Materials and Methods

Adenoviral vectors (AdV-HBV)

The lysate of Adenovirus (AdV)-HBV and AdV-GFP (MOCK) constructs were kindly provided by
Martin Kristian Thomsen (Department of Clinical Medicine, Aarhus University, Denmark). The AdV-
HBV DNA was prepared and used in the ddPCR for determining the limit of detection (LOD) of the
assay, and as positive controls when quantifying the extracellular HBV DNA in untreated
HepG2.2.15 (10) cells.

LOD of the ddPCR assay

For an accurate quantification of the extracellular HBV DNA in 3TC, Tenofovir, PEAA-3TC treated
HepG2.2.15 cells, an initial experiment was carried out to develop a ddPCR assay determining the
LOD of the assay. Seven five-fold serial dilutions of the AdV-HBV DNA were carried out from a
starting stock of 500x dilution. For each dilution, triplicates were analyzed along with triplicates of
MOCK and negative controls (data not shown). Also, a whole ddPCR TM 96-Well Plate (Bio-Rad,
Hercules, CA, USA) containing only negative controls were analyzed determining the rate in which
false positives occur (data not shown). Based on evidence from this in-house experiment, samples
were, following these criteria, considered positive if: ≥1 droplet in 1 triplicates with an amplitude
threefold higher than the negative droplets. Samples were considered positive if meeting

ddPCR assay

The quantification of the extracellular HBV DNA was done using the QX100 TM Droplet DigitalTM
PCR System (Bio-Rad, Hercules, CA, USA) according to the instructions provided by the
manufacturer. In brief, 2X ddPCR Supermix (Bio-Rad, Hercules, CA, USA), 102,86 nmol/L HBV-
forward [5’-TTCCGGAAACTACTGTTGTTAGAC-3] and 91,05 nmol/L HBV-reverse [5’-
GCAACATACCTTGATAGTCCAGAAGAA-3’] (LGC Biosearch Technologies, Petaluma, CA, USA),
100 nmol/L HBV probes [5’-6FAM-CCCTAGAAGAAGAACTCCCTCGCCTC-3’] (ThermoFisher,
Waltham, Massachusetts, USA), nuclease-free water and finally the DNA template comprised the
20 μL ddPCR reaction mixture.

Droplets were generated using the QX100 Droplet Generator (Bio-Rad, Hercules, CA, USA), by
adding 65 μL of DG-oil to the DG8 cartridge wells for each ddPCR reaction. The generated
droplets were then transferred, by aspirating 40 μL, from the DG8 cartridge to corresponding wells
of a ddPCR 96-Well Plate (Eppendorf, Hamburg, Germany). Subsequently, the ddPCR 96-Well
Plate was heat-sealed with foil at 180 °C using the PX1™ PCR plate sealer (Bio-Rad, Hercules,
CA, USA), and amplified in the C1000 Touch™ thermal cycler (Bio-Rad, Hercules, CA, USA) under
following cycling conditions: 1) an initial denaturation cycle of 10 min at 95 °C, 2) 40 cycles of
denaturation for 30 s at 94 °C, extension for 60 s at 58 °C (ramping rs), and a final incubation for
10 min at 98 °C, ending with cooling at 4 °C.

After amplification, the ddPCR™ 96-Well Plate was transferred to the QX100 Droplet Reader (Bio-
Rad, Hercules, CA, USA) in which the readouts were analyzed using the QuantaSoft™ analysis
software version xxx. Thus, to determine the final concentration of templates, the results were
multiplied by the dilution factor.

Cell line

HepG2.2.15 cells were used to evaluate the effect of the antiviral drugs in vitro, and was kindly
provided by Prof. Dr. Ulrike Protzer (Institute of Virology, Technical University of Munich, Germany).
In brief, the cells were cultured and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM;
Biowest, Nuaillé, France) High Glucose w/ L-Glutamine, w/ Sodium Pyruvate, and non-essential
amino acids (NEAA), supplemented with 10% Fetal Calf Serum (FCS), and 1% Penicillin-
Streptomycin (Biowest, Nuaillé, France). The cells were subcultured twice weekly and incubated at
37°C, 5% CO2.

Antiviral drug treatments

All antiviral drugs used (3TC, Tenofovir, and PEAA-3TC) were kindly provided by Dr. Alexander N.
Zelikin (Department of Chemistry, Aarhus University, Denmark). To determine the concentration
and time points in which the HepG2.2.15 cells had to be treated with the antiviral drugs, an initial
experiment was carried out. The cells were seeded at three different densities (6*10 3, 104, and
3*104 cells/100 μL/well) in a TC Plate 96 Well,Cell+,F (Sarstedt AG & Co., Nümbrecht, Germany).
The supernatants of the cells were harvested after 1-, 2-, 3-, and 6 days, and the extracellular HBV
DNA concentrations were analyzed.

Based on this experiment the cells were seeded (3*104 cells/100 μL/well) for 24 h prior to 3TC- and
Tenofovir treatment of four concentrations (10 μM, 10 μM, 1 μM and 0,1 μM ) for 14 days, and
were fed with a fresh drug-containing medium every 2-3 days. The HBV DNA was extracted from
the supernatants and assayed by ddPCR at day 8,11, and 14. Untreated cells were used as
controls.
HBV DNA extraction

The extracellular HBV DNA was extracted from the supernatant of HepG2.2.15 using the AllPreb
DNA/RNA mini Kit (Qiagen, Hilden, Germany) following the instructions of the manufacturer. Thus,
the extracellular HBV DNA was eluted in 50 μL of EB buffer. The DNA template was used to
amplify the HBV S-region when performing the ddPCR.

Cell viability assay

The supernatants of the cells were removed, and each well was washed with 180 µl of phosphate-
buffered saline (PBS) before adding 20 µl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide), (5g/L). The plates were incubated in 37°C for 2,5 h. After incubation,
the supernatant of each well was removed, and 100 µl of dimethylsulfoxide (DMSO) + 96 %
Ethanol was added. The final measurement (endpoint) of the absorption was performed with a
spectrophotometer (xxxxx) at 490 nm and 650 nm.

Statistical analysis

All data were analyzed using the GraphPad Prism version 7.04 (GraphPad Software Inc., San
Diego, CA, USA), and all graphs are expressed as the mean and 95% CI (Poissons confidens
limits) provided by QuantaSoft™ analysis software version (xxx).