Confluent cultures of 2.2.15 cells maintained on 48-well flat-bottomed tissue culture plates are treated as described for the primary assay. HBV virion DNA levels in the culture medium and cytotoxicity are assessed as described for the primary assay.  In addition, intracellular HBV DNA replication is measured by quantitative Southern blot hybridization analysis (Korba & Gerin, 1992, Antivir.Res.19:55). EC₅₀, EC₉₀ and S.I. values are calculated for both virion DNA and intracellular HBV DNA replication intermediates.

Tertiary assays

In addition to standard antiviral assays, several other types of anti-HBV activities can also be assessed.

Drug combination

Compounds are mixed at approximately equipotent concentrations and this molar ratio is maintained during serial dilution (Korba, 1996, Antivir. Res. 29:49; Iyer et al., 2004).  To compensate for potential unforeseen interactions (e.g. changes in uptake, metabolism, etc.), the concentration of one compound is altered approximately 3-fold higher of lower relative to the second compound so that three different ratios are used in one experiment.  Cultures are treated with 6-8 serial dilutions of the mixtures, as with the corresponding monotherapies, as described for the primary assay.  Evaluation of drug interactions in the combination treatments is conducted against the corresponding monotherapies in the same experiments using the Combostat® (Biosoft, Inc.) analysis software.  For combination treatments, EC50, EC90, CC50 and S.I. (CC50/EC90) are presented for the first compound listed.  The molar ratio of the compounds in each combination is also indicated.

Drug-resistant HBV

Activity against recombinant HBV carrying clinically relevant mutations that confer resistance to licensed drugs is performed using transient transfection of HBV DNA (Tatti, et al., 2002, Antivi. Res.55:27; Iyer, et al., 2004, AAC 48:2199).  Cultures are transfected in 48-well culture plates with Lipofectamine 2000™ (Gibco, Inc) following the manufacturer's procedure.  Beginning three days post-transfection, cultures are treated for 5 days with antiviral compounds.  Antiviral activity is determined by quantitative Southern Blot hybridization of intracellular HBV DNA replication intermediates.  Currently the following mutants are available: lamviudine (LMV)-resistant, polM204V, polM204I, polL180M, polM204V/L180M (Allen et al., 1998, Hepatology 27:1670); adefovir dipovoxil (ADV)-resistant, polN236T (Angus, et al., 2003, Gastroenterology 125:292).  Standardized nomenclature is used for HBV polymerase assignment (Stuyver, et al., 2001, Hepatology 33:751).  Additional mutants (TNF^R, ETV^R) are under construction.

HBV protein production and RNA transcription

Semi-quantitative ELISA-based analysis of HBV proteins is performed (Korba & Gerin, 1995, Antivir. Res. 28:225; Korba, et al., 2008, Antivir. Res. 77:56) using samples diluted (2 to 10-fold) to bring levels into the dynamic response ranges of the assays.  Qualitative analysis of HBV proteins is also performed using standard Western blot techniques (Muller, et al., 1992, J. Infect. Dis. 165:929).  HBV surface (HBsAg) and HBV e (HBeAg) antigens are analyzed from culture medium samples and HBV core (HBcAg) is analyzed from intracellular lysates (normalized for total cell protein content in each culture sample).  Intracellular HBV RNA (normalized to the level of cellular B-actin RNA in each culture sample) is assessed by quantitative northern blot hybridization (Korba & Gerin, 1995, Antivir. Res. 28:225).

Southern Research Institute Details

Anti-Hepatitis B Virus (HBV) Evaluation

1. 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 real time quantitative PCR (TaqMan) assay to directly and accurately measure HBV DNA copies. The analysis of this data allows us to calculate:

*        Antiviral activity

*        Compound Cytotoxicity

*        Therapeutic index (toxicity/antiviral activity)

2. Secondary Antiviral Evaluations
   A different HepG2 stable cell containing an HBV adr1 strain genome is employed in the assay. The activity of compounds acting in HepG2 2.2.15 can be confirmed in the same manner using this cell line.
3. Combination Studies
   After the antiviral activity of test compounds against HBV is confirmed, the interactions of the compounds with 3TC, IFN alpha 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.
4. Toxicity Panel
   To evaluate compounds for toxicity against a large panel of established cell lines, PBMCs and human primary hepatocyte cultures.
5. 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
ELISAs are used 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.

6. HBV Drug Resistance Evaluation
   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