cells than had been previously reported for cell lines. Importantly, dual-color competition assays demonstrated comparable or higher fitness for these variants despite their reduced T20 sensitivity. We conclude that reduced sensitivity to T20 is compatible with high viral fitness in the absence of selection pressure. Thus, simultaneously measuring both resistance and viral fitness using this newly described dual-color competition assay will likely provide important information about resistant viral variants that emerge during therapy with entry inhibitors.

Determinants of human immunodeficiency virus type 1 baseline susceptibility to the fusion inhibitors enfuvirtide and T-649 reside outside the peptide interaction site.

The peptide fusion inhibitor (PFI) enfuvirtide is the first of a new class of entry inhibitors to receive FDA approval. We previously determined the susceptibility of 55 PFI-naïve-patient isolates to enfuvirtide and a second peptide inhibitor, T-649. Seven of the 55 viral isolates were insusceptible to enfuvirtide, T-649, or both inhibitors in the absence of prior exposure. To determine the molecular basis of the insusceptible phenotypes, we PCR amplified and cloned five PFI-insusceptible and one PFI-susceptible, full-length, biologically functional env genes and characterized viruses pseudotyped with the Env proteins in a single-round drug sensitivity assay. Overall, the mean 50% inhibitory concentrations of enfuvirtide and T-649 for the PFI-insusceptible Env pseudotypes were 1.4 to 1.7 log(10) and 1.2 to 1.8 log(10) greater, respectively, than those for a PFI-susceptible lab strain, NLHX; however, all of the PFI-insusceptible Env proteins conserved the sequence of a critical enfuvirtide interaction site (residues 36 to 38 of gp41, GIV) in HR-1. In contrast, multiple amino acid changes were observed C-terminal to HR-1, many of which were located in regions of HR-2 corresponding to the PFI. Nevertheless, peptides based on patient-derived HR-2 sequences were not more potent inhibitors than enfuvirtide or T-649, arguing that the basis of PFI susceptibility is not a higher-affinity, competitive HR-1/HR-2 interaction. These results demonstrate that regions of Env outside the enfuvirtide interaction site can significantly impact the PFI susceptibility of patient-derived Env, even prior to drug exposure. We hypothesize that both gp120 gene- and gp41 gene-encoded determinants that minimize the window of opportunity for PFI to bind provide a growth advantage and possibly a predisposition to resistance to this new class of drugs in vivo.

Envelope-constrained neutralization-sensitive HIV-1 after heterosexual transmission.

Heterosexual transmission accounts for the majority of human immunodeficiency virus-1 (HIV-1) infections worldwide, yet the viral properties that determine transmission fitness or outgrowth have not been elucidated. Here we show, for eight heterosexual transmission pairs, that recipient viruses were monophyletic, encoding compact, glycan-restricted envelope glycoproteins. These viruses were also uniquely sensitive to neutralization by antibody from the transmitting partner. Thus, the exposure of neutralizing epitopes, which are lost in chronic infection because of immune escape, appears to be favored in the newly infected host. This reveals characteristics of the envelope glycoprotein that influence HIV-1 transmission and may have implications for vaccine design.