Introduction

Viral hepatitis infections are a major public health challenge globally. According to the World Health
Organization (WHO) Global Hepatitis Report, 2017, an estimated 257 million and 71 million people
suffer from chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, respectively. (1)
Chronic hepatitis infections may eventually lead to liver cirrhosis (LC) and hepatocellular carcinoma
(HCC), responsible for approximately 1.4 million deaths per year globally, making hepatitis-related
LC and HCC deaths comparable to mortality from HIV and tuberculosis (1,2). Of those deaths, 47%
are attributable to HBV, and 48% to HCV. However, these numbers are increasing. (2)

As a response to this public health threat, WHO adopted a global hepatitis strategy in
2015 that aimed to drastically reduce viral hepatitis by 2030, with the goal defined as a “reduction in
incidence by 90% and mortality by 65%.” (1) To meet WHO’s goal by 2030, development of new
diagnostics and therapeutics are urgently needed. (1,3)

HBV is an enveloped partially double-stranded deoxyribonucleic acid (DNA) virus with

a genome of 3,200 bp comprising four partially overlapping open reading frames (ORFs). These
ORFs encode the envelope (pre-S/S region), core (precore/C region), polymerase, and X proteins.
HBV polymerase, which acts as both reverse transcriptase and DNA-dependent DNA polymerase,
is critical for the replication of the HBV genome, making it an ideal target for nucleoside reverse
transcriptase inhibitors (NRTIs). (4,5) Lamivudine (2′,3′ Dideoxy 3′ thiacytidine [3TC]) is one such
NRTI, and the first of its kind to be approved to treat chronic HBV infections. 3TC has been shown
to be beneficial in various categories of patients. (6–8) However, it has also shown some severe side
effects and development of resistance. (Here I need a ref.)

Recently, Zelikin et al., (2017) made the unexpected discovery, that the synthetic
polymer, poly(ethylacrylic acid) (PEAA), binds to albumin without protein aggregation, causing it to
form deposits in the liver with pharmacological properties, namely anti-inflammatory and anti-HCV
activity, in hepatic cells. (9) This discovery raises the question of whether PEAA could also be used
as an agent for drug delivery of 3TC (lamivudine) in the treatment of HBV infections.

The objective of this study is to investigate the effect of 3TC conjugated to PEAA
(PEAA-3TC) in vitro on HepG2.215 cells (10) through an accurate quantification of HBV DNA using
the emerging technique of droplet digital polymerase chain reaction (ddPCR).
Results

1. Determination of HepG2.2.15 density and time point of antiviral treatment.

To determine the density and time points in which the HepG2.2.15 cells had to be seeded and treated
with the antiviral-drugs, an initial experiment was carried out.

As shown in Figure 1, the cells seeded at a density of 3*104 cells/100 μL/well show a consistent
increase (approx. twofold each day) of the extracellular HBV DNA secretion compared to the cells
seeded at different densities. At day six the cells seeded at 3*104 cells/100 μL/well have an
extracellular HBV DNA concentration, which is approx. 400% and 63% higher than the
corresponding cells seeded at a density of 6*103 and 104 cells/100 μL/well, respectively. Therefore,
we believe to have found the sweet spot in which the cells had to be seeded and treated. An
Adenovirus (AdV)-HBV was used as a positive control, and a non-template control (NTC) as negative
control.

2. Effect of antiviral treatment with 3TC and Tenofovir for fourteen days.

Based on the results in Figure 1, and experiments performed with different concentrations of 3TC
(data not shown), the cells were seeded (3*104 cells/100 μL/well) for 24 h prior to 3TC- and TFV
treatment of four concentrations (10 μM, 1 μM, 0,1 μM and 0,01 μM ) for fourteen days as shown in
Figure 2A. The 3TC treatment shows an effect, which is seen specifically on day 11 and 14. As
shown, the high concentration (10 uM) of treatment, shows a consistent higher secretion of HBV
DNA, which we believed due to a toxic effect that leads to release. Contrary, the TFV treatment (Fig.
2B) shows a higer effect and potency compared to that of the 3TC treatment, which is seen
throughout the experiment. Thus, the wells on day 14 for both treatments, were assessed by
microscope, and here an overgrowth and detachment of large patches of the cells floating in the
supernatant was observed. This might explain the result (outlier) seen at the lowest 3TC
concentration (0.01 uM) on day 14. Likewise, dying cells in the well of the high TFV concentration
(10 uM) was observed, which explains the low extracellular HBV DNA secretion throuhout the
experiment . The viability of the cells on day fourteen is shown in Figure/Table (xxx).

These observations lead to the fractionation of the samples of 1 uM of both treatments together with
an untreated sample of day 14 (data not shown) to asses whether intracellular HBV DNA was also
being analyzed, which the results showed no evidens of.
3. Dose-response (EC50) of 3TC, TFV and PEAA-3TC.

The antiviral treatment of the cells was repeated for only eight days, this time including the PEAA-
3TC with a drug load of 4.6 in two different titrations (2.17 µM-0,00217 µM and 10 µM-0,01 µM).
Bibliography

1. World Health Organization, World Health Organization, Global Hepatitis Programme. Global
hepatitis report, 2017 [Internet]. 2017 [cited 2018 Jan 16]. Available from:
http://apps.who.int/iris/bitstream/10665/255016/1/9789241565455-eng.pdf?ua=1

2. Stanaway JD, Flaxman AD, Naghavi M, Fitzmaurice C, Vos T, Abubakar I, et al. The global
burden of viral hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study
2013. Lancet Lond Engl. 2016 Sep 10;388(10049):1081–8.

3. Lok AS, Zoulim F, Dusheiko G, Ghany MG. Hepatitis B cure: From discovery to regulatory
approval. J Hepatol. 2017 Oct 1;67(4):847–61.

4. Ganem D, Varmus HE. The Molecular Biology of the Hepatitis B Viruses. Annu Rev Biochem.
1987;56(1):651–93.

5. Ganem D, Pollack JR, Tavis J. Hepatitis B virus reverse transcriptase and its many roles in
hepadnaviral genomic replication. Infect Agents Dis. 1994 Jun;3(2–3):85–93.

6. Lai C-L, Chien R-N, Leung NW, Chang T-T, Guan R, Tai D-I, et al. A one-year trial of
lamivudine for chronic hepatitis B. N Engl J Med. 1998;339(2):61–68.

7. Dienstag JL, Schiff ER, Wright TL, Perrillo RP, Hann H-WL, Goodman Z, et al. Lamivudine
as initial treatment for chronic hepatitis B in the United States. N Engl J Med.
1999;341(17):1256–1263.

8. Liaw Y-F, Sung JJ, Chow WC, Farrell G, Lee C-Z, Yuen H, et al. Lamivudine for patients with
chronic hepatitis B and advanced liver disease. N Engl J Med. 2004;351(15):1521–1531.

9. Riber CF, Andersen AHF, Rolskov LA, Zuwala K, Gajda P, Løvschall KB, et al. Synthetic
Polymer with a Structure-Driven Hepatic Deposition and Curative Pharmacological Activity in
Hepatic Cells. ACS Macro Lett. 2017 Sep 19;6(9):935–40.

10. Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in Hep G2 cells
transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A. 1987
Feb;84(4):1005–9.

11. Sprinzl MF, Oberwinkler H, Schaller H, Protzer U. Transfer of Hepatitis B Virus Genome by
Adenovirus Vectors into Cultured Cells and Mice: Crossing the Species Barrier. J Virol. 2001
Jun 1;75(11):5108–18.