Med Microbiol Immunol (2015) 204:95–102 DOI 10.1007/s00430-014-0378-6
REVIEW
Mouse models for therapeutic vaccination against hepatitis B virus Claudia Dembek · Ulrike Protzer
Received: 25 July 2014 / Accepted: 2 October 2014 / Published online: 19 December 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract A mouse model for persistent HBV infection is essential for the development of a therapeutic vaccine against HBV. Because HBV cannot infect mouse hepatocytes, even if the HBV receptor is introduced, surrogate models are used. A suitable model needs to establish persistent HBV replication and must allow the establishment of HBV-specific adaptive cellular and humoral immune responses. Therefore, an immunocompetent mouse model is needed in which one can break HBV-specific tolerance and ideally eliminate the HBV transcription template. The most widely used model for chronic HBV infection is the HBV transgenic mouse. Although HBV replicates from an integrated transgene, HBV-specific immune tolerance can be broken upon adequate immune stimulation because antigen expression only starts shortly before birth. Alternative mouse models of chronic HBV infection are generated by introducing HBV genomes either using viral vectors or using hydrodynamic injection. In these alternative models, the HBV transcription template is introduced into a proportion of hepatocytes and stays extra-chromosomal. It thus mimics the natural HBV transcription template, the HBV cccDNA in humans. Unlike an HBV transgene, however, it can be cleared upon appropriate treatment or
This article is part of the special issue “Therapeutic vaccination in chronic hepatitis B—approaches, problems, and new perspectives”. C. Dembek · U. Protzer (*) Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Trogerstr. 30, 81675 Munich, Germany e-mail: [email protected] C. Dembek · U. Protzer German Center for Infection Research (DZIF), Brunswick, Germany
immune stimulation. Human hepatocyte chimeric mice in which murine hepatocytes are widely replaced by human hepatocytes represent another important mouse model for persistent HBV infection. These mice are susceptible for HBV infection, but need to be severely immune deficient to accept human hepatocytes. In conclusion, a variety of mouse models for persistent HBV infection are available suitable for preclinical efficacy evaluations of therapeutic vaccination strategies against HBV. Keywords Chronic hepatitis B · Mouse model · Therapeutic vaccination · Adaptive cellular immune response · Transgenic mice · Human hepatocyte chimeric mice
Introduction Hepatitis B virus (HBV) infects the human liver and often causes inflammatory liver disease (hepatitis B). HBV infection during adulthood is resolved spontaneously in more than 95 % of cases. In contrast, vertical transmission of HBV from hepatitis B e antigen (HBeAg)-positive mothers to their neonates results in up to 90 % of infants in chronic infection. Chronic HBV infection is defined by HBV persistence for more than 6 months with detectable hepatitis B surface antigen (HBsAg) in the blood. It is typically characterised by high circulating HBV titres >2,000 international units (IU)/mL HBV DNA (corresponding to 104 HBV DNA copies/mL blood), and a risk to develop liver cirrhosis and hepatocellular carcinoma [1]. The failure to control HBV infection and subsequent establishment of chronicity often results in exhaustion of virus-specific T-cell responses. HBV-specific T-cell tolerance has been suggested to be induced mainly by high viral
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antigen load, which would be regarded as peripheral tolerance, but this has not formally been proven yet. In contrast to central tolerance that eliminates immature self-reactive lymphocytes in the thymus, peripheral tolerance (the status of HBV-specific T-cell tolerance) prevents mature, circulating lymphocytes from reacting to self- or chronically persisting antigens through clonal anergy and clonal deletion and thereby prevents chronic systemic inflammation. It usually takes place in peripheral lymphoid organs like lymph nodes, tonsils, spleen and mucosal-associated lymphoid tissues. In chronic HBV infection, two mechanisms have been suggested to be responsible for HBV-specific peripheral tolerance. (1) The liver environment supports tolerance induction [2]. As a metabolic organ, the liver removes gut-derived microbial antigens from the blood. Parenchymal and nonparenchymal antigen-presenting cells (APCs) in the liver induce peripheral tolerance towards such antigens to avoid chronic activation of the immune system [3]. (2) Neonatal tolerance [4, 5] can additionally explain the inability of perinatally infected chronic HBV carriers to sufficiently respond to HBsAg, HBeAg and HBV core protein. The idea of therapeutic vaccination is to overcome HBV-specific peripheral tolerance in chronic carriers and activate a potent and timely restricted cytotoxic T lymphocyte (CTL) response that eliminates HBV with minimized hepatocyte damage. A small animal model for HBV infection is a prerequisite for antiviral compound development and preclinical testing of novel therapeutic vaccination strategies. Ideally such animal models allow establishing chronic HBV infection and the induction of peripheral tolerance in order to break this tolerance by therapeutic vaccination. HBV can infect only humans, apes, tree shrews (Tupaia belangeri) and recently discovered macaques [6]. A series of HBV-related hepadnaviruses were discovered in the past (in ducks, geese, herons, woodchucks, squirrels and in woolly monkeys) [7]. Amongst the hosts, woodchucks and Pekin ducks represent established animal models for investigating HBV-related hepadnaviruses. Both models provided essential knowledge about HBV-related disease [8, 9] and served in preclinical efficacy studies to investigate therapeutic vaccines in chronic hepatitis B [10, 11]. Recently, a new hepadnavirus species related to HBV was discovered in bats [12]. The main weakness of woodchucks or ducks as models for vaccine development is that their respective hepadnaviruses species is genetically distinct from human HBV. Thus, hepatitis B vaccines cannot be tested in these animals, and vaccine proteins and constructs need to be adapted to the respective virus strain. Other difficulties are that our understanding of the immunobiology of these hosts is limited, and basic reagents and tools required for immunologic investigations are restricted. Therefore, working with these animals is tedious and the analysis of immune responses to therapeutic vaccination strategies is
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limited. The newly discovered macaque HBV, however, is 99 % identical to human HBV [6], and macaques are well established in preclinical studies. So this may open a new avenue in the coming years. Currently, the mouse is the most widely used experimental animal due to various reasons. A short generation time of 6–8 weeks makes them a cost-effective and efficient tool for rapid screening and research. The mouse genome is easy to manipulate and thereby provides a powerful tool to model specific diseases. Due to the wide use of laboratory mice, all standard tools and methods (e.g. detection of immune responses) are readily available. HBV cannot naturally infect mice; however, different surrogate murine infection models have been generated in the past years, which proved to be useful for vaccine studies, but all have distinct limitations. By now, it is still not possible to fulfil all requirements towards an animal model of chronic HBV infection within one mouse model, and the complementary use of appropriate models can be considered for preclinical efficacy tests. The most important mouse models for acute and chronic HBV infection are described below, and their significance for the establishment of a therapeutic vaccine against HBV is discussed.
HBV transgenic mice Embryo microinjection technology enabled the insertion of a part or a complete copy of the HBV genome into mice and allows studying HBV immunobiology and pathogenesis. A series of HBV transgenic mouse lines have been engineered, expressing single HBV gene products (reviewed in [13]) or replicating HBV [14–16]. A widely used model until today is the HBV1.3 transgenic mouse [16]. From an integrated 1.3 over-length HBV transgene, this lineage produces HBsAg, hepatitis B core antigen (HBcAg) and HBeAg. HBV replicates in the liver and releases infectious virus into the blood at levels comparable to chronic HBV infection in humans, but the virus cannot spread. Like in chronically HBV-infected humans, serum HBeAg levels in HBV1.3 transgenic mice vary significantly between different animals and directly correlate with HBsAg and serum HBV DNA levels. Thus, serum HBeAg level serves as a parameter, which indicates efficacy of transcription of the HBV pregenome and of resulting virus replication. A major limitation of HBV transgenic mice is that HBV replicates from an integrated transgene that cannot be eliminated. Furthermore, mouse hepatocytes do not establish cccDNA, the natural template for HBV transcription [13, 16] limiting the use of mice for natural HBV infection. Consequently, the proof-of-concept of therapeutic vaccination, viral clearance and eradication of HBV cccDNA, cannot be
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achieved in this model. Thus, alternative endpoints are used: (1) the induction of a strong and timely restricted HBV-specific CTL response, (2) the induction of neutralizing antiHBs antibody responses and (3) safety of the immunization scheme with limited liver damage. T-cell responses as well as anti-HBs seroconversion are expected to accompany HBV clearance as they were reported in HBV carriers before clearance of the virus, either spontaneously or by antiviral drugs [17]. In addition, maximally a temporal rise of transaminase levels indicating hepatocyte damage would be tolerated. The main advantage of HBV1.3 transgenic mice is that they serologically and immunologically resemble chronic HBV infection after neonatal transmission and thereby provide a well-accepted model for vertically transmitted chronic HBV infection. This makes them not only suitable to study pathogenesis during HBV infection but also allows establishing immunotherapeutic strategies that modulate the host immune response in order to prevent hepatitis B. While HBV replication varies significantly between different animals, intraindividual variation of HBV replication is low. Antigen expression during embryo- and fetogenesis is prevented by using HBV1.3-fold over-length constructs as, e.g. in HBV1.3.32 transgenic mice, because HBV RNA transcription is driven exclusively by HBV promoters and due to its dependence on sufficient levels of the hepatocyte-specific transcription factor HNF4α only starts around birth ([16], and unpublished data from Quasdorff and Protzer). Therefore, the lacking immune response against HBV in HBV1.3 transgenic mice is, similar as in chronically HBV-infected patients, mainly due to peripheral tolerance. That this tolerance can be broken is exemplified by the spontaneous appearance of antiHBs as well as anti-HBc antibodies in older mice. Indeed, we and others have proven HBV transgenic mice suitable for the development of therapeutic immunization strategies and confirmed that tolerance against HBV antigens can be broken, resulting in the induction of HBV-specific CD8+ and also CD4+ T-cell responses, as well as seroconversion from HBsAg to anti-HBs positivity [18, 19]. In conclusion, the HBV transgenic mouse model has not only contributed substantially to our understanding of different aspects of HBV immunopathogenesis but also represents a suitable small animal model to investigate novel immunotherapeutic strategies for chronic HBV infection. It currently represents the most widely used animal model for HBV infection.
HBV genome transfer into mice HBV genome delivery through hydrodynamic injection Hydrodynamic injection (HDI) is a high-volume tail vein injection specifically suited for the delivery of DNA into
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hepatocytes. The principle relies on rapidly injecting a large volume (8–10 % of the body weight; injection time: 5–8 s) of physiological salt solution containing the DNA to be delivered [20]. This procedure constitutes a significant burden to the mice, but ensures liver targeting of injected DNA and allows efficient gene transfer and expression in approximately 40 % of hepatocytes. HDI of a transposon vector containing replication-competent 1.3 over-length HBV genome in mice initiates the production of viral antigens and replicative intermediates and induces an acute, self-limiting hepatitis B [21]. Viral replication and virus release into the blood were observed. By hydrodynamic injection, anti-HBV antibodies and HBV-specific T-cells are induced 7 and 15 days post injection, respectively [21, 22]. The rapid uptake of liquid into hepatocyte causes significant hepatocyte damage and an ALT peak directly after injection [22]. HBV antigen expression and replication after classical HDI is transient and terminates 2-week post injection [20, 21]. Persistent HBV infection after HDI was only achieved after transfection of a transposon vector encoding an 1.3 over-length HBV genome into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice [21]. Since these mice lack functional B cells, T-cells and natural killer cells, they are not suited to investigate therapeutic vaccination strategies. To overcome the limitation of acute, self-limiting infection, an HDI model for chronic hepatitis B virus infection in immune competent mice was designed taking advantage of adeno-associated virus (AAV) vector plasmids for HBV genome delivery [23]. Two factors were described to be crucial for the establishment of HBV persistence [23]: (1) the use of AAV vector for the delivery of HBV genome as AAV favours long-term transgene expression in hepatocytes and (2) the genetic background (C57BL/6) of the recipient mouse, which is responsible for the strength of the immune response during primary activation. In C57BL/6 mice, Huang et al. [23] found that impaired HBcAg-specific immunity can impede anti-HBs formation and thereby lead to HBV persistence. However, efficiency is variable, and high levels of HBV replication, characteristic for patients with chronic HBV infection, cannot be detected. Very likely due to the initial liver damage, peripheral tolerance at the T-cell level is lacking [22]. In conclusion, HDI can be applied to deliver replicationcompetent HBV constructs and initiate HBV antigen expression and replication. Persistent HBV replication though at low levels has been described. Immune tolerance is limited by the initial damage caused. Advantages of HDI are that it is readily available and can be easily applied to genetically modified mice. Disadvantages are the demanding injection causing significant stress for the mice and resulting in high inter-individual and inter-laboratory variations.
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Adenoviral vector mediated HBV genome transfer To establish HBV genome delivery methods less stressful for the animals, easier to use and resulting in a more reproducible transfer, viral vectors were developed. Adenoviral vectors enable efficient and reproducible transfer of foreign DNA into livers of immune competent experimental animals. They accommodate large segments of DNA (up to 7.5 kilobase pairs), and their genome does not undergo significant rearrangements, ensuring that the inserted foreign genes are maintained without alterations. Adenoviral vectors are relatively easy to manipulate using recombinant DNA techniques. Taking advantage of these properties, replication-competent full-length HBV genome was inserted into adenoviral vectors, which then were used to transfer hepadnaviral genomes across the species barrier [22, 24, 25]. Sprinzl et al. [24] inserted replication-competent 1.3 over-length human or duck HBV genomes into first-generation adenoviral vectors (AdHBV). Thus, HBV proteins are expressed under the control of endogenous hepadnaviral promoter/enhancer elements driving liver-specific HBV expression. Efficient hepadnavirus replication was initiated after infection of hepatoma cell lines or primary hepatocytes from different species (e.g. mouse, rat and tupaia). In hepatoma cell lines and cultured hepatocytes, cccDNA was established after AdHBV infection from newly produced HBV capsids recycling to the nucleus, proving that an intracellular replication cycle independent of the transferred linear viral genome was established [24]. Virions were released at high titres into the culture medium of hepatoma cells and primary hepatocytes. After injection of 1–2 × 109 infectious units of AdHBV into the tail vein of C57BL/6 mice, HBV proteins, pregenomic RNA, all replicative DNA intermediates and HBV release into the bloodstream were found [24]. Based on this, a mouse model for acute hepatitis B was established by AdHBV vector injection that matches many serological and immunological features of acute HBV infection in humans such as the dynamics of T-cell responses and liver damage during virus clearance and HBsAg/anti-HBs seroconversion [25]. Characteristic initial peaks of HBsAg and HBeAg were observed. After AdHBV transfer, HBV efficiently replicates in mouse hepatocytes, with HBV transcripts detectable for more than 3 months. A two-phase moderate liver inflammation with elevated ALT levels was reported in parallel to the appearance of HBVspecific T-cells. Inflammation and liver damage are controlled by regulatory T-cells in this model [26]. HBV replication is terminated when HBsAg/anti-HBs seroconversion is observed. This mouse model of acute, self-limiting hepatitis B proved very valuable to analyse the immunological mechanisms of HBV clearance, and it was used to
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characterize immune determinants involved such as regulatory T-cell [26], NKT cells [27] and intrahepatic expansion of CTL [28]. Because a self-limiting HBV infection model is not well suited to develop immunotherapeutic intervention strategies, a mouse model for persistent HBV infection was established by preventing initial hepatocyte damage. This was achieved by injecting AdHBV at a relatively low dose [1 × 108 infectious doses (ID) per mouse] into immune competent C57BL/6 mice [22]. An advantage of the HBV genome transfer by an adenoviral vector is that the HBV genome, used for viral transcription, is in an extra-chromosomal organization and can serve as a surrogate of HBV cccDNA in infected human hepatocytes since mice do not establish cccDNA. Due to persistent HBsAg and HBeAg expression, low-dose AdHBV-infected mice neither underwent HBsAg/anti-HBs seroconversion until day 98 nor developed effective HBsAg- and HBcAg-specific CTL responses even after HBV DNA vaccination. In this “chronic HBV infection”, as well as in chronic LCMV infection, tolerance could be broken, when so-called iMATES were induced through additional immune stimuli applied in a timely fashion together with a vaccine (see also article from Knolle et al. in this issue) [28]. In conclusion, HBV genome transfer by adenoviral vectors at low doses results in a persistent infection that serologically and immunologically resembles chronic HBV infection in humans. The tolerance to HBV induced by low-dose AdHBV injection into wild-type mice shares many immunological features of HBV-specific peripheral tolerance in chronically infected humans. This nontransgenic mouse model therefore is particularly suited to expand our knowledge about the mechanisms of HBV-specific peripheral tolerance and to develop therapeutic vaccination approaches to clear HBV infection bypassing this tolerance. AAV vector mediated HBV genome transfer Recently, also recombinant adeno-associated virus (AAV) was used to transfer replication-competent HBV genomes. Transduction with 5 × 1010 viral genome equivalents of an AAV serotype 2/8 chimera established a chronic HBV infection in a mouse strain carrying human leucocyte antigen A2/DR1 (HLA-A2/DR1) transgenes. In all AAV2/8HBV-transduced mice, HBsAg, HBeAg and HBV DNA persisted in serum for at least 1 year [29]. Sixty percent of hepatocytes expressed the HBcAg and viral replication intermediates and transcripts were detected in the livers of the AAV2/8-HBV-injected mice, but no significant liver inflammation was observed. This was linked to a higher number of regulatory T-cells in livers of AAV2/8HBV-transduced mice than in controls and a defect in
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HBV-specific functional T-cell responses indicating tolerance resulting from expression of HBV antigens in hepatocytes [29]. Using a DNA vaccine, this tolerance could be broken while this requires additional immune stimuli in low-dose AdHBV-infected mice [28]. In a second report, development of neonatal and adult mouse models with sustained HBV viremia by infection with AAV/HBV was described [30]. In this model, the AAV/HBV was injected intravenously into adult mice or intrahepatically into neonatal mice. AAV-/HBV-infected mice were, similar to chronic HBV-infected patients, seronegative for antibodies against HBsAg and did not elicit HBV-specific immune responses upon immunization with a conventional HBV vaccine. The AAV/HBV mouse represents a suitable animal model for chronic HBV infection. Similar to low-dose AdHBV-transduced mice, AAV-/HBV HBV-transduced mice develop immunotolerance against HBV antigens and can be used for preclinical evaluations of immunotherapeutic interventions. Which model is better suited needs to be evaluated in a careful side-by-side comparison.
Human liver chimeric mice for HBV infection Human hepatocyte chimeric mice Immunodeficient mice that undergo continuous hepatocyte death can be repopulated with human hepatocytes and are referred to as human hepatocyte chimeric mice. Mice with livers efficiently repopulated with human hepatocytes [31] become susceptible for HBV infection and allow the formation of HBV cccDNA. These chimeric mice therefore represent interesting models for chronic HBV and HCV infection [32]. Unfortunately, human hepatocyte chimeric mice are complex to generate, expensive and complicated to maintain [33]. The two major human hepatocyte chimeric mouse models are introduced in the following. (1) The “uPA” mouse model is complex, and its generation requires multiple steps. First, transgenic mice were generated in which the albumin promoter/enhancer is linked to the murine urokinase-type plasminogen activator (uPA) gene. These AlbuPA transgenic mice showed accelerated hepatocyte death [33]. In a next step, Alb-uPA transgenic mice were crossed with immune-deficient recombinant activation gene-2 (RAG-2) knock-out or SCID mice. Isolated primary human hepatocytes are then transplanted into these immune deficient, uPA transgenic mice, resulting in partial repopulation of the liver (up to 15 %) [34]. The chimeric scid/Alb-uPA mouse in homozygous form, however, is a challenging model. Immunodeficiency and the bleeding tendency inherent in the transgene result in significant risk from infection
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and death due to perinatal trauma. Optimal repopulation requires transplantation with high-quality human hepatocytes within a short period after birth and so creates significant challenge for many laboratories due to difficulty in accessing suitable tissue. (2) Another option is the use of mice that specifically lack fumaryl acetoacetate hydrolase (Fah), RAG-2, and the γ-chain of the receptor for IL-2 (Il2rg) (Fah−/−Rag2−/−Il2rg−/− mice) for generation of human liver chimeric mice [31, 35]. The lack of Fah causes accumulation of toxic tyrosine catabolites and results in loss of murine hepatocytes. Transplanted human hepatocytes stay vital due to the human Fah homolog, resulting in a growth disadvantage for mouse hepatocytes and positive selection for transplanted human hepatocytes. Inoculation of human hepatocyte chimeric mice with HBV led also in the Fah−/−Rag2−/−Il2rg−/− mouse to the establishment of productive HBV infection [31, 34] with sustained HBV propagation for more than 6 months [31]. Viral transfer from one chimeric mouse to another was proven for HCV with viral titres and dynamics similar to those achieved with inoculation with patient serum [31] and is similarly observed for HBV. Human hepatocyte chimeric mice are an important model to study HBV infection in a living organism and proved useful for antiviral drug testing. Studies of immune pathology, however, are limited by the fact that adaptive immune cells are lacking in addition to the fact that the cross-talk between (human) hepatocytes and (murine) nonhepatocytes is only very limited [33]. This prevents the use of this model to address immunological questions and makes it unsuitable for the development of therapeutic vaccines for chronic HBV infection. HBV‑Trimera mouse A Trimera mouse is a humanized mouse model that comprises tissues from three genetically distinct sources. Its generation involves a number of successive steps [36]. First, a wild-type mouse host is rendered immune-incompetent by lethal irradiation. Transplantation of bone marrow cells from a SCID mouse donor substitutes the myeloid and erythroid lineages. A transplantation compatible environment is thereby established. The mouse is then transplanted with human cells or tissues. A Trimera mouse model for hepatitis B virus infection (HBV-Trimera) was generated by transplantation of ex vivo HBV-infected human liver fragments under the kidney capsule or in the ear pinna [37]. Sustained, circulating viremia was observed in HBV-Trimera mice for approximately 1 month. The HBV-Trimera system suits for the development of immune therapeutic strategies and was already used as model for chronic HBV infection in preclinical studies to investigate protein vaccines [38], cell-based immunotherapies [39] or the therapeutic effects of polyclonal anti-HBs
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antibodies [40]. The possibility to transplant human liver tissue together with lymphocytes from the same donor into one Trimera mouse allows creating a unique in vivo system for human HBV infection, exceptional for the development of immunotherapies against chronic HBV infection. The generation of HBV-Trimera mice is extremely time- and cost-consuming and therefore difficult to apply for larger-scale preclinical studies. The major drawback of this model is, however, that implanted human hepatocytes remain functional just for 1 month allowing only shortterm anti-HBV therapies. Human immune system and liver chimera A humanized mouse model engrafted with both human immune and human liver cells is needed to study infection and immunopathogenesis of HBV/HCV infection in vivo. Recent developments aim at generating such humanized mouse models with both human immune system and human liver cells. This can be achieved by reconstituting the immunodeficient NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice with a human HLA-A2 transgene (A2/NSG mice) with human hematopoietic stem cells (HSC) and liver progenitor cells named A2/NSG-hu HSC/Hep mice [41]. The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75 % of HBV-infected mice established persistent infection for at least 4 months [42]. The human immune responses, albeit impaired in the liver, induced chronic liver inflammation and liver fibrosis in infected animals. Such models will allow testing of vaccine strategies in a preclinical setting. However, they are much more complex than the models described before. This and the variability between individual mice will only allow the usage for very specific aspects.
Summary HBV cannot naturally infect mouse hepatocytes, but a mouse model for persistent HBV infection is essential for the preclinical development of a therapeutic hepatitis B vaccine. To fulfil the requirements of a suitable mouse model for preclinical evaluation of candidate therapeutic vaccines, three challenges have to be addressed: First, the induction of a persistent HBV infection in a host that naturally cannot be infected by HBV, second, the establishment of HBV-specific peripheral tolerance and third, the possibility to break HBV-specific tolerance by therapeutic vaccination, resulting in induction of an effective HBV-specific immune response and ideally clearance of HBV. A series of mouse models that aim to fulfil these requirements has been developed in the past. The simplest mouse model for
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chronic HBV infection is the HBV transgenic mouse. HBV replicates from an integrated transgene, which naturally cannot be eliminated. Due to their HBV-specific peripheral tolerance that can be broken upon adequate treatment, HBV transgenic mice proved to be suitable for the development of therapeutic vaccines against HBV. Another approach is to transiently or stably transfect mouse hepatocytes with the HBV genome. These models allow the eradication of HBV carrying hepatocytes; however, HBV cccDNA, the natural transcription template of HBV cannot establish in mouse hepatocytes. Human hepatocyte chimeric mice with mouse livers repopulated with human hepatocytes currently represent the most effective mouse model for chronic HBV infection establishing cccDNA. For vaccine research, however, these mice need also to be reconstituted with a human immune system. Currently, such mice can only be generated in low numbers, and immune responses are skewed limiting their application, Currently, many preclinical studies are ongoing in order to immunotherapeutically cure chronic HBV infection. The vast majority of such studies are performed in HBV transgenic mice, because this model is cheap, easy to bread and answers most relevant questions of efficacy. Limitations of HBV transgenic mice are the inability to clear HBV and the lack of formation of HBV cccDNA. These limitations can be compensated by the complementary use of other mouse models for persistent HBV infection, like HBVgenome-transduced mice or human hepatocyte chimeras. The identification of an essential HBV receptor and technical advances will further improve the availability of murine preclinical models for hepatitis B vaccine research in the future. Acknowledgments This work was supported by the HelmholtzAlberta Initiative—Infectious Disease Research (HAI-IDR). Conflict of interest None.
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REVIEW
Mouse models for therapeutic vaccination against hepatitis B virus Claudia Dembek · Ulrike Protzer
Received: 25 July 2014 / Accepted: 2 October 2014 / Published online: 19 December 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract A mouse model for persistent HBV infection is essential for the development of a therapeutic vaccine against HBV. Because HBV cannot infect mouse hepatocytes, even if the HBV receptor is introduced, surrogate models are used. A suitable model needs to establish persistent HBV replication and must allow the establishment of HBV-specific adaptive cellular and humoral immune responses. Therefore, an immunocompetent mouse model is needed in which one can break HBV-specific tolerance and ideally eliminate the HBV transcription template. The most widely used model for chronic HBV infection is the HBV transgenic mouse. Although HBV replicates from an integrated transgene, HBV-specific immune tolerance can be broken upon adequate immune stimulation because antigen expression only starts shortly before birth. Alternative mouse models of chronic HBV infection are generated by introducing HBV genomes either using viral vectors or using hydrodynamic injection. In these alternative models, the HBV transcription template is introduced into a proportion of hepatocytes and stays extra-chromosomal. It thus mimics the natural HBV transcription template, the HBV cccDNA in humans. Unlike an HBV transgene, however, it can be cleared upon appropriate treatment or
This article is part of the special issue “Therapeutic vaccination in chronic hepatitis B—approaches, problems, and new perspectives”. C. Dembek · U. Protzer (*) Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Trogerstr. 30, 81675 Munich, Germany e-mail: [email protected] C. Dembek · U. Protzer German Center for Infection Research (DZIF), Brunswick, Germany
immune stimulation. Human hepatocyte chimeric mice in which murine hepatocytes are widely replaced by human hepatocytes represent another important mouse model for persistent HBV infection. These mice are susceptible for HBV infection, but need to be severely immune deficient to accept human hepatocytes. In conclusion, a variety of mouse models for persistent HBV infection are available suitable for preclinical efficacy evaluations of therapeutic vaccination strategies against HBV. Keywords Chronic hepatitis B · Mouse model · Therapeutic vaccination · Adaptive cellular immune response · Transgenic mice · Human hepatocyte chimeric mice
Introduction Hepatitis B virus (HBV) infects the human liver and often causes inflammatory liver disease (hepatitis B). HBV infection during adulthood is resolved spontaneously in more than 95 % of cases. In contrast, vertical transmission of HBV from hepatitis B e antigen (HBeAg)-positive mothers to their neonates results in up to 90 % of infants in chronic infection. Chronic HBV infection is defined by HBV persistence for more than 6 months with detectable hepatitis B surface antigen (HBsAg) in the blood. It is typically characterised by high circulating HBV titres >2,000 international units (IU)/mL HBV DNA (corresponding to 104 HBV DNA copies/mL blood), and a risk to develop liver cirrhosis and hepatocellular carcinoma [1]. The failure to control HBV infection and subsequent establishment of chronicity often results in exhaustion of virus-specific T-cell responses. HBV-specific T-cell tolerance has been suggested to be induced mainly by high viral
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antigen load, which would be regarded as peripheral tolerance, but this has not formally been proven yet. In contrast to central tolerance that eliminates immature self-reactive lymphocytes in the thymus, peripheral tolerance (the status of HBV-specific T-cell tolerance) prevents mature, circulating lymphocytes from reacting to self- or chronically persisting antigens through clonal anergy and clonal deletion and thereby prevents chronic systemic inflammation. It usually takes place in peripheral lymphoid organs like lymph nodes, tonsils, spleen and mucosal-associated lymphoid tissues. In chronic HBV infection, two mechanisms have been suggested to be responsible for HBV-specific peripheral tolerance. (1) The liver environment supports tolerance induction [2]. As a metabolic organ, the liver removes gut-derived microbial antigens from the blood. Parenchymal and nonparenchymal antigen-presenting cells (APCs) in the liver induce peripheral tolerance towards such antigens to avoid chronic activation of the immune system [3]. (2) Neonatal tolerance [4, 5] can additionally explain the inability of perinatally infected chronic HBV carriers to sufficiently respond to HBsAg, HBeAg and HBV core protein. The idea of therapeutic vaccination is to overcome HBV-specific peripheral tolerance in chronic carriers and activate a potent and timely restricted cytotoxic T lymphocyte (CTL) response that eliminates HBV with minimized hepatocyte damage. A small animal model for HBV infection is a prerequisite for antiviral compound development and preclinical testing of novel therapeutic vaccination strategies. Ideally such animal models allow establishing chronic HBV infection and the induction of peripheral tolerance in order to break this tolerance by therapeutic vaccination. HBV can infect only humans, apes, tree shrews (Tupaia belangeri) and recently discovered macaques [6]. A series of HBV-related hepadnaviruses were discovered in the past (in ducks, geese, herons, woodchucks, squirrels and in woolly monkeys) [7]. Amongst the hosts, woodchucks and Pekin ducks represent established animal models for investigating HBV-related hepadnaviruses. Both models provided essential knowledge about HBV-related disease [8, 9] and served in preclinical efficacy studies to investigate therapeutic vaccines in chronic hepatitis B [10, 11]. Recently, a new hepadnavirus species related to HBV was discovered in bats [12]. The main weakness of woodchucks or ducks as models for vaccine development is that their respective hepadnaviruses species is genetically distinct from human HBV. Thus, hepatitis B vaccines cannot be tested in these animals, and vaccine proteins and constructs need to be adapted to the respective virus strain. Other difficulties are that our understanding of the immunobiology of these hosts is limited, and basic reagents and tools required for immunologic investigations are restricted. Therefore, working with these animals is tedious and the analysis of immune responses to therapeutic vaccination strategies is
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limited. The newly discovered macaque HBV, however, is 99 % identical to human HBV [6], and macaques are well established in preclinical studies. So this may open a new avenue in the coming years. Currently, the mouse is the most widely used experimental animal due to various reasons. A short generation time of 6–8 weeks makes them a cost-effective and efficient tool for rapid screening and research. The mouse genome is easy to manipulate and thereby provides a powerful tool to model specific diseases. Due to the wide use of laboratory mice, all standard tools and methods (e.g. detection of immune responses) are readily available. HBV cannot naturally infect mice; however, different surrogate murine infection models have been generated in the past years, which proved to be useful for vaccine studies, but all have distinct limitations. By now, it is still not possible to fulfil all requirements towards an animal model of chronic HBV infection within one mouse model, and the complementary use of appropriate models can be considered for preclinical efficacy tests. The most important mouse models for acute and chronic HBV infection are described below, and their significance for the establishment of a therapeutic vaccine against HBV is discussed.
HBV transgenic mice Embryo microinjection technology enabled the insertion of a part or a complete copy of the HBV genome into mice and allows studying HBV immunobiology and pathogenesis. A series of HBV transgenic mouse lines have been engineered, expressing single HBV gene products (reviewed in [13]) or replicating HBV [14–16]. A widely used model until today is the HBV1.3 transgenic mouse [16]. From an integrated 1.3 over-length HBV transgene, this lineage produces HBsAg, hepatitis B core antigen (HBcAg) and HBeAg. HBV replicates in the liver and releases infectious virus into the blood at levels comparable to chronic HBV infection in humans, but the virus cannot spread. Like in chronically HBV-infected humans, serum HBeAg levels in HBV1.3 transgenic mice vary significantly between different animals and directly correlate with HBsAg and serum HBV DNA levels. Thus, serum HBeAg level serves as a parameter, which indicates efficacy of transcription of the HBV pregenome and of resulting virus replication. A major limitation of HBV transgenic mice is that HBV replicates from an integrated transgene that cannot be eliminated. Furthermore, mouse hepatocytes do not establish cccDNA, the natural template for HBV transcription [13, 16] limiting the use of mice for natural HBV infection. Consequently, the proof-of-concept of therapeutic vaccination, viral clearance and eradication of HBV cccDNA, cannot be
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achieved in this model. Thus, alternative endpoints are used: (1) the induction of a strong and timely restricted HBV-specific CTL response, (2) the induction of neutralizing antiHBs antibody responses and (3) safety of the immunization scheme with limited liver damage. T-cell responses as well as anti-HBs seroconversion are expected to accompany HBV clearance as they were reported in HBV carriers before clearance of the virus, either spontaneously or by antiviral drugs [17]. In addition, maximally a temporal rise of transaminase levels indicating hepatocyte damage would be tolerated. The main advantage of HBV1.3 transgenic mice is that they serologically and immunologically resemble chronic HBV infection after neonatal transmission and thereby provide a well-accepted model for vertically transmitted chronic HBV infection. This makes them not only suitable to study pathogenesis during HBV infection but also allows establishing immunotherapeutic strategies that modulate the host immune response in order to prevent hepatitis B. While HBV replication varies significantly between different animals, intraindividual variation of HBV replication is low. Antigen expression during embryo- and fetogenesis is prevented by using HBV1.3-fold over-length constructs as, e.g. in HBV1.3.32 transgenic mice, because HBV RNA transcription is driven exclusively by HBV promoters and due to its dependence on sufficient levels of the hepatocyte-specific transcription factor HNF4α only starts around birth ([16], and unpublished data from Quasdorff and Protzer). Therefore, the lacking immune response against HBV in HBV1.3 transgenic mice is, similar as in chronically HBV-infected patients, mainly due to peripheral tolerance. That this tolerance can be broken is exemplified by the spontaneous appearance of antiHBs as well as anti-HBc antibodies in older mice. Indeed, we and others have proven HBV transgenic mice suitable for the development of therapeutic immunization strategies and confirmed that tolerance against HBV antigens can be broken, resulting in the induction of HBV-specific CD8+ and also CD4+ T-cell responses, as well as seroconversion from HBsAg to anti-HBs positivity [18, 19]. In conclusion, the HBV transgenic mouse model has not only contributed substantially to our understanding of different aspects of HBV immunopathogenesis but also represents a suitable small animal model to investigate novel immunotherapeutic strategies for chronic HBV infection. It currently represents the most widely used animal model for HBV infection.
HBV genome transfer into mice HBV genome delivery through hydrodynamic injection Hydrodynamic injection (HDI) is a high-volume tail vein injection specifically suited for the delivery of DNA into
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hepatocytes. The principle relies on rapidly injecting a large volume (8–10 % of the body weight; injection time: 5–8 s) of physiological salt solution containing the DNA to be delivered [20]. This procedure constitutes a significant burden to the mice, but ensures liver targeting of injected DNA and allows efficient gene transfer and expression in approximately 40 % of hepatocytes. HDI of a transposon vector containing replication-competent 1.3 over-length HBV genome in mice initiates the production of viral antigens and replicative intermediates and induces an acute, self-limiting hepatitis B [21]. Viral replication and virus release into the blood were observed. By hydrodynamic injection, anti-HBV antibodies and HBV-specific T-cells are induced 7 and 15 days post injection, respectively [21, 22]. The rapid uptake of liquid into hepatocyte causes significant hepatocyte damage and an ALT peak directly after injection [22]. HBV antigen expression and replication after classical HDI is transient and terminates 2-week post injection [20, 21]. Persistent HBV infection after HDI was only achieved after transfection of a transposon vector encoding an 1.3 over-length HBV genome into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice [21]. Since these mice lack functional B cells, T-cells and natural killer cells, they are not suited to investigate therapeutic vaccination strategies. To overcome the limitation of acute, self-limiting infection, an HDI model for chronic hepatitis B virus infection in immune competent mice was designed taking advantage of adeno-associated virus (AAV) vector plasmids for HBV genome delivery [23]. Two factors were described to be crucial for the establishment of HBV persistence [23]: (1) the use of AAV vector for the delivery of HBV genome as AAV favours long-term transgene expression in hepatocytes and (2) the genetic background (C57BL/6) of the recipient mouse, which is responsible for the strength of the immune response during primary activation. In C57BL/6 mice, Huang et al. [23] found that impaired HBcAg-specific immunity can impede anti-HBs formation and thereby lead to HBV persistence. However, efficiency is variable, and high levels of HBV replication, characteristic for patients with chronic HBV infection, cannot be detected. Very likely due to the initial liver damage, peripheral tolerance at the T-cell level is lacking [22]. In conclusion, HDI can be applied to deliver replicationcompetent HBV constructs and initiate HBV antigen expression and replication. Persistent HBV replication though at low levels has been described. Immune tolerance is limited by the initial damage caused. Advantages of HDI are that it is readily available and can be easily applied to genetically modified mice. Disadvantages are the demanding injection causing significant stress for the mice and resulting in high inter-individual and inter-laboratory variations.
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Adenoviral vector mediated HBV genome transfer To establish HBV genome delivery methods less stressful for the animals, easier to use and resulting in a more reproducible transfer, viral vectors were developed. Adenoviral vectors enable efficient and reproducible transfer of foreign DNA into livers of immune competent experimental animals. They accommodate large segments of DNA (up to 7.5 kilobase pairs), and their genome does not undergo significant rearrangements, ensuring that the inserted foreign genes are maintained without alterations. Adenoviral vectors are relatively easy to manipulate using recombinant DNA techniques. Taking advantage of these properties, replication-competent full-length HBV genome was inserted into adenoviral vectors, which then were used to transfer hepadnaviral genomes across the species barrier [22, 24, 25]. Sprinzl et al. [24] inserted replication-competent 1.3 over-length human or duck HBV genomes into first-generation adenoviral vectors (AdHBV). Thus, HBV proteins are expressed under the control of endogenous hepadnaviral promoter/enhancer elements driving liver-specific HBV expression. Efficient hepadnavirus replication was initiated after infection of hepatoma cell lines or primary hepatocytes from different species (e.g. mouse, rat and tupaia). In hepatoma cell lines and cultured hepatocytes, cccDNA was established after AdHBV infection from newly produced HBV capsids recycling to the nucleus, proving that an intracellular replication cycle independent of the transferred linear viral genome was established [24]. Virions were released at high titres into the culture medium of hepatoma cells and primary hepatocytes. After injection of 1–2 × 109 infectious units of AdHBV into the tail vein of C57BL/6 mice, HBV proteins, pregenomic RNA, all replicative DNA intermediates and HBV release into the bloodstream were found [24]. Based on this, a mouse model for acute hepatitis B was established by AdHBV vector injection that matches many serological and immunological features of acute HBV infection in humans such as the dynamics of T-cell responses and liver damage during virus clearance and HBsAg/anti-HBs seroconversion [25]. Characteristic initial peaks of HBsAg and HBeAg were observed. After AdHBV transfer, HBV efficiently replicates in mouse hepatocytes, with HBV transcripts detectable for more than 3 months. A two-phase moderate liver inflammation with elevated ALT levels was reported in parallel to the appearance of HBVspecific T-cells. Inflammation and liver damage are controlled by regulatory T-cells in this model [26]. HBV replication is terminated when HBsAg/anti-HBs seroconversion is observed. This mouse model of acute, self-limiting hepatitis B proved very valuable to analyse the immunological mechanisms of HBV clearance, and it was used to
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characterize immune determinants involved such as regulatory T-cell [26], NKT cells [27] and intrahepatic expansion of CTL [28]. Because a self-limiting HBV infection model is not well suited to develop immunotherapeutic intervention strategies, a mouse model for persistent HBV infection was established by preventing initial hepatocyte damage. This was achieved by injecting AdHBV at a relatively low dose [1 × 108 infectious doses (ID) per mouse] into immune competent C57BL/6 mice [22]. An advantage of the HBV genome transfer by an adenoviral vector is that the HBV genome, used for viral transcription, is in an extra-chromosomal organization and can serve as a surrogate of HBV cccDNA in infected human hepatocytes since mice do not establish cccDNA. Due to persistent HBsAg and HBeAg expression, low-dose AdHBV-infected mice neither underwent HBsAg/anti-HBs seroconversion until day 98 nor developed effective HBsAg- and HBcAg-specific CTL responses even after HBV DNA vaccination. In this “chronic HBV infection”, as well as in chronic LCMV infection, tolerance could be broken, when so-called iMATES were induced through additional immune stimuli applied in a timely fashion together with a vaccine (see also article from Knolle et al. in this issue) [28]. In conclusion, HBV genome transfer by adenoviral vectors at low doses results in a persistent infection that serologically and immunologically resembles chronic HBV infection in humans. The tolerance to HBV induced by low-dose AdHBV injection into wild-type mice shares many immunological features of HBV-specific peripheral tolerance in chronically infected humans. This nontransgenic mouse model therefore is particularly suited to expand our knowledge about the mechanisms of HBV-specific peripheral tolerance and to develop therapeutic vaccination approaches to clear HBV infection bypassing this tolerance. AAV vector mediated HBV genome transfer Recently, also recombinant adeno-associated virus (AAV) was used to transfer replication-competent HBV genomes. Transduction with 5 × 1010 viral genome equivalents of an AAV serotype 2/8 chimera established a chronic HBV infection in a mouse strain carrying human leucocyte antigen A2/DR1 (HLA-A2/DR1) transgenes. In all AAV2/8HBV-transduced mice, HBsAg, HBeAg and HBV DNA persisted in serum for at least 1 year [29]. Sixty percent of hepatocytes expressed the HBcAg and viral replication intermediates and transcripts were detected in the livers of the AAV2/8-HBV-injected mice, but no significant liver inflammation was observed. This was linked to a higher number of regulatory T-cells in livers of AAV2/8HBV-transduced mice than in controls and a defect in
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HBV-specific functional T-cell responses indicating tolerance resulting from expression of HBV antigens in hepatocytes [29]. Using a DNA vaccine, this tolerance could be broken while this requires additional immune stimuli in low-dose AdHBV-infected mice [28]. In a second report, development of neonatal and adult mouse models with sustained HBV viremia by infection with AAV/HBV was described [30]. In this model, the AAV/HBV was injected intravenously into adult mice or intrahepatically into neonatal mice. AAV-/HBV-infected mice were, similar to chronic HBV-infected patients, seronegative for antibodies against HBsAg and did not elicit HBV-specific immune responses upon immunization with a conventional HBV vaccine. The AAV/HBV mouse represents a suitable animal model for chronic HBV infection. Similar to low-dose AdHBV-transduced mice, AAV-/HBV HBV-transduced mice develop immunotolerance against HBV antigens and can be used for preclinical evaluations of immunotherapeutic interventions. Which model is better suited needs to be evaluated in a careful side-by-side comparison.
Human liver chimeric mice for HBV infection Human hepatocyte chimeric mice Immunodeficient mice that undergo continuous hepatocyte death can be repopulated with human hepatocytes and are referred to as human hepatocyte chimeric mice. Mice with livers efficiently repopulated with human hepatocytes [31] become susceptible for HBV infection and allow the formation of HBV cccDNA. These chimeric mice therefore represent interesting models for chronic HBV and HCV infection [32]. Unfortunately, human hepatocyte chimeric mice are complex to generate, expensive and complicated to maintain [33]. The two major human hepatocyte chimeric mouse models are introduced in the following. (1) The “uPA” mouse model is complex, and its generation requires multiple steps. First, transgenic mice were generated in which the albumin promoter/enhancer is linked to the murine urokinase-type plasminogen activator (uPA) gene. These AlbuPA transgenic mice showed accelerated hepatocyte death [33]. In a next step, Alb-uPA transgenic mice were crossed with immune-deficient recombinant activation gene-2 (RAG-2) knock-out or SCID mice. Isolated primary human hepatocytes are then transplanted into these immune deficient, uPA transgenic mice, resulting in partial repopulation of the liver (up to 15 %) [34]. The chimeric scid/Alb-uPA mouse in homozygous form, however, is a challenging model. Immunodeficiency and the bleeding tendency inherent in the transgene result in significant risk from infection
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and death due to perinatal trauma. Optimal repopulation requires transplantation with high-quality human hepatocytes within a short period after birth and so creates significant challenge for many laboratories due to difficulty in accessing suitable tissue. (2) Another option is the use of mice that specifically lack fumaryl acetoacetate hydrolase (Fah), RAG-2, and the γ-chain of the receptor for IL-2 (Il2rg) (Fah−/−Rag2−/−Il2rg−/− mice) for generation of human liver chimeric mice [31, 35]. The lack of Fah causes accumulation of toxic tyrosine catabolites and results in loss of murine hepatocytes. Transplanted human hepatocytes stay vital due to the human Fah homolog, resulting in a growth disadvantage for mouse hepatocytes and positive selection for transplanted human hepatocytes. Inoculation of human hepatocyte chimeric mice with HBV led also in the Fah−/−Rag2−/−Il2rg−/− mouse to the establishment of productive HBV infection [31, 34] with sustained HBV propagation for more than 6 months [31]. Viral transfer from one chimeric mouse to another was proven for HCV with viral titres and dynamics similar to those achieved with inoculation with patient serum [31] and is similarly observed for HBV. Human hepatocyte chimeric mice are an important model to study HBV infection in a living organism and proved useful for antiviral drug testing. Studies of immune pathology, however, are limited by the fact that adaptive immune cells are lacking in addition to the fact that the cross-talk between (human) hepatocytes and (murine) nonhepatocytes is only very limited [33]. This prevents the use of this model to address immunological questions and makes it unsuitable for the development of therapeutic vaccines for chronic HBV infection. HBV‑Trimera mouse A Trimera mouse is a humanized mouse model that comprises tissues from three genetically distinct sources. Its generation involves a number of successive steps [36]. First, a wild-type mouse host is rendered immune-incompetent by lethal irradiation. Transplantation of bone marrow cells from a SCID mouse donor substitutes the myeloid and erythroid lineages. A transplantation compatible environment is thereby established. The mouse is then transplanted with human cells or tissues. A Trimera mouse model for hepatitis B virus infection (HBV-Trimera) was generated by transplantation of ex vivo HBV-infected human liver fragments under the kidney capsule or in the ear pinna [37]. Sustained, circulating viremia was observed in HBV-Trimera mice for approximately 1 month. The HBV-Trimera system suits for the development of immune therapeutic strategies and was already used as model for chronic HBV infection in preclinical studies to investigate protein vaccines [38], cell-based immunotherapies [39] or the therapeutic effects of polyclonal anti-HBs
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antibodies [40]. The possibility to transplant human liver tissue together with lymphocytes from the same donor into one Trimera mouse allows creating a unique in vivo system for human HBV infection, exceptional for the development of immunotherapies against chronic HBV infection. The generation of HBV-Trimera mice is extremely time- and cost-consuming and therefore difficult to apply for larger-scale preclinical studies. The major drawback of this model is, however, that implanted human hepatocytes remain functional just for 1 month allowing only shortterm anti-HBV therapies. Human immune system and liver chimera A humanized mouse model engrafted with both human immune and human liver cells is needed to study infection and immunopathogenesis of HBV/HCV infection in vivo. Recent developments aim at generating such humanized mouse models with both human immune system and human liver cells. This can be achieved by reconstituting the immunodeficient NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice with a human HLA-A2 transgene (A2/NSG mice) with human hematopoietic stem cells (HSC) and liver progenitor cells named A2/NSG-hu HSC/Hep mice [41]. The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75 % of HBV-infected mice established persistent infection for at least 4 months [42]. The human immune responses, albeit impaired in the liver, induced chronic liver inflammation and liver fibrosis in infected animals. Such models will allow testing of vaccine strategies in a preclinical setting. However, they are much more complex than the models described before. This and the variability between individual mice will only allow the usage for very specific aspects.
Summary HBV cannot naturally infect mouse hepatocytes, but a mouse model for persistent HBV infection is essential for the preclinical development of a therapeutic hepatitis B vaccine. To fulfil the requirements of a suitable mouse model for preclinical evaluation of candidate therapeutic vaccines, three challenges have to be addressed: First, the induction of a persistent HBV infection in a host that naturally cannot be infected by HBV, second, the establishment of HBV-specific peripheral tolerance and third, the possibility to break HBV-specific tolerance by therapeutic vaccination, resulting in induction of an effective HBV-specific immune response and ideally clearance of HBV. A series of mouse models that aim to fulfil these requirements has been developed in the past. The simplest mouse model for
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chronic HBV infection is the HBV transgenic mouse. HBV replicates from an integrated transgene, which naturally cannot be eliminated. Due to their HBV-specific peripheral tolerance that can be broken upon adequate treatment, HBV transgenic mice proved to be suitable for the development of therapeutic vaccines against HBV. Another approach is to transiently or stably transfect mouse hepatocytes with the HBV genome. These models allow the eradication of HBV carrying hepatocytes; however, HBV cccDNA, the natural transcription template of HBV cannot establish in mouse hepatocytes. Human hepatocyte chimeric mice with mouse livers repopulated with human hepatocytes currently represent the most effective mouse model for chronic HBV infection establishing cccDNA. For vaccine research, however, these mice need also to be reconstituted with a human immune system. Currently, such mice can only be generated in low numbers, and immune responses are skewed limiting their application, Currently, many preclinical studies are ongoing in order to immunotherapeutically cure chronic HBV infection. The vast majority of such studies are performed in HBV transgenic mice, because this model is cheap, easy to bread and answers most relevant questions of efficacy. Limitations of HBV transgenic mice are the inability to clear HBV and the lack of formation of HBV cccDNA. These limitations can be compensated by the complementary use of other mouse models for persistent HBV infection, like HBVgenome-transduced mice or human hepatocyte chimeras. The identification of an essential HBV receptor and technical advances will further improve the availability of murine preclinical models for hepatitis B vaccine research in the future. Acknowledgments This work was supported by the HelmholtzAlberta Initiative—Infectious Disease Research (HAI-IDR). Conflict of interest None.
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