Towards novel immunocompetent animal models for hepatitis B virus infection

Hepatitis B (HBV) and delta (HDV) viruses are major causes of chronic hepatitis leading to the development of fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Given the high prevalence of HBV and the associated liver disease within the global population, those viruses pose major challenges for public health. Current treatments can effectively control HBV replication, however viral cure is rarely achieved and the risk of HCC still persists in patients with treatment induced-suppression of viral DNA (reviewed in [1]).

HBV and HDV share the same envelope proteins to enter into human hepatocytes, but the underlying molecular mechanisms are still only partially understood. Previously, the human sodium taurocholate cotransporting polypeptide (hNTCP) has been identified as a key receptor for HBV and HDV entry into human hepatocytes [2, 3]. This landmark discovery, more than 40 years after the identification of HBV, allowed the development of robust cell culture models enabling the study of the full HBV life cycle. However a major unmet need for a research tool remains: an immunocompetent animal model for the study of HBV infection. Currently, humanized mice engrafted with human hepatocytes provide one elegant approach to study of HBV and HDV infection, however these animals lack an immune system and therefore do not allow the study of antiviral immune responses, immune mediated pathogenesis or immunotherapeutic approaches [4]. Immunocompetent mice are resistant to HBV/HDV infection and over the past decades, the study of HBV/HDV infection in vivo had largely relied on chimpanzees - a model that cannot be used anymore due to ethical considerations. Unfortunately, while hNTCP transgenic or knock-in mice become to a limited extend susceptible to HDV infection, they still remain resistant to HBV infection [5, 6]. These disappointing findings encouraged the field to search for additional host factors [7] and to investigate the reasons for the different susceptibility of mouse hepatocytes to HBV and HDV.

In this issue of Hepatology, Lempp and colleagues from Stephan Urban’s laboratory at the University of Heidelberg/German Center for Infection Research (DZIF) further advance the knowledge of the function of NTCP as a species-specific HBV/HDV entry factor by studying how overexpression of hNTCP promotes HBV and HDV infection in primary hepatocytes from different animal species [8]. First, the authors demonstrate that hNTCP overexpression allows efficient binding of Myrcludex-B, a myristoylated peptide derived from the pre-S1 domain of the HBV large surface protein. This peptide specifically blocks the NTCP-receptor function and is presently in phase II clinical development. Furthermore, they show that hNTCP-overexpressing hepatocytes from macaque and pig became susceptible to both HBV and HDV infection while those from rat, mouse and dog remained refractory to HBV infection (Table 1). These results suggest that hNTCP is the only/major limiting factor for infection of macaque and pig hepatocytes. At the same time the results indicate the lack of an important host factor or the presence of a restriction factor in rat, mouse and dog hepatocytes. To further address this question, the authors studied the effect of the respective NTCP orthologues on HBV and HDV infection in the Huh7 human hepatoma cell line, naturally resistant to viral infection. Human NTCP as well as the rat, mouse and dog orthologues efficiently bound Myrcludex-B, while macaque and pig NTCP did not. However, only hNTCP was able to confer efficient HBV and HDV infection [8]. Taken together these data suggest that only hNTCP is able to confer HBV and HDV entry and that hepatocytes from distinct species lack additional essential entry factor or express a species-specific restriction factor. Since mouse, rat and dog NTCP strongly bind Myrcludex-B, while hepatocytes from those species cannot be infected when complemented with hNTCP, it could be speculated that the non-human NTCP orthologues might serve as a decoy for HBV and HDV. According to the authors this hypothesis can be excluded given that in their experimental set-up endogenous NTCP is not expressed anymore 3 days after cell seeding. In this regard it is of interest to note that the partial humanization of mouse NTCP by only a few amino acids promoting HDV and HBV infection of resistant human cell line [9] also triggers HDV, but not HBV infection, in a knock-in mouse model [5]. The HBV genome being able to replicate in mouse hepatocytes despite the lack of clear evidence for the presence of robust cccDNA levels [4], the different behavior of these two viruses sharing the same envelope proteins for entry is very intriguing. This difference could be also explained by the absence of a yet unknown factor important for HBV entry, or on the presence of a restriction factor preventing viral entry. It could also be the result of an impeded proper interaction between the virus and the hepatocytes of the respective species. In light of recently published data from the same group, it is likely that mouse hepatocytes rather lack a host dependency factor or do not interact properly with the virus than express a restriction factor [10]. Further studies are needed to identify these pathways and investigate whether the entry or post-entry block is mediated by the same factors in mouse, rat and dog hepatocytes.

Table 1. Features of HBV and HDV infection in primary hepatocytes from different species expressing hNTCP according to [8].

Primary hepatocytes transcomplemented with hNTCP
Myrcludex-B binding	+++	+++	+++	+++	+++
HDV infection HDAg positive cells	+++	++	+/-	+/-	+/-
HBV infection HBs positive cells	+++	++	-	-	-
HBc positive cells	+++	++	-	-	-
HBe secretion	+++	++	-	-	-
HBV RNA detection	+++	++	-	-	-
HBV cccDNA detection	+++	++	-	-	-
HBV genome secretion	++	++	-	-	-

HDAg, Hepatitis Delta Antigen, cccDNA, covalently closed circular DNA; HBs, hepatitis B surface antigen; HBc, hepatitis B core antigen; HBe, hepatitis B e antigen; +++, strong; ++, moderate; +/-, weak; -, negative

A key finding of this study is the observation that primary hepatocytes from macaque (cynomolgus and rhesus) and pig become fully susceptible to both HDV and HBV infection when transcomplemented with hNTCP [8]. Although viral replication appeared to be lower than in human hepatocytes, it was robustly detectable including formation of cccDNA [8]. This discovery is of high impact since it opens the perspective to develop a fully immunocompetent animal model for HBV infection based on macaques or pigs engineered to express hNTCP in the liver through adenovirus-associated virus-mediated expression or after genetic modification. The validity of this concept is further supported by an earlier study demonstrating that macaque hepatocytes can efficiently replicate HBV after baculovirus-mediated transfer of the HBV genome (reviewed in [4]). Given their close genetic relationship with humans, macaques are widely used for the preclinical development of human therapeutics including in vivo proof-of-concept and safety studies. Importantly, during the last decades a large body of knowledge has been obtained in the characterization of the macaque innate and adaptive immune responses (for example see [11]). Thus, a macaque-based HBV model would be ideal to study the efficacy and safety of novel immunotherapeutic approaches for HBV cure. However, at the same time the close genetic relationship of this species with humans is associated with ethical concerns. Furthermore, animal experimentation with non-human primates requires special care and infrastructure, is highly regulated and cost- and labor intensive. Thus, the perspective of a permissive pig-based model for HBV infection provides an interesting alternative as a more widely accessible and less costly animal model. Although the characterization of the pig immune system is less advanced compared to macaques, recent efforts in the application of this model for transplantation provide new tools to study virus-host interactions including the study of innate and adaptive immune responses [12]. A potential hurdle for both animal models could be lower levels of viral replication or strong virus suppressive innate immune responses limiting long-term persistence as observed in a humanized small animal model for hepatitis C virus infection [13] or Tupaia belangeri, a non-natural host for HBV infection [4].

Taken together, the study by Lempp et al. significantly advances our knowledge of HBV cell entry and species tropism. Although additional technical hurdles may still have to be taken, the approach of Lempp et al. provides a viable perspective for the development of robust immunocompetent animal models for the full HBV life cycle. A hNTCP-transgenic macaque or pig animal model will be a milestone to facilitate and accelerate the development of novel therapeutic strategies for HBV cure.