Globally, around 250 million people are infected with hepatitis B virus (HBV) and more than 1 million more are estimated to become infected yearly. Researching the HBV viral life cycle and identifying potential novel therapeutics is challenging as only human and chimpanzee hepatocytes are permissive to HBV. However, due to the cost and ethical restrictions, alternative models are being explored. In vivo, rodent models including humanized liver chimeric mice (for example PXB-mice) and the AAV-HBV mouse model are being utilized. While these models are useful, each has their own pitfalls. The AAV-HBV model fails to replicate the complete HBV lifecycle and while humanized liver chimeric mice are permissive to HBV, they lack an adaptive immune response. Whereas, in vitro primary human hepatocytes (PHH) are the gold standard for HBV research. However, due to the inconsistencies between donor lots and limited availability, using PHH often requires researchers to devote time and resources to find a suitable donor human hepatocyte.
Introducing In Vitro Tools for Viral Hepatitis
PXB-cells® are mature and fully functional primary human hepatocytes which are freshly isolated from the PXB-mouse®, a humanized liver chimeric mouse model. Propagating human hepatocytes through the PXB-mouse model allows for a stable supply of fresh ready-to-use PHHs from the same source of donor human hepatocytes minimizing the search & optimization time for researchers. 
.png?width=350&height=201&name=rect431%20(1).png)
Figure 1: PXB-cells remain susceptible to de novo HBV infection at 28 days post-HBV infection (dpi). Western blot analysis shows robust sodium taurocholate co-transporting polypeptide (NTCP) expression up to 28 dpi in PXB-cells. Additionally, other hepatocyte markers including cytochrome P450 3A4 (CYP3A4), aldehyde dehydrogenase 2 (ALDH2), ornithine transcarbamylase (OCT), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) are maintained throughout long-term infection in PXB-cells. (Chida, T et al., 2023, from Supplemental Figure 1)
Compared to cryopreserved primary human hepatocytes and other in vitro tools (including human cell lines Huh7, HepG2, HepaRG, and iPSCs), PXB-cells maintain stability, both morphologically and in terms of their gene expression profile, long-term1. Their stable expression of phase I and II metabolic enzymes and transporters offer a reliable model for drug metabolism testing. They are susceptible to a wide range of HBV inoculums including different genotypes and patient-derived inoculums2. Furthermore, their robust long-term expression of sodium taurocholate co-transporting polypeptide (NTCP), an HBV entry receptor, demonstrates that PXB-cells remain susceptible to HBV throughout long-term cultures3, unlike hepatocyte cell lines which either lack NTCP or have low endogenous levels (Figure 1). Furthermore, HBV-infected PXB-cells express viral proteins and support viral replication allowing researchers to develop therapeutics that target the natural lifecycle of HBV2.
Targeting cccDNA In Vitro Using PXB-cells
For example, Dr. Liudi Tang and colleagues recently published a paper utilizing the PXB-cells model to show that inhibition of sirtuin 2 (SIRT2), a cellular deacetylase, disrupts the conversion of viral relaxed circular DNA to covalently closed circular DNA (cccDNA), ultimately blocking de novo synthesis of cccDNA4. In their study, they used an allosteric inhibitor of SIRT2, FLS-359, to investigate how SIRT2 modulation can impact HBV infection. They found that in addition to blocking de novo synthesis of cccDNA (Figure 2), SIRT2 inhibition significantly decreased HBV RNA, HBsAg, and HBeAg levels demonstrating its potential to reduce viral replication to a similar level as MyrcludexB (MyrB) (Figure 3). Pre-treating PXB-cells short-term with SIRT2 inhibitor prior to infection significantly reduced the levels of cccDNA, albeit it was unable to reduce pre-existing cccDNA levels. Taken together, FLS-359 treatment limits HBV infection markers.
Figure 2: Sirtuin 2 inhibition decreases de novo synthesis of cccDNA in PXB-cells (A) but has no effect on the existing cccDNA levels (B). (Tang, L et at., 2024, from Figures 1 and 3)
Figure 3: FLS-359 treatment significantly reduces levels of HBV RNA, HBsAg, and HBeAg in HBV-infected PXB-cells to similar levels as Myrcludex B (MyrB) treatment. (Tang, L et al., 2024, from Figure 1)
Current HBV therapeutics often fail to achieve a functional cure primarily due to their inability to completely clear hepatic cccDNA. Although this novel SIRT2 inhibitor does not reduce the existing cccDNA pool, it offers a means to block new formation of cccDNA. With the intrahepatic turnover of cccDNA, blocking de novo synthesis will ultimately lead to reduced cccDNA levels. Given this, SIRT2 inhibition in combination with antiviral therapeutics may lead to a functional cure of HBV.
PXB-cells isolated from humanized livers allow the maintenance of hepatocyte differentiation and HBV replication for up to 30 days.
Other strategies to target cccDNA have also been tested in PXB-cells. Smekalova et al., used cytosine base editing, a gene editing approach that leads to deamination and the conversion of cytosine to thymine in target sequences5. Thus, stop codons can be introduced at specific loci in the in the HBV genome. This study found that these strategies could be used successfully to reduce viral markers in HBV-infected PXB-cells. Moreover, no rebound was observed 2-weeks after the end of treatment using this gene editing approach, unlike treatment with lamivudine (3’-thiacytidine, 3TC) which had a rebound in HBV DNA levels (Figure 4). Further experiments showed that the base editor had successfully silenced cccDNA and integrated HBV DNA, and this was also observed in vivo.
Figure 4: Cytosine base editing targeting the HBV genome (red line/bars - gRNAs(37+40)) successfully reduced viral parameters including HBsAg, HBeAg, total HBV DNA, and 3.5kb RNA in PXB-cells with no observed rebound in HBV DNA levels. (Smeklova, EM et al., 2023, from Figure 3)
Together, these studies show that PXB-cells provide a new gold standard for studying viral hepatitis in vitro. Longevity in culture, coupled with their ability to support the entire life cycle of HBV (including cccDNA) provide a valuable platform for investigating a wide range of novel therapeutics, from small molecules through to new modalities including gene editing.
References
- Yamasaki, C et al., (2020) Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: Novel, long-term, functional two-dimensional in vitro tool for developing new drugs. PLoS ONE, 15(9):e0237809.
- Ishida, Y et al., (2015) Novel robust in vitro hepatitis B virus infection model using fresh human hepatocytes isolated from humanized mice. Am J Pathol., 185(5):1275-85.
- Chida, T et al., (2023) Persistent hepatic IFN system activation in HBV-HDV infection determines viral replication dynamics and therapeutic response. JCI Insight, 8(9):e162404. Supplemental Figure 1
- Tang, L et al., (2024) An allosteric inhibitor of sirtuin 2 blocks hepatitis B virus covalently closed circular DNA establishment and its transcriptional activity. Antiviral Research, 226:105888.
- Smekalova EM, et al., (2023) Cytosine base editing inhibits hepatitis B virus replication and reduces HBsAg expression in vitro and in vivo. Mol Ther Nucleic Acids. 2023 Dec 27;35(1):102112.