Infectious Disease Research

Virology
Hepatitis Virus Research and Development

Anti-Hepatitis B Virus (HBV) Evaluation

• Primary Antiviral Evaluations in the HepG2 cell line 2.2.15
  HepG2-2.2.15 is a stable cell line containing the hepatitis B virus (HBV) ayw strain genome. Antiviral compounds blocking any late step of viral replication such as transcription, translation, pregenome encapsidation, reverse transcription, particle assembly and release can be identified and characterized using this cell line. Initially we test whether a compound will reduce the production of secreted HBV from cells utilizing our real time quantitative PCR (TaqMan) HBV DNA assay. The analysis of this data allows us to calculate:
• Antiviral activity
• Compound toxicity
• Therapeutic index (toxicity/antiviral activity)
• Secondary Antiviral Evaluations
  We have a different HepG2 stable cell containing an HBV adr1 strain genome. The activity of compounds acting in HepG2 2.2.15 can be confirmed in the same manner using this cell line.
• Combination Studies
  After the antiviral activity of test compounds against HBV is confirmed, the interactions of the compounds with 3TC, IFNá and other compounds in terms of efficacy (synergy, additivity, antagonism) and toxicity (combination toxicity) are evaluated with the 2.2.15 cells using the quantitative HBV TaqMan PCR assay.
• Toxicity Panel
  We can test compounds for toxicity against a large panel of established cell lines, PBMCs and human primary hepatocyte cultures.
• HBV Mechanism of Action Studies
  A variety of assays are used to pinpoint the MOA of antiviral compounds.
• Extracellular HBV virions
  In addition to the quantitative PCR analysis we may perform a Southern blot of the HBV particles secreted from drug-treated cells.
• Intracellular HBV particles
  HBV particles can be isolated from the treated 2.2.15 cells and the pregenomic RNA examined by Southern blot analysis. This may help us identify the site of action of a late-acting compound.
• Intracellular HBV replicative intermediates Nucleic acids isolated from the cells can be examined by Southern blots to examine the distribution of circular partially double-stranded HBV DNA, linear partially double-stranded DNA and single stranded HBV DNA.
• HBV transcription
  Effects on HBV genomic and subgenomic viral RNA synthesis are studied by Northern blot and primer extension analysis.
• HBsAg and HBeAg release assay
  We use ELISAs to quantify the amounts of the HBV envelope protein, surface antigen (HBsAg), and of secreted e-antigen (HBeAg) released from cultures.
• Western blot analysis
  To study HBV core and envelope protein expression.
• Novel MOA studies
  Specific effects on HBV transcription and replication may arise from alterations in DNA-protein interactions, sometimes affected by cellular growth factors, at the HBV enhancers, promoters or through the transcriptional transactivator X-protein.
• Endogenous Polymerase Assay
  Extracellular HBV virions contain partially double-stranded circular DNA genomes. We use purified virions to assay the ability of antiviral drugs to inhibit the endogenous polymerase activity of HBV. Normally this activity functions to complete (+)strand synthesis following the infection of new cells by HBV virions.
• HBV Drug Resistance Evaluation
  We have the ability to evaluate the ability of compounds to inhibit the known 3TC- and penciclovir-resistant mutants of HBV. We have stable cell lines with control wild-type HBV and the following mutations known to be associated with resistance of HBV to these agents:
• L526M (rtL180M) of Domain B & YMDD M550V (rtM204V) of Domain C (The most common mutation pattern observed during HBV breakthrough viremia)
• L526M alone (The most common mutation associated with penciclovir resistance; also associated with some resistance against 3TC).
• Control wild-type HBV
• Animal Studies
  We have a competitive arrangement with various subcontractors who perform the actual in vivo work. They provide us with the biological samples which we analyze for virologic and pharmacokinetic end-points. The following model systems may be used:
• Woodchucks & WHV
• HBV Transgenic mice

---

Anti-Hepatitis B Virus Evaluation
Anti-Hepatitis C Virus Evaluation

---