Infectious Diseases

In Vitro Evaluations

Antiviral Testing Capabilities

Anti-Hepatitis C Virus (HCV) Evaluations

  • Evaluations with HCV replicon. Compounds are evaluated for their antiviral activities using HCV subgenomic replicon 1b (Con1), and assessment of cytotoxicity is conducted in parallel. The replicon has a stable luciferase (Luc) reporter and three cell culture-adaptive mutations. Both luciferase and qRT-PCR/TaqMan endpoints are available. Other HCV replicons (e.g., full-length HCV) are available upon request.
  • Evaluation for cross genotype spectrum. Assays are performed using stable human hepatoma cell lines harboring HCV 1a or 2a replicons. HCV RNA replication is measured directly using TaqMan endpoints.
  • Range of action studies. Bovine viral diarrhea virus (BVDV/NADL strain), yellow fever virus (17D strain), West Nile virus, and dengue virus are distantly related members within the Flaviviridae family of viruses that are used to evaluate anti-flavivirus activity in virus-based assays. The endpoint in these assays is inhibition of virus-induced cytopathic effects (CPE). Analysis of this data allows calculation of antiviral activity, compound toxicity, and therapeutic index (toxicity/antiviral activity). Other non-related DNA and RNA viruses are available for evaluation of test articles for broad-spectrum activity or specificity.
  • In vitro combination activity. Combination therapy is the current standard of care for treatment of HCV infections. As such, it is critical to evaluate the activity of compounds in development in combination with clinically relevant agents being used in the treatment of HCV-infected patients. Data are evaluated using the Prichard and Shipman MacSyngery II software; however, other methods, such as Chou and Talalay median dose effect equation, as well as ComboSTAT can be used upon request. The interactions of compounds are evaluated in terms of antiviral efficacy and cytotoxicity to identify synergistic, additive, or antagonistic activities in the HCV replicon assay.
  • Cytotoxicity and therapeutic indices. Understanding the cytotoxic activity of a test compound is an important aspect of understanding the compound's antiviral activity, since virus replication depends on the cell. Therefore, all cell-based antiviral assays conducted at Southern Research include a parallel cytotoxicity assay to allow the determination of a compound's in vitro therapeutic index (TI). TI is calculated as the ratio of the compound's 50% toxic concentration to the compound's 50% inhibitory concentration against virus RNA replication (TI=TC50/IC50).
  • Comprehensive cytotoxicity evaluation. Testing compounds for toxicity against a panel of established cell lines, human PBMCs, and human primary hepatocyte cultures is available upon request.
  • HCV molecular target assays, including:
    • IRES assay. Specific inhibition of the HCV IRES-mediated translation is measured in a cell-based dual luciferase assay format.
    • NS5B assay. Specific inhibition of the HCV RNA-dependent RNA-polymerase NS5B is measured using an in vitro biochemical assay. Similar assays to measure cellular polymerase activity/inhibition are available as controls.
    • NS3 protease assay. Specific inhibition of the HCV NS3 protease activity is measured using an in vitro biochemical assay to measure substrate cleavage inhibition.
    • HCV NS3 helicase assay. Specific inhibition of the HCV NS3 helicase activity is measured in an in vitro biochemical assay using fluorescence resonance energy transfer (FRET)-based methodology.
  • Resistance profiling with the HCV replicon. Genotypic and phenotypic analyses of drug resistance can be performed using the HCV replicon. These studies include: 1. Selection of resistant replicons under dual selection pressure of G418 and the study compound; 2. Identification of mutations in the target regions of the replicon; and 3. Reeingeineering mutation(s) back into the parental replicon to confirm resistance. Results of these studies also confirm a compound's mechanism of action. In addition, a panel of HCV replicons resistant to representative IND drugs has been established and can be used to evaluate a novel compound's ability to inhibit HCV mutants resistant to compounds in clinical development. Dose escalating and single high-dose models are used for selection of resistant replicons.

Anti-Hepatitis B Virus (HBV) Evaluations

  • Antiviral activity, cytotoxicity, and therapeutic index (toxicity/antiviral activity). The HepG2 cell line 2.2.15 (a stable cell line containing the Hepatitis B virus (HBV) ayw strain genome) is employed as a primary tool for antiviral evaluation. Using this cell line, antiviral compounds blocking any late-step of viral replications can be identified and characterized. Evaluations also include testing a compound's ability to reduce secreted HBV from cells utilizing the real-time quantitative PCR (TaqMan) HBV DNA assay.
  • In vitro combination assays. Combination drug therapy is the current paradigm for treatment of HBV infections. Therefore, it is critical to evaluate novel compounds in development in combination with drugs already being used in the treatment of HBV-infected patients. The interactions of compounds are evaluated in terms of antiviral efficacy and cytotoxicity to identify synergistic, additive, or antagonistic interactions using the HepG2 2.2.15 cells and the quantitative HBV TaqMan PCR assay. Data are evaluated using the Prichard and Shipman MacSynergy II software; however, other methods, such as Chou and Talalay median dose effect equation as well as ComboSTAT can be used upon request.
  • Cytotoxicity and therapeutic indices. Understanding the cytotoxic activity of a test compound is an important aspect of understanding the compound's antiviral activity, since virus replication depends on the cell. Therefore, all cell-based antiviral assays conducted at Southern Research include a parallel cytotoxicity assay to allow the determination of a compound's in vitro therapeutic index (TI). TI is calculated as the ratio of the compound's 50% toxic concentration to the compound's 50% inhibitory concentration against virus RNA replication (TI=TC50/IC50).
  • Comprehensive cytotoxicity evaluation. Testing compounds for toxicity against a panel of established cell lines, human PBMCs, and human primary hepatocyte cultures is available upon request.
  • Other anti-HBV and HBV mechanism-based assays. A variety of assays have been developed in our laboratories to address special requirements of our clients. These assays are used to pinpoint the MOA of antiviral compounds and are used to determine the compound's activity to:
    • Inhibit viral budding (formation of extracellular virions)
    • Inhibit formation of intracellular HBV replicative intermediates
    • Inhibit HBV transcription (Northern blot and primer extension analysis)
    • Inhibit HBsAG and HBeAg release (ELISA analyses)
    • HBV core and envelope protein expression analysis using western blotting
    • Novel MOA studies to evaluate specific effects on HBV transcription and replication that may result from alterations in DNA-protein interactions
    • Endogenous polymerase assay utilizing purified HBV virions to evaluate the activity of antiviral drugs to inhibit endogenous HBV polymerase activity
  • Inhibition of drug-resistant virus. Antiviral drug resistance is an important factor in treatment failure in HBV-infected people treated with nucleotide analogs. Evaluating the capability of a novel compound to inhibit know nucleotide analogs (3TC), Lamivudine- and penciclovir-resistant mutants of HBV is available through the development of stable cell lines with the following mutations known to be associated with resistance of HBV to these agents:
    • L526M (rtL180M) of Domain B and YMDD M55OV (rtM204V) of Domain C (the most common mutation pattern observed during HBV breakthrough viremia)
    • L526M alone (the most common mutation associated with penciclovir resistance and also associated with some resistance to 3TC)

Custom Assay Development

In addition to our comprehensive range of standardized assays, we also offer the capability to develop customized assays to meet our clients' specific anti-hepatitis research needs.

Contact Us

For more information about our capabilities, contact us at:
BusDev@SouthernResearch.org
888-322-1166 (U.S.)
1-205-581-2830 (International)