Hepatitis B is an infectious illness caused by hepatitis B virus (HBV) which infects the human liver and causes an inflammation called hepatitis. Originally known as “serum hepatitis”, the disease has caused epidemics in parts of Asia and Africa, and it is endemic in . About a third of the world’s population, more than 2 billion people, have been infected with the hepatitis B virus. This includes 350 million chronic carriers of the virus. Transmission of hepatitis B virus results from exposure to infectious blood or body fluids.
The acute illness causes liver inflammation, vomiting, jaundice and, rarely, death. Chronic hepatitis B may eventually cause liver cirrhosis and liver cancer—a fatal disease with very poor response to current chemotherapy.
Hepatitis B virus is an hepadnavirus—“hep” from “hepar” (liver in Latin) and “dna” because it is a DNA virus – and it has a small circular genome of partially double-stranded DNA. The viruse replicates through an RNA intermediate form by reverse transcription, and in this respect it is similar to retroviruses. Although replication takes place in the liver, the virus spreads to the blood where virus-specific proteins and their corresponding antibodies are found in infected people. Blood tests for these proteins and antibodies are used to diagnose the infection.
We offer the following assays for the HBV drug discovery and development programs:
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 to calculate:
Therapeutic index (toxicity/antiviral activity)
Drug Combination Studies
After the antiviral activity of test compounds against HBV is confirmed, the interactions of the compounds with 3TC, IFNs and other compounds in terms of efficacy (synergy, additivity, antagonism) and toxicity (combination toxicity) are evaluated in the 2.2.15 cells using the quantitative HBV TaqMan PCR assay.
We can test compounds for toxicity against a large panel of established cell lines and also primary cell cultures, including PBMCs and human primary hepatocytes.
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.
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 drug-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)
Control wild-type HBV
ChemDiv has 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: