Toward novel immunocompetent animal models for hepatitis B virus infection
Authors: Laurent Mailly, Mirjam B. Zeisel, Thomas F. Baumert
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article On Page 703 Supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (U19AI123862‐01), the European Union (ERC‐2014‐AdG‐671231‐HEPCIR, FP7 HepaMAb, EU H2020 HepCAR), the French Cancer Agency (ARC IHU201301187), Agence Nationale de la Recherche (LABEX ANR‐10‐LAB‐28), and France Recherche Nord & Sud Sida‐hiv Hépatites (Infect‐ERA HBVccc). Hepatitis B virus (HBV) and hepatitis delta virus (HDV) are major causes of chronic hepatitis leading to the development of fibrosis, cirrhosis, and hepatocellular carcinoma. Given the high prevalence of HBV and the associated liver disease within the global population, these viruses pose major challenges for public health. Current treatments can effectively control HBV replication; however, viral cure is rarely achieved, and the risk of hepatocellular carcinoma persists in patients with treatment‐induced suppression of viral DNA (reviewed in Baumert et al.1). HBV and HDV share the same envelope proteins to enter into human hepatocytes, but the underlying molecular mechanisms are still only partially understood. The human sodium taurocholate cotransporting polypeptide (hNTCP) has been identified as a key receptor for HBV and HDV entry into human hepatocytes.2 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 the 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 has 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 extent susceptible to HDV infection, they remain resistant to HBV infection.5 These disappointing findings encouraged the field to search for additional host factors7 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 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 NTCP receptor function and is presently in phase 2 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 address this question, the authors studied the effect of the respective NTCP orthologs on HBV and HDV infection in the Huh7 human hepatoma cell line, which is naturally resistant to viral infection. Human NTCP as well as the rat, mouse, and dog orthologs 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 an additional essential entry factor or express a species‐specific restriction factor. Because 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 nonhuman NTCP orthologs might serve as a decoy for HBV and HDV. According to the authors, this hypothesis can be excluded given that in their experimental setup 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 a resistant human cell line9 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 covalently closed circular DNA 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 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 postentry 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 Lempp et al.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 ++ ++ − − − cccDNA, covalently closed circular DNA; HBc, hepatitis B core antigen; HBe, hepatitis B e antigen; HBs, hepatitis B surface antigen; HDAg, hepatitis delta 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 covalently closed circular DNA.8 This discovery is of high impact because 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 Allweiss and Dandri4). 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 recent decades a large body of knowledge has been obtained on the characterization of the macaque innate and adaptive immune responses (for example, see Snyder‐Mackler et al.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 nonhuman primates requires special care and infrastructure and is highly regulated, cost‐intensive, 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 that of 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 infection13 or Tupaia belangeri, a non‐natural host for HBV infection.4 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 overcome, the approach of Lempp et al. provides a viable perspective for the development of robust immunocompetent animal models for the full HBV life cycle. An hNTCP‐transgenic macaque or pig animal model will be a milestone to facilitate and accelerate the development of novel therapeutic strategies for HBV cure. Author names in bold designate shared co‐first authorship.