Review New insights into how HBV manipulates the innate immune response to establish acute and persistent infection

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1 Antiviral Therapy 2013; 18:1 15 (doi: /IMP2542) Review New insights into how HBV manipulates the innate immune response to establish acute and persistent infection Peter Revill 1 *, Zhenghong Yuan 2 * 1 Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia 2 Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, Shanghai Medical College, Fudan University, Shanghai, China *Corresponding author s: peter.revill@mh.org.au; zhyuan@shaphc.org Both authors made an equal contribution to this work The mechanisms by which HBV establishes and maintains chronic infection are poorly understood. Although adult acquired HBV is generally cleared by a robust immune response, most individuals infected at childbirth or in very early childhood develop lifelong chronic infection. In addition, acute infections are unresolved in approximately 5% of individuals infected in adulthood. The host cell mechanisms that ensure establishment and resolution of acute infection and persistent infection remain unclear. Currently, two schools of thought suggest that either HBV is a stealth virus, which initially establishes infection by avoiding host innate immune responses, or that HBV facilitates initial infection and progression to persistence by actively manipulating the host innate immune response to its advantage. There is increasing evidence that activation of innate host cell signalling pathways plays a major role in limiting adult acquired HBV infection and that, in turn, HBV has evolved numerous strategies to counteract these defence mechanisms. In this review, we summarize current knowledge regarding innate immune responses to HBV infection and discuss how HBV regulates cell signalling pathways to its advantage, particularly in the setting of chronic HBV infection. In turn, we show how an intimate knowledge of innate immune responses is driving development of novel therapeutic agents to treat chronic HBV infection. HBV lifecycle and replication Following infection of a hepatocyte by an as yet unidentified mechanism, viral nucleocapsids disassemble and genomic HBV DNA is transported to the cell nucleus, where the partially double-stranded genomic viral DNA is converted to covalently closed circular DNA and minichromosomes that act as the major transcriptional template for the virus [1] (Figure 1). HBV DNA replication occurs within cytoplasmic viral nucleocapsids, meaning that viral replicative intermediates are protected from digestion by cellular nucleases and in turn do not themselves trigger cellular responses. These replicative intermediates include the greaterthan-genome-length pregenomic RNA (pgrna) molecule, which is the template for synthesis of single-stranded DNA, which, in turn, is the template for second-strand DNA synthesis, producing the circular partially doublestranded DNA genome. The 3.2 kb HBV genome is organized into four overlapping open- reading frames, which encode the hepatitis B core antigen (HBcAg; or nucleocapsid protein, translated from the 3.5 kb pgrna), the polymerase protein (also translated from the pgrna), the soluble and secreted hepatitis B e antigen (HBeAg; translated from an additional 3.5 kb messenger RNA [mrna] the precore RNA [pcrna]), the viral envelope proteins, (hepatitis B surface antigen [HBsAg]; translated from 2.4 and 2.1 kb RNAs) and the hepatitis B X protein (HBx; translated from a 0.7 kb RNA). Importantly, experimental evidence has indicated that each of these viral proteins can regulate the innate immune response, although the mechanisms and outcomes of this interaction differ markedly for each HBV protein. HBV natural history HBV is one of the most successful human pathogens, with almost 2 billion people having been infected 2013 International Medical Press (print) (online) 1

2 P Revill & Z Yuan Figure 1. The HBV lifecycle? Cytoplasm Secreted HBeAg Dane particle 0.7 kb 2.1 kb pcrna 2.4 kb 3.5 kb pgrna 3.5 kb 2.0 kb Attachment and entry Nucleus Conversion of RC DNA to cccdna and formation of minichromosome mrna transcription intron splicing p17 HBeAg ER/Golgi complex? Release of virus p22 harbouring genomic length or splicederived DNA Transport of non-spliced mrna mediated by PRE and LA Nuclear export of spliced RNA Nucleocapsid Translation recycled to nucleus HBSP Precore p25 protein HBx S, M, L (envelope proteins) Translation Packaging of spliced or pgrna and Pol Translation HBcAg Pol Envelopment HBsAg Packaging for export Synthesis of genomic-length or splice-derived DNA Secreted HBsAg cccdna, covalently closed circular DNA; ER, endoplasmic reticulum; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBSP, hepatitis B splice protein; HBx, hepatitis B X protein; L, large; LA, La protein; M, medium; mrna, messenger RNA; pcrna, precore RNA; pgrna, pregenomic RNA; Pol, HBV polymerase; PRE, post-transcriptional regulatory element; p17, precore 17 kda variant; p22, precore 22 kda variant; p25, precore 25 kda variant; RC DNA, relaxed circular DNA; S, small. worldwide. Chronic hepatitis B (CHB) infection causes progressive liver disease, including cirrhosis and hepatocellular carcinoma and is the direct cause of death in approximately 25% of afflicted individuals. Although a small proportion of individuals with CHB will spontaneously clear HBV infection, CHB in most individuals is lifelong unless they are responsive to treatment with interferon (IFN)-a or one of a small panel of nucleoside/ nucleotide analogues. The primary end point of therapy is the development of antibodies to HBeAg and ultimately HBsAg, leading to viral clearance. However, the continued presence of covalently closed circular DNA in the hepatocyte nucleus means that, although episomal virus has been removed from the cytoplasm, there is a reservoir of transcriptional template for production of viral mrnas, including the replication competent pgrna. Consequently, in some settings, such as immuosuppressive drug therapy for cancer treatment, infection can re-emerge in patients previously thought to have been cured [2]. Although such reactivation may itself be cleared, continued immunosuppression can see the re-emergence of CHB [2]. Although HBV is cleared and controlled by host immune responses in approximately 95% of individuals infected during adulthood, the virus has established chronic infection in over 400 million individuals [3], this being most prevalent in those infected at birth or during early childhood. Yet the reasons why HBV establishes persistent infection so readily following transmission at birth or in early childhood, and so poorly in adulthood, International Medical Press

3 New insights into HBV pathogenesis remain undetermined. Does it relate simply to the immature immune system in infants and young children, or do HBV genotype and other viral factors play a role? Nor is it clear how HBV manages to evade host responses to establish persistent infection in 5% of individuals infected during adulthood. Again, does this relate to host factors, viral factors or a combination thereof? Each of these questions is explored throughout this review. Is there an innate immune response to HBV infection? Although it has been clearly identified that the adaptive immune response is required for efficient control of HBV infection [4], the role of the innate response is more difficult to analyse, in part because HBV-infected patients are almost universally diagnosed weeks post-infection [5]. Studies with acute HBVinfected chimpanzees showed that HBV did not modify host cellular gene transcription early in infection and did not induce any innate antiviral responses in hepatocytes and the liver [6]. Indeed, HBV did not enter the logarithmic phase of amplification until 5 weeks after infection [7,8] and no host transcripts, including IFN-a/b stimulated genes, were uniformly induced or repressed during this period [6]. Although it is possible that gene expression was below the limit of detection, this phenomenon could also be explained by the following scenarios: HBV in chimpanzees is a stealth virus that cannot be detected by the innate immune response early in the infection or HBV efficiently suppresses the innate immune response very early following infection. Since only 5% of adult humans progress to chronicity following an acute infection, it is also possible that a study using a larger number of chimpanzees may have identified innate antiviral responses in additional animals. Although aspects of the HBV life cycle, such as viral replication occurring within nucleocapsids, may effectively hide viral nucleic acid from the cell, HBV proteins are expressed in the cytoplasm. Numerous studies have shown direct interaction between these HBV-encoded proteins and innate immune responses, suggesting that HBV has developed multiple mechanisms to counteract cellular defences. The human liver is an organ formed by two types of cells, non-parenchymal cells (for example, Kupffer cells and sinusoidal endothelial cells) and parenchymal cells (for example, hepatocytes and modified epithelial cells), each with unique immunological characteristics. The innate immune response to HBV infection is driven by an early response to specific pathogenassociated molecular patterns, and drives the subsequent adaptive immune response that is crucial for viral clearance [9]. Although HBV almost exclusively infects hepatocytes, it is becoming increasingly evident that other cells within the liver and peripheral blood play an important regulatory role in the innate immune response, which in turn regulates the adaptive immune response to infection. Factors contributing to the establishment and resolution of acute HBV infection The resolution of adult acquired HBV infection in approximately 95% of cases is generally attributed to robust T-cell-mediated clearance of HBV-infected hepatocytes. However, it is becoming increasingly apparent that innate immune responses also play an important role in this process. Natural killer (NK) and natural killer T (NKT) cells are crucial for host antiviral innate immune responses, bridging host innate and adaptive immune responses in order to play a central role in HBV clearance [10]. HBV infection has been shown to directly activate NK cells, through the induction of cytokines such as IFN-a, IFN-b and interleukin (IL)-12, or indirectly, via the activation of dendritic cells (DCs) and macrophages to produce IL-12, IL-18 and CXCR3 and other chemokines [11]. Activated NK cells destroy virusinfected target cells through direct cytotoxic effects and also inhibit HBV replication by producing IFN-g, tumour necrosis factor (TNF)-a, transforming growth factor-b, granulocyte macrophage colony- stimulating factor and IL-10 [12 14]. The development of NK and NKT cell responses in two HBV- seronegative blood donors who became HBsAg- and HBV-DNA-positive, but had persistent normal alanine aminotransferase level, indicates that the innate immune system was able to sense HBV infection at a very early stage [15]. Indeed a study quantifying kinetics of the related woodchuck hepatitis virus (WHV) replication and gene activation (a model of acute hepatitis B infection) 1 h post-inoculation onward revealed significant augmented intrahepatic transcription of IFN-g and IL-12, 3 6 h after infection [16]. In the early stages of human HBV infection, NK and NKT cells were shown to up-regulate IFN-g and TNF-a production in accordance with the peak of HBV replication, when the HBV-specific T-cell response was still very weak [15]. Within h, NK and NKT cells were activated and virus replication was transiently reduced, indicating a robust innate immune response. However, T-cells were only activated 4 5 weeks later. These data implied that early innate responses were indeed activated but were unable to prompt a timely adaptive T-cell response [16]. These observations were consistent with studies that showed the highest frequency of circulating NK cells occurred at the earliest point in the incubation period of natural HBV infection, with NK cell numbers reduced in accordance with declining HBV DNA [5]. However, Antiviral Therapy

4 P Revill & Z Yuan the role of NK cells during acute infection remains unclear, as it has also been recently shown that NK cell activation and IFN-g-producing capacity were reduced at peak viraemia, and cytokines including type-i IFNs, IL-15 and IFN-l, were not appropriately induced during acute HBV infection [17]. In fact, the transient inhibition of an NK response coincided with high levels of IL-10, which is an anti-inflammatory cytokine that inhibits NK and T-cell function [17]. Transgenic mouse models of acute HBV infection have shown that NKT cells are activated in response to hepatocytes expressing hepatitis B viral antigens [18]. NK and NKT cells were activated by antigen-presenting cells, which produce IL-18 that stimulates NK and NKT cells to secrete IFN-g and IFN-a/b [19,20]. Induction of IFN-g, IFN-a/b and TNF-a in the livers of HBV transgenic mice inhibited HBV replication by eliminating pgrna-containing viral capsids from the hepatocyte, or inhibiting HBV gene expression by destabilizing viral mrna [21,22]. In HBV transgenic mice deficient of IFN-g and IFN-a/b receptors, HBV replicates at higher levels than those observed in control mice [23]. In an in vitro model, viral replication in hepatocytes from HBV transgenic mice was also efficiently controlled by IFNa/b and IFN-g, eliminating pgrna-containing capsids from the cells, as observed in the liver. Furthermore, IFN-g inhibits HBV gene expression, especially when it acts synergistically with TNF-a [24]. In summary, studies using animal models and most studies using human patients have shown that in self-limited acute infection, activation of NK and NKT cells leads to IFN-g and IFN-a/b production by these NK cells, causing rapid inhibition of HBV replication, recruitment of virusspecific and non-specific cells and assists in the activation of adaptive immune responses [25]. However, the finding of Dunn et al. [17], that NK cell activation and IFN-g-producing capacity was reduced at peak viraemia during acute infection of human subjects, warrants further investigation. Perhaps these seemingly incongruent findings reflect differences in the human subjects tested, or HBV genotype, which could be clarified by additional studies. Rapid innate immune responses leading to viral clearance are not limited to non-parenchymal cells, such as NK cells. Indeed, in vitro studies using differentiated HepaRG cells transduced with recombinant baculovirus encoding the complete HBV genome, showed that HBV elicited a strong and specific innate antiviral response in this cell line that resulted in a non-cytopathic clearance of HBV DNA, with up-regulation of IFN-b as well as other IFN-stimulated genes [26]. It remains to be determined if these findings are truly reflective of what occurs in the liver. If HBV can elicit such a strong antiviral response in hepatocytes, it begs the question, how does infection establish in the first place? Factors contributing to the establishment and resolution of persistent HBV infection Although individuals infected with HBV during adulthood predominantly clear the infection, a high proportion of individuals infected at or shortly after birth, or during early childhood, develops lifelong chronic infection. Indeed, >80% of individuals infected at birth develop chronic infection, decreasing to 20 30% in those infected between 1 and 5 years of age [27]. Clearance rates approach adult levels in children infected beyond 5 years of age. The mechanisms by which HBV establishes and maintains chronic infection are yet to be resolved, but in adults it is becoming increasingly apparent that a complex array of immune responses in a variety of cells determines whether HBV is cleared or establishes persistent infection, including expression of CD4 + and CD8 + T-cells, NK cells, Fas, IFN-g, IFN-a/b receptor 1, and TNF receptor 1 [28,29] (Figure 2). HBV viral load Development of persistent infection is associated with an absence of the acute phase response and the failure to prime an adequate adaptive immune response. In the related WHV model, animals that developed chronic infection were characterized by increasing initial viral load in liver and plasma, and a detectable but diminished acute hepatic inflammation [30]. By contrast, the self-limited outcome was characterized by decreased viral load in acute-phase liver and plasma and a generally robust acute hepatic inflammatory response [30]. In other experiments using the WHV model, neonatal infected woodchucks that progressed to chronicity showed no significant or less frequent and incomplete acute-phase virus-specific cell-mediated immunity compared to that of resolved woodchucks [31], and diminished inflammation by CD31 T-cells, plasma cells and macrophages, as well as markedly deficient intrahepatic expression of IFN-g and TNF-a mrnas [32]. These data suggested that the onset of chronic infection may be associated with deficiencies in the primary T-cell response, but could also reflect reduced NK and NKT cell responses. In-line with these animal model observations, clinical data showed that chronicity is often associated with absent or mild symptoms of acute hepatitis [5]. Thus, activation of innate components of the immune system seems to represent a key element in the control of the initial HBV burst. Studies of HCV infection have shown that virus replication kinetics may be an important factor influencing the duration of virus persistence, with the rate of virus replication directly influencing the triggering of NK cells and the subsequent induction of an effective T-cell response. Viruses with lower growth rates may persist in the host because they trigger a International Medical Press

5 New insights into HBV pathogenesis Figure 2. Host factors involved in HBV clearance TLR7/9 pdc FasL NK IFN-γ IL-6 IL-12 FasL IFN-γ CD8 + T-cell CD4 + T-cell APC/DC Type I IFNs KC/M HBV LSEC TNF-α IFN-αR1 Antivirals Fas TNFR1 Hepatocyte APC, antigen-presenting cells; DC, dendritic cells; FasL, Fas ligand; IFN, interferon; IFN-aR1, interferon-a receptor-1; IL, interleukin; KC/M, Kupffer cells/macrophages; LSEC, liver sinusoidal endothelial cells; NK, natural killer cells; pdc, plasmacytoid dendritic cells; TLR, Toll-like receptor; TNFR1, tumour-necrosis factor receptor-1. weaker immune response [33], with studies of nine adult chimpanzees inoculated with different concentrations of infectious HBV virions showing that viral clearance or progression to persistent infection was influenced by the size of the initial inoculum [34]. In a surprising finding, chimpanzees inoculated with high (10 10 ) or low (10 0 ) genome equivalents demonstrated rapid spread of virus to all hepatocytes and delayed clearance, whereas persistent infection was readily established in the two chimpanzees inoculated with 10 1 genome equivalents [34]. Viral persistence in this study was attributed to variant CD4 + and CD8 + T-cell responses [34]; however, it would be interesting to determine whether NK cell responses also contributed to this response. Innate immune responses in specific cell types during CHB infection Natural killer cells In HBV infection, the ratio of activated circulating NK cells is lower in patients with chronic infection than healthy controls and NK cell cytolytic and IFNg-producing activity is also reduced, suggesting that impairment of NK cell function may contribute to viral persistence [35]. This impairment may, in part, be due to up-regulation of immunosuppressive cytokines, such as IL-10 and TGF-β, which suppress NK cell IFN-γ expression. Indeed, blocking expression of these cytokines restores the capacity of NK cells in both the liver and periphery to produce IFN-γ [36]. In an important finding, Tjwa et al. [37] showed that impairment of HBV replication improved NK cell IFN-γ production. Since IFN-γ is a key antiviral cytokine, HBV-mediated regulation of NK-cell-driven IFN-γ production may be contributing to viral persistence. Kupffer cells In addition to NK cells, liver-specific macrophages may also play an important role in HBV persistence. Hösel et al. [38] showed that recognition of the HBV envelope protein by primary human liver-specific macrophages (Kupffer cells) activates the transcriptional regulator NF-kB, leading to the release of IL-6 and other proinflammatory cytokines (IL-8, TNF-a and IL-1b), although it did not induce an IFN response. Antiviral Therapy

6 P Revill & Z Yuan This response was rapid, occurring within 3 h of hepatocyte infection. Importantly, IL-6 released by Kupffer cells controlled HBV gene expression and replication in hepatocytes through activating the mitogen- activated protein kinase signalling pathway, and as a consequence, inhibited expression of hepatocyte nuclear factor-1a and -4a, both of which are essential for HBV gene expression and replication [38]. It has also been reported that IFN-b produced by Toll-like receptor (TLR)3- and TLR4-stimulated murine Kupffer cells controls HBV replication in HBV-infected cells in a myeloid differentiation primary response gene (MyD88)-independent manner [39], although stimulation of other TLR receptors had no such effect, despite the fact that Kupffer cells do respond to a wide range of TLR ligands [40]. In vitro studies have shown that human THP-1 macrophages produce proinflammatory and regulatory cytokines in response to HBV capsid protein, suggesting that Kupffer cells could also respond to HBV proteins other than envelope [41]. Full-length HBV capsids efficiently bound differentiated THP-1 macrophages and strongly induced TNF-a, IL-6 and IL-12p40. Cytokine induction by HBV capsid particles was dependent on TLR2 expression and the subsequent activation of NF-kB, extracellular signal-regulated protein kinase (ERK)-1/2 and p38 mitogen-activated protein kinase [41]. Conversely, it has also been shown that HBV down-regulates TNF-a expression in differentiated human THP-1 cells [42], suggesting that HBV regulation of cytokine production is finely tuned and we are only beginning to understand its complexities. Although it is possible, indeed likely, that impairment of macrophage-mediated regulation of HBV replication in hepatocytes would contribute to viral persistence, this is yet to be shown experimentally. Dendritic cells The role of DCs is being increasingly recognized. DCs are major producers of type I IFN, and circulating myeloid DCs (mdcs) and plasmacytoid DCs (pdcs) the two main subsets of human DCs play an important role in antiviral immune responses, although they represent only % and % of peripheral blood mononuclear cells (PBMCs), respectively. mdcs are antigen-presenting cells that strongly activate T-cells and promote Th1 cell differentiation, leading to IFN-g production and viral clearance. pdcs produce type I IFN but are poor T-cell activators [43]. Type I IFN produced by pdcs not only plays a direct antiviral role, but can also prime other antiviral immune responses [44]. Viruses directly activate DCs by two mechanisms, either through TLRs [45] or through C-type lectins [46]. Although mdcs respond to only a limited number of TLR ligands [40], DCs can also be indirectly activated by cytokines produced following viral infection [47]. Activated DCs themselves produce a wide range of cytokines, such as IL-15, IL-12 and IFN-a, which in turn activate NK cells and influence T-cell survival and differentiation [48]. Therefore, DCs play an important role in bridging innate and adaptive immune responses. Both mdcs and pdcs play a key role in HBV disease progression during chronic infection, with the degree of liver inflammation inversely proportional to mdc and pdc numbers in peripheral blood [49,50]. Changes in peripheral blood DC populations may be directly due to migration from peripheral blood to the liver, leading to accumulation of DCs in the liver inflammatory activity area during chronic infection. There is increasing evidence that DC responses are impaired in the setting of CHB infection. DCs from patients with CHB have reduced allostimulatory capacity and exhibit decreased IL-6, TNF-a and IFN-a production in response to TLR ligand stimulation [51 56]. Murine studies show that the cytokine-producing and T-cell proliferation-inducing capacities of liver DCs in HBV transgenic mice are significantly decreased compared with normal mice [57]. In contrast to healthy controls, mdcs of patients with CHB infection produce less IFN-b and TNF-a, whereas production of the proinflammatory cytokines IL-6, IL-1b and IL-12 secretion is not affected [55]. Additionally, mdcs isolated from patients with CHB infection are less efficient in inducing T-cell proliferation in vitro than mdcs from healthy controls [58]. Similarly, IFN-a production was impaired in pdcs from CHB patients following herpes simplex virus and CpG stimulation [9]. In patients who have undergone HBeAg seroconversion to HBeAg antibody (anti-hbe), the number and function of peripheral pdc rebounds as the level of serum HBeAg decreases [51], suggesting the HBeAg may play an as yet unidentified role in pdc regulation. HBV-mediated down-regulation of innate immune responses Since the innate immune response regulates HBV infection, it is not surprising that HBV has evolved mechanisms to subvert innate immune responses. Stimulation of TLRs and IL signalling pathways leads to production of a range of proinflammatory and/or antiviral cytokines, including type I IFNs (TLR3, 4, 7, 8 and 9) [59] and TNF-a (TLR2). Furthermore, we have shown using an in vitro cell culture model that stimulation of TLR2 and IL-1 signalling pathways in hepatocytes decreases HBV replication [60], providing further evidence that the innate immune response in hepatocytes plays an important role in regulatory of infection [26] International Medical Press

7 New insights into HBV pathogenesis Numerous studies have shown that TLR expression is lower in CHB patients compared with healthy controls, although some experimental findings are contradictory. Generally, however, TLR1, 2, 4 and 6 expression is lower in PBMCs from CHB patients, with impaired cytokine production in PBMCs following challenge with TLR2 and TLR4 ligands correlating with the levels of plasma HBsAg [61]. Since TLR2 and TLR4 in peripheral blood are mainly expressed on monocytes [62] it is possible that HBV persistent infection directly influences monocyte function. HBV infection regulates expression of a broad range of TLRs and related receptors, including TLR1, 2, 4, 6 [61,63 67], 7 and 9 [68] in PBMCs of HBVinfected patients. It has also been shown that TLR2, 3, 4, 5, 6, 7, 9 and 10 expression was up-regulated during the active stage of CHB and CHB-related liver failure and, although TLR9 mrna expression negatively correlated with a number of clinical parameters including serum alanine aminotransferase, it positively correlated with HBV viral load [69]. The reasons for these incongruent findings are unclear. Wu et al. [70] used a murine model to demonstrate that HBV suppresses TLR innate immune responses in hepatocytes and non-parenchymal Kuppfer cells and liver sinusoidal endothelial cells. Pretreatment of cells with HBV virions, HBeAg or HBsAg almost completely abrogated TLR antiviral activity, due to suppression of IFN-b and subsequent induction of IFN-stimulated genes (ISGs) [70]. Taken together, these findings suggest HBV has evolved mechanisms to suppress TLR responses. In fact, numerous studies now show that HBV-encoded proteins regulate key innate immunity signalling pathways in both the peripheral blood and hepatocytes that may in turn facilitate establishment and/or maintenance of persistent infection. In the following section, we describe some of the mechanisms by which individual HBV proteins subvert innate immunity signalling (Table 1). Hepatitis B surface antigen The inhibitory effect of HBsAg on monocyte/ macrophage function was explored by Vanlandschoot et al. [71], who showed that recombinant HBsAg particles that contain the S protein only bound almost exclusively to monocytes. Attachment of recombinant HBsAg to the THP-1 premonocytic cell line occurred upon 1,25-dihydroxyvitamin D3-induced differentiation. This binding in turn suppressed lipopolysaccharide (LPS)- and IL-2-induced cytokine production. We and others have shown that the HBsAg also inhibits DC function, most likely through interacting with pdcs surface inhibitory receptor BDCA-2 [72,73] (Figure 3). HBsAg purified from Chinese patients inhibited TLR9-mediated activation and Table 1. Summary of innate immune responses directly manipulated by HBV genes HBV gene Cellular target References HBeAg/p25/p22 TLR2/MAL [67,78,80] IL-1 signalling [88,134] HBcAg IFN-ß [97] MxA [98] Il-18 [135] HBsAg ERK/JNK [74,75] IL-12/IL-18 [76] TLR-9/IFN-a [73] HBSP T-cell responses [101] Polymerase RIG-I/TLR-3 [103,104] HBx RIG-I/MDA5 [94,95] ERK, extracellular signal-regulated protein kinase; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBSP, hepatitis B splice protein; HBx, hepatitis B x protein; IFN, interferon; IL, interleukin; JNK, c-jun N-terminal kinase; MAL, MyD88 adaptor-like protein; MxA, myxovirus resistance A; p22, precore 22 kda variant; p25, precore 25 kda variant; TLR, Toll-like receptor. IFN-a production in pdcs [73]. HBsAg up-regulated SOCS1 and bound to BDCA-2 receptors on the plasma membrane to inhibit IFN-a production, but had no effect on TLR7 mediated IFN-a secretion. Although this was the first report showing a direct interaction between HBV and pdcs, it is one potential mechanism by which HBV could establish persistent infection. The HBsAg was also reported to bind to CD14 and interfere with LPS-induced activation of ERK- 1/2 and c-jun N-terminal kinase-1/2 in monocytes [74,75] (Figure 3). Recombinant HBsAg also inhibits LPS-induced COX-2 expression and IL-12/IL-18 production in THP-1 cells via interfering with the NF-kB pathway [76]. Since IL-12 and IL-18 are key regulators of IFN-g production in NK, NKT and Th1 CD4 cells, this may represent an important mechanism by which circulating HBV manipulates production of a key antiviral cytokines. Murine studies showed that IL-18 inhibits HBV replication through induction of IFN-g in NK/NKT cells and IFN-a/b in the liver [77]. Hepatitis B e antigen TLR expression is also regulated by HBeAg, with TLR2 expression on Kupffer cells, peripheral monocytes and hepatocytes significantly reduced in the setting of HBeAg-positive CHB compared with patients with HBeAg-negative CHB [67]. This finding is supported by in vitro cell culture studies, which suggest that HBeAg directly inhibits TLR2 expression. Transfection of a hepatoma cell line with complementary DNA (cdna) plasmids encoding replicationcompetent wild-type HBV down-regulated TLR2 expression [67]. This was in contrast to HBV cdna Antiviral Therapy

8 P Revill & Z Yuan Figure 3. HBV impairs function of immune cells Macrophage Monocyte pdc TLR2 HBeAg CD14 TLR4 HBsAg BDCA-2 MyD88 MyD88 HBeAg MAL HBsAg MAL Endosome CpG DNA IκB NF-κB MAPK IκB NF-κB MAPK TLR9 MyD88 IRF7 NF-κB AP1 NF-κB AP1 IRF7 HBsAg IL-12, IL-18 HBsAg IFN-α Hepatitis B e antigen (HBeAg) suppresses Toll-like receptor (TLR)2 expression in macrophages and monocytes. Cell culture studies using Hek293T cells show that the N terminus of the HBeAg interacts with membrane proximal proteins MyD88 adaptor-like protein (MAL) and TRIF-related adapter molecule (TRAM) [78], although the mechanism for internalization of HBeAg by macrophages and/or monocytes has yet to be determined. Hepatitis B surface antigen (HBsAg) inhibits lipopolysaccharide (LPS)-induced cytokine production in monocytes through interfering with nuclear factor (NF)-kB and the mitogen-activated protein kinase (MAPK) pathway. HBsAg also inhibits TLR9-induced interferon (IFN)-a production in plasmacytoid dendritic cells (pdcs) through interaction with the surface receptor blood dendritic cell antigen 2 (BDCA-2). AP1, activation protein 1; IRF7, interferon regulatory transcription factor-3; IkB, IkB kinase. plasmids encoding the G1896A precore stop codon mutation (which abrogates production of HBeAg), that had no effect on TLR2 expression [67]. More recently it has been shown that HBeAg directly interacts with key adapters in the TLR2 pathway [78], particularly the MAL adapter, providing one possible mechanism for the suppressive action of the HBeAg on TLR2 expression in monocytes/macrophages, although internalization of the HBeAg by these cells is yet to be determined (Figure 3). The region of the HBeAg that interacts with the MAL adapter for TLR2 signalling [78] has recently been mapped to the unique 10-mer amino acid sequence located at the HBeAg N terminus (SKLCLGWLWG). This sequence is highly conserved in all orthohepdnaviruses [79], and shares sequence similarity with sequences encoded by Box 2 of TIR domains that are encoded by TLRs and numerous adapters [78]. The importance of the N-terminal HBeAg sequence in regulation of TLR2 signalling was confirmed by Lang et al. [78] using site-directed mutagenesis studies, which showed that mutations in this sequence disrupted the interaction with MAL. HBeAg-mediated down-regulation of cytokine and IFN responses has also been reported by Wu et al. [80]. By contrast, recent studies by Zhang et al. [81] have observed the opposite effect, with exogenous HBeAg treatment of PBMCs resulting in up-regulation of TLR2 expression. The authors suggest that differences in HBV genotype may explain the different observations compared with the findings of Visvanathan et al. [67], which was based in Oceania (Australia), and suggest that most of the patients analysed would be subtype A2. We think this unlikely, as most HBV in Australia is in fact genotype B and C (from Asian immigrants) and genotype D (from Mediterranean immigrants) [82]. The A2 subtype is not common in HBV monoinfection in Australia, although it is detected in patients who are coinfected with HIV [83,84]. Human hepatocytes are the primary functional cells in the liver and the only host cells supporting the complete HBV life cycle during acute and CHB infection. Although not an immune cell, a growing body of evidence has shown that numerous molecules essential for innate immune response are expressed in hepatocytes, International Medical Press

9 New insights into HBV pathogenesis which allows them the limited ability to defend against viral infection [85,86]. Hepatocyte cell lines respond to stimulation with IL-1 and TLR2 ligands, leading to activation of the transcription factor NF-kB that in some cell lines (HepG2 and PH5CH8 cells) mounts an IFN response [86], although this response was lacking from Huh7 hepatoma cells [86]. Indeed we have shown that stimulation of TLR2 (and IL-1) receptors in hepatocytes triggers signalling cascades that inhibit HBV replication, although the mechanism remains unclear [60]. TLR2 stimulation drives production of TNF-a, which has direct antiviral effect on HBV by interfering with capsid formation [87]. However, in our study TNF-a levels following TLR2 stimulation were below the level of detection, suggesting down-regulation of HBV replication was mediated by an unidentified mechanism [60]. We have also recently used our in vitro cell culture model to show that the HBV precore p22 protein down-regulates IL-1-mediated signalling in hepatocytes [88]. The p22 protein is a precursor to the HBeAg and indicates that the HBV precore protein itself, in addition to HBeAg, is a potent regulator of the innate immune response. Together these and previously described findings [26,70] suggest that regulation of innate immune responses in the hepatocyte plays an important role in the establishment and/or maintenance of HBV infection. Down-regulation of type I interferon responses The type I IFN response is a major component of the innate immune response and is essential for viral clearance. To establish persistent infection, sabotage of type I IFN induction is an effective strategy adopted by many viruses [89 93]. Numerous studies have shown that HBV infection directly inhibits type I IFN production. For example, the HBsAg, HBeAg and virion particles significantly abrogate TLR-induced IFN-b induction and subsequent ISG expression [70], and the HBx protein prevents IFN-b induction by degradation of adaptor protein IPS1 (MAVS/Cardif/VISA) in HepG2 cells [94,95]. In CHB infection, HBV inhibits STAT1 methylation and disrupts STAT1 transcription function, in JAK-STAT signalling triggered by IFN-a [96]. Additional interactions between individual HBV proteins and IFN signalling pathways are described below. HBV core protein and hepatitis B splice protein The first evidence for HBV-mediated inhibition of type I IFN responses was provided by Twu et al. [97] who showed that the HBV core protein interfered with IFN-b expression through binding to IFN-b promoter as a transacting silencer in murine fibroblasts. Subsequently, Rosmorduc et al. [98] showed that HBV core protein, encoded by spliced pgrna down-regulated IFN-inducible myxovirus resistance A (MxA) protein expression, MxA being an important antiviral protein kinase. Further to these findings, Soussan et al. [99] showed that in addition to the core and precore protein, the most frequently detected spliced pgrna also encodes a novel protein termed the hepatitis B splice protein (HBSP). Although it is possible that the down-regulation of MxA observed by Rosmorduc et al. [98] was due in part to HBSP expression, Fernández et al. [100] subsequently showed that MxA down-regulation was directly attributable to the precore/ core protein. However, the HBSP has subsequently been shown to enhance T-cell responses in HBV-infected individuals [101] and its role in HBV pathogenesis requires further investigation. Hepatitis B e antigen In addition to the aforementioned murine studies showing that HBV suppresses IFN-b and subsequent induction of ISGs [70], Wu et al. [80] showed that expression of IFN-a and IFN-b mrna was down-regulated in stably transformed HBeAg-positive HepG2 cells, compared to an HBeAg-negative HepG2 cell line. Together these studies support a role for the HBeAg in regulation of type I IFN, although further studies using primary human hepatocytes may be necessary to tease out the mechanism. HBV polymerase The IFN response is also regulated by the HBV polymerase. Foster et al. [102] showed that expression of the terminal protein domain of the HBV polymerase alleviated cellular responses to double-stranded RNA and IFN-a. We and others have shown that the HBV polymerase suppresses IFN-b induction via disturbance of interaction between TBK1/IKKe and DDX3 and interference with interferon regulatory transcription factor-3 activation, in the primary hepatocyte cell line PH5CH8 and hepatocellular carcinoma cell line HepG2 [103,104]. Thus the HBV polymerase interferes with both type I IFN induction and signalling (Figure 4). In addition to the above roles, the HBV polymerase has the capacity to inhibit MyD88 expression by blocking STAT1 nuclear translocation [105]. These findings suggest HBV can manipulate IFN-a signalling through modification of key molecules in the JAK-STAT pathway to favour persistent infection. Indeed, a recent study using human hepatocytes injected into immune-deficient mice showed that HBV prevented the induction of IFN-a signalling by inhibiting nuclear translocation of STAT1 [106]. The authors propose that this may contribute to the limited effectiveness of endogenous and therapeutic IFN-a in patients and promote viral persistence. Since the mice in this study were infected with HBV genotype D, and response to IFN treatment is affected by HBV genotype [107], it would be interesting to observe Antiviral Therapy

10 P Revill & Z Yuan responses in mice infected with other HBV genotypes, such as genotype A2, which responds best to exogenous IFN treatment [107]. Hepatitis B X protein The HBx protein down-regulates IFN-b expression in hepatocytes, via suppression of the RIG-I MDA5 pathway. Wei et al. [94] showed that HBx, but not core or envelope proteins, suppressed poly(deoxyadenylate-thymidylate)-activated IFN-b production in hepatocytes by direct interaction with the mitochondrial antiviral signalling (MAVS) protein, an essential component of the virus-activated signalling pathway that activates NF-kB and IFN regulatory factor-3 to induce the production of type I IFN. HBx promoted the degradation of MAVS via Lys136 ubiquitin, thereby preventing the induction of IFN-b. In summary, all of these experimental studies provide evidence that HBV has developed multiple strategies to down-regulate IFN production, all of which Figure 4. HBV suppression of type I interferon response in hepatocytes Type I IFNs HBV Endosome dsrna RNA Pol III dsdna Tyk2 IFNAR1/2 JAK1 TLR3 Mitochondria Antivirals RIG-I OAS-1 TRIF MAVS MxA HBV TBK1 IKK-ε IKK-α IKK-β DDX3 Pol HBx IRF9 STAT1 STAT2 Core IRF3 IκB NF-κB ISGF3 Pol Type I IFNs ISGs IRFs NF-κB ISRE Core HBV interferes with both type I interferon (IFN) induction and signalling. HBV proteins and the region they affect in host signalling pathways are indicated in red. DDX3, DEAD (Asp-Glu-Ala-Asp) box polypeptide; dsdna, double-stranded DNA; dsrna, double-stranded RNA; HBx, hepatitis B X protein; IFN-aR1/2, interferon-a receptor-1/2; IKK, inhibitor of nuclear factor-kb kinase; IRF, interferon regulatory transcription factor; ISG, interferon-stimulated gene; ISGF3, interferon-stimulated gene factor 3; ISRE, interferon-sensitive response element; IkB, IkB kinase; JAK, Janus kinase; MAVS, mitochondrial antiviral signalling complex; MxA, myxovirus resistance A; NF-kB, nuclear factor-kb; OAS-1, 2-5 -oligoadenylate synthetase 1; Pol, HBV polymerase; RIG-I, retinoic acid-inducible gene-i; STAT, signal transducer and activator of transcription; TBK1, TANK-binding kinase 1; TRIF, Toll/interleukin-1 receptor domain-containing adaptor inducing interferon; Tyk, tyrosine kinase International Medical Press

11 New insights into HBV pathogenesis may contribute to the establishment and maintenance of persistent infection. Collectively, these studies show the importance of inhibition of type I IFN induction in the context of HBV infection and shed light on related molecular mechanisms responsible for HBV persistence. However, the differential responses to exogenous IFN therapy observed for each of the HBV genotypes [107] suggests that regulation of IFN responses may also differ throughout the globe and the role of HBV genotype needs to be further explored in experimental and clinical studies. Recent advances in manipulation of the innate immune response to treat CHB infection Since HBV infection is cleared by a robust innate and adaptive immune response in approximately 95% of individuals infected during adulthood, can the innate immune response be manipulated to eradicate HBV from those with persistent infection? IFN-a used for treating patients infected with HBV is a product of the innate immune system, produced by pdcs in response to TLR7 and TLR9 ligands stimulation [108]. As discussed previously, treatment with exogenous IFN is only effective in a minority of HBV patients, due in part to inhibition of IFN-signalling by up-regulation of protein phosphatase 2A, an inhibitor of STAT1 methylation, which is required for IFN-a signal transduction [109]. For reasons that remain unclear, the effectiveness of exogenous IFN-a therapy for treatment of HBV infection appears to be influenced by viral genotype and host polymorphisms. For HBV, therapy is most effective for patients infected with HBV genotype A2 (compared with genotype D) and least effective for patients infected with HBV genotype C (compared with genotype B), irrespective of whether the IFN used was conventional or pegylated [ ]. The reasons for this are unknown, but differences in IFN response of HBV genotype A2 and the Asian genotypes B and C may relate to the fact that genotype A2 is adult acquired whereas in Asia, HBV genotypes B and C are transmitted at birth or in very early childhood. However, the differences in IFN response observed within Asian genotypes B and C are more difficult to reconcile as the timing of transmission is similar. Perhaps HBV subtype plays a role in this differential IFN response, as it is becoming increasingly apparent that HBV genotype and subtype influences HBV pathogenesis and natural history [107]. Although it is possible that host genetic factors may contribute to the differential response to IFN therapy for HBV in different geographic regions, as recently identified for HCV genotypes [116], this does not explain the differential IFN responses observed for the Asian genotypes. In addition to IFN-a, the type III IFN, IFN-l, shows promise for treatment of HBV and HCV infection. Initial studies by Robek et al. [117] showed that IFN-l inhibited HBV replication in cell culture. This group has more recently shown that IFN-l manifests its antiviral activity by inhibiting nucleocapsid assembly, similar to IFN-a/b [118]. Since we and others have demonstrated that stimulation of the IL-1 and TLR receptors inhibits HBV replication [60,119], could approaches such as the TLR7 agonist Imiquimod used for treatment of human papillomavirus infection [120] have relevance for treatment of CHB infection? Agonists and antagonists targeting TLRs and adapter molecules such as MyD88 [121] are under investigation for treatment of infection with a range of different pathogens [122,123]. Indeed agonists for TLR7 and TLR9 have proven useful as adjuvants to improve immunogenicity of the preventative HBV vaccine [124,125]. A number of antagonistic molecules targeting TLR4 are in various phases of investigation [126,127] and, since TLR4 signalling is important in the setting of liver inflammation, these approaches may have direct relevance for HBV infection. However, since stimulation of TLR receptors reduces HBV replication [38,60], treatments that reduce TLR expression need to be carefully assessed. Conversely, although it may seem logical to enhance TLR responses in the setting of HBV infection, the potential for induction of conditions such as sepsis using this approach means great caution needs to be taken. These approaches are further complicated by the observation that HBV has developed numerous mechanisms to subvert TLRmediated immunity. However, in a recent development, the TLR-7 agonist GS-9620 has been shown to have anti-hbv activity in chronically infected chimpanzees, reducing viral load by 2 logs, with corresponding decreases in serum HBsAg and HBeAg of 50 61% and 58 93%, respectively [128]. This exciting finding, together with the rapid advances in our understanding of the innate immune system and the varied methods used by HBV to circumvent this intricate defence mechanism, provide hope that specific therapies targeting innate immune responses will soon become a valuable part of our armoury against this important chronic disease. Innate immune responses in children with CHB infection Although CHB infection is most readily established at birth or in early childhood [27], thought largely to be due to immature immune responses [129], there have been very few studies on innate immune responses in children, particularly in the setting of chronic viral infections. A study of 20 children with chronic HCV infection Antiviral Therapy

12 P Revill & Z Yuan identified elevated TLR2 and TLR4 expression in neutrophils following LPS stimulation of whole blood compared with healthy controls [130], but there have been no similar studies in the setting of HBV infection. Studies of immune responses in children with CHB infection have been limited to HBcAg-specific T-cells, with studies of 36 and 20 children showing that CD4 + T-cells activate Th1-specific responses [131,132]. The most comprehensive analysis of innate immune responses in children undertaken to date (36 children under the age of 2 years), showed that TLR responses driving antiviral defense mechanisms (IFN-g and IFN-a) in human blood mononuclear cells were much lower in the first 2 years of life than in adults [133]. This has important implications for HBV infection, where chronicity is established much more readily in early childhood than adulthood. This observation is further clouded by the fact that the mode of transmission differs in childhood and adulthood, and different HBV genotypes are associated with the different modes of transmission and have different propensities to establish chronic infection. For example, transmission at birth or in early childhood is mainly restricted to Asian genotypes B and C (vertical transmission at birth or very early thereafter) and the African A1, D and E genotypes (horizontal, early childhood) [27,107]. By contrast, the Western A2 genotype is almost exclusively transmitted horizontally in adulthood. As a consequence, chronic infection establishes more readily in the Asian and African genotypes than the Western A2 genotype. Do differences in the propensity for chronic HBV relate to the child s immature immune system (innate and adaptive), mode of transmission, HBV genotype, or unidentified host and virological factors? Since we and others have shown that innate immune responses are down-regulated in chronic adult HBV infection, it will be important to repeat many of the studies described in this review using PBMCs or cell lines derived from children infected with different HBV genotypes. Then we may truly determine the mechanisms driving HBV persistence in different regions of the globe. Summary In summary, although advances in this field have been hampered by the lack of a reliable infection model, the weight of evidence from multiple studies using animal models, primary human cells, cell lines and human subjects, shows that the innate immune response is an important regulator of HBV infection and that HBV has evolved numerous mechanisms to subvert innate immune responses. It is likely that this subversion contributes to the establishment and maintenance of persistent infection. It is hoped that through further understanding the intricacies of the interactions between HBV and the innate immune response, we will be able to develop effective therapies for the cure of CHB infection, providing new hope for the 400 million individuals worldwide living with CHB. Disclosure statement The authors declare no competing interests. References 1. Beck J, Nassal M. Hepatitis B virus replication. World J Gastroenterol 2007; 13: Hoofnagle JH. Reactivation of hepatitis B. Hepatology 2009; 49 Suppl 5:S156 S Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat 2004; 11: Bertoletti A, Gehring AJ. The immune response during hepatitis B virus infection. J Gen Virol 2006; 87: Webster GJ, Reignat S, Maini MK, et al. Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms. Hepatology 2000; 32: Wieland S, Thimme R, Purcell RH, Chisari FV. 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