Introduction

Precursors of the hepatitis B virus (HBV) can be traced back up to 82 million years till the Mesozoic when one of them was integrated in the genome of prehistoric birds that are likely to have been the ancestral hosts of Hepadnaviridae [1]. Their evolutionary origin has also been studied by analyzing endogenous avian hepadnavirus DNA in the genome of zebra finches [2, 3]. Phylogenetic studies suggest that HBV has existed in humans since at least 33,600 years ago, and it is supposed to have co-migrated with human populations thereafter [4]. The oldest human virus isolate known so far was obtained by laparoscopic liver biopsy from a Korean mummy from the sixteenth century [5]. Today, more than 240 million people worldwide are chronically infected with hepatitis B, and despite the availability of an effective vaccine, the virus causes about 780,000 deaths every year [6].

Virological features

Viral structure

HBV is a partially double-stranded DNA virus that belongs to the family of Hepadnaviridae. As a hepatotropic agent, it infects mostly liver cells in a non-cytopathic manner. It forms small, enveloped virions of 42 nm, also known as Dane particles, that consist of an outer envelope of lipoproteins, the core antigen or viral nucleocapsid protein, the viral genome and a polymerase [7]. In addition, budding of HBV surface (HBs) proteins at intracellular membranes can also lead to the formation and secretion of empty, spherical or filamentous subviral particles (SVPs) of 20 nm in diameter [8]. These non-infectious particles are secreted into the blood in a 1,000- to 1,000,000-fold excess over virions depending on the phase of infection [9, 10].

The 3.2-kb large genome encodes seven proteins in four overlapping open reading frames (ORF). Two of them include more than one in-frame initiation codon in order to allow differential translation. The viral envelope consists of three transmembrane hepatitis B surface (HBs) proteins of different sizes called S, M and L, all encoded by the S ORF. The smallest of them is the S protein, while the M protein is extended by a preS2 domain using an up-stream start codon in the same ORF. For the largest form of the lipoprotein, called L, an extra preS1 domain is added to the previous ones in the same manner [11].

The precore/core gene in the second ORF encodes the viral capsid protein or core antigen (HBcAg) and the hepatitis B e antigen (HBeAg). HBeAg is a soluble polypeptide of about 16 kDa [12]. Its secretion is a marker of viral replication, but it is not required for reproduction of the virus [13, 14]. In fact, mutations in the precore regions occur frequently during infection [15]. However, their effect on the clinical course or outcome is discussed controversially [16] and might depend on the time point of mutation [17].

The third open reading frame contains the polymerase gene coding for the N-terminal primer, the reverse transcriptase and RNase H domains [10]. Finally, the X gene encodes the small regulatory X protein that is mandatory for viral replication in vivo [18].

There are at least eight genotypes of HBV (A–H) in humans and nine serologically defined HBs antigen (HBsAg) subtypes [19]. While the most common genotypes in Europe and the USA are A and D, there is a dominance of genotypes B and C in China and Southeast Asia [20, 21]. The clinical course and the response to interferon therapy differ between genotypes, C and D being less responsive compared with A and B [22, 23].

Viral replication cycle

After entering the blood flow, the virus first attaches to hepatocytes through heparan sulfate proteoglycans [24]. Thereafter, the preS1 domain of the large envelope protein binds to a Na+ bile acid symporter called sodium taurocholate cotransporting polypeptide (NTCP) in order to enter the cell [25, 26]. NTCP is a multiple transmembrane transporter located on the basolateral plasma membrane of hepatocytes [27]. Physiologically, it contributes to the maintenance of the enterohepatic circulation of bile acids [28, 29].

The following steps of viral entry are largely unknown. Once inside, the nucleocapsid is released from the virus particle and transferred via the microtubular transport system to the nuclear pore. Here, the core particle is loosened up and the non-covalently closed, so-called relaxed circular DNA (rcDNA) is translocated to the nucleoplasm and converted to covalently closed circular DNA (cccDNA) [30]. These episomal minichromosomes serve as genome reservoir for active propagation of the virus and for persistence even after serologic clearance of HBsAg [31].

The cccDNA is transcribed by the cellular RNA polymerase II into viral pregenomic RNA (pgRNA) and several subgenomic mRNAs. The pgRNA serves as a template for reverse transcription, the core protein and the viral polymerase, whereas the subgenomic mRNAs code for the envelope proteins and the X protein [11]. All these essential viral mRNAs are transported without splicing to the cytoplasm where they are translated.

Next, binding of the viral polymerase to pgRNA leads to a selective encapsidation into core particles [32]. Inside the freshly assembled immature nucleocapsids, reverse transcription is initiated. First, a complete minus-DNA strand is synthesized, followed by an incomplete plus strand. Finally, the mature nucleocapsids can either be transported back to the nucleus and contribute to the maintenance of a stable intranuclear pool of cccDNA or are enveloped by HBs protein containing membranes and flush out into the blood via exocytosis [33, 34].

Natural history of HBV infection

Acute infection

HBV can be transmitted both vertically and horizontally. Particularly in highly endemic regions as sub-Saharan Africa and East Asia, the most common route of infection is perinatal or during early childhood, leading to a rate of chronicity of up to 90 %. In contrast, most patients in Western countries are infected as adults by sexual transmission or contaminated drug needles. Their risk of developing chronic infection is less than 5 % [6]. Interestingly, the progression of chronicity is influenced by the size of the viral inoculum, as could be shown in a chimpanzee study [35].

Acute infection can be symptomatic with clinical features as jaundice, fatigue, nausea, vomiting and abdominal pain [6]. In most cases, however, infected patients are primarily asymptomatic despite a high-titer viremia of about 1010 virions per milliliter [34, 36] and an infection rate of 75–100 % of all hepatocytes [37]. Interestingly, the virus does not activate the innate immune response during the early phase of infection. Indeed, a lack of type I interferon response was observed during acute infection in chimpanzees as well as in humans [38, 39]. These findings have led to the description of HBV as a stealth virus. Different strategies have been proposed for the early evasion of HBV from the immune system, e.g., that viral replication is invisible to the host cells innate sensing machinery [40] or that the immunosuppressive cytokine interleukin-10 (IL-10) is induced [39].

Persistent infection

Chronic hepatitis B infection is a state with dynamic alterations of disease. Serological and clinical parameters such as viral load or hepatic inflammation may change over time [41]. For reasons of clarity, three phases of chronic HBV infection have been defined: the immune tolerant phase, the immune active phase and the inactive hepatitis B phase [42].

Perinatal transmission of HBV often leads to the development of immune tolerance. This phase is characterized by the presence of HBeAg, high levels of serum HBV DNA, normal serum aminotransferases and minimal or no liver inflammation [43]. The latter is due to the absence of any cytolytic T-cell response [44]. Patients may remain in the immune tolerant phase for more than 30 years, suffering only from minimal disease progression [45]. However, over time, the stochastic integration of HBV replicative DNA intermediates into the host cell’s genome increases. This silent activity leads to an elevated risk of developing hepatocellular carcinoma even without preexisting fibrosis [41].

The immune active phase is characterized by high levels of alanine aminotransferase (ALT), still high HBV DNA levels and active liver inflammation [44]. HBeAg may be present in these patients, but some patients may become HBeAg negative. The rate of HBe seroconversion is influenced by factors such as older age, high levels of liver enzymes, acute exacerbations, HBV genotype B and ethnicity [43]. The development of anti-HBe antibodies can be preceded by hepatic flares [46].

The inactive phase of chronic hepatitis B is characterized by the presence of HBsAg and anti-HBe, normal levels of aminotransferases, low or undetectable HBV DNA in the serum and mild or inactive liver disease [43] and can last for decades [47]. The average clearance rate of HBsAg is 0.5 % per person per year [48]. However, a reactivation of replication and subsequent inflammatory disease can occur at any time following immunosuppression, e.g., cancer, infection with human immunodeficiency virus (HIV), autoimmune diseases, transplantation and treatment with corticosteroids or rituximab [49].

Reactivation can also be observed in patients with occult HBV infection (OBI) which was defined as the “presence of HBV DNA in the liver of individuals testing HBsAg negative by currently available assays” by the Taormina Consensus Conference in 2008 [50]. In these patients, stable cccDNA molecules persist in hepatocytes, but viral replication is inhibited by genetic, epigenetic and co- or post-transcriptional mechanisms as well as by multispecific T-cell responses [51]. Importantly, the cccDNA molecules are fully replication competent and can even lead to acute HBV infection in liver transplantation recipients from OBI-positive donors [52, 53].

Long-term complications of chronic HBV infection include liver fibrosis, cirrhosis and hepatocellular carcinoma. About 20 % of chronically infected patients develop cirrhosis [34], and their risk of HCC is elevated 100 times compared with healthy controls [54]. The risk of development of HCC is further increased by decompensated cirrhosis, male sex, age, alcohol, exposure to aflatoxines, HBV genotype C and viral co-infection with HCV or HIV [41, 55].

Role of immune response

HBV does not act by itself in a cytopathic manner. Thus, liver inflammation, cirrhosis and fibrosis are largely consequences of the host’s immune response. As discussed above, the virus evades the innate immune system during early infection. The pathogenesis of the infection is therefore mainly mediated by adaptive immunity.

Humoral immune response

Neutralizing antibodies can be detected only late after acute HBV infection and probably play no dominant role in early viral clearance. They are thought to form complexes with circulating viral particles and thereby prevent viral spread [40]. By this means, antibodies protect from reinfection as has been shown in chimpanzees [56]. Furthermore, a high titer of anti-HBs in humans after vaccination correlates with protection from viral contagion [6]. Indeed, after the constitution of a universal hepatitis B vaccination program in Taiwan, in 1984, the incidence of both chronic infection and HCC could be reduced [57, 58].

Cellular immune response

The impact of CD4+ T cells in determining the outcome of HBV infection has been investigated in the chimpanzee model. Although depletion of CD4+ T cells in week 6 after inoculation had no effect on duration or outcome of acute HBV infection [59], immuno-depletion of CD4+ T cells before inoculation with the virus led to persistent disease [35]. These results indicate that CD4+ T cells do not act primarily as effector cells but are essential for priming of other immune cells. The timing of CD4+ T-cell priming may be crucial for their contribution to the induction and maintenance of virus-specific B and CD8+ T-cell responses [40].

CD8+ T cells play a central role during the resolution of HBV infection. Indeed, during acute infection, virus-specific CD8+ T cells emerge even before the development of symptomatic liver disease, concomitant with a massive reduction in viral load [60]. Furthermore, the fundamental role of CD8+ T cells in viral clearance has been supported by depletion studies in chimpanzees. Animals that were deprived of CD8+ T cells could not clear the infection until the reappearance of these very same cells [59]. In patients who clear the infection spontaneously, the virus-specific CD8+ T-cell response is vigorous, polyfunctional and multispecific [61, 62].

On the contrary, chronic hepatitis B infection is characterized by a weak and functionally impaired virus-specific CD8+ T-cell response, with a reduced proliferation rate and low production of cytokines such as IL-2 or IFN-γ [61, 63]. The loss of functionality is inversely correlated with the level of HBV DNA, as a high viral load could be shown to suppress multispecific CD8+ T-cell responses [64]. Indeed, ongoing exposure to viremia combined with the tolerogenic milieu of the liver gives rise to exhausted CD8+ T cells. The state of exhaustion is defined as poor effector function, expression of inhibitory receptors such as PD-1 and an intermediate state of T-cell differentiation [65–67].

Many factors are likely to contribute to this impairment. Among them, the immunosuppressive cytokine IL-10 might play a vital role in the establishment of chronic HBV infection [68]. Of note, IL-10 is known to be upregulated during acute and chronic HBV infection in humans [39, 69]. Its importance is supported furthermore by data from LCMV clone 13 infected mice, in which blockade of the cytokine contributed to the restoration of a virus-specific CD8+ T-cell response and elimination of infection [70, 71]. Besides, deprivation of the essential amino acid l-arginine, caused by dying hepatocytes releasing arginase, impairs T-cell receptor signaling due to the down-regulation of the CD3ζ-chain on T cells [72, 73]. Further determinants of CD8+ T-cell failure are the lack of CD4+ T-cell help as well as inhibition of virus-specific CD8+ T cells by other immune cells, e.g., natural killer (NK) cells [74, 75].

During the last years, NK cells have entered the limelight as another important immune cell subset during acute and chronic HBV infection. NK cells do not only represent a vital part of innate immunity but also shape adaptive immunity by modulating T cells and dendritic cells [76]. In the liver, there is an enrichment of NK cells compared with the blood [77]. However, this cell population is kept in a functionally hyporesponsive state characterized by a high expression of the inhibitory receptor NKG2A and a loss of MHC class I-binding Ly49 receptors. This is at least partly caused by the influence of Kupffer cell-derived IL-10, as blockade of the IL-10 receptor in mice was able to increase the proportion of active NK cells [78, 79]. While NK cell suppression is thought to contribute to the tolerogenic liver milieu under physiological conditions, it might be exploited by hepatotropic pathogens to evade immune control.

NK cells exert both direct cytolytic and cytokine-mediated effector functions. During chronic HBV infection, a bias of NK cell effector function toward cytotoxicity has been reported [80]. Possible reasons for this functional shift are on one side the immunosuppressive cytokine environment in the liver with high levels of IL-10 and TGF-β [81] and on the other side an inhibitory influence of HBV on the cross talk between NK cells and plasmacytoid dendritic cells [82]. As the non-cytolytic antiviral effects are suppressed, NK cells act mainly via killing of hepatocytes, thereby contributing to hepatic damage and inflammation [83]. However, NK cells might also play a protective role in liver disease, limiting hepatic fibrosis by killing stellate cells [84]. The dual role of NK cells in chronic hepatitis B is underlined furthermore by their suppression of virus-specific CD8+ T-cell responses which may prevent both liver inflammation and viral clearance [85, 86].

Conclusion

In summary, HBV remains a major health burden from a global point of view and a principal cause of hepatocellular carcinoma. Acting as a stealth virus, it evades the innate immune system by using a replication strategy invisible to the host cell. While acute infection is mostly asymptomatic, chronic infection often leads to active liver inflammation, fibrosis and cirrhosis. Different phases of chronicity have been described, dependent on the level of HBV DNA in the blood, aminotransferases as a marker of hepatic cell death and the HBeAg status. CD4+ and CD8+ T cells as well as natural killer cells play important roles in the resolution of infection. However, the presence of the virus leads to immune failure and viral persistence. Understanding HBV biology and the impairment of immune response is essential for the development of new therapeutic approaches.