Journal of Viral Hepatitis, 2014, 21, 305–313

doi:10.1111/jvh.12255

REVIEW

Adaptive response in hepatitis B virus infection E. Loggi,1,2 N. Gamal,2 F. Bihl,3 M. Bernardi2 and P. Andreone2

1

Institute for Research in Biomedicine,

2

Bellinzona, Switzerland; Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy; and 3Hepatology unit, Ospedale Regionale di Bellinzona, Bellinzona, Switzerland Received February 2014; accepted for publication March 2014

SUMMARY. Hepatitis B virus (HBV) is a major cause of acute

and chronic liver inflammation worldwide. The immune response against the virus represents a key factor in determining infection outcome, in terms of both viral clearance and the perpetuation of liver damage. Significant advances have recently been achieved regarding the functions of antiviral CD8+ T cells, leading to a better understanding of their abnormalities during chronic infection as well as the pathways to be manipulated to reverse the immune

INTRODUCTION Worldwide, two billion people have been infected with the hepatitis B virus (HBV), more than 370 million currently carry chronic HBV infection, and approximately one million die each year from HBV-related liver diseases. HBV is a noncytopathic DNA virus belonging to the family of hepadnaviridae, with a marked hepatotropism, for which a receptor has recently been identified [1]. HBV causes liver disease of varying severity, ranging from acute self-limiting infection, occurring in the vast majority of cases in adulthood, to chronic infection, which occurs when the infection persists for more than 6 months. The latter condition exhibits different phenotypes, ranging from mild hepatitis to liver cirrhosis, eventually leading to liver cancer and end-stage liver failure [2,3]. The outcome of HBV infection is believed to be primarily determined by the host immune response against the virus, which is necessary to control the virus. Nevertheless, the

Abbreviations: Anti-HBc, Hepatitis B core antigen antibody; AntiHBs, Hepatitis B surface antigen antibody; APCs, antigen-presenting cells; CTL, cytotoxic T cell; CTLA-4, Cytotoxic T-Lymphocyte Antigen 4; FCRL4, Fc-receptor-like-4; HBcAg, Hepatitis B core antigen; HBeAg, Hepatitis B e antigen; HBsAg, Hepatitis B surface antigen; ICOS, inducible costimulator; MHC, major histocompatibility complex; PD-1, programmed cell death 1; TCR, T cell receptor. Correspondence: Pietro Andreone, Department of Medical and Surgical Sciences - UO Semeiotica Medica, Via Massarenti 9, 40138 Bologna, Italy. E-mail: [email protected]

© 2014 John Wiley & Sons Ltd

impairment of chronic infection. In this review, we aimed to analyse the patterns of adaptive immunity that develop during acute infection and the profiles in chronic infection. In addition to CD8+ T cells, which are the best-described subset to date, we reviewed and commented on the direct and indirect roles of CD4+ T cells and B cells. Keywords: B lymphocytes, CD4 lymphocytes, CD8 lymphocytes, Hepatitis B, immune response.

immune response is also responsible for liver damage, as HBV is not a direct cytopathic virus. Therefore, both viral clearance and disease pathogenesis result from the same mechanisms that consequently require a tight regulation. Here, we review the pattern of adaptive immunity, being aware that innate immunity also plays a key role in the infection, primarily in its first phase. These aspects, however, have been already covered by recent, excellent reviews [5,6]. We shall briefly describe the profile and function of antiviral CD8+ T cells, which have received the greatest attention, and analyse more exhaustively the important roles of CD4+ and B cells, which have been far less studied to date.

HUMORAL RESPONSE The current knowledge of the humoral response and its meaning emerge from the routine monitoring of serological patterns of patients with acute or chronic HBV infection. This, in turn, allows their allocation into distinct clinical profiles on the basis of the humoral response evoked against the structural antigens of the virus, namely the core (HBcAg) and envelope antigens (HBsAg). Anti-core (antiHBc)-specific IgM is the earliest marker of infection, whereas antibodies against HBsAg and HBeAg, a circulating nonparticulate form of HBcAg, appear much later and indicate a favourable outcome of the infection [3,7]. Anti-HBc IgG develops during the acute phase and persists lifelong after clinical recovery. Thus, anti-HBc IgG represents the only reliable hallmark of past exposure to HBV,

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as the anti-envelope (anti-HBs) antibodies may decline overtime. Consequently, a significant proportion of patients with signs of a resolved HBV infection may be negative for these antibodies. Anti-HBs antibodies are virus neutralizing antibodies and mediate protective immunity by both complexing with free viral particles and preventing their uptake by uninfected hepatocytes [8]. Typically, neutralizing antibodies only develop after HBsAg disappearance from the serum and recovery from clinical symptoms. Moreover, they are usually absent in the clinical, symptomatic phase of infection as well as in the chronic stage. For this reason, no specific role can be attributed to anti-HBs-positive B cells in the resolution of infection. The causes of this delayed development of neutralizing antibodies are still undefined, even though this feature appears to be unique to viral infections caused by poorly or noncytopathic viruses [9]. It should be underlined that the scarcity of studies on the memory B cell repertoire at the cellular level in HBV infection does not allow the measurement of the actual antiHBs production: soluble HBsAg is present in large excess as subviral but not infectious particles and exceeds the number of virions by a factor of 102–105 [10]. Thus, the dynamics of serum antibodies do not reflect the dynamics of specific memory B cells, and the timing of HBsAg production cannot therefore be addressed. Similarly, the extent of HBsAg production in chronic infection cannot be established because it is masked by HBsAg excess. This is confirmed by the coexistence of both HBsAg and anti-HBs in patients found to harbour escape mutations of HBsAg [11]. In this setting, a potential contribution by ‘hidden’ antiHBs production against different levels of circulating HBsAg that characterize the various serovirological profiles of HBV infection cannot be excluded; this would represent a most interesting area for future investigation. Even if not primarily involved in the infection control during the acute phase, the importance of B cells in the resolution phase of infection is remarkable. At this stage, despite serological negativity for HBsAg, HBV is still able to synthesize minute amounts of antigens, which are undetectable by available assays but sufficient to maintain an HBV-specific immune response and to be controlled by anti-HBs. This hypothesis appears to be supported by the recent report of HBV reactivation triggered by B-cell-depleting anti-CD20 treatments [12]. In addition to the unexplored aspects of antibody function in HBV infection, B cells can have an important, albeit indirect, role in HBV infection control, acting as antigen-presenting cells (APCs) for antiviral CD4+ T cells [13]. In this context, murine core antigen-specific B cells have been demonstrated to process and present HBcAg to na€ıve Th cells and to T cell hybridomas in a manner that is largely more efficient than that of ‘classical’ APCs (macrophages and dendritic cells) [14,15]. This preferential presentation by B cells in part explains the HBcAg immunogenicity, which is essen-

tial for the development of an anti-HBc CD4 response. This has important implications, as the anti-HBc response has been demonstrated to play a crucial role in both acute and chronic infection [16,17]. However, some detrimental effects of preferential B cell presentation should be considered: the high amount of serum anti-HBc antibodies in chronic HBV patients can potentially compete with B cell Ig receptor uptake and, consequently, inhibit or skew Th activation [14]. Moreover, some lines of evidence indicate that interactions between B and T cells can favour immunotolerance in mouse models in which the predominant APCs are B cells in a context of chronic antigenic stimulation [18], as they are able to trigger the expression of negative costimulatory molecules such as programmed cell death 1 (PD-1), CTLA-4, and B and T lymphocyte attenuator (BTLA) [19]. However, the lack of anti-HBs in chronic infection can be attributed to a selective exhaustion of specific B cells. The phenomenon of B cell exhaustion, with a consequent inadequate antibody response, has recently been described in different models of chronic antigen exposure [20,21]. As far as HBV infection is concerned, a recent study aimed to address the extent of B cell activation and differentiation in patients with chronically persisting HBV and HCV infections showed a higher proportion of B cells with an activated phenotype than in healthy control groups. However, these cells showed a reduced proliferative capacity [22], and interestingly, they expressed low levels of the inhibitory receptor Fc-receptor-like-4 (FCRL4), an inhibitory receptor that is overexpressed on a subset of putative exhausted memory B cells [20]. The last aspect to be considered regarding the role of B cells in HBV infection emerges from recent evidence that has been provided on the role of humoral immunity in the pathogenesis of HBV-related liver disease. Indeed, a subset of regulatory, IL-10-producing B cells has been found to be enriched in chronic HBV patients; this subset is correlated with hepatic flares and can suppress the HBV-specific CD8 response in an IL-10-dependent manner [23]. Lastly, a direct primary pathogenetic role of the antibody response has been identified in HBV-associated acute liver failure, due to a massive intrahepatic accumulation of anti-HBc IgG- and IgM-secreting plasma cells [24]. To summarize, it is becoming progressively evident that B cell biology in HBV infection has been largely neglected to date and that it should now represent a main research focus to better understand the mechanisms underlying the interaction between the cellular and humoral arms of the immune system and to highlight the potential role played by B cells in the pathogenesis and outcome of HBV-related liver disease.

CELLULAR RESPONSE The basic concept underlying cellular HBV immunology is represented by the well-recognized dichotomy between © 2014 John Wiley & Sons Ltd

HBV and the adaptive immune response patients who are able to control the acute infection and subjects with established chronicity, who present a clear difference in CD4 and CD8 responses to the virus. Indeed, both the frequency and the intensity of the immune response are significantly higher in patients with self-limiting infection [5,25,26]. The control of infection after the acute phase is widely attributed to the development of an efficient T cell memory, which can function properly even in special circumstances, such as in immunosuppressed subjects with serological signs of previous exposure to the virus who are transplanted with HBV-infected grafts [27,28]. Thus, HBV infection can evoke varied cell-mediated immune responses, which are characterized by several serovirological profiles, indicating a variable host reactivity against the virus. Therefore, many aspects need to be considered as the immune reactivity against HBV cannot be easily categorized in two functional profiles, particularly in the context of chronic infection. For instance, the heterogeneous profiles of the T cell response are influenced by the extent of HBV replication [29,30] as well as the amounts of circulating HBsAg [17], with a progressive impairment of the specific response up to suppression in cases with high levels of viremia [29]. This results in different immune profiles according to patients’ clinical profiles [17].

CD4 T-cell response A conventional concept in antiviral immunity is that CD8+ T cells play a major role in infection control. This holds particularly true for HBV, a typical ‘single’-cell targeting infection, where successful control is guaranteed by the elimination of virus-infected hepatocytes expressing viral antigens associated with MHC class I on their cell surface. However, recent observations led to a renewed interest in the role of CD4+ T cells as contributors to the antiviral immune response [31]. As far as HBV is concerned, the study of the role of CD4+ T cells in the final control of HBV infection has generated some inconsistent data. In fact, while CD4 depletion does not influence the outcome of acute HBV infection in HBV-infected chimpanzees [32], the CD4-mediated response has been reported to be completely undetectable in the acute phase in patients who subsequently developed chronic infection [33]. Similar to the CD8+ T cells, the CD4 response in the acute phase of self-limiting infection is significantly more frequent, strong and multispecific than that observed in the chronic phase [30,33–35]. The characterization of CD4+ T cells has identified a preferential Th-1 profile, a significantly more frequent targeting of the core antigen than HBsAg [16,35– 37], and the ability to persist for decades after the resolution of infection [38,39] as a long-term memory response, as well as a response triggered by minute amounts of the circulating virus. We are now completely aware of the fact © 2014 John Wiley & Sons Ltd

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that a T cell response can be detected to the various forms of previous exposure to HBV, defined by different serological profiles, and collectively grouped as ‘occult hepatitis’, even if at different extents and with different mechanisms [40]. The proper activation of all players involved in this type of immune response requires a complex interplay that has to be tightly coordinated. Among the many factors influencing the immune response, the timing of CD4+ T cell priming has a strong influence on the outcome of HBV infection. In fact, early CD4 priming is required to obtain a synchronized influx of HBV-specific CD8+ T cells that ultimately results in viral clearance [26]. The importance of the response timing has been confirmed by a recent study performed during a very early phase of infection, before the development of the humoral response and the appearance of symptoms [41]. In the infected host, CD4- and CD8-mediated responses have a similar profile of expansion, reaching the maximal frequency at a similar time, but the CD4 response declines earlier than the CD8 response, which persists up to the peak of the antiviral response. Likewise, cytokine production by CD4+ T cells also showed a time-dependent regulation, starting with secretion of Th2-type cytokines and then followed by that of Th-1 cytokines. Although not involved in the ultimate control of infection, it has become clear that a tight quantitative and qualitative regulation of the CD4 response is required to allow the antiviral CD8+ T cells to eliminate the infection. The complexity of the CD4+ T cell population has recently been increased by the identification of additional memory T helper subsets, defined according to cytokine production and the expression of certain transcription factors and homing receptors [42]. The memory CD4+ T cells specific for different pathogens cluster quite consistently in different functional modules, identifying a type of pathogen-based signature [43]. In the case of viral infection, specific CD4+ T cells predominantly belong to the Th1 subset [43] and express CXCR3 [44]. In agreement with these observations, many intrahepatic cells such as hepatocytes, stellate cells, sinusoidal endothelial cells and CD4 lymphocytes in the inflammatory infiltrate are potential sources of CXCR3 ligands in liver disease, suggesting that these chemokines play a role in effector cell recruitment to the liver [45,46]. Furthermore, CXCR3 ligands are upregulated in the serum and liver tissue in chronic liver infection [47,48], and their expression varies in different stages of liver injury [49]. While a univocal chemokine receptor for liver homing has not been identified as yet, other chemokine receptors expressed on Th1 or activated effector cells, such as CCR1, CCR5, CXCR1 and CXCR6, have been proven to contribute to the recruitment and retention of CD4+ T cells in liver inflammation and chronic infection [45,46,50]. In chronic infection, the significance of the new additional memory T helper subsets lies not in the mechanisms of

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antiviral immunity but more likely in the non-virus-specific inflammatory infiltration. Th17 is a proinflammatory subset involved in the defence against fungi and extracellular bacteria [51] and in the pathogenesis of autoimmunity [52]. Although not enriched among HBV-specific T cells, neither in acute nor chronic infection (Loggi, unpublished data), several studies have reported an increased frequency of circulating Th17 cells in HBV infection and correlated this to the degree of liver injury, even in advanced stages of disease [53–56]. Recently, it has been shown that Th17-related liver damage can be triggered directly by HBV via intrahepatic IL-17A/IL-23 production but not by generic inflammation [57]. The role of IL-22, another cytokine produced not only by Th17 cells but also by a distinct autonomous Th subset defined as Th22, is more controversial [58]. Much evidence supports the hepatoprotective role of IL-22 in several conditions [59], although it does not have direct antiviral effects, as IL-22 does not suppress HBV replication in cell culture [60] or in HBV- transgenic mice [61]. Regulatory CD4+ T cells (Tregs) represent a specialized T cell subpopulation able to suppress autoreactive T cell responses and maintain immunological tolerance. They are characterized by the constitutive surface expression of the interleukin-2 receptor alpha CD25 and the transcription factor Foxp3 [62]. The significance of this cell subset in chronic infection has largely been investigated for several reasons. First, the liver is a naturally tolerizing immunological environment, which necessarily developed due to the continuous exposure to large amounts of food antigens [63]; this ‘naturally pro-tolerogenic’ milieu is exacerbated in the context of persisting infection due to the need to dampen immunopathology from the liver itself and also as a consequence of attempts by microbes to impair the immune response. Although the strong rationale potentially underlying a primary role for Tregs in HBV infection, the study of their contribution to T-cell dysfunction has provided conflicting results [64–67]. In fact, while most studies have reported a higher frequency of Tregs in both the periphery and the liver of HBV-infected patients, their effect on antiviral immunity has not been defined. A recent study performed in a murine model of acute HBV infection highlighted this controversial role; it showed that Tregs are recruited early in the liver to prevent viral damage and delay viral clearance, acting on both the innate and adaptive immune systems. However, Tregs do not influence the late development of HBV-specific CD8+ T cells or memory T cells [68]. Interestingly, Tregs appear to be recruited in response to CXCR3, similar to effector cells, further emphasizing the need of a synchronized efflux of different cell types to maintain the appropriate homeostasis between response, activation and regulation. Among the various Th subsets, T follicular helper cells (Tfhs) deserve further investigation. Indeed, this subset, which is defined by the expression of CXCR5, inducible

costimulator (ICOS) and PD-1, is specialized to aid the development of B cells into Ab-producing cells in germinal centres [69,70]. The magnitude of the Tfh response is related to persistent antigen stimulation, resulting in polyclonal B cell activation [71], and appears to be critical for achieving the control of chronic infection [72]. These findings are consistent with the report that IL-21, the hallmark cytokine produced by Tfhs, is essential for sustaining the CD8 response in models of chronic infection [73]. Patients with chronic HBV infection have a higher frequency of CXCR5+ CD4+ T cells than healthy controls [74,75], and this could play a role in favouring HBeAg seroconversion [75]. However, these data are limited by the study of the circulating CXCR5+ compartment. Although these cells share some properties with Tfhs [76], their exact relationship with follicular T cells is not well-established. To summarize, it is clear that the evaluation of the CD4+ T cell contribution to the immune response against HBV should be extended to both specific and nonspecific contributors. All of these cells are recruited to the liver by a complex, largely unknown and most likely redundant chemokine network that is produced by liver components and driven by both inflammation and direct viral modulation. Discriminating between cell classes promoting inflammation and fibrogenesis and those exerting antiviral activity is a major challenge to understanding the mechanisms potentially leading to the control of HBV infection. This can be achieved by a wide multi-parameter evaluation that incorporates all of the involved cell subsets.

CD8+ T-cell response It is widely believed that the CTL response clears viral infections by killing infected cells. The same above mentioned depletion experiments [32] in HBV-infected chimpanzees have provided strong support for the concept that CD8+ T cells are the main cellular subset responsible for viral clearance. Many studies support the centrality of this role: (i) patients with acute hepatitis show a vigorous polyclonal CD8+ T cell response that is readily detectable in peripheral blood, while patients with chronic infection tend to have transient or narrowly focused T-cell responses [30,33,34,39]; (ii) at the peak of the antiviral response, the frequency of virus-specific CD8+ T cells has been reported to dominate the response [41,77]; (iii) CD8+ T cells are the only cells detectable in conditions of immunosuppression, following transplantation for HBV-related disease [78]. In addition to quantitative differences, qualitative differences have also been demonstrated to further distinguish between the successful and unsuccessful control of infection. MHC/peptide tetramer staining methodologies have helped to identify some crucial frequently targeted epitopes among the CD8 repertoire, including the highly immunodominant core sequence © 2014 John Wiley & Sons Ltd

HBV and the adaptive immune response 18–27 as well as pol 455–63, env 183–91 and env 335– 43, which are largely targeted in the great majority of A2+ patients with self-limited hepatitis [29,79]. However, the latter approach is limited to a few sequences that are HLA-A2 restricted, and HBV-specific T-cell repertoires tend to diverge according to ethnicity because of HLA polymorphisms [80]. Nevertheless, a more comprehensive approach using overlapping peptides of proper length to stimulate a response regardless of HLA did not increase the rate of detectable responses in a chronic infection [17,30]. Similar to CD4+ T cells, nucleocapsid-specific CD8+ T cells appear to represent an effective correlate of protection; the core and polymerase proteins are the most immunogenic proteins, while the envelope specificities have the ability to persist in the face of high levels of HBV replication, although with an apparent inability to exert antiviral function [81]. Interestingly, it is widely recognized that CTLs are responsible for both liver damage and viral clearance, but these two events are not dependent on each other and are driven by different mechanisms. In an HBV transgenic mouse model, HBV gene expression and replication in the liver were abolished without killing the hepatocytes, by IFN gamma and TNF alpha secretion, or by the apoptosis of hepatocytes physically engaged by CTLs [82,83]. There is a growing body of evidence on the mechanisms underlying the typical CD8 dysfunction in chronic infection, resulting in complete deletion of HBV-specific T cells or in their functional inability to exert antiviral function, a status called T cell exhaustion. Since its first description more than a decade ago, T cell exhaustion has been demonstrated in a wide variety of animal models and in human chronic viral and bacterial infections, as well as in human cancer [84]. Recent advancements in this setting have demonstrated that exhaustion reflects a unique state of gene expression, as it differs from na€ıve, effector and memory cells [85]. It is regulated by extrinsic negative pathways (immunoregulatory cytokines) and intrinsic cell-mediated mechanisms, which overall result in a large variety of cellular abnormalities: overexpression of multiple inhibitory co-receptors, altered development and maintenance of memory, modification of cytokine production, and metabolic and bioenergetic deficiencies. This model of functional T cell exhaustion, applied to multiple types of viral diseases, particularly holds true in chronic HBV due to the peculiarities of such an infection. The first of these peculiarities is the large amount of viral antigens. Second, the unique features of the intrahepatic milieu and its tolerizing signals are notable. Lastly, antigen presentation by hepatocytes results in an increased propensity to apoptosis [86], which is relatively inefficient because of the low expression of MHC molecules and consequent higher requirement of peptides to prime an equivalent number of CD8+ T cells [87]. This results in an insufficient or altered stimulation, occurring in the context of an out© 2014 John Wiley & Sons Ltd

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weighed excess of co-inhibitory signals that drives the cells to exhaustion. Among the co-inhibitory signals, one of the best characterized in chronic HBV is mediated through the PD-1 pathway. Similar to other models of viral infection, PD-1 is already upregulated in the acute phase of infection, but it declines with the clearance of infection, concomitant with the emergence of memory T cells [88]. Conversely, circulating HBV-specific CD8+ T cells in chronic HBV patients are mainly PD-1 positive, and the percentage of tetramerpositive CD8+ T cells is even higher in the intrahepatic compartment. Blockade of the PD-1 interaction with one of its ligands, PDL1, by a specific monoclonal antibody, antiPDL1, increases CD8 cell proliferation and cytokine production in both circulating and intrahepatic lymphocytes [30,89]. In this same view, as exhaustion is a complex event that involves a large series of nonredundant molecules that can be differently manipulated, blocking an inhibitory pathway associated with positive signal stimuli can provide better results: the functional restoration of intrahepatic HBV-specific T cells was greater when combining a PD-1 inhibitor with a trigger of CD137, a pathway with an anti-apoptotic effect [90]. Similarly, CTLA-4, another coinhibitory receptor that is not constitutively expressed on effector T cells and is rapidly upregulated upon their activation [91], acts synergistically with PD-1 in promoting T cell exhaustion and is also over-expressed on HBV-specific CD8+ T cells [92]. The blocking of CTLA-4 rescues the antiviral function of antigen-specific CD8+ T cells [92]. Indeed, the antiviral dysfunction of CD8+ T cells should be included in a broader scenario, in which these cells face the liver microenvironment. For instance, persistent inflammation has been proven to deplete essential amino acids such as arginine, which in turn results in the downregulation of CD3f, a component of the TCR signalling complex, and in reduced capacities of T cells to proliferate and produce cytokines [93]. This can exacerbate the intrinsic pro-apoptotic propensity of intrahepatic HBV-specific CD8+ T cells [94]. From this, the elucidation of different mechanisms and of dysregulated expression molecules has led to a ‘measure’ of CD8 dysfunctionality, and it has formed the basis for new therapeutic perspectives. Despite the encouraging data, breaking immunological tolerance is not easy and requires careful evaluation of the risk/benefit ratio. This should be considered foremost because of the multiple, and most likely synergistic, components of antiviral dysfunction in chronic HBV; the modification of just one of these components is most likely not sufficient to improve viral control. For instance, the longterm viral suppression induced by antiviral treatment is not able itself to restore antiviral immunity [17].

CONCLUDING REMARKS The adaptive anti-HBV response represents the key element affecting the outcome of HBV infection. It is currently

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accepted that the dysfunction of the antigen-specific CD8 compartment is the primary cause of a lack of antiviral control in persistent infection, although to different extents according to the clinical stage of infection. To date, the possibility to reverse it by manipulating the different pathways involved represents the most attractive challenge for a therapeutic approach to chronic infection [95,96], which will hopefully be able to substitute or complement the currently available therapeutic strategies in the near future. Although the rationale is supported by accumulating evidence, this perspective needs to face the complexities, even

those that are inapparent, of (i) HBV infection models; (ii) the environment in which the infection occurs and (iii) the countless strategies of this indirectly cytopathic virus, which can survive within an infected host for decades and carefully balance its survival with damage to the host. Thus, the possibility of immunologically improving the control of HBV infection requires in-depth knowledge of both HBV-specific and nonspecific cellular networks as well as their interplay. This knowledge is required to better define the pathways that should be blocked and those that should be triggered.

REFERENCES 1 Yan H, Zhong G, Xu G et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B an, nd D virus. Elife 2012; 1: e00049. 2 McMahon BJ. Epidemiology and natural history of hepatitis B. Semin Liver Dis 2005; 25: 3–8. 3 European Association for the Study of the Liver. EASL clinical practice guidelines: management of chronic hepatitis B virus infection. J Hepatol 2012; 57: 167–185. 4 Guidotti LG, Chisari FV. Immunobiology and pathogenesis of viral hepatitis. Annu Rev Pathol 2006; 1: 23– 61. 5 Bertoletti A, Gehring AJ. The immune response during hepatitis B virus infection. J Gen Virol 2006; 87: 1439–1449. 6 Bertoletti A, Ferrari C. Innate and adaptive immune responses in chronic hepatitis B virus infections: towards restoration of immune control of viral infection. Gut 2012; 61: 1754–1764. 7 McMahon BJ. The influence of hepatitis B virus genotype and subgenotype on the natural history of chronic hepatitis B. Hepatol Int 2009; 3: 334–3428. 8 Alberti A, Diana S, Sculard GH, Eddleston AL, Williams R. Detection of a new antibody system reacting with Dane particles in hepatitis B virus infection. Br Med J 1978; 2: 1056–1058. 9 Hangartner L, Zinkernagel RM, Hengartner H. Antiviral antibody responses: the two extremes of a wide spectrum. Nat Rev Immunol 2006; 6: 231–243.

10 Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev 2000; 64: 51–68. 11 Lada O, Benhamou Y, Poynard T, Thibault V. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of “a” determinant variants. J Virol 2006; 80: 2968–2975. 12 Matsue K, Kimura S, Takanashi Y et al. Reactivation of hepatitis B virus after rituximab-containing treatment in patients with CD20positive B-cell lymphoma. Cancer 2010; 116: 4769–4776. 13 Mitchison NA. T-cell-B-cell cooperation. Nat Rev Immunol 2004; 4: 308–312. 14 Milich DR, Chen M, Schodel F, Peterson DL, Jones JE, Hughes JL. Role of B cells in antigen presentation of the hepatitis B core. Proc Natl Acad Sci U S A 1997; 94: 14648–14653. 15 Lee BO, Tucker A, Frelin L et al. Interaction of the hepatitis B core antigen and the innate immune system. J Immunol 2009; 182: 6670– 6681. 16 Ferrari C, Bertoletti A, Penna A et al. Identification of immunodominant T cell epitopes of the hepatitis B virus nucleocapsid antigen. J Clin Invest 1991; 88: 214–222. 17 Loggi E, Bihl FK, Cursaro C et al. Virus-specific immune response in HBeAg-negative chronic hepatitis B: relationship with clinical profile and HBsAg serum levels. PLoS ONE 2013; 8: e65327. 18 Raimondi G, Zanoni I, Citterio S, Ricciardi-Castagnoli P, Granucci F. Induction of peripheral T cell

19

20

21

22

23

24

25

tolerance by antigen-presenting B cells. II. Chronic antigen presentation overrules antigen-presenting B cell activation. J Immunol 2006; 176: 4021–4028. Frommer F, Heinen TJ, Wunderlich FT et al. Tolerance without clonal expansion: self-antigen-expressing B cells program self-reactive T cells for future deletion. J Immunol 2008; 181: 5748–5759. Moir S, Ho J, Malaspina A et al. Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIVinfected viremic individuals. J Exp Med 2008; 205: 1797–1805. Weiss GE, Crompton PD, Li S et al. Atypical memory B cells are greatly expanded in individuals living in a malaria-endemic area. J Immunol 2009; 183: 2176–2182. Oliviero B, Cerino A, Varchetta S et al. Enhanced B-cell differentiation and reduced proliferative capacity in chronic hepatitis C and chronic hepatitis B virus infections. J Hepatol 2011; 55: 53–60. Das A, Ellis G, Pallant C et al. IL10-producing regulatory B cells in the pathogenesis of chronic hepatitis B virus infection. J Immunol 2012; 189: 3925–3935. Farci P, Diaz G, Chen Z et al. B cell gene signature with massive intrahepatic production of antibodies to hepatitis B core antigen in hepatitis B virus-associated acute liver failure. Proc Natl Acad Sci U S A 2010; 107: 8766–8771. Rehermann B. Immune responses in hepatitis B virus infection. Semin Liver Dis 2003; 23: 21–38.

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HBV and the adaptive immune response 26 Chisari FV, Isogawa M, Wieland SF. Pathogenesis of hepatitis B virus infection. Pathol Biol (Paris) 2010; 58: 258–266. 27 Loggi E, Bihl F, Chisholm JV et al. Anti-HBs re-seroconversion after liver transplantation in a patient with past HBV infection receiving a HBsAg positive graft. J Hepatol 2009; 50: 625–630. 28 Loggi E, Micco L, Ercolani G et al. Liver transplantation from hepatitis B surface antigen positive donors: a safe way to expand the donor pool. J Hepatol 2012; 56: 579– 585. 29 Webster GJ, Reignat S, Brown D et al. Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy. J Virol 2004; 78: 5707–5719. 30 Boni C, Fisicaro P, Valdatta C et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. J Virol 2007; 81: 4215–4225. 31 Sant AJ, McMichael A. Revealing the role of CD4(+) T cells in viral immunity. J Exp Med 2012; 209: 1391–1395. 32 Thimme R, Wieland S, Steiger C et al. CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol 2003; 77: 68–76. 33 Urbani S, Boni C, Amadei B et al. The impairment of CD8 responses limits the selection of escape mutations in acute hepatitis C virus infection. J Immunol 2005; 175: 7519–7529. 34 Ferrari C, Penna A, Bertoletti A et al. Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection. J Immunol 1990; 145: 3442–3449. 35 Jung MC, Spengler U, Schraut W et al. Hepatitis B virus antigen-specific T-cell activation in patients with acute and chronic hepatitis B. J Hepatol 1991; 13: 310–317. 36 Penna A, Del Prete G, Cavalli A et al. Predominant T-helper 1 cytokine profile of hepatitis B virus nucleocapsid-specific T cells in acute

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46

47

self-limited hepatitis B. Hepatology 1997; 25: 1022–1027. Bocher WO, Herzog-Hauff S, Schlaak J. Meyer zum Buschenfeld KH, Lohr HF. Kinetics of hepatitis B surface antigen-specific immune responses in acute and chronic hepatitis B or after HBs vaccination: stimulation of the in vitro antibody response by interferon gamma. Hepatology 1999; 29: 238–244. Penna A, Artini M, Cavalli A et al. Long-lasting memory T cell responses following self-limited acute hepatitis B. J Clin Invest 1996; 98: 1185–1194. Rehermann B, Ferrari C, Pasquinelli C, Chisari FV. The hepatitis B virus persists for decades after patients’ recovery from acute viral hepatitis despite active maintenance of a cytotoxic T-lymphocyte response. Nat Med 1996; 2: 1104–1108. Zerbini A, Pilli M, Boni C et al. The characteristics of the cell-mediated immune response identify different profiles of occult hepatitis B virus infection. Gastroenterology 2008; 134: 1470–1481. Fisicaro P, Valdatta C, Boni C et al. Early kinetics of innate and adaptive immune responses during hepatitis B virus infection. Gut 2009; 58: 974–982. Zhu J, Yamane H, Paul WE. Differentiation of effector CD4 T cell populations (*). Annu Rev Immunol 2010; 28: 445–489. Zielinski CE, Corti D, Mele F, Pinto D, Lanzavecchia A, Sallusto F. Dissecting the human immunologic memory for pathogens. Immunol Rev 2011; 240: 40–51. Sallusto F, Lanzavecchia A. Heterogeneity of CD4+ memory T cells: functional modules for tailored immunity. Eur J Immunol 2009; 39: 2076–2082. Oo YH, Shetty S, Adams DH. The role of chemokines in the recruitment of lymphocytes to the liver. Dig Dis 2010; 28: 31–44. Wasmuth HE, Tacke F, Trautwein C. Chemokines in liver inflammation and fibrosis. Semin Liver Dis 2010; 30: 215–225. Zeremski M, Petrovic LM, Chiriboga L et al. Intrahepatic levels of CXCR3associated chemokines correlate with

48

49

50

51

52

53

54

55

56

57

58

311

liver inflammation and fibrosis in chronic hepatitis C. Hepatology 2008; 48: 1440–1450. Zeremski M, Dimova R, Brown Q, Jacobson IM, Markatou M, Talal AH. Peripheral CXCR3-associated chemokines as biomarkers of fibrosis in chronic hepatitis C virus infection. J Infect Dis 2009; 200: 1774– 1780. Tan AT, Koh S, Goh W et al. A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B. J Hepatol 2010; 52: 330–339. Heydtmann M, Adams DH. Chemokines in the immunopathogenesis of hepatitis C infection. Hepatology 2009; 49: 676–688. Sallusto F, Zielinski CE, Lanzavecchia A. Human Th17 subsets. Eur J Immunol 2012; 42: 2215–2220. O’Quinn DB, Palmer MT, Lee YK, Weaver CT. Emergence of the Th17 pathway and its role in host defense. Adv Immunol 2008; 99: 115–1163. Zhang JY, Zhang Z, Lin F et al. Interleukin-17-producing CD4(+) T cells increase with severity of liver damage in patients with chronic hepatitis B. Hepatology 2010; 51: 81–91. Zhang GL, Xie DY, Ye YN et al. High level of IL-27 positively correlated with Th17 cells may indicate liver injury in patients infected with HBV. Liver Int 2014; 34: 266–273. Li J, Qiu SJ, She WM et al. Significance of the balance between regulatory T (Treg) and T helper 17 (Th17) cells during hepatitis B virus related liver fibrosis. PLoS ONE 2012; 7: e39307. Wang LY, Meng QH, Zou ZQ et al. Increased frequency of circulating Th17 cells in acute-on-chronic hepatitis B liver failure. Dig Dis Sci 2012; 57: 667–674. Wang Q, Zhou J, Zhang B et al. Hepatitis B virus induces IL-23 production in antigen presenting cells and causes liver damage via the IL23/IL-17 axis. PLoS Pathog 2013; 9: e1003410. Duhen T, Geiger R, Jarrossay D, Lanzavecchia A, Sallusto F. Production of interleukin 22 but not interleukin 17 by a subset of human

312

59

60

61

62

63 64

65

66

67

68

69

E. Loggi et al.

skin-homing memory T cells. Nat Immunol 2009; 10: 857–863. Cobleigh MA, Robek MD. Protective and pathological properties of IL-22 in liver disease: implications for viral hepatitis. Am J Pathol 2013; 182: 21–28. Wang B, Zhao XP, Fan YC, Zhang JJ, Zhao J, Wang K. IL-17A but not IL-22 suppresses the replication of hepatitis B virus mediated by overexpression of MxA and OAS mRNA in the HepG2.2.15 cell line. Antiviral Res 2013; 97: 285–292. Zhang Y, Cobleigh MA, Lian JQ et al. A proinflammatory role for interleukin-22 in the immune response to hepatitis B virus. Gastroenterology 2011; 141: 1897–1906. Josefowicz SZ, Lu LF, Rudensky AY. Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol 2012; 30: 531–564. Crispe IN. Liver antigen-presenting cells. J Hepatol 2011; 54: 357–365. Stoop JN, van der Molen RG, Baan CC et al. Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection. Hepatology 2005; 41: 771–778. Franzese O, Kennedy PT, Gehring AJ et al. Modulation of the CD8+T-cell response by CD4+ CD25+ regulatory T cells in patients with hepatitis B virus infection. J Virol 2005; 79: 3322–3328. Xu D, Fu J, Jin L et al. Circulating and liver resident CD4+ CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B. J Immunol 2006; 177: 739–747. Peng G, Li S, Wu W, Sun Z, Chen Y, Chen Z. Circulating CD4+ CD25+ regulatory T cells correlate with chronic hepatitis B infection. Immunology 2008; 123: 57–65. Stross L, Gunther J, Gasteiger G et al. Foxp3+ regulatory T cells protect the liver from immune damage and compromise virus control during acute experimental hepatitis B virus infection in mice. Hepatology 2012; 56: 873–883. Vinuesa CG, Linterman MA, Goodnow CC, Randall KL. T cells and follicular dendritic cells in germinal

70

71

72

73

74

75

76

77

78

79

center B-cell formation and selection. Immunol Rev 2010; 237: 72– 89. Crotty S. Follicular helper CD4 T cells (TFH). Annu Rev Immunol 2011; 29: 621–663. Baumjohann D, Preite S, Reboldi A et al. Persistent antigen and germinal center B cells sustain T follicular helper cell responses and phenotype. Immunity 2013; 38: 596–605. Fahey LM, Wilson EB, Elsaesser H, Fistonich CD, McGavern DB, Brooks DG. Viral persistence redirects CD4 T cell differentiation toward T follicular helper cells. J Exp Med 2011; 208: 987–999. Yi JS, Du M, Zajac AJ. A vital role for interleukin-21 in the control of a chronic viral infection. Science 2009; 324: 1572–1576. Feng J, Lu L, Hua C et al. High frequency of CD4+ CXCR5+ TFH cells in patients with immune-active chronic hepatitis B. PLoS ONE 2011; 6: e21698. Li Y, Ma S, Tang L et al. Circulating chemokine (C-X-C Motif) receptor 5 (+) CD4(+) T cells benefit hepatitis B e antigen seroconversion through IL-21 in patients with chronic hepatitis B virus infection. Hepatology 2013; 58: 1277–1286. Morita R, Schmitt N, Bentebibel SE et al. Human blood CXCR5(+)CD4 (+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity 2011; 34: 108–121. Maini MK, Gudgeon N, Wedderburn LR, Rickinson AB, Beverley PC. Clonal expansions in acute EBV infection are detectable in the CD8 and not the CD4 subset and persist with a variable CD45 phenotype. J Immunol 2000; 165: 5729–5737. Bihl F, Loggi E, Chisholm JV et al. Sustained and focused hepatitis B virus nucleocapsid-specific T-cell immunity in liver transplant recipients compared to individuals with chronic and self-limited hepatitis B virus infection. Liver Transpl 2008; 14: 478–485. Webster GJ, Reignat S, Maini MK et al. Incubation phase of acute hepatitis B in man: dynamic of

80

81

82

83

84 85

86

87

88

89

cellular immune mechanisms. Hepatology 2000; 32: 1117–1124. Tan AT, Loggi E, Boni C et al. Host ethnicity and virus genotype shape the hepatitis B virus-specific T-cell repertoire. J Virol 2008; 82: 10986–10997. Reignat S, Webster GJ, Brown D et al. Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection. J Exp Med 2002; 195: 1089–1101. Guidotti LG, Ishikawa T, Hobbs MV, Matzke B, Schreiber R, Chisari FV. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity 1996; 4: 25– 36. Guidotti LG, Ando K, Hobbs MV et al. Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice. Proc Natl Acad Sci U S A 1994; 91: 3764–3768. Wherry EJ. T cell exhaustion. Nat Immunol 2011; 12: 492–499. Wherry EJ, Ha SJ, Kaech SM et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 2007; 27: 670– 684. Bowen DG, Zen M, Holz L, Davis T, McCaughan GW, Bertolino P. The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity. J Clin Invest 2004; 114: 701– 712. Gehring AJ, Sun D, Kennedy PT et al. The level of viral antigen presented by hepatocytes influences CD8 T-cell function. J Virol 2007; 81: 2940–2949. Boettler T, Panther E, Bengsch B et al. Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection. J Virol 2006; 80(7): 3532–3540. Fisicaro P, Valdatta C, Massari M et al. Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B. Gastroenterology 2010; 138: 682–693, 693 e1-4.

© 2014 John Wiley & Sons Ltd

HBV and the adaptive immune response 90 Fisicaro P, Valdatta C, Massari M et al. Combined blockade of programmed death-1 and activation of CD137 increase responses of human liver T cells against HBV, but not HCV. Gastroenterology 2012; 143 (1576–1585): e4. 91 Walunas TL, Bakker CY, Bluestone JA. CTLA-4 ligation blocks CD28dependent T cell activation. J Exp Med 1996; 183: 2541–2550. 92 Schurich A, Khanna P, Lopes AR et al. Role of the coinhibitory receptor

© 2014 John Wiley & Sons Ltd

cytotoxic T lymphocyte antigen-4 on apoptosis-Prone CD8 T cells in persistent hepatitis B virus infection. Hepatology 2011; 53: 1494– 1503. 93 Das A, Hoare M, Davies N et al. Functional skewing of the global CD8 T cell population in chronic hepatitis B virus infection. J Exp Med 2008; 205: 2111–2124. 94 Lopes AR, Kellam P, Das A et al. Bim-mediated deletion of antigenspecific CD8 T cells in patients

313

unable to control HBV infection. J Clin Invest 2008; 118: 1835–1845. 95 Maini MK, Schurich A. The molecular basis of the failed immune response in chronic HBV: therapeutic implications. J Hepatol 2010; 52: 616–619. 96 Michel ML, Deng Q, Mancini-Bourgine M. Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: perspectives and challenges. J Hepatol 2011; 54: 1286–1296.