The natural history of CHB

The immunopathogenesis of hepatitis B depends on a complex interplay of host and viral factors. Factors such as age, gender and immune status are important. Perinatal and childhood infection results in chronic infection in 90–95% and 50% of cases respectively, whereas greater than 95% of immunocompetent adults with acute HBV spontaneously clear the infection. Furthermore, CHB infection is more common in immunocompromised hosts (eg, HIV infection). Viral factors such as HBeAg status are also important. Under normal circumstances viral replication is not cytopathic, and it is the host's immune response, which is typically ineffective and inappropriate, that causes much of the damage associated with CHB.

The natural history of CHB is generally regarded as consisting of four phases36: immune tolerant; HBeAg-positive CHB (immune clearance); immune control (low or non-replicative); and HBeAg negative CHB (immune escape). These phases have been identified on the basis of specific biochemical, serological and virological characteristics, including serum alanine aminotransferase (ALT) levels, HBeAg serostatus, HBV DNA titre, and more recently, HBsAg level (figure 2). It is important to note that these phases do not occur in all individuals with CHB and do not always occur sequentially.37 It is patients in either the immune clearance or immune escape phases that are potential candidates for the currently approved antiviral and immune modulatory treatments.

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Figure 2

Phases of chronic hepatitis B (CHB) infection: serum and liver compartment. ALT, alanine aminotransferase; cccDNA, covalently closed circular DNA; HBeAg, hepatitis B secreted e-antigen; HbsAg, hepatitis B surface antigen; HBV, hepatitis B virus; ORF, open reading frame; PC/C, precore/core; PE, Paul Ehrlich Institute; rcDNA, relaxed circular partially double-stranded DNA.

The immune tolerant phase is characterised by positivity for serum HBeAg, high levels of HBV DNA, normal serum ALT levels and normal/near normal histological profile in the liver. The levels of HBV DNA typically exceed 20 000 IU/ml while HBsAg levels range from 4.5 to 5.0 log IU/ml.38 39 Patients in the immune tolerant phase are usually young and were infected early in life. The immune tolerant phase can last for more than 20 years. In contrast, individuals progressing to chronic infection following adulthood infection do not typically undergo a prolonged immune tolerant phase, and may instead enter directly into the immune clearance phase.

When CHB infection is acquired from an early age, the immune clearance phase usually occurs between ages 20 and 40 years, and is characterised by HBeAg positivity, elevated serum ALT levels, fluctuating HBV DNA levels (>20 000 IU/ml), lower levels of HBsAg (approx 4.3 log IU/ml) and histological damage. During this phase, the virus is under intense immune pressure and the emergence of dominant PC and/or basal core promoter (BCP) variants may occur, with eventual HBeAg seroconversion (SC) and control of HBV replication. Alternatively, HBV escapes from immune clearance with the person then developing HBeAg-negative CHB (figure 3). Following successful HBeAg SC, the majority of patients enter an immune control or low/non-replicative phase, characterised by low/undetectable viral replication and normal ALT levels. A serum HBV DNA of <2000 IU/ml and a quantitative HBsAg level of <1000 IU/ml have been suggested to differentiate the immune control and escape phases.40 41 A subset of patients can enter into the HBeAg-negative hepatitis phase, whereby HBV DNA levels >2000 IU/ml and increased quantitative HBsAg (3.5 log IU/ml) are found with increasing serum ALT levels and consequent further histological damage (figure 2).

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Figure 3

Proposed interactions between hepatitis B virus (HBV) proteins and components of the innate immune system. The diagram shows the intracellular effects of the precore/hepatitis B secreted e-antigen (HBeAg) on hepatocyte signalling via the MyD88-dependent (toll-like receptor (TLR)-2) and MyD88-independent (TLR-4) pathways. The terminal protein of the HBV polymerase was also reported to hinder the type I interferon (IFN)-mediated transcription of IFN-stimulated genes by impairing nuclear translocation of signal transducer and activator of transcription (STAT) proteins, while HBx expression was shown to inhibit the retinoic acid inducible gene I (RIG-I)–MAVS signalling. Circulating subviral particles (SVPs) were shown to repress TLR-9 signalling by enhancing SOCS expression in dendritic cells (pDCs). Furthermore, secreted HBeAg was shown to interact and downregulate TLR-2 expression on hepatocytes. In HBeAg-negative chronic hepatitis B (CHB), the absence of HBeAg results in upregulation of TLR-2 and secretion of the proinflammatory cytokines, including tumour necrosis factor α (TNFα). IRF, interferon regulatory factor; ISG, interferon-stimulated gene; MDA5, melanoma differentiation associated gene 5; NF-κB, nuclear factor κB; TRAM, TRIF-related adaptor molecule; MAVS, mitochondrial antiviral signaling; TRIF, TIR-domain containing adaptor protein inducing interferon–β; TRAF, TNF receptor-associated factor MAL; MyD88-adapter-like protein.

It is generally accepted that the extent of chronic liver disease is directly linked to the frequency, duration and severity of hepatic flares occurring in either the HBeAg-positive (immune clearance) and/or HBeAg-negative phases. HBeAg SC is predictive of a sustained multi-log reduction in HBV DNA (>3 log₁₀ IU/ml), a decrease in intrahepatic inflammatory activity and an improved prognosis.42 For these reasons, HBeAg SC has been traditionally used as an important therapeutic endpoint in the management of HBeAg-positive CHB. Not surprisingly, then, delayed HBeAg SC, particularly in patients >40 years old, is associated with higher fibrosis scores on liver biopsy. HBsAg SC is associated with a favourable long-term clinical prognosis,43 but unfortunately, spontaneous HBsAg loss and/or SC, now regarded as an important immune control endpoint, is rare in CHB with approximately a 1% annual rate.44

Spontaneous HBV reactivation in the low/non-replicative phase is clinically significant, since this is typically followed by increased hepatitis activity and ALT flares.45 The annual incidence of hepatic flare has been reported to be over 10% in HBeAg-negative patients.45 These hepatic flares are due to the resurgence of host immunity against HBV-infected hepatocytes and severe hepatic flares can be complicated by hepatic decompensation in 2–3% of patients. These adverse events typically drive the development of advanced fibrosis, which in time, eventually leads to cirrhosis.

Major virological events during the natural history of CHB

Viral evolution is important in the pathogenesis of chronic viral diseases such as hepatitis C,46 HIV47 and HBV.48 For example, during the immune clearance phase, Lim et al demonstrated that before HBeAg SC actually occurred, the viral sequence diversity in the PC/BCP region was significantly higher in HBeAg seroconverters compared with non-seroconverters.48 The selection pressure driving this diversity was found to be in effect at least 3 years before HBeAg SC actually occurred, and interestingly, the viral diversity increased further after viral load reduction. These results extended previous work demonstrating that viral mutation rates are increased in the immunoelimination phase compared with patients in the immunotolerant phase of CHB.49 Lim and colleagues also demonstrated that nucleotide substitution rates persisted at high levels after HBeAg SC, with viral diversity increasing further.48 Thus, it appears that this phase is far from being a ‘non-replicative state’, and that there is an ongoing dynamic post-SC process, albeit at lower levels of viral load, with viral diversity likely being driven by an immune-based selection pressure such as significant production of anti-HBe antibody.

Occult HBV infection: the final phase of CHB?

Occult HBV infection (OBI) can be defined as the presence of HBV DNA in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing HBsAg negative by currently available assays. When detectable, the amount of serum HBV DNA is typically very low (<200 IU/ml). The molecular basis of OBI is related to the long-lasting persistence of HBV cccDNA in hepatocyte nuclei.20 Although some HBV isolates from OBI cases may harbour ‘a’/α determinant mutations that prevent HBsAg detection by antibodies against the wild type virus, almost all OBI cases are infected with replication-competent HBV rather than escape variants, and so the lack of HBsAg in the peripheral compartment is regarded as a possible reflection of ‘strong immune control’ or suppression of viral gene expression of the infecting virus.50 51 The role of OBI in the development of chronic liver disease and as a risk factor for HCC development appears significant50 and further molecular studies are needed to elucidate the underlying pathogenetic mechanisms.51

Immuno-pathogenesis of HBV infection: general considerations

The innate immune system represents an immediate, first-line defence against foreign pathogens and plays a fundamental role in regulating the adaptive immune response. While the role of the adaptive immune system in the resolution of HBV infection has been studied extensively,77–79 knowledge of the innate immune processes occurring in HBV infection has been limited by the restricted availability of in vitro and in vivo infection systems and technical difficulties in recruiting patients at the earlier stages of infection. Despite these limitations, studies in acute infected patients80 and chimpanzees81 documented a lack of type I IFN induction and proinflammatory cytokine production in acute HBV infection.81 In contrast to these observations, recent in vitro studies indicated that the innate immune response of the hepatocytes may sense the infection and limit the spread of HBV.82–84 A modest activation of IFN-stimulated genes was also shown in human hepatocytes after HBV infection in chimeric mice.30 Induction of the antiviral state and production of type 1 IFN (α/β) can be mediated by pathogen recognition receptors, such as toll-like receptors (TLRs).84 In turn, type 1 IFN production stimulates antigen-presenting cells such as dendritic cells and Kupffer cells (resident intrahepatic macrophages) to produce interleukins (IL-8, IL-12, IL-18) and other cytokines.85 IL-12 and IL-18 subsequently induce natural killer (NK) and natural killer T (NKT) cells.86 NK cells can also be activated by the major histocompatibility complex I molecules on the surface of infected hepatocytes or by recognition of stress-induced molecules.87 Although NK and NKT cells can promote liver damage through direct cytotoxic effects,88 their role in HBV persistence and mechanisms regulating NK cell function in HBV infection are still controversial.89

The hepatocellular injury caused by HBV infection is predominately immune mediated.90 91 The immune attack by the host against HBV is mainly mediated by a cellular response to small epitopes of HBV proteins, especially HBcAg, presented on the surface of liver cells. HLA class I restricted CD8 cells recognise HBV peptide fragments derived from intracellular processing and presentation on the liver cell surface by class I molecules. This process leads to direct cell killing by the CD8 cytotoxic T lymphocytes. It is important to note that B and T lymphocytes are only activated following recognition of HBV by the innate immune system. Ultimately, it is the breadth and strength of this specific adaptive immune response which determines the outcome of acute and chronic HBV infection. Animal and clinical studies demonstrated that in acute self-limited HBV infection, the CD8 T-cell and CD4 T-cell responses to HBV proteins92 are strong, polyclonal and multispecific, while in chronic HBV infection, the responses are weak and narrowly focused.89 Besides having a direct cytolytic effect, as discussed above, CD8 T cells also have a non-cytolytic effect through the production of IFNγ and tumour necrosis factor α (TNFα) (also produced by NK and NKT cells), which are known to elicit antiviral effects via multiple mechanisms.93 Indeed, studies have shown that IFNα and IFNβ significantly suppress HBV gene expression in vivo, indicating that non-cytolitic direct antiviral effects elicited by the immune response may play a determinant role in controlling HBV infection in immunological normal adults.30 57 75 Thus the current concept is that non-cytolytic and cytolytic mechanisms are requisite for successful HBV control and eventual clearance.

HBV interactions with innate immune responses of the hepatocytes

The limited induction of innate immune responses determined upon HBV infection may also be due to recently recognised abilities of HBV to evade innate immune recognition. TLRs are evolutionary conserved germline-encoded pattern recognition receptors that play a crucial role in early host defences by recognising pathogen-associated molecular patterns and also serve as an important bridge between the innate and the adaptive immune response. HBeAg was shown to act as an immune decoy for the core antigen by depleting HBeAg-specific and HBcAg-specific T-helper cells and exerting a tolerogenic effect in utero.94 Furthermore, HBeAg has been shown to potentially downregulate genes involved in cell signalling, RNA transport and processing, cytosol-nuclear trafficking and innate immune responses.95 Lang et al12 confirmed the presence of a Box-2-like TIR motif within the unique N-terminal 10-amino-acid sequence of PC/HBeAg protein which could antagonise TLR signalling pathways via blocking the adapter proteins Mal, TRAM and TRIF but not MyD-88, thereby suppressing TIR-induced nuclear factor κB and IFNβ activation (table 1). These findings provide a possible mechanism of the ‘HBeAg immunomanipulation’ of the innate immune response, including the induction of ‘tolerance’ in the first phase of CHB, especially following perinatal HBV transmission. In patients with HBeAg-positive CHB, the expression of TLR-2 on hepatocytes, Kupffer cells and peripheral blood monocytes was shown to be significantly downregulated, while upregulation of the TLR-2 pathway and increased TNFα production was found during HBeAg-negative chronic hepatitis96 (Table 1). Although these clinical studies have indicated a role for HBeAg in downregulating immune surveillance of HBV, loss of HBeAg synthesis commonly occurs during chronic HBV infection and the emergence of HBeAg-negative variants may present selective advantages, possibly by limiting the cytotoxic T-lymphocyte (CTL)-mediated clearance of infected hepatocytes.97 However, the selection of HBeAg variants is associated with increased viral activity,26 more severe liver disease and worse prognosis.44

In support of the concept that HBV has evolved mechanisms to sabotage pathways of the IFN response, a recent study in human-liver chimeric mice showed that administration of regular IFNα failed to promote detectable nuclear translocation of signal transducer and activator of transcription 1 (STAT-1) in HBV-infected human hepatocytes, although STAT-1 nuclear accumulation and enhancement of antiviral defence mechanisms were promptly induced in uninfected human hepatocytes.30 Furthermore, in vitro studies indicated that HBV may affect STAT methylation98 and activity of cellular DNA methyltransferases (DNMT).99 100 The non-structural HBx protein has been shown to also interfere with the innate immunity by downregulating mitochondrial antiviral signalling protein101 by suppressing the retinoic acid inducible gene I (RIG-I)–melanoma differentiation associated gene 5 (MDA5) pathway in vitro. Interactions between HBx, mitochondrial antiviral signalling and members of the cellular epigenetic machinery have been also reported.101 Furthermore, plasma-derived HBsAg SVPs were reported to inhibit TLR-9 mediated IFNα production by plasmacytoid dendritic cells, probably via stimulation of suppressor of cytokine signalling 1 expression.102 Although most of these analyses were performed in systems that overexpressed HBx and further analyses are needed in more relevant infection systems, taken together, these studies suggest that distinct virus-mediated mechanisms may contribute to the limited effectiveness of the innate immune responses and subsequent adaptive immune responses in HBV infection and thereby may significantly contribute to viral persistence.

Mutual interplays influencing HBV activity and cellular pathways

The incidence of HCC development has been shown to correlate with the levels of HBV replicative activity.103 This highlights the importance of elucidating the mechanisms modulating viral activity because these factors will affect both cccDNA loads and cellular pathways, thus influencing the clinical course of infection.

Numerous transcription factors implicated in the activation of hepatic metabolic processes, such as hepatocyte nuclear factor, cAMP responsive element binding protein, retinoid X receptor and peroxisome proliferator-activated receptors, are known to bind the HBV genome and the recruitment of liver-enriched transcription factors on the cccDNA minichromosome appears essential for controlling viral gene expression.6 104 However, interactions between viral components and cellular factors may also impact the liver metabolism. Glucocorticoids are steroid hormones playing major roles in glucose homeostasis and the presence of a glucocorticoid response element on HBV genome can explain the so-called HBV reactivation observed in patients receiving steroid-based immunosuppressive agents. Although a clinical association between HBV and glucose homeostasis is lacking,104 HBV was shown to promote lipogenesis and fatty acid accumulation in HBV-replicating transgenic mice105 and in the liver of HBV-infected patients.106 Such metabolic alterations are likely to contribute to disease progression. Furthermore, a multitude of in vitro transfection experiments showed that HBx can modify cellular pathways by altering the activity of numerous transcription factors, kinases of the Src family, of the Ras-Raf-mitogen-activated protein kinase cascade and members of the protein kinase C pathway, and by affecting mitochondria function and protein degradation.107 108 Whether HBx, expressed at levels comparable to those present in infected human livers, also similarly affects these cellular functions awaits further investigation.

CccDNA-based chromatin immunoprecipitation (ChIP) assays indicated a correlation between viraemia levels and acetylation status of cccDNA-bound histones,109 suggesting that epigenetic modifications may affect the cccDNA transcriptional status in the different phases of chronic infection. Using ChIP assays HBx was shown to be recruited to the cccDNA minichromosome,110 where by favouring cccDNA-bound histone acetylation, HBx appears to play an essential role in initiating the cccDNA-driven transcription of the HBV RNAs (figure 1).8 However, epigenetic modifications, such as DNA hypermethylation, may induce suppression of cccDNA transcription, since increased expression of DNMTs has been correlated with increased cccDNA methylation and decreased HBV transcription in advanced HBV infection.111 Moreover, HBx was shown to act as a potent epigenetic modifying factor in human liver by modulating the transcription of DNMT required for maintenance of hypomethylation of tumour suppressor genes.99 The HBx-promoted hypermethylation of tumour suppressor genes suggests a novel mechanism by which this promiscuous transactivating protein may accelerate hepatocarcinogenesis.

Numerous cellular factors, such as microRNAs, may affect HBV productivity by binding directly to HBV transcripts or by targeting cellular transcription factors relevant for cccDNA transcription.112 Further studies based on a combination of bioinformatic and miRNA expression screenings are needed to determine whether specific patterns of miRNA expression could serve as diagnostic biomarkers for liver disease prediction.

A number of studies identified host variations that may contribute to the outcome of HBV infection. Genome-wide association studies determined, for instance, polymorphisms in the human leucocyte antigen (HLA) allele DRB1,113 HLA-DP114 and the oestrogen receptor115 that were associated with the susceptibility to HBV clearance or persistence. Similarly, naturally occurring sequence variations in the promoter region of CXCL10 gene were shown to result in the altered expression of the CXCL10 protein, which is important for the intrahepatic recruitment of inflammatory cells, thereby impacting disease progression of patients with chronic HBV infection.116

Emergence of virus quasi-species and infection with other viruses

The selective pressure induced by host immune responses and/or during antiviral therapy with polymerase inhibitors promotes the emergence of mutations. For instance, single amino acid substitutions have been shown to impair HBsAg recognition and lead to the development of acute or chronic HBV infection despite immunoprophylaxis. The region of the genome encoding for the rt domain of the HBV polymerase overlaps completely with the envelope; therefore, mutations emerging during nucleos(t)ide analogue treatment may lead to changes in the viral envelope. In vitro studies have shown that the rtA181T mutation, which introduces a nonsense mutation in the overlapping envelope gene, leads to intracellular retention of the truncated surface proteins and viral particles.72 As discussed above, during the immune competent HBeAg-positive phase, the host immune system may set the stage for the selection of PC/BCP mutations with reduced or absent HBeAg secretion.25 117 Sequencing of cccDNA amplicons indicated that the replacement of wild type cccDNA with populations of BCP mutations was associated with the re-establishment of high virion productivity26 (figure 3). In agreement with these observations, studies in chimpanzees indicated that HBeAg-negative viruses induce more severe hepatitis118 and the accumulation of BCP mutations have been shown to be associated with worsening of clinical manifestations and development of HCC.119

HBV/HCV and HBV/hepatitis D virus (HDV) co-infection are known to lead to a rapid and more severe progression of chronic liver disease. Although the virological profiles in the setting of co-infection appears to be widely divergent and fluctuating levels of viraemia120 are observed, information about the mechanisms responsible for such variations are scant. Although immune-mediated mechanisms may play a major role in suppressing HBV replication in the setting of co-infection,69 121 122 recent studies in chimeric mice31 showed that HDV lowered HBV productivity even in the absence of adaptive immune responses, indicating that complex virus–virus and virus–host interactions may affect replication levels and disease progression in the course of HBV/HDV co-infection.