Adaptive immunity in HBV infection

Review

Adaptive immunity in HBV infection

Summary During hepatitis B virus (HBV) infection, the presence of HBV-specific antibody producing B cells and functional HBV-specific T cells (with helper or cytotoxic effects) ultimately determines HBV infection outcome. In this review, in addition to summarizing the present state of knowledge of HBV-adaptive immunity, we will highlight controversies and uncertainties concerning the HBV-specific B and T lymphocyte response, and propose future directions for research aimed at the generation of more efficient immunotherapeutic strategies. Ó 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Review

Antonio Bertoletti1,2,⇑, Carlo Ferrari3

Keywords: Antiviral immunity; B lymphocytes; T lymphocytes; Immunodominance; T cell exhaustion. Received 3 December 2015; received in revised form 12 January 2016; accepted 25 January 2016

1

Introduction Adaptive immunity forms a sophisticated branch of the immune system. Its ‘‘adaptability” consists in the capacity to undergo changes in response to the varying challenges imposed by different pathogens. During an infection, microbe-specific T and B lymphocytes increase in both number and fitness to fight the infection. Following disease resolution, immunological memory of the pathogen is maintained allowing a stronger and more rapid immune defense to be mounted should the same pathogen be encountered subsequently. Immunological memory is linked to the other fundamental feature of the adaptive immune system: antigen specificity. Through a complex mechanism of genetic recombination of genes coding for the variable regions of antigen receptors (i.e. antibodies and T cell receptors; TCR), B and T lymphocytes are generated with vast numbers of specificities that are then selected by the specific antigens. Antibodies secreted by B cells recognize conformational antigen, either binding the pathogen directly or binding the proteins secreted or expressed on the surface of infected cells. The TCR of the T lymphocyte provides critical recognition of pathogen protein fragments, known as epitopes, expressed on the surface of cells in association with major histocompatibility complex (MHC) class I or class II molecules [1]. These epitopes may be formed during the processing of pathogenic proteins that are synthesized within the cells (e.g., as it occurs for example in virusinfected cells) for presentation on MHC class I

molecules (HLA-class I in humans) to the TCR of CD8+ T cells [2]. Epitopes derived from the internalization of pathogens by specialized antigen presenting cells (i.e. dendritic cells, B cells, monocytes/macrophages) are presented by MHC class II molecules to the TCR of CD4 helper T cells [3] (Fig. 1). There are exceptions to this rule (i.e. a specialized sub-populations of dendritic cells can cross-present internalized antigen through MHC class-I molecules to CD8 T cells) [4], but the existence of two segregated pathways of antigen presentation ensure that CD8 T cells can discriminate between infected cells and cells that have only internalized pathogen’s proteins [5]. During hepatitis B virus (HBV) infection, the process of generating a complex repertoire of virus-specific B and T cells is considered of paramount importance. While the innate branch of immunity (reviewed in the companion article of M. Maini and A. Gehring [148]) is designed for rapid viral replication control, the presence of HBV-specific antibody producing B cells and functional HBV-specific T cells (with helper or cytotoxic effects) ultimately determines HBV infection outcome. Such concepts have already been analyzed in several reviews and readers are directed to these works for a broad discussion of the causes of such differences and their clinical implications [6–9]. Nevertheless, further clarification is necessary to understand the mechanism through which HBV is capable of eluding the sophisticated adaptive immune system and persists unchecked throughout a patient’s lifespan.

Journal of Hepatology 2016 vol. 64 j S71–S83

Emerging Infectious Diseases (EID) Program, Duke-NUS Medical School, Singapore; 2 Viral Hepatitis Laboratory, Singapore Institute for Clinical Sciences, Agency of Science Technology and Research (A*STAR), Singapore; 3 Divisione Malattie Infettive, Ospdale Maggiore Parma, Parma, Italy

⇑ Corresponding author. Address: Emerging Infectious Diseases Program, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore. Tel.: +65 66011372. E-mail address: [email protected] (A. Bertoletti).

Review

A

Target protein “antigen”

Ab variable regions

B

Peptide generated by the processing of endogenous antigen

Peptide generated by the processing of exogenous antigen Antigen presenting cell

Review

Target cell

Light chain MHC-class I

T cell receptor

MHC-class II

Peptide

TCR variable regions

T cell receptor

Heavy chain CD8 T cell

Antibody

CD4 T cell

T cell receptor

Fig. 1. Schematic representation of antibody and T cell receptor structures and their recognition ability. A schematic representation of the recognition of conformational antigen by an antibody (A) or of endogenous synthesized or exogenous captured antigen by a CD8 (cytotoxic) or CD4 (helper) T cells (B).

Key point HBV neutralizing antibodies have a role in protection and modulation of chronic HBV infection.

Key point Frequency and function are less characterized for HBV-specific B cells than T cells.

S72

B cells, neutralizing antibodies and more Over the last few decades, research into the HBVspecific B cell response has been largely neglected. Following the demonstration that distinct serum antibodies specific for different HBV proteins can be used to define different clinical groups of HBV-infected patients (reviewed in [10]), most of the research on the role of HBVspecific lymphocytes in protection and liver damage has focused on T cells instead. Antibody responses can be elicited to the different HBV proteins (core, e, envelope, polymerase and x) and the presence or absence of these antibodies, particularly those specific for the envelope (anti-HBs) and nucleocapsid antigens (anti-HBc, anti-HBe), have been used to distinguish different clinical phases of HBV infection [10]. During an acute HBV infection, anti-HBs and anti-HBc antibodies are produced with different kinetics (Fig. 2A) but only anti-HBs detection is associated with disease resolution and virus control, while anti-HBc can coexist with a high level of HBV replication [11]. As such, while anti-HBs antibodies are considered to be protective, antiHBc antibodies are used as a marker of ongoing or prior HBV contact. An overwhelming antiHBc response has also been associated with acute liver damage [12]. However, the protective ability of anti-HBV antibodies was not fully elucidated until the recent discovery of the sodium-taurocholate cotransporting polypeptide (NTCP) as the HBV receptor [13,14], along with the establishment of easily infectable in vitro cell lines. This allowed precise mapping of HBV regions essential for infectivity to the preS1 domain and the antigenic loop region (also known as the ‘‘adeterminant”) of the HBs antigen (Fig. 2B and

reviewed in [15]). The preS1 domain (in particular amino acids 2–48) interacts directly with NTCP [13,14], whereas the HBs antigenic loop (residues 104–163, located between the HBs transmembrane regions I and II of the S protein) interacts with heparin sulfate proteoglycans on hepatocytes, increasing the concentration of HBV virions on the cell surface and aiding NTCP receptor interaction [16]. Antibodies against these two regions are capable of blocking HBV infection [17,18], while antibodies specific to other HBV regions not involved in HBV infectivity, such as the PreS2 region, do not show any neutralizing ability [19]. It is clear that neutralizing antibodies have a role in prevention: the presence of antibodies against the ‘‘a-determinant” region confers the protective efficacy of immunoglobulins in liver transplant patients [20] and HBV vaccination. However, because current HBV vaccines only consist of HBsAg and do not contain the preS1 region (with the enclosed 2-48 region interacting with the NTCP receptor; Fig. 2B), antibodies capable of blocking the direct interaction of HBV with its receptor cannot be produced. This is likely to be behind the induction of nonsterilizing immunity reported in vaccinated human subjects [21] and chimpanzees [22] despite the high presence of anti-HBs antibody titers. In combination with the increased detection of mutations in the a-determinant region of several HBV isolates (reviewed in [10]), this fact raises concerns regarding the rationale of targeting a single infectivity region (the adeterminant) for prophylaxis and vaccination. These concerns are heightened by a recent large epidemiological study, which revealed a substantially lower than expected vaccination efficacy in hyperendemic areas of Taiwan [23].

Journal of Hepatology 2016 vol. 64 j S71–S83

JOURNAL OF HEPATOLOGY

A

Kinetic of HBV-specific antibodies and T cells detection after acute HBV infection

B

311

109

HBV-T cells

10

Anti-HBc 1

HBV

2 HBV infection

1 2

Anti-HBs 4

6 8 10 12 Weeks after infection

164

I

48

265

328

II

III

389

IV

Review

Virions 106/ml

100

2-48 region PreS1 “a determinant region” putative interaction (288-311) 288 with NTCP receptor

Outside Viral membrane

Inside

14

PreS1 PreS2 109

55

S 226

HBV envelope protein (large)

Fig. 2. Kinetic of HBV-specific adaptive immunity and large envelope protein structure. (A) Emergence of HBV-specific antibodies and T cell responses during acute HBV infection in relation to the kinetics of HBV replication. (B) Schematic representation of the conformation of HBV large envelope protein in the HBV particles. PreS1 domain is facing the outside of the viral membrane. The PreS1 receptor is likely to be buried in the viral membrane. For a more detailed description see Urban S et al., [15].

Another question is whether neutralizing antibodies can not only prevent acute HBV infection but can modulate the development of chronic HBV infection. This is certainly a possibility since HBV spreads to non-infected hepatocytes through an HBV receptor dependent mechanism. This was clearly demonstrated in UPA-SCID mice reconstituted with normal human hepatocytes, where blockage of HBV receptors with the Myrcludex B peptide blocked further HBV spread in infected livers [24]. The contribution of neutralizing antibodies to the control of HBV spread in chronic HBV (CHB) has not yet been fully investigated, and robust data regarding the frequency of neutralizing antibody producing B cells during chronic HBV is lacking. Patients with CHB do not have global defects in antibody production and are reported to display an increased frequency of activated B cells of memory and naïve phenotype which were functionally intact [25,26]. Furthermore, gene expression profiling performed in the peripheral blood of HBV-infected patients with different clinical and virological profiles of diseases identified a gene-signature consistent with robust B cell activation in patients with chronic active hepatitis [27], although the functional significance of this finding is unknown. The quantitative dichotomy of HBV-specific T cell responses in acute vs. chronic patients may be mirrored by HBV-specific B cell responses. As reported for HBV-specific T cells, anti-HBsproducing B cells are more commonly detected in the periphery of acute patients compared with CHB patients [26,28–31]. A defect in anti-HBV antibody production in CHB has also been elegantly suggested by studies in HBV transgenic mice. It was shown that T regulatory cells educated in the liver could block maturation of HBV-specific B and T follicular helper cells in the lymph node germinal center [32]. Anti-HBs production is thought to be T cell dependent [33] and a defect in T helper follicular

cells has also been suggested in CHB patients [34]. These are intriguing data but they do not explain the apparently robust presence of HBsAg/anti-HBs immune complexes in CHB patients [35] that mask the detection of free anti-HBs antibodies and it is indicative of the persistence of anti-HBs-producing B cells [36]. Furthermore, the detection of a reduced antiHBs producing B cells frequency in the periphery might not be fully informative. Memory B cells and plasma cells mainly home to the inflamed sites and to the bone marrow [37] and HBVspecific B cells have been reported to localize to the intrahepatic environment in HBV acute liver failure [12]. Chronic viral hepatitis is characterized by the formation of B cell-enriched ectopic lymphoid tissue in the liver parenchyma [38]; however, the function of these intrahepatic B cells in CHB is unknown. While a proportion of these cells have been shown to exert an antiinflammatory function through IL-10 secretion [39], further investigations are required into the possibility that anti-HBV-specific B cells localized to the liver are involved in the increase of HBs/ anti-HBs immune complex formation during CHB exacerbations [40,41]. Indeed, B cell studies analyzing the behavior of HBV-neutralizing antibodies during the natural history of HBV infection are now warranted. Should specific defects in HBV-neutralizing antibody production occur in CHB patients, this might indicate a therapeutic potential for neutralizing antibodies in chronic infection. Recently, the therapeutic effect of a single neutralizing anti-HBs antibody was tested in a number of HBV mouse models [42]. Antibody treatment not only reduced HBV dissemination in human chimeric mice but also suppressed HBsAg load and facilitated HBV T cell responses in HBV transgenic mice. This suggests that, in addition to their role in reducing HBV spread, neutralizing antibodies might recognize HBVinfected hepatocytes and either induce

Journal of Hepatology 2016 vol. 64 j S71–S83

S73

Review

Review

antibody-mediated cellular cytotoxicity or mediate direct lysis through complement activation. The presence of such mechanisms needs to be demonstrated in the context of natural HBV infection, but it is compelling that a therapeutic effect of antibody administration has been demonstrated in cancer as well as in other chronic viral diseases [43,44]. Furthermore, although HBV-specific T cells are generally viewed as the main cells responsible for keeping under control minute amounts of virus that persist in a few hepatocytes after resolution of acute hepatitis B [45,46], also antibodies should play a role in long-term HBV control. The progressive decline of HBV-specific CD8 T cell responses after HBsAg clearance, which become barely detectable decades after infection [47] and the possible occurrence of HBV reactivation upon B cell depletion with rituximab [48,49] are consistent with this conclusion which requires however further investigation. The potential importance of B cells in HBV infection is not only limited to their ability to produce neutralizing antibodies. B cells also act as potent antigen presenting cells, particularly for helper T cells [33] and have been shown to play a regulatory role. IL-10 producing B cells are enriched, particularly during hepatic flares of chronic hepatitis B, and have been shown to modulate not only inflammatory events as well as HBV-specific T cell responses [39].

Not all T cells are equal: frequency, specificity and immunodominance of HBV T cells HBV-specific T lymphocytes act as the principal effector mechanism of viral clearance and liver inflammation. HBV-specific CD8 T are enriched within the infected liver [50–52], lyse HBVinfected hepatocytes [50,52] and secrete cytokines (mainly interferon (IFN)-c) that trigger a process of non-cytolytic HBV clearance [53] and recruitment of inflammatory immune cells [54,55]. HBV-specific CD4 T lymphocytes regulate the intensity of these processes [56] and their frequency and function correlates with

HBV clearance [46,57]. The kinetics of HBVspecific CD8 T cell appearance and frequency have been well-characterized in adult patients with acute HBV infection [58,59] and in chimpanzees [52]. Unlike in other viral infections (such as HIV and HCMV), where virus-specific CD8 T cells can be detected within 1–2 weeks, circulating HBV-specific CD8 T cells are only apparent 6–8 weeks after infection [59]. The peak of their appearance is often concomitant with the peak of liver damage, but the quantity of HBV-specific CD8 T cells is low, with frequencies against any single HBV epitope rarely exceeding 1–3% of the total CD8 T lymphocyte pool [58–60] (see also Table 1). The peak of the HBV-specific T cell response is also concomitant with a robust bystander activation of CD8 T cells against heterologous viruses (HCMV, Epstein Barr virus), possibly contributing to inflammatory events in the liver [61]. The HBV-specific T cell response is modulated during acute hepatitis by the presence of distinct regulatory mechanisms, including PD-1 expression, IL-10, arginase, myeloid suppressor cells, and T regulatory cells [62–66]. HBV-specific T cell frequency (both CD8+ and CD4+) declines after serum HBV clearance, but a memory T cell response [45,46,60] is detectable for 20–30 years after infection [45–47,67]. The frequency of HBVspecific CD8 and CD4 T cells in chronic patients [68–73] is usually lower. Average frequency of HBV-specific T cells in different clinical settings is illustrated in Table 1. The knowledge of frequency and function of HBV-specific T cell responses associated with HBV infection resolution or chronicity in adult patients (discussed later in the paragraph ‘‘Chronic HBV and T cell exhaustion”) is not however matched by a similar understanding of what is occurring after vertical infection, which is a major cause of HBV chronicity. However, the effect of HBV infection at birth [74] and its impact on the maturation of innate and adaptive immunity has been discussed recently [9,75]. Instead, here we will discuss further important aspects regarding the protective effect of T

Table 1. Frequency of HBV-specific CD8 T cells in HBV infected subjects (% total CD8 T cells).⁄

Acute HBV Acute phase

Chronic HBV

Occult HBV

Recovery phase**

Immune tolerant

Chronic active

Chronic inactive

Anti-HBc Pos

Anti-HBc Neg

0.03-0.3%

~0-0.02%

~0.01-0.08%

~0.01-0.2%

~0.01-0.2%

~0.01

++

+/-

+/-

+

++

Neg

Ex vivo 0.2-2% In vitro expansion +/⁄

Frequency detected by individual tetramers/dextramers containing HBV epitopes originally identified in self-limited acute infections or by ex vivo Direct Elispot [58– 60,67,70,72,73,77,81,87,89,90,123]. ⁄⁄ Frequency detected 6 to 12 months after the acute phase of infection [45,46,58–60].

S74

Journal of Hepatology 2016 vol. 64 j S71–S83

JOURNAL OF HEPATOLOGY MHC-class I/Viral peptide complexes Processing

Trafficking of MHC-class I

Immunodominant CD8 T response

Review

Transport Viral proteins

Viral genome Nucleus

Sub-dominant CD8 T responses

ER

Viruses CD8 T cells specific for different viral epitopes

Infected cell

Fig. 3. Schematic representation of the hierarchy of antiviral CD8 T cell response. CD8 T cells specific for different MHC class I restricted viral epitopes are induced at different frequencies. CD8 T cells with higher frequency within an individual are defined as immunodominant (vertical immunodominance). The CD8 T cell response more frequently found in individuals expressing the same MHC class I is instead defined as horizontal immunodominance.

cells relate to HBV-specific T cell immunodominance and the distinct functions and protective values of T cells specific for different HBV determinants. CD4 and CD8 T cells specific to the nucleocapsid [68,69,76,77], envelope [78,79], polymerase [77,80] and x [81] proteins can all be induced after HBV infection. Although multi-specificity has been associated with resolution [82], CD4 and CD8 T cells recognizing different viral determinants are present in different quantities and establish a hierarchy of dominant and subdominant epitopes [70,83] (Fig. 3), which vary in their antiviral capacity [84]. Understanding immunodominance is not just an academic question, but it is necessary for the rational design of vaccines capable of eliciting T cell responses in sufficient quantity and with sufficiently broad specificity to contain viral replication and avoid selection of virus escape variants [5]. Data regarding immunodominance and protective efficacy are scarce in human viral infections. T cell epitopes able to induce the most frequent T cell clone within an individual (vertical immunodominance, see Fig. 3) or in the population (horizontal immunodominance) are not necessarily those with the highest antiviral efficacy [5]. In addition, methods to define T cell epitopes in humans are cumbersome. Human cells express up to six different MHC class I molecules capable of presenting CD8 T cell epitopes with different requirements for peptide binding [5]. Furthermore, HBV-specific T cells are scarce, and algorithms that help to define T cell epitope specificity are often designed for use in particular ethnicities (such as Caucasian), which are not the

most relevant in HBV [85]. Although evidence of some level of degeneracy in MHC class I peptide binding allowing presentation of some HBV CD8 T cell epitopes by different alleles exists [86], subtle differences even in closely related MHC class I molecules can affect the presentation of specific epitopes or change the conformation of the complexes [5]. This means that individuals with different MHC class I profiles (but also MHC class II for CD4 T cells) focus the response toward different CD8 T cell epitopes. A further variable is imposed by the fact that single amino acid variations characterizing for example different HBV subtypes can affect processing and presentation of T cell epitopes [78,87]. These factors combine to affect the relative immunodominance of different peptides across a population. Indeed, an HBV-specific T cell study performed in patients of different ethnicities and infected with different HBV genotypes, demonstrated that virological and clinical parameters determined the overall quantity of HBV-specific T cells (i.e. irrespective of ethnicities and HBV genotypes, patients who resolve HBV have a more robust response than chronic patients) but the immunodominance of different epitopes was very different [87] even in subjects who expressed closely related HLA-A2 subtypes (Fig. 4). Consequently, knowledge of HBV-specific CD8 T cells acquired in Caucasian populations (mainly infected by HBV genotypes A and D) cannot be translated directly to patients of Asian/Australian ethnicities (mainly infected by HBV genotypes B and C). Characterization of HBV-specific T cells in these highly affected pop-

Journal of Hepatology 2016 vol. 64 j S71–S83

S75

Review A*02:01

A α1

A*02:03

α2

α2

α1

Review

α3

Location of AA substitutions on α2 or α1 chain in comparison to A*02:01

B Key point HLA-class I profile and HBV genotypes heavily influence HBV-specific T cell immunodominance.

Key point Knowledge of HBV-specific T cell immunodominance and protective efficacy are still scarce during natural HBV infection.

S76

Core 18-27 Env 183-91 Pol 455-63 Enx 338-47

A*02:06

A*02:07

α2

α1

α3

α1

α2

α3

α3

α2:142, 156, 159

α1:9

α2:99

0/3 0/3 0/3 0/3

2/5 0/5 0/5 1/5

3/8 0/8 0/8 2/8

17/19 16/19 5/7 0/7

CD8 T cell response in acute HBV patients with distinct A*02 subtypes Fig. 4. Minimal differences in HLA class I molecules profoundly alter the profile of T cell response against HBV. (A) HLAA⁄02:06/02:07 and A⁄02:03 (HLA-A⁄02 subtypes characteristic of Asian ethnicities) differ from HLA-A⁄02:01 (more frequent in Caucasian) for one and three amino acids only. Locations of AA substitutions is indicated. (B) Proportion of acute HBV patients carrying distinct HLA-A⁄02 subtypes with a positive CD8 T cell response against the indicated HLA-A⁄02:01 epitope. See Tan et al., [87].

ulations is still limited [77,87–89], but a predominance of HLA-B and HLA-C class I molecules in the presentation of dominant HBV epitopes has been demonstrated [79,90]. The implication of these ethnic/genotypebased differences in epitope recognition is that, at least for CD8 T cell response, immunodominance is both HLA and HBV genotype specific and not dependent from the HBV protein of origin. This leads to another central and, in our opinion, still unresolved question on T cell adaptive immunity in HBV infection. Do HBV antigens (core, envelope, polymerase and X) intrinsically possess differential abilities to induce a protective/dominant T cell response? This concept was suggested by a study in HBV transgenic mice which compared the relative immunogenicity of the HBV envelope and polymerase proteins [84]. The two antigens induced a quantitatively comparable CD8 T cell response; however, envelope-specific CD8 T cells were more functionally protective, being of higher affinity and more capable of recognizing HBVproducing hepatocytes. Interestingly, they were also more subject to tolerization/deletion in the HBV transgenic mouse. While these observations are intriguing, it is unlikely that they can be fully translated to the setting of natural infection. Only two epitopes were compared (one for polymerase and one for envelope), which were induced by immunization with a single protein, not whole virus. As such, the model does not recapitulate the simultaneous processing and presentation of multiple epitopes by different MHC class I molecules, as occurs in natural infection. In HBV-infected individuals, different envelope-specific CD8+ T cells vary in their TCR affinity and relative immun-

odominance [90], and in some patients, polymerase-specific CD8+ T cells represent the dominant response [73,80,81]. However, HBV envelope, core, and polymerase proteins are produced in different quantities in an HBV-infected cell (i.e., the predicted ratio of Env: polymerase protein is 100:1) [91]. Such differential production supports the scenario suggested by the HBV transgenic mouse model [84]. Theoretically, envelope- (or core-) and polymerase-specific CD8+ T cells with identical TCR affinity should be able to recognize an HBV-infected target equally. However, because the quantity of antigen presented by hepatocytes modulates T cell function [92], envelope- or core-specific T cells should exert a more robust antiviral effect. On the other hand, less strongly activated polymerase-specific T cells might persist for longer under chronic HBV conditions. Data supporting this scenario are available: analysis of the global T cell repertoire in CHB patients treated with nucleos(t)ide analogue revealed a non-compromised proliferative capacity of polymerase-specific T cell responses (Rivino and Bertoletti, manuscript in preparation), while envelope and core-specific CD8+ T cells are often affected by HBV chronicity [93]. Similarly, a recent study of the global HBV-specific T response in patients with different clinical profile of chronic HBV infection have show that polymerase-specific T cells are more likely to be detectable than core or envelope-specific T cells [94]. Furthermore, although HBV mutations within T cell epitopes are not commonly found [95], they are detected more frequently in epitopes derived from the core [79,96–98], or envelope [99,100] proteins than in polymerase epitopes [79]. Consistent with this, polymerase

Journal of Hepatology 2016 vol. 64 j S71–S83

antigen has been shown to undergo less frequent mutations [79,99]. We consider the question of T cell immunodominance and hierarchy of the protective T cell response in HBV open to future dedicated studies. The protective ability of distinct HBVCD8 T cell specificities appears to be proportional to their affinity to the targeted antigen [84] and the quantity of HBV protein produced in an infected target cell may dictate the immunogenicity of the different HBV proteins. However, knowledge in this area is still too limited to reach solid conclusions. Only recently, HBV-infected targets have become available in the analysis of CD8 T cell function [101]. Such cell lines could be finally used to study how infection chronicity and/or inflammation can alter the kinetics and efficiency of HLA-class I presentation of distinct HBV proteins. This is a relevant issue because the immunoproteasome is required for the generation of some HBV T cell epitopes [102] implying that the HBV epitope repertoire can be modulated by the inflammatory conditions. The issue of immunodominance and protective ability also affects HBV-specific CD4-helper T cells, which have not been extensively covered as part of this review. In adult patients with clinically evident acute hepatitis B, the core antigen can induce a stronger HBV-specific CD4 T cell response than envelope antigens (HBs, PreS1 and PreS2) [57,76] and data in CHB patients who received bone marrow transplantation suggested that core-specific T cells are the principal effector of HBV clearance [103]. However, bone marrow transplantation of vaccinated subjects (who primarily have HBsAg-specific B and T cells) results in identical clinical efficiency [104,105]. In conclusion, the indistinct definition of the hierarchy of HBV T cell specificities correlated with defined protective effects impinges on our ability to design rational immune therapeutic strategies, and in our opinion, robust research efforts should be undertaken to resolve this area. How do T cells control HBV? Location and effector mechanisms T cells exert their anti-HBV function through a variety of mechanisms. Effector CD8 T cells directly lyse or induce apoptosis of infected hepatocytes through secretion of lytic granules (perforin/granzyme A and B) or by Fas/Fas ligand mediated mechanisms [106]. Lysis of HBV-infected hepatocytes by CD8 T cells clearly occurs during the acute symptomatic hepatitis B, and depletion of CD8 T cells reduces liver damage and HBV decline [52]. To exert their effector function, CD8 T cells must first reach their target location. During acute hepatitis B

infection, when the majority of hepatocytes are infected and the liver anatomy is intact, HBVspecific T cells can easily access the infected organ. Sophisticated intrahepatic in vivo imaging performed in HBV transgenic mice have illustrated the ability of HBV-specific CD8 T cells to detect and interact with HBV+ hepatocytes through the protrusion of cellular villi in the fenestrated endothelial barrier of the hepatic sinusoids [107]. This direct interaction between target cells and CD8 T cells is peculiar to the liver, which lacks the classical continuous endothelial cell layer and basement membrane that physically separate circulating immune cells and parenchymal cells [108]. The mechanisms by which effector T cells traffic within the liver involve expression of CD161 and the CXCR6 receptor [109], with a critical role of platelets in the docking of virus-specific CD8+ T cells to the liver parenchyma, has been extensively discussed elsewhere [108]. The liver is however an organ indispensable for life, and perhaps also to compensate for the effortless access of CD8 T cells to the liver parenchyma, specific biological mechanisms are in place to reduce the CD8 T cell mediated lysis of hepatocytes [110] and to achieve an efficient HBV suppression without substantial liver damage. The rapid decline in HBV load during acute HBV infection indicates that HBV replication can be suppressed in part by cytokine-mediated immune mechanisms [111]. Seminal work in HBV transgenic mice has shown that HBVspecific CD8 T cells can abolish HBV replication by non-cytopathic mechanisms mediated by IFN-c and TNF-a [53]. This direct contribution of IFN-c and TNF-a to HBV suppression has been therefore well documented both in vitro [112] and in vivo [53], but some controversies remain. For example, although recent work has shown the ability of IFN-c/TNF-a to reduce cccDNA content in HBVinfected hepatocytes [113], the bulk of HBV suppression mediated by CD8 T cells requires contact between effector and target cells [101], while non-cytolytic HBV suppression is observed only with long incubation times and high cytokine doses [112,113]. Notably, experiments in a woodchuck model of hepatitis B [114,115], in which intrahepatic IFN-c production was induced by gene therapy, did not find evidence of an HBV antiviral effect. One explanation is that IFN-c and TNF-a secreted by CD8+ T cells might not activate antiviral mechanisms in the infected hepatocytes directly but instead act through intermediate mediators. Experiments in HBV transgenic mice demonstrate that the antiviral activity of IFN-c is largely a result of its ability to trigger the release of nitric oxide from monocytes and hepatocytes [116]. Other

Journal of Hepatology 2016 vol. 64 j S71–S83

Review

JOURNAL OF HEPATOLOGY

Key point The protective activity of HBVspecific T cells can be modulated by the inflammatory state of the liver microenvironment.

S77

Review

Review

Key point Multiple co-inhibitory molecules are up-regulated in HBVspecific T cells of chronic patients: functional restoration might require therapeutic strategies that simultaneously target multiple regulatory pathways.

recently discovered antiviral mechanisms, not related to IFN-c and TNF-a, might also contribute to non-cytolytic T cell-mediated HBV control. For example, the activation of nuclear deaminases (APOBEC3B), triggered by the engagement of the lymphotoxin-beta (LTB) receptor on HBV-infected hepatocytes, was shown to efficiently reduce HBV-DNA and destabilize cccHBV DNA [117]. Activated T cells are known to express LIGHT and LTB2LTA heterotrimers, the physiological ligands of the LTB receptor [118]. The possibility that HBVspecific T cells can clear HBV using a noncytopathic mechanism via activation of the LTB is currently under investigation. Until now, the activation of this pathway in HBV infection has been associated with inflammation and the development of hepatocellular carcinoma [119]. One important consideration regarding the ability of T cells to suppress HBV directly is that most experimental systems used to study the inhibition of HBV replication (both in vivo and in vitro) are devoid of chronic inflammatory events. However, chronic inflammation can not only alter the access and function of HBVspecific T cells in the liver parenchyma, but also the ability of secreted cytokines to activate antiviral mechanisms. SOCS-3, a negative regulator of cytokine signaling, is upregulated in patients [120] and woodchucks [121] during chronic HBV infection and might therefore suppress direct cytokine inhibition of HBV replication in infected hepatocytes.

Chronic HBV and T cell exhaustion: viral control or persistence of liver inflammation? What leads to the failure of the adaptive T cell response in chronic HBV infection? As mentioned above, chronic inflammatory events in the liver can suppress the ability of HBV-specific T cells to access the infected liver parenchyma [107] or suppress hepatocyte responsiveness to antiviral cytokines [121]. However, it is clear that quantitative and functional defects of the HBV-specific T cell response are a major contributing factor to viral persistence. Multifactorial mechanisms, acting simultaneously, underlie these defects, with varying effects on individual patients according to the viral load and the presence or absence of liver inflammatory events. The end result is a deep metabolic and energetic impairment of HBV-specific T cells, revealed by genome-wide expression profiling studies of HBV-specific CD8 T cells that show a prevalent transcriptional phenotype of T cell exhaustion, with a dysregulation of various core cellular processes [71,122].

S78

Persistent exposure of T cells to HBV antigens is crucial for maintaining depressed T cell functionality. In other models of chronic viral infection, viral persistence causes a hierarchical loss of antiviral T cell functions, with cytotoxicity and IL-2 production generally being the first to go, followed by TNF-a and IFN-c production, and ultimately T cell deletion [123]. Direct evidence of this hierarchical impairment is difficult to be provided in natural HBV infection settings. Available studies have tried to correlate the strength and quality of T cell responses with levels of serum antigens, but these do not fully reflect overall antigen expression within the liver environment. For example, studies conducted in nucleoside analogue treated chronic HBV patients who achieve a complete control of virus replication with either clearance of HBsAg or variable levels of antigen in their serum, reported varying degrees of functional T cell restoration, which improved as a function of therapy duration. However, T cell function was not always significantly correlated with serum levels of antigen [73,81] and could persist after HBsAg seroconversion [81]. Negative co-inhibitory molecules are highly expressed on functionally exhausted HBVspecific T cells [124], especially within the liver [125] where the vast majority are PD-1 positive. Additional co-inhibitory molecules, including CLTA4, lymphocyte activation gene-3 (LAG-3), CD160, T cell immunoglobulin domain and mucin 3 (TIM-3), and 2B4, are also upregulated on HBV-specific T cells during chronic infection, with a clear hierarchy of expression [71,126,127]. PD-1 and 2B4 have been detected in over 90% of liver-infiltrating HBV-specific CD8 T cells, followed by LAG3 and CD160 [71,126,127]. Exhausted T cells also upregulate the TRAIL-death receptor, TRAIL-2, making them susceptible to TRAIL-dependent NK cell lysis [128,129], a mechanism that might further contribute to the suppression or even deletion of HBV-specific T cells. The simultaneous expression of multiple coinhibitory molecules on individual T cells may therefore require therapeutic strategies that simultaneously target multiple pathways. Indeed, individual pathway blockade using antiPD-L1 antibodies was only able to induce partial restoration of HBV-specific T cell function in vitro [71,81]. Restoration is often more efficient in intrahepatic T cells than in the periphery, but is always incomplete and detectable only in a proportion of chronic patients [125]. Interestingly, PD-1 mediated inhibition of T cell function in exhausted HBV-specific T cells can be overcome in transgenic mice through activation of CD40 on myeloid dendritic cells. This results in the induction of intrahepatic antigen presentation

Journal of Hepatology 2016 vol. 64 j S71–S83

and is thought to provide positive stimulatory signals capable of rescuing PD-1-inhibited CD8 T-cell effector function [130]. A role in the reprogramming of functionally exhausted T cells has also been suggested for IL-12 [131], which can induce a downregulation of PD-1 expression on HBV-specific T cells and improve the effect of co-inhibitory pathway blockade on T cell function. In addition to T cell exhaustion, the inflammatory events present in chronically HBVinfected livers seem to preferentially activate a series of suppressive mechanisms within the liver that are unfavorable for T cell function. While the inflammatory milieu can increase the sensitivity of CD8+ T cells for their target [132], damaged hepatocytes release enzymes [133], such as arginase, tryptophan 2,3-dioxygenase, and indoleamine 2,3-dioxygenase, causing a depletion of amino acids, including arginine and tryptophan, essential for T cell function [134]. Arginine-deprived T cells downregulate the CD3z signaling molecule with subsequent TCRsignaling dysfunction [135]. Other cell types infiltrating the liver, e.g., myeloid suppressor cells, can also produce arginase [65], while T regulatory cells, B cells [39], and stellate cells [136] can secrete suppressor cytokines, such as IL-10 and TGF-b. These inhibitory mechanisms are important to reduce damaging levels of liver inflammation, but can also further dent the function of the already antigen-exhausted HBV-specific T cell. Therefore, it is important to understand whether the design of therapies aimed at restoring HBVspecific T cell adaptive immunity should be guided only by virological factors (high/low quantity of HBV antigens) or also by the level of liver inflammation. The use of alanine aminotransferase levels as an indirect measurement of the potency of HBV-specific T cell responses may be appropriate in acute disease but not in chronic infection. Among the overall chronic patient population, HBV-specific T cell responses in the active disease phase are not necessarily less exhausted than those of patients in the so called ‘‘immune tolerant phase”, since liver inflammatory events are primarily proportional to the magnitude of the non-antigen specific immune cell infiltration [51]. In support of this concept, a comparative study of T cell responses in young immunotolerant and adult patients with active chronic hepatitis showed that HBVspecific T cells are less compromised in the former group of younger patients [137]. Similarly, a recent study in a large population of chronic HBV patients didn’t find any HBV-specific T cell differences among patients with different clinical classification of CHB [94]. A liver environment in which T cell access, T cell function and responsiveness of hepatocytes

to the antiviral cytokine activity is restored, is expected to create ideal conditions for more efficient immune therapy with antibodies against check point inhibitors (anti-PD-1, anti-PD-L1 or anti-CTLA4), therapeutic vaccines or transfer of engineered T cells. Thus, conditioning the chronically infected liver with nucleoside analogue therapy that has a negligible effect on antigen load but can reduce liver inflammation [138] will probably remain for a long time the basis for future immune modulatory therapies, also in the possible perspective of treating the so called ‘‘immune tolerant” young patients [139,140]. Notably, successful immune therapy in chronically infected woodchucks with IFN-a, vaccines, or anti-PD-1 was observed in animals with normal alanine aminotransferase levels and low inflammatory liver biopsy scores [141], or in animals treated with nucleotide analogues [142,143]. The final question is whether the recovery of the HBV-specific T cell response in persistent HBV infection would be curative or might actually exacerbate inflammation, as has been shown following adoptive transfer of suboptimal doses of HBV-specific CD8 T cells in HBV transgenic mice [144]. Although the functional recovery of virus-specific T cell responses is typically seen in patients and animal models able to control infection either spontaneously or following therapies [81,141–143,145], there is a possibility that a partially recovered immune response might lead to an aggravation of liver inflammation. As such, the use of anti-inflammatory drugs capable of preferentially inhibiting the intrahepatic recruitment of inflammatory cells [146] should be considered as an alternative strategy to treat HBV-related disease.

Review

JOURNAL OF HEPATOLOGY

Key point Several intrahepatic inhibitory mechanisms modulate liver damage but also suppress T cell function.

Conclusions Many lines of research must still be clarified in order to understand the best therapeutic approach for HBV. Nevertheless, new strategies targeting HBV antigen production (RNAi, CRISPR genome editing) as well as HBV replication, and both antigen-specific (anti-PD-1, vaccination, T cell engineering) and non-antigen-specific strategies (TLR agonists) (reviewed in [9,147]), represent an important opportunity to further our understanding of the immune pathogenesis of hepatitis B and finally achieve a curative goal.

Financial support This work was supported by a Singapore Translational Research (STaR) Investigator Award (NMRC/STaR/013/2012) and a Translational Research Grant (NMRC/TCR/014/NUHS-2015)

Journal of Hepatology 2016 vol. 64 j S71–S83

S79

Review

Review

from the National Medical Research Council Singapore to AB and by a grant (PRUa1RI-2012-006) from Regione Emilia-Romagna, Italy, Programma di Ricerca Regione-Università 2010-2012, by a grant (2012.0033) from Fondazione Cassa di Risparmio di Parma, Italy, and by a FIRB grant from the Italian Ministry of the University and Research, Protocol RBAP10TPXK to CF.

Conflict of interest Antonio Bertoletti (AB) declares the following relationship with commercial entities developing therapeutics for HBV treatment. He collaborates and receives research support from Gilead Sciences to test the effect of HBV antigens on

References Author names in bold designate shared co-first authorship [1] Oldstone MBA, Whitton JL. Fields virology. 4 ed. Philadelphia: Lippincott Williams & Wilkins; 2001. [2] Rock KL, Goldberg AL. Degradation of cell proteins and the generation of MHC class I-presented peptides. Annu Rev Immunol 1999;17:739–779. [3] Roche PA, Furuta K. The ins and outs of MHC class II-mediated antigen processing and presentation. Nat Rev Immunol 2015;15:203–216. [4] Haniffa M, Shin A, Bigley V, McGovern N, Teo P, See P, et al. Human tissues contain CD141hi cross-presenting dendritic cells with functional homology to mouse CD103+ nonlymphoid dendritic cells. Immunity 2012;37:60–73. [5] Yewdell JW. Confronting complexity: real-world immunodominance in antiviral CD8+ T cell responses. Immunity 2006;25:533–543. [6] Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995;13:29–60. [7] 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. [8] Schuch A, Hoh A, Thimme R. The role of natural killer cells and CD8(+) T cells in hepatitis B virus infection. Front Immunol 2014;5:258. [9] Gish RG, Given BD, Lai C-L, Locarnini SA, Lau JYN, Lewis DL, et al. Chronic hepatitis B: Virology, natural history, current management and a glimpse at future opportunities. Antiviral Res 2015;121:47–58. [10] Gerlich WH. Medical virology of hepatitis B: how it began and where we are now. Virol J 2013;10:239. [11] Hoofnagle JH, Gerety RJ, Barker LF. Antibody to hepatitis-B-virus core in man. Lancet 1973;2:869–873. [12] Farci P, Diaz G, Chen Z, Govindarajan S, Tice A, Agulto L, 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. [13] Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. eLife 2012;1:e00049–e00059. [14] Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Fälth M, et al. Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology 2014;146:1070–1083. [15] Urban S, Bartenschlager R, Kubitz R, Zoulim F. Strategies to inhibit entry of HBV and HDV into hepatocytes. Gastroenterology 2014;147:48–64. [16] Jaoudé GA, Sureau C. Role of the antigenic loop of the hepatitis B virus envelope proteins in infectivity of hepatitis delta virus. J Virol 2005;79:10460–10466. [17] Glebe D, Aliakbari M, Krass P, Knoop EV, Valerius KP, Gerlich WH. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus. J Virol 2003;77:9511–9521.

S80

immune cell function. He acted as a consultant and served on the advisory boards of Gilead Sciences, Jansseen-Cilag, Hoffman-La Roche Ltd, Novartis, ISIS, Medimmune, Abivax. AB is also a co-founder of LION TCR pte. ltd. a biotech company developing T cell receptors for treatment of virus-related cancers and chronic viral diseases. Carlo Ferrari acted as a consultant and served on the advisory boards of Gilead Sciences, Hoffmann La Roche Ltd., Janssen-Cilag, MSD Italy, AbbVie, Arrowhead Research Corporation, Transgene, Abivax, MedImmune, Peregrine Pharmaceutical Inc. He was awarded research grants, paid to his Institution, from Gilead Sciences, Janssen-Cilag S.p.A., Merck Sharp & Dohme Corp, Hoffmann La Roche Ltd.

[18] Ryu CJ, Gripon P, Park HR, Park SS, Kim YK, Guguen-Guillouzo C, et al. In vitro neutralization of hepatitis B virus by monoclonal antibodies against the viral surface antigen. J Med Virol 1997;52:226–233. [19] Le Seyec J, Chouteau P, Cannie I, Guguen-Guillouzo C, Gripon P. Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity. J Virol 1998;72:5573–5578. [20] Samuel D, Muller R, Alexander G, Fassati L, Ducot B, Benhamou JP, et al. Liver transplantation in European patients with the hepatitis B surface antigen. N Engl J Med 1993;329:1842–1847. [21] Werner JM, Abdalla A, Gara N, Ghany MG, Rehermann B. The hepatitis B vaccine protects re-exposed health care workers, but does not provide sterilizing immunity. Gastroenterology 2013;145:1026–1034. [22] Rybczynska J, Campbell K, Kamili S, Locarnini S, Krawczynski K, Walker CM. CD4+ T cells are not required for suppression of hepatitis B virus replication in the liver of vaccinated chimpanzees. J Infect Dis 2015. http://dx.doi.org/ 10.1093/infdis/jiv348. [23] Chen C-L, Yang J-Y, Lin S-F, Sun C-A, Bai C-H, You S-L, et al. Slow decline of hepatitis B burden in general population: Results from a population-based survey and longitudinal follow-up study in Taiwan. J Hepatol 2015;63:354–363. [24] Petersen J, Dandri M, Mier W, Lütgehetmann M, Volz T, Weizsäcker von F, et al. Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein. Nat Biotechnol 2008;26:335–341. [25] Oliviero B, Cerino A, Varchetta S, Paudice E, Pai S, Ludovisi 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. [26] Xu X, Shang Q, Chen X, Nie W, Zou Z, Huang A, et al. Reversal of B-cell hyperactivation and functional impairment is associated with HBsAg seroconversion in chronic hepatitis B patients. Cell Molec Immunol 2015;12:309–316. [27] Vanwolleghem T, Hou J, van Oord G, Andeweg AC, Osterhaus ADME, Pas SD, et al. Re-evaluation of hepatitis B virus clinical phases by systems biology identifies unappreciated roles for the innate immune response and B cells. Hepatology 2015;62:87–100. [28] Dusheiko GM, Hoofnagle JH, Cooksley WG, James SP, Jones EA. Synthesis of antibodies to hepatitis B virus by cultured lymphocytes from chronic hepatitis B surface antigen carriers. J Clin Invest 1983;71:1104–1113. [29] Barnaba V, Valesini G, Levrero M, Zaccari C, Van Dyke A, Falco M, et al. Immunoregulation of the in vitro anti-HBs antibody synthesis in chronic HBsAg carriers and in recently boosted anti-hepatitis B vaccine recipients. Clin Exp Immunol 1985;60:259–266. [30] Böcher WO, Herzog-Hauff S, Herr W, Heermann K, Gerken G, Meyer Zum Büschenfelde KH, et al. Regulation of the neutralizing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vaccine recipients and patients with acute or chronic hepatitis B virus (HBV) infection. Clin Exp Immunol 1996;105:52–58. [31] Böcher WO, Galun E, Marcus H, Daudi N, Terkieltaub D, Shouval D, et al. Reduced hepatitis B virus surface antigen-specific Th1 helper cell frequency

Journal of Hepatology 2016 vol. 64 j S71–S83

[32]

[33]

[34]

[35] [36] [37] [38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

of chronic HBV carriers is associated with a failure to produce antigenspecific antibodies in the trimera mouse. Hepatology 2000;31:480–487. Xu L, Yin W, Sun R, Wei H, Tian Z. Liver type I regulatory T cells suppress germinal center formation in HBV-tolerant mice. Proc Natl Acad Sci U S A 2013;110:16993–16998. Milich DR, McLachlan A, Thornton GB, Hughes JL. Antibody production to the nucleocapsid and envelope of the hepatitis B virus primed by a single synthetic T cell site. Nature 1987;329:547–549. Li Y, Ma S, Tang L, Li Y, Wang W, Huang X, 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. Madalin´ski K, Bragiel I. HBsAg immune complexes in the course of infection with hepatitis B virus. Clin Exp Immunol 1979;36:371–378. Gerlich WH. The enigma of concurrent hepatitis B surface antigen (HBsAg) and antibodies to HBsAg. Clin Infect Dis 2007;44:1170–1172. Kunkel EJ, Butcher EC. Plasma-cell homing. Nat Rev Immunol 2003;3:822–829. Mohamadkhani A, Naderi E, Sotoudeh M, Katoonizadeh A, Montazeri G, Poustchi H. Clinical feature of intrahepatic B-lymphocytes in chronic hepatitis B. Int J Inflam 2014;2014:896864–896865. Das A, Ellis G, Pallant C, Lopes AR, Khanna P, Peppa D, et al. IL-10-producing regulatory B cells in the pathogenesis of chronic hepatitis B virus infection. J Immunol 2012;189:3925–3935. Araki K, Nagashima H, Tsuji T. Detection and characterization of circulating immune complexes during acute exacerbation of chronic viral hepatitis. Clin Exp Immunol 1982;47:520–526. Maruyama T, Schödel F, Iino S, Koike K, Yasuda K, Peterson D, et al. Distinguishing between acute and symptomatic chronic hepatitis B virus infection. Gastroenterology 1994;106:1006–1015. Zhang T-Y, Yuan Q, Zhao J-H, Zhang Y-L, Yuan L-Z, Lan Y, et al. Prolonged suppression of HBV in mice by a novel antibody that targets a unique epitope on hepatitis B surface antigen. Gut 2015. http://dx.doi.org/10.1136/gutjnl2014-308964, [Epub ahead of print]. Barouch DH, Whitney JB, Moldt B, Klein F, Oliveira TY, Liu J, et al. Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIVinfected rhesus monkeys. Nature 2013;503:224–228. Shingai M, Nishimura Y, Klein F, Mouquet H, Donau OK, Plishka R, et al. Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia. Nature 2013;503:277–280. 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. Penna A, Artini M, Cavalli A, Levrero M, Bertoletti A, Pilli M, et al. Long-lasting memory T cell responses following self-limited acute hepatitis B. J Clin Invest 1996;98:1185–1194. Kefalakes H, Jochum C, Hilgard G, Kahraman A, Bohrer AM, Hindy El N, et al. Decades after recovery from hepatitis B and HBsAg clearance the CD8+ T cell response against HBV core is nearly undetectable. J Hepatol 2015;63:13–19. Dervite I, Hober D, Morel P. Acute hepatitis B in a patient with antibodies to hepatitis B surface antigen who was receiving rituximab. N Engl J Med 2001;344:68–69. Evens AM, Jovanovic BD, Su Y-C, Raisch DW, Ganger D, Belknap SM, et al. Rituximab-associated hepatitis B virus (HBV) reactivation in lymphoproliferative diseases: meta-analysis and examination of FDA safety reports. Ann Oncol 2011;22:1170–1180. Moriyama T, Guilhot S, Klopchin K, Moss B, Pinkert CA, Palmiter RD, et al. Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science 1990;248:361–364. Maini MK, Boni C, Lee CK, Larrubia JR, Reignat S, Ogg GS, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 2000;191:1269–1280. Thimme R, Wieland S, Steiger C, Ghrayeb J, Reimann KA, Purcell RH, et al. CD8 (+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol 2003;77:68–76. 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. Ando K, Moriyama T, Guidotti LG, Wirth S, Schreiber RD, Schlicht HJ, et al. Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med 1993;178:1541–1554.

[55] Kakimi K, Lane TE, Wieland S, Asensio VC, Campbell IL, Chisari FV, et al. Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes. J Exp Med 2001;194:1755–1766. [56] Yang PL, Althage A, Chung J, Maier H, Wieland S, Isogawa M, et al. Immune effectors required for hepatitis B virus clearance. Proc Natl Acad Sci U S A 2010;107:798–802. [57] Ferrari C, Penna A, Bertoletti A, Valli A, Antoni AD, Giuberti T, 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. [58] Maini MK, Boni C, Ogg GS, King AS, Reignat S, Lee CK, et al. Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection. Gastroenterology 1999;117:1386–1396. [59] Webster GJ, Reignat S, Maini MK, Whalley SA, Ogg GS, King A, et al. Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms. Hepatology 2000;32:1117–1124. [60] Boettler T, Panther E, Bengsch B, Nazarova N, Spangenberg HC, Blum HE, et al. Expression of the interleukin-7 receptor alpha chain (CD127) on virusspecific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection. J Virol 2006;80:3532–3540. [61] Sandalova E, Laccabue D, Boni C, Tan AT, Fink K, Ooi EE, et al. Contribution of herpesvirus specific CD8 T cells to anti-viral T cell response in humans. PLoS Pathog 2010;6 e1001051. [62] Zhang Z, Zhang J-Y, Wherry EJ, Jin B, Xu B, Zou Z-S, et al. Dynamic programmed death 1 expression by virus-specific CD8 T cells correlates with the outcome of acute hepatitis B. Gastroenterology 2008;134:1938–1949, 1949.e1–e3. [63] Dunn C, Peppa D, Khanna P, Nebbia G, Jones M, Brendish N, et al. Temporal analysis of early immune responses in patients with acute hepatitis B virus infection. Gastroenterology 2009;137:1289–1300. [64] Sandalova E, Laccabue D, Boni C, Watanabe T, Tan A, Zong HZ, et al. Increased levels of arginase in patients with acute hepatitis B suppress antiviral T cells. Gastroenterology 2012;143:78–87, e3. [65] Pallett LJ, Gill US, Quaglia A, Sinclair LV, Jover-Cobos M, Schurich A, et al. Metabolic regulation of hepatitis B immunopathology by myeloid-derived suppressor cells. Nat Med 2015;21:591–600. [66] Franzese O, Kennedy PTF, Gehring AJ, Gotto J, Williams R, Maini MK, 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. [67] Zerbini A, Pilli M, Boni C, Fisicaro P, Penna A, Di Vincenzo P, et al. The characteristics of the cell-mediated immune response identify different profiles of occult hepatitis B virus infection. Gastroenterology 2008;134:1470–1481. [68] Penna A, Chisari FV, Bertoletti A, Missale G, Fowler P, Giuberti T, et al. Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen. J Exp Med 1991;174:1565–1570. [69] Bertoletti A, Ferrari C, Fiaccadori F, Penna A, Margolskee R, Schlicht HJ, et al. HLA class I-restricted human cytotoxic T cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen. Proc Natl Acad Sci U S A 1991;88:10445–10449. [70] Webster GJM, Reignat S, Brown D, Ogg GS, Jones L, Seneviratne SL, 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. [71] Bengsch B, Martin B, Thimme R. Restoration of HBV-specific CD8+ T cell function by PD-1 blockade in inactive carrier patients is linked to T cell differentiation. J Hepatol 2014;61:1212–1219. [72] Kurktschiev PD, Raziorrouh B, Schraut W, Backmund M, Wachtler M, Wendtner CM, et al. Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction. J Exp Med 2014;54:167. [73] Loggi E, Bihl FK, Cursaro C, Granieri C, Galli S, Brodosi L, 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. [74] Hong M, Sandalova E, Low D, Gehring AJ, Fieni S, Amadei B, et al. Trained immunity in newborn infants of HBV-infected mothers. Nat Commun 2015;6:6588. [75] Bertoletti A, Hong M. Age-dependent immune events during HBV infection from birth to adulthood: an alternative interpretation. Front Immunol 2014;5:441.

Journal of Hepatology 2016 vol. 64 j S71–S83

S81

Review

JOURNAL OF HEPATOLOGY

Review

Review [76] Jung MC, Spengler U, Schraut W, Hoffmann R, Zachoval R, Eisenburg J, et al. Hepatitis B virus antigen-specific T-cell activation in patients with acute and chronic hepatitis B. J Hepatol 1991;13:310–317. [77] Sobao Y, Sugi K, Tomiyama H, Saito S, Fujiyama S, Morimoto M, et al. Identification of hepatitis B virus-specific CTL epitopes presented by HLAA⁄2402, the most common HLA class I allele in East Asia. J Hepatol 2001;34:922–929. [78] Nayersina R, Fowler P, Guilhot S, Missale G, Cerny A, Schlicht HJ, et al. HLA A2 restricted cytotoxic T lymphocyte responses to multiple hepatitis B surface antigen epitopes during hepatitis B virus infection. J Immunol 1993;150:4659–4671. [79] Abbott WGH, Tsai P, Leung E, Trevarton A, Ofanoa M, Hornell J, et al. Associations between HLA class I alleles and escape mutations in the hepatitis B virus core gene in New Zealand-resident Tongans. J Virol 2010;84:621–629. [80] Rehermann B, Fowler P, Sidney J, Person J, Redeker A, Brown M, et al. The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis. J Exp Med 1995;181:1047–1058. [81] Boni C, Laccabue D, Lampertico P, Giuberti T, Viganò M, Schivazappa S, et al. Restored function of HBV-specific T cells after long-term effective therapy with nucleos(t)ide analogues. Gastroenterology 2012;143:963–969. [82] Chisari FV. Cytotoxic T cells and viral hepatitis. J Clin Invest 1997;99:1472–1477. http://dx.doi.org/10.1172/JCI119308. [83] Ferrari C, Bertoletti A, Penna A, Cavalli A, Valli A, Missale G, et al. Identification of immunodominant T cell epitopes of the hepatitis B virus nucleocapsid antigen. J Clin Invest 1991;88:214–222. [84] Kakimi K, Isogawa M, Chung J, Sette A, Chisari FV. Immunogenicity and tolerogenicity of hepatitis B virus structural and nonstructural proteins: implications for immunotherapy of persistent viral infections. J Virol 2002;76:8609–8620. [85] Rivino L, Tan AT, Chia A, Kumaran EAP, Grotenbreg GM, Macary PA, et al. Defining CD8+ T cell determinants during human viral infection in populations of Asian ethnicity. J Immunol 2013;191:4010–4019. [86] Bertoni R, Sidney J, Fowler P, Chesnut RW, Chisari FV, Sette A. Human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis. J Clin Invest 1997;100:503–513. [87] Tan AT, Loggi E, Boni C, Chia A, Gehring AJ, Sastry KSR, et al. Host ethnicity and virus genotype shape the hepatitis B virus-specific T-cell repertoire. J Virol 2008;82:10986–10997. [88] Tsai SL, Chen MH, Yeh CT, Chu CM, Lin AN, Chiou FH, et al. Purification and characterization of a naturally processed hepatitis B virus peptide recognized by CD8+ cytotoxic T lymphocytes. J Clin Invest 1996;97:577–584. [89] Chen X, Wang W, Wang S, Meng G, Zhang M, Ni B, et al. An immunodominant HLA-A⁄1101-restricted CD8+ T-cell response targeting hepatitis B surface antigen in chronic hepatitis B patients. J Gen Virol 2013;94:2717–2723. [90] Tan AT, Sodsai P, Chia A, Moreau E, Chng MHY, Tham CYL, et al. Immunoprevalence and immunodominance of HLA-Cw⁄0801-restricted T cell response targeting the hepatitis B virus envelope transmembrane region. J Virol 2014;88:1332–1341. [91] Hirsch RC, Lavine JE, Chang LJ, Varmus HE, Ganem D. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature 1990;344:552–555. [92] Gehring AJ, Sun D, Kennedy PTF, Nolte-’t Hoen E, Lim SG, Wasser S, et al. The level of viral antigen presented by hepatocytes influences CD8 T-cell function. J Virol 2007;81:2940–2949. [93] Maini MK, Reignat S, Boni C, Ogg GS, King AS, Malacarne F, et al. T cell receptor usage of virus-specific CD8 cells and recognition of viral mutations during acute and persistent hepatitis B virus infection. Eur J Immunol 2000;30:3067–3078. [94] Park J-J, Wong DK, Wahed AS, Lee WM, Feld JJ, Terrault N, et al. Hepatitis B virus-specific and global T-cell dysfunction in chronic hepatitis B. Gastroenterology 2015. http://dx.doi.org/10.1053/j.gastro.2015.11.050. [95] Rehermann B, Pasquinelli C, Mosier SM, Chisari FV. Hepatitis B virus (HBV) sequence variation of cytotoxic T lymphocyte epitopes is not common in patients with chronic HBV infection. J Clin Invest 1995;96:1527–1534. [96] Bertoletti A, Costanzo A, Chisari FV, Levrero M, Artini M, Sette A, et al. Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope. J Exp Med 1994;180:933–943. [97] Bertoletti A, Sette A, Chisari FV, Penna A, Levrero M, De Carli M, et al. Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells. Nature 1994;369:407–410.

S82

[98] Kefalakes H, Budeus B, Walker A, Jochum C, Hilgard G, Heinold A, et al. Adaptation of the hepatitis B virus core protein to CD8(+) T-cell selection pressure. Hepatology 2015;62:47–56. [99] Desmond CP, Gaudieri S, James IR, Pfafferott K, Chopra A, Lau GK, et al. Viral adaptation to host immune responses occurs in chronic hepatitis B virus (HBV) infection, and adaptation is greatest in HBV e antigen-negative disease. J Virol 2012;86:1181–1192. [100] Liu H-G, Fan Z-P, Chen W-W, Yang H-Y, Liu Q-F, Zhang H, et al. A mutant HBs antigen (HBsAg)183–191 epitope elicits specific cytotoxic T lymphocytes in acute hepatitis B patients. Clin Exp Immunol 2008;151:441–447. [101] Hoh A, Heeg M, Ni Y, Schuch A, Binder B, Hennecke N, et al. Hepatitis B virusinfected HepG2hNTCP cells serve as a novel immunological tool to analyze the antiviral efficacy of CD8+ T cells in vitro. J Virol 2015;89:7433–7438. [102] Sijts AJ, Ruppert T, Rehermann B, Schmidt M, Koszinowski U, Kloetzel PM. Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes. J Exp Med 2000;191:503–514. [103] Lau GKK, Suri D, Liang R, Rigopoulou EI, Thomas MG, Mullerova I, et al. Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen. Gastroenterology 2002;122:614–624. [104] Ilan Y, Nagler A, Adler R, Naparstek E, Or R, Slavin S, et al. Adoptive transfer of immunity to hepatitis B virus after T cell-depleted allogeneic bone marrow transplantation. Hepatology 1993;18:246–252. [105] Lindemann M, Koldehoff M, Fiedler M, Schumann A, Ottinger HD, Heinemann FM, et al. Control of hepatitis B virus infection in hematopoietic stem cell recipients after receiving grafts from vaccinated donors. Bone Marrow Transplant 2015. http://dx.doi.org/10.1038/bmt.2015.253. [106] Balkow S, Kersten A, Tran TT, Stehle T, Grosse P, Museteanu C, et al. Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection. J Virol 2001;75:8781–8791. [107] Guidotti LG, Inverso D, Sironi L, Di Lucia P, Fioravanti J, Ganzer L, et al. Immunosurveillance of the liver by intravascular effector CD8(+) T cells. Cell 2015;161:486–500. [108] Iannacone M. Hepatic effector CD8(+) T-cell dynamics. Cell Mol Immunol 2014. http://dx.doi.org/10.1038/cmi.2014.78. [109] Billerbeck E, Kang YH, Walker L, Lockstone H, Grafmueller S, Fleming V, et al. Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties. Proc Natl Acad Sci U S A 2010;107:3006–3011. [110] Knolle PA, Thimme R. Hepatic immune regulation and its involvement in viral hepatitis infection. Gastroenterology 2014;146:1193–1207. [111] Guidotti LG, Rochford R, Chung J, Shapiro M, Purcell R, Chisari FV. Viral clearance without destruction of infected cells during acute HBV infection. Science 1999;284:825–829. [112] Phillips S, Chokshi S, Riva A, Evans A, Williams R, Naoumov NV. CD8+ T cell control of hepatitis B virus replication: direct comparison between cytolytic and noncytolytic functions. J Immunol 2009;184:287–295. [113] Xia Y, Stadler D, Lucifora J, Reisinger F, Webb D, Hösel M, et al. Interferon-c and tumor necrosis factor-a produced by T cells reduce the HBV persistence form, cccDNA, without cytolysis. Gastroenterology 2015. http://dx.doi.org/ 10.1053/j.gastro.2015.09.026. [114] Fiedler M, Rodicker F, Salucci V, Lu M, Aurisicchio L, Dahmen U, et al. Helperdependent adenoviral vector-mediated delivery of woodchuck-specific genes for alpha interferon (IFN-) and IFN-: IFN- but Not IFN- reduces woodchuck hepatitis virus replication in chronic infection in vivo. J Virol 2004;78:10111–10121. [115] Jacquard AC, Nassal M, Pichoud C, Ren S, Schultz U, Guerret S, et al. Effect of a combination of clevudine and emtricitabine with adenovirus-mediated delivery of gamma interferon in the woodchuck model of hepatitis B virus infection. Antimicrob Agents Chemother 2004;48:2683–2692. [116] Guidotti LG, McClary H, Loudis JM, Chisari FV. Nitric oxide inhibits hepatitis B virus replication in the livers of transgenic mice. J Exp Med 2000;191:1247–1252. [117] Lucifora J, Xia Y, Reisinger F, Zhang K, Stadler D, Cheng X, et al. Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science 2014;343:1221–1228. [118] Wolf MJ, Seleznik GM, Zeller N, Heikenwalder M. The unexpected role of lymphotoxin b receptor signaling in carcinogenesis: from lymphoid tissue formation to liver and prostate cancer development. Oncogene 2010;29:5006–5018.

Journal of Hepatology 2016 vol. 64 j S71–S83

[119] Haybaeck J, Zeller N, Wolf MJ, Weber A, Wagner U, Kurrer MO, et al. A lymphotoxin-driven pathway to hepatocellular carcinoma. Cancer Cell 2009;16:295–308. [120] Koeberlein B, zur Hausen A, Bektas N, Zentgraf H, Chin R, Nguyen LT, et al. Hepatitis B virus overexpresses suppressor of cytokine signaling-3 (SOCS3) thereby contributing to severity of inflammation in the liver. Virus Res 2010;148:51–59. [121] Fletcher SP, Chin DJ, Ji Y, Iniguez AL, Taillon B, Swinney DC, et al. Transcriptomic analysis of the woodchuck model of chronic hepatitis B. Hepatology 2012;56:820–830. [122] Lopes AR, Kellam P, Das A, Dunn C, Kwan A, Turner J, et al. Bim-mediated deletion of antigen-specific CD8+ T cells in patients unable to control HBV infection. J Clin Invest 2008;118:1835–1845. [123] Wherry EJ, Ha S-J, Kaech SM, Haining WN, Sarkar S, Kalia V, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 2007;27:670–684. [124] Boni C, Fisicaro P, Valdatta C, Amadei B, Di Vincenzo P, Giuberti T, et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. J Virol 2007;81:4215–4225. [125] Fisicaro P, Valdatta C, Massari M, Loggi E, Biasini E, Sacchelli L, et al. Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B. Gastroenterology 2010;138: 682–684. [126] Raziorrouh B, Schraut W, Gerlach T, Nowack D, Grüner NH, Ulsenheimer A, et al. The immunoregulatory role of CD244 in chronic hepatitis B infection and its inhibitory potential on virus-specific CD8+ T-cell function. Hepatology 2010;52:1934–1947. [127] Schurich A, Khanna P, Lopes AR, Han KJ, Peppa D, Micco L, et al. Role of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 on apoptosis-Prone CD8 T cells in persistent hepatitis B virus infection. Hepatology 2011;53:1494–1503. [128] Peppa D, Gill US, Reynolds G, Easom NJW, Pallett LJ, Schurich A, et al. Upregulation of a death receptor renders antiviral T cells susceptible to NK cellmediated deletion. J Exp Med 2013;210:99–114. [129] Boni C, Lampertico P, Talamona L, Giuberti T, Invernizzi F, Barili V, et al. Natural killer cell phenotype modulation and natural killer/T-cell interplay in nucleos(t)ide analogue-treated hepatitis e antigen-negative patients with chronic hepatitis B. Hepatology 2015;62. http://dx.doi.org/10.1002/ hep.28155. [130] Isogawa M, Chung J, Murata Y, Kakimi K, Chisari FV. CD40 activation rescues antiviral CD8+ T cells from PD-1-mediated exhaustion. PLoS Pathog 2013;9 e1003490. [131] Schurich A, Pallett LJ, Lubowiecki M, Singh HD, Gill US, Kennedy PT, et al. The third signal cytokine IL-12 rescues the anti-viral function of exhausted HBVspecific CD8 T cells. PLoS Pathog 2013;9 e1003208. [132] Richer MJ, Nolz JC, Harty JT. Pathogen-specific inflammatory milieux tune the antigen sensitivity of CD8. Immunity 2013;38:140–152.

[133] Chisari FV, Nakamura M, Milich DR, Han K, Molden D, Leroux-Roels GG. Production of two distinct and independent hepatic immunoregulatory molecules by the perfused rat liver. Hepatology 1985;5:735–743. [134] Bronte V, Zanovello P. Regulation of immune responses by L-arginine metabolism. Nat Rev Immunol 2005;5:641–654. [135] Das A, Hoare M, Davies N, Lopes AR, Dunn C, Kennedy PTF, et al. Functional skewing of the global CD8 T cell population in chronic hepatitis B virus infection. J Exp Med 2008;205:2111–2124. [136] Mann DA, Marra F. Fibrogenic signalling in hepatic stellate cells. J Hepatol 2010;52:949–950. [137] Kennedy PTF, Sandalova E, Jo J, Gill U, Ushiro-Lumb I, Tan AT, et al. Preserved T-cell function in children and young adults with immune-tolerant chronic hepatitis B. Gastroenterology 2012;143:637–645. [138] Wursthorn K, Jung M, Riva A, Goodman ZD, Lopez P, Bao W, et al. Kinetics of hepatitis B surface antigen decline during 3 years of telbivudine treatment in hepatitis B e antigen-positive patients. Hepatology 2010;52:1611–1620. [139] Bertoletti A, Kennedy PT. The immune tolerant phase of chronic HBV infection: new perspectives on an old concept. Cell Mol Immunol 2015;12:258–263. [140] Zoulim F, Mason WS. Reasons to consider earlier treatment of chronic HBV infections. Gut 2012;61:333–336. [141] Fletcher SP, Chin DJ, Gruenbaum L, Bitter H, Rasmussen E, Ravindran P, et al. Intrahepatic transcriptional signature associated with response to interferon-a treatment in the woodchuck model of chronic hepatitis B. PLoS Pathog 2015;11:e1005103–e1005131. [142] Kosinska AD, Zhang E, Johrden L, Liu J, Seiz PL, Zhang X, et al. Combination of DNA prime – adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis B in the woodchuck model. PLoS Pathog 2013;9 e1003391. [143] Liu J, Zhang E, Ma Z, Wu W, Kosinska A, Zhang X, et al. Enhancing virusspecific immunity in vivo by combining therapeutic vaccination and PD-L1 blockade in chronic hepadnaviral infection. PLoS Pathog 2014;10 e1003856. [144] Sitia G, Aiolfi R, Di Lucia P, Mainetti M, Fiocchi A, Mingozzi F, et al. Antiplatelet therapy prevents hepatocellular carcinoma and improves survival in a mouse model of chronic hepatitis B. Proc Natl Acad Sci U S A 2012;109:E2165–E2172. [145] Rehermann B, Lau D, Hoofnagle JH, Chisari FV. Cytotoxic T lymphocyte responsiveness after resolution of chronic hepatitis B virus infection. J Clin Invest 1996;97:1655–1665. [146] Sitia G, Iannacone M, Muller S, Bianchi ME, Guidotti LG. Treatment with HMGB1 inhibitors diminishes CTL-induced liver disease in HBV transgenic mice. J Leuk Biol 2006;81:100–107. [147] Bertoletti A, Rivino L. Hepatitis B: future curative strategies. Curr Opin Infect Dis 2014;27:528–534. [148] Maini MK, Gehring AJ. The role of innate immunity in the immunopathology and treatment of HBV infection. J Hepatol 2016;64:S60–S70.

Journal of Hepatology 2016 vol. 64 j S71–S83

S83

Review

JOURNAL OF HEPATOLOGY