Despite the availability of a vaccine against hepatitis B virus (HBV), over 350 million people worldwide are chronically infected with this virus and over 600,000 people die per year as a result of chronic HBV infection. The persistence of the HBV is due to the formation of covalently closed circular DNA (cccDNA) which acts as the template for viral transcription. Treatment with nucleosides or nucleotide analogues does not cure HBV due to the cccDNA; therefore, much effort has been invested in models that can elucidate the mechanisms of viral replication and prevent it with non-toxic therapies.
Very few cell types are available to investigate the entire process of HBV infection - from entry to replication and re-infection. HuH7 or HepG2 cells do have the apparatus to allow replication once transfected but like all other cell lines, they lack the receptors allowing their entry into the cells. By contrast, HepaRG™ cells express the membrane receptors to allow HBV infection and can also be used to investigate the regulation of gene transcription. For this reason, there have been multiple publications using HepaRG™ cells as a model to investigate HBV (Go to Publications). The ability of HBV to replicate is species-specific and only occurs in polarised and differentiated hepatocytes. Since the basolateral NTCP transporter has been demonstrated as theHBV receptor (Yan et al., eLife, 2012), HepaRG ™ cells must be completely differentiated to allow virus entry. The extent of HBV infection of HepaRG™ cells is dependent on the concentration of the virus added to the culture and the time of virus inoculation
Although IFN-γ and TNF -α can control HBV, they cannot be used in its therapy because they cause severe side-effects. Moreover, when treatment of HBV with nucleoside analogues such as lamivudine is discontinued, there is a rebound of HBV replication. Lucifora et al. (Science, 2014) used HepaRG™ cells to identify a pathway that could be exploited to induce a persistent antiviral effect. This pathway is regulated by the lymphotoxin β receptor (LTβR –TNFs are the natural ligands) and its activation leads to HBV cccDNA-specific degradation by deamination in HBV-infected cells. Importantly, LTβR agonists (e.g. antibodies) not only reduce HBV cccDNA, HBV surface antigen secretion, and HBV-DNA replication, when the agonist treatment was stopped, there was no rebound of HBV replication. Moreover, the antiviral effects of LTβR agonists were also demonstrated - and shown to be non-toxic - in HBV-transgenic mice, supporting the use of HepaRG™ cells as a relevant in-vitro model for investigating HBV infection pathways and potential therapies.
The original hepatic cells from which HepaRG™ were derived were isolated from a donor who was infected with Hepatitis C (HCV), which was why the researchers at Inserm were interested in this particular batch of cells (Go to Origins of the HepaRG cell line). However, as the cells were passaged, they lost all the HCV markers such that HepaRG™ cells are not infected with HCV. As with HBV, HepaRG™ cells have also been shown to be a good model for HCV infection for use in developing antiviral therapies. For example, the uptake of HCV into cells occurs via hepatic cholesterol uptake pathways and this process can be recapitulated in HepaRG™ cells and used to demonstrate that drugs inhibiting lipid transport also inhibit uptake of HCV (Lucifora et al., Antiviral Res., 2014).
The HVC envelope protein, E1E2, is essential for virus binding to host cells (Figure 2); therefore, agents that inhibit this binding could be promising anti-HCV agents. Ndongo-Thiam et al. (Hepatology, 2011) demonstrated that an E1E2-specific monoclonal antibody (D32.10) inhibits ~90% entry of serum-derived HCV particles in HepaRG™ cells. Notably, unlike HBV, both proliferating progenitor and differentiated HepaRG™ cells were susceptible to HCV infection. After infection of proliferating progenitor cells with serum derived HCV particles, approximately 50-60% of the resulting differentiated HepaRG™ were infected with HCV and this persisted for 28 days; thus, these cells support long-term production of infectious lipoprotein-associated enveloped HCV articles.
Infection by hepatitis E (HEV) is not as serious as HBV or HCV but, unlike HBV and HCV, this zoonotic virus can be transmitted from animals (the swine population being a major HEV reservoir) by consumption of raw or undercooked contaminated meat. Rogée et al. (J General Virology, 2013) investigated the application of differentiated HepaRG™ cells to better understand the life cycle of HEV. They demonstrated the complete replication of HEV and the release of encapsidated RNA into the cell culture medium over a month and that HEV infection could be inhibited by anti-HEV antibodies, suggesting specific virus-cell interactions. HepaRG™ cells are thus a promising cell type to investigate the pathways involved in HEV entry, replication and exit in host cells and to identify the zoonotic potential of different HEV strains.
Until recently, only in-vivo models were available to investigate the processes involved in malaria infection and relapse. Some success was achieved by culturing primary monkey hepatocytes and infecting them with P. cynomolgi sporozoites; however, the short life-span of the hepatocytes, particularly once infected with sporozoites, prevented comprehensive measurements and the optimisation of screening assay for treatments (Dembélé et al., PLoS ONE, 2011). Further work by Dembélé et al. resulted in a modified culture procedure in which monkey primary hepatocytes were co-cultured with HepaRG™ cells. The HepaRG™ cells represent only 1/30 of the culture and are themselves not infected, but protect the monkey hepatocytes and plug gaps (Dembélé et al., Protocol Exchange, 2014). The HepaRG™ cells were fluorescently-labelled with GFP in order to discriminate them from the primary hepatocytes. The resulting co-cultures could be maintained for approximately one month and allowed enough time to enrich hypnozoites (uninucleate hepatic forms of sporozoites that persist for months in a dormant state) before resuming their development into mature hepatic schizonts. This technology has been extended to model the infection of human primary hepatocytes using Matrigel overlay and subsequent development of antimalarials with lower side-effects than the currently used drug, quinidine (Figure 3, Dembélé et al., Nature Medicine, 2014).
Figure 1. HBV infection of HepaRG™ cells.
(A) HBV RNA levels ± PEG after infection at a multiplicity of infection of 200, 400, 800, or 1,600 genome equivalents per cell (Geq/cell). M = cell mock-infected cells. The relative amounts of subgenomic RNA detected in infected cells are indicated below the figure (%). (B) intracellular HBV DNA over 10 day post-infection (pi), RC = relaxed circular form, CCC = covalently closed circular DNA, (C) HBV surface antigen (HBsAg) secreted into the supernatant over 15 days after infection. HepaRG™ cells () or HepG2 cells ();
Adapted from Gripon et al., PNAS, 2002.
Figure 2. Detection of HCV E1E2 in HepaRG™ cells 28 days after infection with serum-derived HCV particles.
Adapted from Petit et al., 2012
|(B) HCV infected
28 days post-infection
Figure 3. Activation of hypnozoites () and sporozoites () in (A) human hepatocytes cultured with (+) or without (-) HepaRG™ cells and (B) in co-cultures of HepaRG™ cells and monkey hepatocytes treated with solvent control (-) or the anitmalarial drug, atovaquone (AQ - kills sporozoites but not hypnozoites) with or without a Matrigel overlay.
Pf PE forms = preerythrocytic forms.
Adapted from Dembélé et al., Nature Medicine, 2014.
(A) Human hepatocytes (B) Monkey hepatocytes