Hepatitis B Virus

Hepatitis B Virus 16 Antonio Bertoletti and Hongming Huang Key Points • • • • • • • • • • HBV epidemiology and geographical distribution HBV morphology HBV viral cycle Host immunity in HBV infection: innate immunity Host immunity in HBV infection: NK and NKT cells Host immunity in HBV infection: adaptive immunity How T and B cells control HBV Immune modulatory role of HBV antigens Natural history of HBV infection Present and future therapies in chronic HBV infection eases, morbidity and mortality rates related to infection with both hepatitis B and C viruses have increased over the last 20 years [4]. However, while new therapies for HCV have delivered remarkable results [5], with more than 90% of patients achieving viral clearance with directly acting antivirals (DAA), the therapy options with curative intent for HBV are still a distant future [6]. In this chapter, after a brief summary of HBV epidemiology, we will mainly describe the virological and immunological features of HBV that make it difficult to eradicate from the infected host. We will also discuss the natural history of infection with a final paragraph focused on the therapeutic strategies that are currently under development to achieve a better cure. Epidemiology and HBV Genotypes Introduction Hepatitis B virus (HBV), a hepatotropic, non-cytopathic DNA virus, apparently present in Homo sapiens from the dawn of its evolution [1, 2], represents a very important health problem worldwide. Despite the availability of an efficient prophylactic vaccine, it is calculated that HBV still infects approximately 300 million people and causes more than half a million death per year for hepatic diseases (HCC and liver cirrhosis) that develop as a consequence of its persistent infection [3]. In contrast to most communicable disA. Bertoletti (*) Department of Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore e-mail: [email protected] H. Huang Department of Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China The global burden of hepatitis B virus (HBV) infection is estimated to be around 260 million people [3]. The prevalence of chronic HBV in the population varies between different countries and appears endemic in most parts of Asia, Pacific Islands, Africa, Southern Europe, and Latin America, but it has been calculated that two thirds of HBV-infected people live in Asia, and in China, the burden of disease is considerable [7, 8]. Chronic HBV infection present in different geographical areas can be classified based on the presence of distinct serotypes or genotypes. Before the advent of molecular methods, HBV was differentiated based on diverse serological reactivities against the envelope protein (HBsAg) of the virus and classified into four serotypes, adw, adr, ayw, and ayr [9, 10]. Nowadays, analysis of the viral sequence differentiates ten major genotypes (A to J) with 4–8% of genetic distinctions [11, 12]. Figure 16.1 shows the geographical distribution of these genotypes: genotype A is highly prevalent in Europe, Africa, India, and America. Genotypes B and C are instead common in the Asia-Pacific region. Genotype D is prevalent in Africa, © Springer Nature Switzerland AG 2020 M. E. Gershwin et al. (eds.), Liver Immunology, https://doi.org/10.1007/978-3-030-51709-0_16 255 256 A. Bertoletti and H. Huang Fig. 16.1 1 HBV prevalence and HBV genotype localization Europe, the Mediterranean region, and India. Genotype E is restricted to West Africa. Genotype F is found in Central South America but also Alaska. Other genotypes like G have been reported in America [13] but also in Europe, while genotype H has been found in Central America [12]. New isolates of genotypes I and J were identified in Vietnam and in Japan, respectively [14]. HBV genotypes, present in different geographical area, are associated with particular infection modalities. For example, HBV genotypes B and C, prevalent in East Asia, are associated with vertical transmission from mother to child, which is the prevalent transmission way in this part of the world. In contrast, genotypes A and D are associated with horizontal transmission (close personal contact between young children, blood, or sexual interactions between adults), which is more frequent in Africa and Europe. A large quantity of studies have also linked HBV genotypes with different courses of infection, rate of chronicity, and response to treatment [15, 16]. The heterogeneity of the infected populations has made it extremely difficult to compare such associations between all the different genotypes. However, data, especially from Asia, where similar populations of patients can be infected by HBV genotypes B or C, have shown that genotype C-infected patients have more severe disease, higher level of HBV replication, and lower response to interferon therapy (reviewed in [16]). HBV Morphology and Genome Organization HBV belongs to the family of hepadnaviruses, a group of para-retroviruses found in different animal species (birds and different mammals like bats, woodchucks, and squirrel) [17], which replicates a DNA genome via reverse transcription of an RNA intermediate [18]. It is a small virus of approximately 42 nm diameter and contains a 3.2 k relaxed circular DNA genome, packaged together with a DNA polymerase (reverse transcriptase) in an icosahedral nucleocapsid of approximately 30 nm diameter that is wrapped by an envelope containing three related proteins and probably lipid [17, 19] (Fig. 16.2). The production of mature infectious virions starts when the pre-genomic HBV RNA (pgRNA) gets packaged with the viral reverse transcriptase (RT, or polymerase) [20] by approximately 240 copies of the viral capsid or core protein (HBcAg) that form the icosahedral nucleocapsid. Inside the nucleocapsid, the pgRNA is converted by the reverse transcriptase into a single-stranded DNA and then in a partial double-stranded circular DNA, called relaxed circular DNA (rcDNA). These nucleocapsid particles can either enter the nucleus of the host cell and replenish the pool of HBV-DNA (cccDNA) or they can be enveloped in the endoplasmic reticulum by the surface proteins (collectively called HBsAg) and form the mature virions that are then secreted [21]. The three related surface proteins forming the viral envelope are called small (S), medium (M), and large (L) and are present at a ratio of 4:1:1 in the complete viral particles [22]. The gene encoding for the S protein constitutes the 3′ end of the envelope gene open reading frame that is divided into Pre-S1, Pre-S2, and S regions. The protein M is encoded by the Pre-S2 and S regions, while the L protein is encoded by Pre-S1, Pre-S2, and S regions [23] (see Figs. 16.2 and 16.3). An important and unique trait of HBV infection is that infected hepatocytes not only secrete complete viral particles (made of envelope proteins and a nucleocapsid which contains a rcDNA) but also a large quantity of incomplete subviral particles (SVP) made of only surface proteins or viral particles with an empty nucleocapsid (without genome) [24]. The schematic representation of these different viral SVP secreted by infected hepatocytes and their relative quantity 257 16 Hepatitis B Virus M L S Relaxed circular DNA RNA core S RT RNase H preS2 4 S-HBsAg : 1 M-HBsAg : 1 L-HBsAg preS1 HBeAg HBcAg P protein Fig. 16.2 HBV morphology: a 42 nm diameter virion contains a 3.2 k relaxed circular DNA genome, packaged together with a DNA polymerase (reverse transcriptase, RT) in an icosahedral nucleocapsid of approximately 30 nm diameter formed by HBcAg dimers and wrapped by an envelope containing three related proteins – small (S), medium (M), and large surface proteins are presented in Fig. 16.3. The most abundant population of SVP is the spherical or filamentous lipoprotein particles of 20 nm diameter, which are present in the serum of infected patients at high quantity (~1014 × ml), and about 100.000fold in excess of complete viral particles. Natural spherical SVP contain mainly S proteins and some M proteins but only small amounts of L, while filamentous SVP carry more L proteins. Historically, they were called Australian antigen due to their initial identification in the blood of Aboriginal Australians [25], and their presence in the serum is quantified as HbsAg [26]. HBV-infected cells also secrete genome-empty viral particles that are quite abundant since they are present in quantities (~1011 × ml) that are about 100 times more than mature complete virions (~109 × ml) (see Fig. 16.3). Less abundant are particles similar to complete virions with nucleocapsid and envelope proteins containing viral RNA instead of DNA (100–1000 lower than complete HBV ~106 × ml) [24] (see Fig. 16.3). The evolutive advantages provided by the production of incomplete viral particles and their effect on immunity will be discussed later [27]. HBV-infected cells secrete also a soluble, dimeric protein called hepatitis B e antigen (HBeAg) (see Fig. 16.3). HBeAg is derived from the so-called PreCore (PreC) protein [23, 28, 29]. Most of its amino acid residues are shared with HBcAg but have a C-terminal deletion of 34 amino acid residues and an N-terminal extension of 10 amino acids (unique to the PreC region). HBeAg is secreted by the infected cells, and it has been shown in animal studies to exert immunoregulatory effects [30, 31]. Historically, serum HBeAg has been used to check viral production, since its level is associated with high levels of HBV-DNA [32]. The production of complete and incomplete virions, the synthesis of the different HBV proteins, the secretion of all HBV particles and proteins, and viral replication are ultimately directed by a viral DNA episome, the covalently closed circular DNA (cccDNA) present in the nucleous of infected hepatocytes. CccDNA is synthesized in the nuclei of infected hepatocytes from rcDNA present in the nucleocapsid, derived from complete virions infecting the hepatocyte or from the nucleocapsid particles produced in the cytoplasm (schematic representation in Fig. 16.4), the so-called intracellular cccDNA amplification pathway [33, 34]. The cccDNA of HBV contains four genes with extensively overlapping open reading frames that can produce different RNAs coding for seven distinct proteins: envelope proteins (with the three forms of large, middle, and small), the nucleocapsid protein (HBcAg and its truncated form HBeAg), the polymerase protein, and the transcriptional transactivator protein X, which controls HBV transcription from cccDNA. A schematic representation of the HBV genome and its open reading frames is presented in Figs. 16.3 and 16.4. HBV Viral Cycle The HBV replication cycle initiates when the complete and mature HBV virion docks to human hepatocytes through binding heparin sulfate proteoglycans located on their membrane (see Fig. 16.4). HBV then enters the cells through high-affinity binding with sodium taurocholate cotransporting polypeptide (NTCP) receptor [35, 36]. The specific binding is mediated by the PRE-S1 protein (present in the large 258 A. Bertoletti and H. Huang RNA virion 10^6/mL Complete virion 10^9/mL Empty virion 10^11/mL Subviral particles (SVP) 10^14/mL L-HBsAg s mRNA 2.1kb M-HBsAg S1 preS2 DR2 R D A HBeAg C preC pgRNA 3.2kb ε S HBcAg Poly s- P s- preS 2 Xm 0.7 RNA kb C pre X 1 +str S-HBsAg preS 1 c A mRN pres 2.4kb -stra RT P S nd and pre RT P P protein Fig. 16.3 Schematic representation of the different vial particles produced during HBV infection and HBV genome organization envelope protein) that is budding out from the envelope of mature virions [37]. Interestingly, the location of PRE-S1 protein is changing during the maturation of HBV. Newly secreted HBV virions have their PRE-S1 protein located in the interior side of the viral envelope, and this might diminish unspecific docking of viral particles immediately after excretion. After entering the hepatocytes, the viral particles are uncoated and the whole nucleocapsid reaches the nucleus, where they release their partially double-stranded rcDNA [38]. The host DNA repair machinery then converts the rcDNA to cccDNA, which gets packaged by histone and nonhistone proteins and forms a viral minichromosome [34]. The cccDNA is the HBV template for the transcription of HBV mRNA producing the different HBV proteins and of the pgRNA. This process is regulated mainly by HBx protein that, through the block of the function of host chromosome protein 5 (SMC5) and SMC6 complexes, governs cccDNA expression. The SMC5/6 complex is a host restriction factor for extracellular DNA that silences cccDNA. HBV X, by destroying this complex, relieves this inhibition and allows gene expression of HBV [39]. As we already described in the paragraph related to HBV morphology, HBV pgRNA is encapsulated by core proteins in the cytoplasm of the cells together with the polymerase protein (see Fig. 16.4). The polymerase protein reverse transcribes pgRNA to rcDNA [18]. This is the process that is inhibited by the nucleoside or nucleotide inhibitors (nucleoside analogs or NA), the drugs currently used to treat HBV [40]. Note, therefore, that NA drugs inhibit the production of HBV-DNA (and therefore the production of new mature virions) but do not alter the quantity of cccDNA already present in the nucleous of infected cells [41] and the quantity of HBV mRNA and HBV protein expression. The HBV viral cycle is terminated by the process of production of enveloped HBV: the mature nucleocapsid (containing rcDNA) gets enveloped by HBV surface proteins (large, medium, and small) and secreted outside [38]. 259 16 Hepatitis B Virus NTC P HBcAg HBeAg PF-rc DNA Subviral particles (SVP)10^14/mL ccc DNA HSP G rc DNA Empty virion 10^11/mL preC/C RN A mRNA pgRNA preS/S mRNA HBx mRNA L,M,S Complete virion 10^9/mL RNA virion 10^6/mL Fig. 16.4 Schematic representation of HBV viral cycle One other important part of the HBV viral cycle is its ability to integrate into the host genome. It is common in all the hepadnaviruses [42], and HBV-DNA integration is detected also in hepatocytes that have cleared HBV cccDNA [43]. While initially HBV-DNA integration was described preferentially in patients with HBV-related HCC [44], it is now clear that HBV-DNA integration is present even in the early phases of HBV infection [45] and occurs very early during HBV replication cycle [46], within 5 days from infection [46], generally in 1 out of 105 infected cells [42]. The mechanism of HBV-DNA integration into the host genome derives from the production of mature particles with double-stranded linear HBV-DNA (dsl DNA) [47]. The production of dslDNA is occurring in about 10% of pgRNA containing capsids. Like the rcDNA containing capsids, dslDNA can recirculate back into the nucleous, and it might more easily integrate into the host genome, particularly in genome zones with double-stranded genomic breaks [48]. Despite woodchuck hepatitis virus (WHV) (a virus of hepadnavirus family infecting woodchucks) integrations were frequently detected in the Myc oncogenes [49], integrated HBV-DNA sequences in hepatocytes are heterogeneous, without any real hot spots of integration sites. They can present high complex rearrangements and deletions, but the whole dslDNA can also be integrated [47]. However, due to its reduced length, the integrated dslDNA cannot produce the entire pgRNA and thus HBV-DNA integration is not a source of whole mature virions [47]. Instead, it can produce whole or fractions of the different HBV proteins. For example, since the ORF of HBsAg is often intact in the integrated HBV-DNA, HBsAg can be produced, and a large part of HBsAg present in sera of adult chronic HBV-infected patients (particularly with anti-HBe+ infection or hepatitis) is produced from HBV-DNA integration [50]. HBx protein can also be produced from HBV integration, but it is classically fused to other host proteins since the stop codon of HBx is lacking in the integrated HBV-DNA form [51]. Other HBV proteins are not produced in the complete form, but short sequences or chimeric proteins can be synthesized [52]. The evolutive advantage for HBV to integrate into host genomes is not clear. However, its conservation in all hepadnaviruses infecting different animals (duck and woodchucks) suggests that it has a natural role in HBV persistence. The pathological consequences of HBV integration are also not clear. Historically, HBV-DNA integration has been studied mainly in the context of its role in hepatocarcinogenesis [44, 53, 54]. The first demonstration of HBV-DNA integration into the host genome was shown in HCC lines and HCC tissue [53–55] and leads to the suggestion that HBV-DNA integration was the cause of tumorigenesis. Many different 260 A. Bertoletti and H. Huang Fig. 16.5 Kinetics of HBV-DNA and host immunity in acute HBV infection NK and T cell expansion WEAK INNATE T-B CELL memory 100 10 Anti-HBc 1000 HBV-DNA 1 ALT 2 4 6 8 10 12 ALT U/L HBV-DNA 106/ml 1500 Anti-HBs 500 14 Weeks mechanisms were reported like insertional mutagenesis, induction of chromosomal instability, or production of aberrant proteins, but the mechanism of HBV carcinogenesis remains still poorly elucidated [56]. Host Immunity in HBV Infection: Innate Immunity Host immunity against pathogens evolved in separate branches defined as innate and adaptive immunity. They perform different tasks to efficiently limit infections. The innate immunity branch has the scope to limit pathogen spread. It is activated by recognition of nucleic acids or proteins of the pathogen or by tissue damage. Activation is mediated by different families of cellular receptors (i.e., TLRs, RIG-1, DHX9, AIM2) located within the infected cells and classically leads to rapid intracellular production of cytokines like IFN-alpha or IFN-beta and to an activation of NK cells [57]. Innate immune activation triggers then adaptive immunity that acts through the maturation and expansion of distinct pathogen-specific B and T cells. One of the peculiar characteristics of HBV in comparison with other viral infections is its poor activation of the innate immune system. The quantity of pro-inflammatory cytokines in the serum of patients with acute HBV infection is of lower magnitude and with delayed kinetics compared, for example, with HCV- and HIV-infected patients [58, 59]. The lower quantity of cytokines present in patients’ sera is in line with the lack of flu-like symptoms experienced by most HBV infection patients. These observations are supported by the demonstration of limited induction of IFN-related genes in experimentally infected chimpanzees during the initial phases of HBV infection [60]. One reason for the inability of HBV to trigger innate immunity might be its delayed viral replication kinetics. Indeed, while after infection most viruses enter a logarithmic phase of propagation (e.g., HCV, HCMV, HIV, influenza), HBV, after acute infection, displays a delayed amplification of virions and spread (Fig. 16.5). HBV-DNA levels reach a maximum only 6–8 weeks after infection [59–62]. The lack of induction of type I IFN genes is, however, not only observed during acute infection but also during chronic viral reactivation [63] and in the livers of woodchucks chronically infected with woodchuck hepatitis virus (WHV) [64]. Whether HBV escapes or actively inhibits innate immune recognition has been highly debated [65–67]. A list of recent reviews discussing in detail the relation between HBV and innate immune is provided [68–70]. However, briefly, even though a minimal level of HBV recognition by RIG-1 [71] and production of type I IFNs were detected in in vitro infection systems [72] and in HBV chimeric mice [73], respectively, more recent data performed in HBV-infected primary hepatocytes [74] and in liver biopsies of HBV-infected patients [75] suggests that HBV mainly escapes intracellular innate immune recognition and does not exert any robust suppressive effect. Such lack of detection could result from the replication strategy of HBV, which uses a limited amount of transcription template (cccDNA/2–4 copies × cell) sequestered within the nucleus of infected cells. This produces polyadenylated viral mRNA that resembles the normal cellular transcripts and transcribes pgRNA to rcDNA within viral capsids shielding this process from the RNA-detection machinery of cells [65]. HBV is however susceptible to innate immune triggering. Intracellular activation of retinoic acid-inducible gene-I (RIG-I) [71] or APOBEC [76] pathways or cytokines such as IFNα [77], IFNγ, TNFα, and IL-1β [78, 79] produced by non-parenchymal cells of the liver suppresses HBV replication and reduces the pool of cccDNA [77]. The efficacy of innate cytokines to inhibit HBV is also supported by data of coinfection with HCV and HDV that activate innate immunity [80, 81]. The antiviral efficiency of type I IFNs against HBV is, however, weak. Only high doses of IFN-alpha are able to directly inhibit/clear HBV in vitro [76], and the antiviral effi- 261 16 Hepatitis B Virus ciency of IFN-alpha therapy is higher in HCV than HBVinfected patients [82]. For example, whereas in HCV-infected patients, IFN-a-based therapy results in a sharp decrease in viremia within the first 48 hours [82], a HBV-DNA reduction is observed only after 3–4 weeks of therapy in CHB patients. In studies conducted in humanized chimeric mice repopulated with human hepatocytes, HBV limited the direct antiviral effect of IFN-alpha by inhibiting nuclear translocation of STAT-1 and thus interfering with transcription of interferonstimulated genes (ISG) [73]. Efficient HBV suppression and even complete clearance can be also mediated by more classical T cell cytokines like IFN-gamma and TNF-alpha [78, 83, 84] or through triggering of lymphotoxin beta/alpha receptor that induces activation of nuclear deaminases capable of destabilizing cccDNA [76]. Yet, it is however important to note that the ability of cytokines to suppress HBV was demonstrated in experimental systems devoid of chronic inflammatory events. The intrahepatic environment of chronic HBV patients is often altered by the presence of IL-10 [85], TGF-beta [86], and arginase [87], and high levels of suppression of cytokine signaling 3 (SOCS3), a negative regulator of cytokine signaling, can be detected [64, 88]. Thus, the efficacy of cytokines might be different in a liver microenvironment characterized by chronic inflammatory events. Host Immunity in HBV Infection: NK and NKT Cells NK and NKT cells are the cellular arm of innate immunity and can recognize and kill viral infected cells. NK cells are activated by target cells that express low level of MHC class I along with upregulation of host- or pathogen-encoded ligands signaling cell stress. NK cells respond also to cytokines induced by viral infections, such as type 1 interferons, IL-12, and IL-18 [89]. Other cells at the crossroad between innate and adaptive immunity that are, together with NKbright cells, extremely abundant in the liver are iNKT cells and MAIT cells. iNKT cells are a lymphocyte population that gets activated after recognition of lipid antigen associated with MHC class I like molecule CD-1. Their impact in HBV control has been shown in elegant models, but their role during natural infection is controversial, since CD-1-restricted NKT cells are abundant in mouse but extremely rare in human livers [90]. In human livers, different types of NKT cells, such as MAIT cells, are abundant [91], but these cells are not activated by lipids. MAIT cells are known to play a major role in antibacterial immunity, and their role in HBV infection is not clear. So far, a specific activation of MAIT cells has been only shown in HDV-HBV coinfection [92]. The impact of NK cells during HBV infection remains controversial, with possible protective or pathogenic roles [93]. The efficacy of IFN-α therapies has been linked with activation of NK cells [94], which can be also detected in patients who controlled acute HBV infection [61, 95]. A possible role of NK cells in the viral control was shown in woodchucks acutely infected with extremely high WHV doses (1011) [96], and activation of NK cells is detectable in acute patients [61, 95]. However, at present it is not clear whether NK cells recognize HBV-infected hepatocytes. HBV has never been demonstrated to induce cellular stress or downregulation of HLA-class molecules. This is why the regulatory capacity of NK cells on HBV-specific T cells has also been analyzed, which showed in chronically infected patients that NK cells can actually promote HBV persistence, since they contribute to HBV-specific T cell deletion by a mechanism of direct T cell killing [97]. Host Immunity in HBV Infection: Adaptive Immunity The adaptive immunity is recognized as a crucial player in the clearance of HBV infection. Numerous reviews have summarized their different aspects [68, 98, 99]. Briefly, while data have shown that CD8+ cytotoxic T cells can clear HBV-infected hepatocytes through cytolytic and noncytolytic mechanisms, CD4+ helper T cells are necessary for the efficient maturation of HBs-specific B cells (producing protective antibodies) and for the induction and maintenance of efficient CD8+ cytotoxic T cells. This coordinated collaboration between the different components of adaptive immunity (T and B cells) is occurring in adults after acute HBV infection and leads to HBV control, but the kinetic of such induction is peculiar in HBV [98] in comparison with other viral diseases. Following the slow HBV expansion after infection (the so-called incubation period), HBV-specific CD4+ and CD8+ T cells are detectable in the blood at around 4–7 weeks after infection at the time of exponential increase in HBV replication [61, 95] (see Fig. 16.5). In other viral infections (i.e., HIV, influenza), activation of adaptive immunity occurs only 1–2 weeks after infection. The detection of HBV-specific T cells in the blood is temporally associated with a decline of more than 90% of the HBV-DNA that precedes the peak detection of liver damage [84, 100, 101] (see Fig. 16.5), a kinetic that suggests that a large quantity of virus elimination is caused by a noncytopathic process mediated by IFN-γ and TNF-α, secreted by CD8 T cells. We also know that, during successive HBV control, intrahepatic recruitment of HBV-specific cytotoxic T cells is facilitated by the secretion of chemokines (i.e., CXCL-10) and by platelet activation [102–105]. Platelets 262 help the docking of HBV-specific T cells to liver endothelial cells and facilitate the recognition/killing of infected hepatocytes. Recruitment of non-antigen-specific cells (monocytes, non-HBV-specific T cells) that amplify hepatocellular damage [106, 107] occurs after. Quantitatively, the frequency of HBV-specific T cells in patients with acute HBV infection is low in comparison to other viral infection, with frequencies in blood that rarely exceed 1–2% of total T cells [108]. However, such numbers might not perfectly represent the size of the HBV-specific T cell repertoire, since HBV-specific T cells are enriched in the liver [109, 110] and an analysis of intrahepatic HBV-specific T cell frequency during acute hepatitis has so far not been performed in humans. Antibodies are also produced during acute HBV infection. Their kinetics of production reveal a difference between antibodies specific for envelope (anti-HBs Ab) and nucleocapsid (anti-HBc Ab) (see Fig. 16.5). Anti-HBc antibodies are detected at very early stage of the infection, while antiHBs antibodies only appear at later time points, after HBVDNA declines [27, 111]. Such differential kinetics are not only explained by the fact that large quantity of HBsAg is produced and secreted into the circulation during acute HBV infection and thus can block anti-HBs detection [27, 112] but also by the maturation defects present in HBsspecific and not HBc-specific B cells in the presence of HbsAg [113, 114]. Such dysfunctionality of HBs-specific B cells was not only demonstrated in chronically infected patients but also during the early phase of acute HBV infection [115]. The profile of adaptive immunity in patients with chronic HBV infection is instead radically different. If we exclude anti-HBc-specific B cells that are functional during chronic HBV infection [114], T cells specific for different HBV proteins and HBs-specific B cells are present in lower frequency, upregulate exhaustion markers (mainly PD-1, but also TIM3, Lag-3, and CD160) [115, 116], and have deep metabolic and energetic impairments [117–119], making them unable to exert antiviral functions and more susceptible to killing by NK cells [97]. Similarly, HBs-specific B cells, both in periphery and within the liver, display defects in the maturation toward antibody-producing cells [113, 120]. The causes of the functional defects of adaptive immunity in chronic HBV infection are heterogeneous. Dose of HBV infection and age and genetic background of the host play roles in the ability of the immune system to control the virus and mount a coordinated activation of T and B cell responses [121–123]. It is however likely the protracted presentation of large quantity of viral antigens can drive T and B cells toward progressive functional exhaustion. In this prospect, it seems logical that preferentially HBsAg, whose quantity exceeds other HBV antigens, appears to affect both B and T cells. Functional impairment is exclusively detected in HBs- and A. Bertoletti and H. Huang not in HBc-specific B cells [114], and envelope-specific T cells (both CD8 and CD4) are preferentially deleted in adult patients with chronic HBV infection [116, 124]. The Antiviral Mechanisms of T and B Cells During HBV infection, B cells can produce antibodies specific for all the different HBV proteins, but only antibodies specific for envelope proteins (S and Pre-S1) have protective values [112]. Antibodies against HBcAg (anti-HBc Ab) have been hypothesized to be responsible for some form of fulminant hepatitis [125], and they are an immune marker of ongoing or progress of HBV infection. Antibodies against HBeAg (anti-HBe Ab) are used to differentiate clinical phases of HBV-induced disease, and their pathogenic role is unknown. It is important to remember that since HBV spreads to noninfected hepatocytes through an HBVreceptor-dependent mechanism [126], protective antibodies (specific for HBs and PreS1) have importance not only in prevention of the infection but can also modulate HBV spread during chronic infection. 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 [35, 36], along with the establishment of easily infectible in vitro cell lines. This allowed precise mapping of HBV regions essential for infectivity, which are the pre-S1 domain and the antigenic loop region (known as the “a-determinant”) of the HBsAg (reviewed in [127]). The pre-S1 domain (in particular amino acids 2–48) interacts directly with NTCP [35, 36], whereas the “a-determinant” mediates the initial docking of HBV to heparin sulfate proteoglycans on hepatocytes [128]. HBV-specific T lymphocytes act instead as the principal effector mechanism of viral clearance and liver inflammation. HBV-specific CD8 T cells recognize directly HBVinfected hepatocytes through recognition of HBV peptides presented at the surface of infected cells [100]. HBV-specific T cells can lyse HBV-infected hepatocytes [100, 129] and secrete cytokines (IFN-gamma, TNF-alpha) that trigger a process of non-cytolytic HBV clearance [83] and recruitment of inflammatory immune cells [106, 107]. HBVspecific CD4 T lymphocytes regulate the intensity of these processes. While it is clear that HBs-specific but not HBc-specific B cell functionality has a protective function in HBV infection, the hierarchy of protective ability of T cells specific for different HBV antigens is still not clear. Multi-specificity has been associated with resolution [130], and CD4+ and CD8+ T cells recognizing different viral determinants are present in different quantities and establish a hierarchy of dominant and subdominant epitopes, but their comparative protective effect is unknown [98]. Instead, it seems established that 16 Hepatitis B Virus core- and polymerase-specific T cells persist more easily in chronic HBV patients, while HBs-specific T cells appear deleted in adult chronic HBV patients, likely caused by the continuous impact of HBsAg presentation [116, 124]. Immunomodulatory Roles of HBV Antigens The persistent production of the soluble forms of HBV surface antigen (HBsAg) and e antigen derived from the core protein (HBeAg) in excessive amounts over whole virions is likely to play a role in the inhibition of host immunity. Data in animal models have elegantly defined the mechanisms that enable HBeAg to suppress HBV-specific T cells in newborns [30, 31]. Moreover, experimental evidences demonstrated the ability of HBsAg to block the protective efficacy of anti-HBs antibodies [131] and to alter HBsspecific B cell function [113, 120]. Other effects are much more controversial. Persistent exposure to circulating HBsAg was suggested to impair the frequency and function of myeloid cells [132, 133] and plasmacytoid dendritic cells [134, 135]. It was also suggested that soluble viral antigens can inhibit antigen presentation, by altering the ability to produce cytokines, and thus prevent the induction of HBV-specific T cells [136]. However, the inhibitory effect of HBsAg on dendritic cell function has not been confirmed [137], and such defects are not compatible with the fact that chronic HBV patients were never shown to be more susceptible to opportunistic infections. In contrast, for example, reports have shown that in patients with malaria, HBsAg positivity is associated with lower parasitemia [138]. There are also data derived from newborns of HBV-infected mothers that show a beneficial effect of maturation of host immunity by HBV infection [139]. It is therefore likely that the effect of HBsAg on host immunity is specific for HBsspecific B and T cells and not for global immunity. The evidences of the preferential persistence of polymerase and core T cells in adults with HBV infection support such conclusions [116, 124]. Natural History of HBV Infection The natural course of HBV infection can be modified by variables such as viral load, virus genotype, route of infection, and the age, sex, and genetics of the infected hosts [15, 123]. The majority of symptomatic acute HBV infections that occur in adults (after sexual or iatrogenic infection) do not develop chronic HBV infection but are able to control the virus and become HBsAg negative with the development of anti-HBs antibodies [6]. Such state is not associated with the presence of liver diseases despite HBV, like other human DNA viruses (HCMV, EBV), is not completely eliminated. 263 Small quantities of infected hepatocytes persist in all subjects that were productively infected by HBV [140]. HBVDNA can be detected in biopsies of individuals after resolution of HBV infection or in patients with chronic hepatitis but with HBsAg negativity with or without presence of anti-HBc antibodies. Such profile has also been defined as “occult HBV infection.” The pathological consequences of the presence of such low quantity of infected hepatocytes are controversial [141, 142]. Patients with “occult HBV infection” are at risk of HBV reactivation after immunosuppressive treatment [143]. Yet the risk of HCC development in patients with “occult HBV infection,” in the absence of other pathological processes of the liver, is low in comparison with chronically infected patients with productive HBV infection and detectable HBsAg and HBV-DNA in the blood [144]. This is also the reason why the current goal of therapies in chronic HBV patients is to obtain “functional HBV cure,” a clinical, virological, and immunological status which coincides with “occult HBV” [145]. A few reviews discussing these definitions and the different pathological consequences of HBV infection are listed [6, 41, 144–147]. HBV infection occurring at birth and during childhood is instead more frequently developing into chronic HBV infection [121]. However, neonates/children are not completely unable to mount an efficient antiviral immunity against HBV, and evidences of vertically infected patients able to control HBV spontaneously or after early treatment are increasing [148]. Nevertheless, a large quantity of patients with chronic HBV infection acquired the viral infection at birth, without any associated symptoms of acute hepatitis. The establishment of chronic HBV infection is then characterized by different levels of HBV replication and occurrence of inflammatory events in the liver that have been used to schematically divide the infection into four clinical phases [147]. Figure 16.6 shows the different phases using the definition accordingly to EASL 2017 [149] and AASLD nomenclatures [147]. The EASL 2017 nomenclature divides clinical phases of natural chronic HBV infection in relation to the presence/absence of HBeAg and serological signs of liver inflammation (hepatitis/infection). AASLD uses a definition based on the concept that the initial phase of infection, characterized by a high level of viral replication and absence of serological signs of liver inflammation, is a more “immunotolerant phase” than later phases when liver inflammatory events are evident (thus defined “immune active” and immune reactivation). Patients with low level of HBV replication and normality of ALT are instead defined as “inactive chronic HBV.” These divisions have been the cornerstone of clinical management of chronic HBV, since also pathological processes (i.e., cirrhosis or HCC) are more likely to develop while patients are in the inflammatory phases [147]. Nevertheless, these divisions do not always provide a correct representation of the clinical or immunological features. For 264 Fig. 16.6 Natural history of hepatitis B. Representation of the four clinical/virological phases (AASLD 2018 or EASL 2017 nomenclatures) of chronic hepatitis B, followed by functional cure A. Bertoletti and H. Huang AASLD EASL Immune tolerant Immune active HBeAg+ infection HBeAg+ hepatitis Inactive chronic HBV Immune reactivation Functional cure HBeAg- infection HBeAg- hepatitis HBV DNA HBsAg ALT HBsAb AntiHBeAg HBeAg Liver histological Mild Moderate/severe example, the demonstration that patients defined as “immunotolerant” have HBV-specific T and B cells at similar frequency and with comparable functionality as patients defined with chronic active hepatitis has challenged the use of his term [150]. Similarly, patients without serological evidences of hepatitis (thus with normal ALT level) have shown the presence of inflammatory events in the liver [151]. Nevertheless, it seems clear that chronic HBV infection is characterized by phases of infection in which levels of HBV replication, production of HBV antigens (HBsAg and HBeAg), and inflammatory responses in the liver vary, and they are differentially associated with pathological consequences. It is, for example, controversial whether treatment of chronic HBV infection should only be started in subjects with elevated ALT levels (thus with clear signs of hepatitis) or whether patients in the initial phase of disease (HBeAg+ infection or immunotolerant phase) should be targeted in order to prevent pathological consequences [152, 153]. Therapy of Chronic HBV Infection The best therapy of HBV infection is its prevention that can be efficiently achieved with a vaccine based on HBsAg protein that elicit high titers of anti-HBs antibodies in the majority of immunized individuals [154]. Vaccination and immunoglobulin enriched with anti-HBs antibodies diminishes also the rate of the vertical infection of newborns from HBV-infected mothers [155]. These preventive strategies have contributed to reduce drastically HBV infection and its pathological complications in countries with high endemicity [156, 157]. activity None/mild Moderate/severe Variable When chronic HBV infection is already established, the current standard therapies are nucleotide/nucleoside analogs, thus drugs that targeted the reverse transcriptase, and IFN-alpha, a cytokine that has both antiviral and immunomodulatory effects. Nucleotide/nucleoside analog (NA) therapies are very effective in reducing both viral replication and liver inflammation. They suppress the development of hepatic failures and hepatocellular carcinoma in chronic HBV patients [158], and they diminish the risk of vertical infection by pregnant women [159]. Inhibition of viral replication is robust, with HBV-DNA becoming undetectable in sera after 2–4 weeks of therapy, and the emergence of resistant strains of HBV is rare particularly with the last generation of compounds [160]. However, since NAs do not target the HBV cccDNA, treatment needs to continue over time to avoid the risk of HBV reactivation and associated hepatic flares. In addition, NAs do not target the production of HBV proteins, and thus HBsAg levels are not altered even after long-term treatments [161]. As such, the frequency of patients achieving functional HBV cure, a condition characterized by sustained HBsAg negativity and anti-HBs positivity, is negligible. IFN-alpha treatment can instead achieve functional HBV cure through mechanisms that involve a direct inhibition of viral replication and a boost of antiviral immunity, but this occurs only in about 5% to 8% of the treated patients and is linked with side effects that can be severe. To increase therapy efficacy, new drugs targeting different steps of viral life cycle or the antiviral immune response (Fig. 16.7) have been developed, and recent reviews have described the current efforts to provide better HBV treatment [6, 40, 69, 99, 162]. 16 Hepatitis B Virus 265 Fig. 16.7 Schematic representation of the therapeutic strategies targeting directing HBV natural cycle in hepatocytes and innate or adaptive immunity The holy grail of HBV treatment is to eliminate HBV cccDNA, either directly or indirectly by affecting intracellular cccDNA recycling or blocking new rounds of infection or silencing its transcriptional activity. Since the release of complete HBV virions results in infection of new hepatocytes with an increase of the cccDNA pool, blocking infection can progressively reduce the pool of infected hepatocytes [163]. Strategies to prevent HBV infection include the use of a peptide (Myrcludex B, also known as bulevirtide) that derived from the sequence of the Pre-S1 domain that binds to the NTCP receptor or monoclonal antibodies [126, 164]. A clinical trial of Myrcludex associated with IFN-alpha therapy showed encouraging results in HBVHDV coinfection [165]. Other compounds such as ezetimibe 266 and cyclosporine derivatives have also been evaluated in experimental models for their ability to inhibit HBV viral entry [166, 167]. Direct inhibition of cccDNA formation using small molecules is attractive, but since cccDNA formation requires the use of many host nuclear enzymes, nuclear histones, and other components of host chromatin [168], such task might have severe side effects and has been so far unsuccessful. cccDNA can possibly be targeted by zinc-finger nucleases or transcription activator-like effector nucleases, which were used with success in vitro [169]. CRISPR and Cas protein endonucleases have been also used to inactivate cccDNA [170]. Nevertheless, before these gene editing approaches can reach the clinic, problems of hepatocytespecific delivery, off-target effects, cleavage of integrated HBV-DNA, and the unpredictable consequences of chromosomal DNA recombination need to be addressed. Other new therapeutic strategies are instead trying to target viral gene expression with the aim to reduce not only mature virion production but also the expression of viral antigens (see Fig. 16.7). Blocking HBx activity that regulates HBV transcription through degradation of the transcriptome repressor SMC5/6 can lead to an inhibition of viral transcription. Nitazoxanide, an anti-protozoa drug, demonstrated, in cultured HBV-infected hepatocytes, to inhibit the HBx-DDB1 interaction and to restore SMC5 levels, therefore obtaining a suppression of viral transcription and protein productions [171]. Numerous nucleic acid-based strategies (RNA interference, antisense oligonucleotides, and ribozymes) to control posttranscriptionally the production of HBV proteins and mature virions were developed [172]. The suppression of HBsAg production derived from both cccDNA and HBVDNA integration might facilitate the recovery of HBVspecific immunity. HBsAg presence is indeed altering HBs-specific B cell function [113, 120], while its general effect on the functionality of global host immunity is controversial. Data in animal models and trials in humans have shown the efficacy of such strategies to reduce HBV protein and HBV replication levels, but there has been no demonstration of a spontaneous recovery of HBV host immunity [50]. Compounds that block HBsAg release have been also developed. Nucleic acid polymers have shown clinical efficacy in combination with IFN-alpha, but the mechanism of action, the specificity of the “release inhibition” to HBV viral protein, and its overall toxicity need to be better evaluated [173]. Finally, a numerous number of small molecules have been developed to interfere with the formation of capsid [40, 174]. Many different therapeutic interventions are instead trying to achieve functional cure through direct stimulation or restoration of antiviral host immunity [69, 99, 175]. A. Bertoletti and H. Huang Therapies targeting the innate immunity exploit the antiviral effect of cytokines but can also restore adaptive immunity. IFN-alpha therapy, for example, acts by directly inhibiting viral replication in HBV-infected hepatocytes and stimulating NK cell activity [94], but a recovery of HBVspecific B and T cell immunity is detected only in patients who reach “functional cure” [176]. Therapies with antiviral cytokines do not only inhibit viral replication but can also clear cccDNA [76]. The cytokines can be delivered in their native form or pegylated to increase their half-life, or they can be bound to antibodies targeting HBV-infected hepatocytes for more targeted delivery [177]. Activation and production of antiviral cytokines able to suppress HBV replication can be also obtained with molecules, which are targeting pattern recognition receptors present in different cell types and that have been modified to have a preferential intrahepatic delivery. TLR (TLR-7, TLR-8) and RIG-I agonists are the major representatives of such class of immune therapy. A Rig-I agonist (Inarigivir) that has been shown to directly inhibit HBV replication and to activate type IFN-I within the hepatocytes has demonstrated efficacy in animal models and in patients [162]. Similarly, a TLR-7 agonist (GS-9620), which preferentially induces IFN-alpha production in liver resident plasmacytoid dendritic cells, induced a very robust but transient antiviral effect in woodchucks [178] and chimpanzees [179]. A recent phase I/II clinical trial in chronic HBV patients has however shown little antiviral efficacy [180], but this is likely due to the fact that the dose used in human was low (~40 times less than in animals) to avoid the triggering of hepatic flares that were detected in some treated animals. The potential induction of inflammatory events in the liver caused by immune-based therapies is however a general problem of all these strategies, which requires to be better rationalized. It is difficult to think that, for example, compounds like TLR-8 agonists will not induce any inflammatory events in the liver. TLR-8 agonists have been designed to activate, through production of IL-18 and IL-12 by intrahepatic myeloid cells, NK and MAIT cells [181], and they can possibly recover exhausted HBV-specific CD8 T cells [182] (see Fig. 16.7). Interesting new TLR7/8 agonists tested in woodchucks have been also suggested to restore HBVspecific B cell responses [183]. The different therapeutic strategies that are designed to restore adaptive immunity are also likely to trigger inflammatory events in the liver in relation to their ability to restore HBV-specific T cell immunity. Strategies like vaccine therapies [184, 185] or the use of antibodies blocking checkpoint inhibitors (anti-PD-1 antibodies) on T and B cells have shown some efficacy in animal models [186] and in few patients [187, 188]. However, the success of anti-PD-1 therapy in chronic HBV patients was linked with the triggering of a hepatic flare that coincides with the recovery of HBV-T 16 Hepatitis B Virus cell responses [188]. Other new strategies are also in development and utilize the possibility to restore HBV immunity through engineering HBV-specific T cells using different constructs able to recognize HBsAg (chimeric antigen receptor, CAR) [189] or classical HBV epitopes (through T cell receptors) [190]. These new therapies are efficient in animal models, where they can achieve even complete HBV clearance, but control of HBV is linked with the triggering of hepatitis [190, 191]. Even though different strategies have been developed to limit such liver inflammation [192], it seems logical that all the therapeutic approaches designed to restore immunity will trigger liver inflammatory events. Even antibody therapies, which have shown success in some mouse models [193], act not only by blocking infection, but they can also facilitate NK cell recognition of HBV-infected hepatocytes through antibody-dependent cellular cytotoxicity. Therefore, one of the next challenges in the development of HBV therapies will be to understand the optimal doses and the combination of therapies that can achieve functional cure with triggering a level of hepatic inflammation that is safe and easily controlled [194, 195]. In addition, the possibility to treat patients at initial phases of infection will be taken into consideration, when immune recovery can be potentially better achieved [194, 196]. It is an exciting time for HBV research and therapies. A better understanding of the HBV life cycle and of the host immunity linked with advancements in biological technologies gives us the opportunity to explore different variables that could, in a not so distant future, achieve a functional cure of HBV in the large worldwide population of infected people. References 1. Mühlemann B, Jones TC, Damgaard P de B, Allentoft ME, Shevnina I, Logvin A, et al. Ancient hepatitis B viruses from the Bronze Age to the Medieval period. Nature. 2018;557(7705):418–23. 2. Krause-Kyora B, Susat J, Key FM, Kühnert D, Bosse E, Immel A, et al. Neolithic and medieval virus genomes reveal complex evolution of hepatitis B. elife. 2018;7:500. 3. Wiktor SZ, Hutin YJ-F. The global burden of viral hepatitis: better estimates to guide hepatitis elimination efforts. Lancet 2016; 388: 1030–31. 4. Stanaway JD, Flaxman AD, Naghavi M, Fitzmaurice C, Vos T, Abubakar I, et al. The global burden of viral hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study 2013. Lancet. 2016;388(10049):1081–8. 5. Foster GR, Afdhal N, Roberts SK, Bräu N, Gane EJ, Pianko S, et al. Sofosbuvir and velpatasvir for HCV genotype 2 and 3 infection. N Engl J Med. 2015;373(27):2608–17. 6. 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. Antivir Res. 2015;121:47–58. 267 7. Wong MCS, Huang JLW, George J, Huang J, Leung C, Eslam M, et al. The changing epidemiology of liver diseases in the Asia– Pacific region. Nat Rev Gastroenterol Hepatol. 2018;16(1):57–73. 8. Liang X, Bi S, Yang W, Wang L, Cui G, Cui F, et al. Epidemiological serosurvey of hepatitis B in China--declining HBV prevalence due to hepatitis B vaccination. Vaccine. 2009;27(47):6550–7. 9. Le Bouvier GL, McCollum RW, Hierholzer WJ, Irwin GR, Krugman S, Giles JP. Subtypes of Australia antigen and hepatitisB virus. JAMA. 1972;222(8):928–30. 10. Tiollais P, Pourcel C, Dejean A. The hepatitis B virus. Nature. 1985;317(6037):489–95. 11. Kurbanov F, Tanaka Y, Mizokami M. Geographical and genetic diversity of the human hepatitis B virus. Hepatol Res. 2010;40(1):14–30. 12. Velkov S, Ott JJ, Protzer U, Michler T. The global hepatitis B virus genotype distribution approximated from available genotyping data. Genes (Basel). Multidisciplinary Digital Publishing Institute. 2018;9(10):495. 13. Chu C-J, Keeffe EB, Han S-H, Perrillo RP, Min AD, Soldevila-Pico C, et al. Hepatitis B virus genotypes in the United States: results of a nationwide study. Gastroenterology. 2003;125(2):444–51. 14. Tatematsu K, Tanaka Y, Kurbanov F, Sugauchi F, Mano S, Maeshiro T, et al. A genetic variant of hepatitis B virus divergent from known human and ape genotypes isolated from a Japanese patient and provisionally assigned to new genotype J. J Virol. 2009;83(20):10538–47. 15. Rajoriya N, Combet C, Zoulim F, Janssen HLA. How viral genetic variants and genotypes influence disease and treatment outcome of chronic hepatitis B. Time for an individualised approach? J Hepatol. 2017;67(6):1281–97. 16. Lin C-L, Kao J-H. Hepatitis B virus genotypes and variants. Cold Spring Harb Perspect Med. 2015;5(5):a021436. 17. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev. 2000;64(1):51–68. 18. Summers J, Mason WS. Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell. 1982;29(2):403–15. 19. Bruss V. Hepatitis B virus morphogenesis. World J Gastroenterol. 2007;13(1):65–73. 20. Porterfield JZ, Dhason MS, Loeb DD, Nassal M, Stray SJ, Zlotnick A. Full-length hepatitis B virus core protein packages viral and heterologous RNA with similarly high levels of cooperativity. J Virol. 2010;84(14):7174–84. 21. Hu J, Seeger C. Hepadnavirus genome replication and persistence. Cold Spring Harb Perspect Med. 2015;5(7):a021386. 22. Heermann KH, Goldmann U, Schwartz W, Seyffarth T, Baumgarten H, Gerlich WH. Large surface proteins of hepatitis B virus containing the pre-s sequence. J Virol. 1984;52(2): 396–402. 23. McLachlan A, Milich DR, Raney AK, Riggs MG, Hughes JL, Sorge J, et al. Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences. J Virol. 1987;61(3):683–92. 24. Hu J, Liu K. Complete and incomplete hepatitis B virus particles: formation, function, and application. Viruses. 2017;9(3):56. 25. Blumberg BS. Australia antigen and the biology of hepatitis B. Science. 1977;197(4298):17–25. 26. Cornberg M, Wong VW-S, Locarnini S, Brunetto M, Janssen HLA, Chan HL-Y. The role of quantitative hepatitis B surface antigen revisited. J Hepatol. 2017;66(2):398–411. 27. Gerlich WH. Medical virology of hepatitis B: how it began and where we are now. Virol J. BioMed Central. 2013;10(1):239. 28. Ou JH, Laub O, Rutter WJ. Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc Natl Acad Sci U S A. 1986;83(6):1578–82. 268 29. Roossinck MJ, Jameel S, Loukin SH, Siddiqui A. Expression of hepatitis B viral core region in mammalian cells. Mol Cell Biol. 1986;6(5):1393–400. 30. Milich DR, Jones JE, Hughes JL, Price J, Raney AK, McLachlan A. Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero? Proc Natl Acad Sci U S A. 1990;87(17):6599–603. 31. Tian Y, Kuo C-F, Akbari O, Ou J-HJ. Maternal-derived hepatitis B virus e antigen alters macrophage function in offspring to drive viral persistence after vertical transmission. Immunity. 2016;44(5):1204–14. 32. Milich D, Liang TJ. Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection. Hepatology. 2003;38(5):1075–86. 33. Tuttleman JS, Pourcel C, Summers J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell. 1986;47(3):451–60. 34. Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut. 2015;64(12):1972– 84. https://doi.org/10.1136/gutjnl-2015-309809. 35. 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. 36. Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Fälth M, et al. Hepatitis B and D viruses exploit sodium taurocholate cotransporting polypeptide for species-specific entry into hepatocytes. Gastroenterology. 2014;146(4):1070–83. 37. Glebe D, Urban S, Knoop EV, Cag N, Krass P, Grün S, et al. Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes. Gastroenterology. 2005;129(1):234–45. 38. Blondot M-L, Bruss V, Kann M. Intracellular transport and egress of hepatitis B virus. J Hepatol. 2016;64(1 Suppl):S49–59. 39. Decorsière A, Mueller H, van Breugel PC, Abdul F, Gerossier L, Beran RK, et al. Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor. Nature. 2016;531(7594):386–0. 40. Zoulim F, Lebossé F, Levrero M. Current treatments for chronic hepatitis B virus infections. Curr Opin Virol. 2016;18:109–16. 41. Liang TJ, Block TM, McMahon BJ, Ghany MG, Urban S, Guo J-T, et al. Present and future therapies of hepatitis B: from discovery to cure. Hepatology. 2015;62(6):1893–908. 42. Yang W, Summers J. Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor. J Virol. 1999;73(12):9710–7. 43. Summers J, Mason WS. Residual integrated viral DNA after hepadnavirus clearance by nucleoside analog therapy. Proc Natl Acad Sci U S A. 2004;101(2):638–40. 44. Sung W-K, Zheng H, Li S, Chen R, Liu X, Li Y, et al. Genomewide survey of recurrent HBV integration in hepatocellular carcinoma. Nat Genet. 2012;44(7):765–9. 45. Mason WS, Gill US, Litwin S, Zhou Y, Peri S, Pop O, et al. HBV DNA integration and clonal hepatocyte expansion in chronic hepatitis B patients considered immune tolerant. Gastroenterology. 2016;151(5):986–98. 46. Tu T, Budzinska MA, Vondran FWR, Shackel NA, Urban S. Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via sodium taurocholate cotransporting polypeptide-dependent uptake of enveloped virus particles. Ou JHJ, editor. J Virol. 2018;92(11):e02007–17. 47. Tu T, Budzinska M, Shackel N, Urban S. HBV DNA integration: molecular mechanisms and clinical implications. Viruses. 2017;9(4):75–52. 48. Bill CA, Summers J. Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration. Proc Natl Acad Sci U S A. National Academy of Sciences. 2004;101(30):11135–40. 49. Wei Y, Fourel G, Ponzetto A, Silvestro M, Tiollais P, Buendia MA. Hepadnavirus integration: mechanisms of activation of the A. Bertoletti and H. Huang 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. N-myc2 retrotransposon in woodchuck liver tumors. J Virol. 1992;66(9):5265–76. Wooddell CI, Yuen M-F, Chan HL-Y, Gish RG, Locarnini SA, Chavez D, et al. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci Transl Med. 2017;9(409):eaan0241. Schlüter V, Meyer M, Hofschneider PH, Koshy R, Caselmann WH. Integrated hepatitis B virus X and 3′ truncated preS/S sequences derived from human hepatomas encode functionally active transactivators. Oncogene. 1994;9(11):3335–44. Ruan P, Dai X, Sun J, He C, Huang C, Zhou R, et al. Different types of viral-host junction found in HBV integration breakpoints in HBV-infected patients. Mol Med Rep. 2019;19(2):1410–6. Edman JC, Gray P, Valenzuela P, Rall LB, Rutter WJ. Integration of hepatitis B virus sequences and their expression in a human hepatoma cell. Nature. 1980;286(5772):535–8. Brechot C, Pourcel C, Louise A, Rain B, Tiollais P. Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma. Nature. 1980;286(5772):533–5. Koshy R, Maupas P, Muller R, Hofschneider PH. Detection of hepatitis B virus-specific DNA in the genomes of human hepatocellular carcinoma and liver cirrhosis tissues. J Gen Virol. 1981;57(Pt 1):95–102. Tu T, Budzinska MA, Shackel NA, Jilbert AR. Conceptual models for the initiation of hepatitis B virus-associated hepatocellular carcinoma. Liver Int. 2015;35(7):1786–800. Janeway CA. How the immune system works to protect the host from infection: a personal view. Proc Natl Acad Sci U S A. 2001;98(13):7461–8. Stacey AR, Norris PJ, Qin L, Haygreen EA, Taylor E, Heitman J, et al. Induction of a striking systemic cytokine cascade prior to peak viremia in acute human immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis B and C virus infections. J Virol. 2009;83(8):3719–33. 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(4):1289–300. Wieland S, Thimme R, Purcell RH, Chisari FV. Genomic analysis of the host response to hepatitis B virus infection. Proc Natl Acad Sci U S A. 2004;101(17):6669–74. 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(5):1117–24. Cote PJ, Toshkov I, Bellezza C, Ascenzi M, Roneker C, Ann Graham L, et al. Temporal pathogenesis of experimental neonatal woodchuck hepatitis virus infection: increased initial viral load and decreased severity of acute hepatitis during the development of chronic viral infection. Hepatology. 2000;32:807–17. Tan AT, Koh S, Goh W, Zhe HY, Gehring AJ, Lim SG, et al. A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B. J Hepatol. 2010;52(3):330–9. 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(3):820–30. Wieland SF, Chisari FV. Stealth and cunning: hepatitis B and hepatitis C viruses. J Virol. 2005;79(15):9369–80. Durantel D, Zoulim F. Innate response to hepatitis B virus infection: observations challenging the concept of a stealth virus. Hepatology. 2009;50(6):1692–5. Lebossé F, Testoni B, Fresquet J, Facchetti F, Galmozzi E, Fournier M, et al. Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B. J Hepatol. 2017;66(5):897–909. Tan A, Koh S, Bertoletti A. Immune response in hepatitis B virus infection. Cold Spring Harb Perspect Med. 2015;5(8):a021428. 16 Hepatitis B Virus 69. Gehring AJ, Protzer U. Targeting innate and adaptive immune responses to cure chronic HBV infection. Gastroenterology. 2019;156(2):325–37. 70. Maini MK, Gehring AJ. The role of innate immunity in the immunopathology and treatment of HBV infection. J Hepatol. 2016;64(1 Suppl):S60–70. 71. Sato S, Li K, Kameyama T, Hayashi T, Ishida Y, Murakami S, et al. The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus. Immunity. 2015;42(1):123–32. 72. Lucifora J, Durantel D, Testoni B, Hantz O, Levrero M, Zoulim F. Control of hepatitis B virus replication by innate response of HepaRG cells. Hepatology. 2010;51(1):63–72. 73. Lütgehetmann M, Bornscheuer T, Volz T, Allweiss L, Bockmann JH, Pollok JM, et al. Hepatitis B virus limits response of human hepatocytes to interferon. Gastroenterology. 2011;140(7):2074– 2083.e2. 74. Mutz P, Metz P, Lempp FA, Bender S, Qu B, Schöneweis K, et al. HBV bypasses the innate immune response and does not protect HCV from antiviral activity of interferon. Gastroenterology. 2018;154(6):1791–804. 75. Suslov A, Boldanova T, Wang X, Wieland S, Heim MH. Hepatitis B virus does not interfere with innate immune responses in the human liver. Gastroenterology. 2018;154(6):1778–90. 76. 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(6176):1221–8. 77. Belloni L, Allweiss L, Guerrieri F, Pediconi N, Volz T, Pollicino T, et al. IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome. J Clin Invest. 2012;122(2):529–37. 78. McClary H, Koch R, Chisari FV, Guidotti LG. Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines. J Virol. 2000;74(5):2255–64. 79. Watashi K, Liang G, Iwamoto M, Marusawa H, Uchida N, Daito T, et al. Interleukin-1 and tumor necrosis factor-α trigger restriction of hepatitis B virus infection via a cytidine deaminase activation-induced cytidine deaminase (AID). J Biol Chem. 2013;288(44):31715–27. 80. Liaw Y-F, Chen Y-C, Sheen I-S, Chien R-N, Yeh C-T, Chu C-M. Impact of acute hepatitis C virus superinfection in patients with chronic hepatitis B virus infection. Gastroenterology. 2004;126(4):1024–9. 81. Sagnelli E, Coppola N, Messina V, Di Caprio D, Marrocco C, Marotta A, et al. HBV superinfection in hepatitis C virus chronic carriers, viral interaction, and clinical course. Hepatology. 2002;36(5):1285–91. 82. Neumann AU, Lam NP, Dahari H, Gretch DR, Wiley TE, Layden TJ, et al. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science. 1998;282(5386):103–7. 83. 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(1):25–36. 84. 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(5415):825–9. 85. Peppa D, Micco L, Javaid A, Kennedy PTF, Schurich A, Dunn C, et al. Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection. Guidotti LG, editor. PLoS Pathog. 2010;6(12):e1001227. 86. Sun C, Fu B, Gao Y, Liao X, Sun R, Tian Z, et al. TGF-β1 downregulation of NKG2D/DAP10 and 2B4/SAP expression on human NK cells contributes to HBV persistence. Walker CM, editor. PLoS Pathog. 2012;8(3):e1002594. 269 87. 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(9):2111–24. 88. Patzwahl R, Meier V, Ramadori G, Mihm S. Enhanced expression of interferon-regulated genes in the liver of patients with chronic hepatitis C virus infection: detection by suppression-subtractive hybridization. J Virol. 2001;75(3):1332–8. 89. Biron CA. Expansion, maintenance, and memory in NK and T cells during viral infections: responding to pressures for defense and regulation. Madhani HD, editor. PLoS Pathog. 2010;6(3):e1000816. 90. Zeissig S, Murata K, Sweet L, Publicover J, Hu Z, Kaser A, et al. Hepatitis B virus–induced lipid alterations contribute to natural killer T cell–dependent protective immunity. Nat Med. 2012;17:1–11. 91. Tang X-Z, Jo J, Tan AT, Sandalova E, Chia A, Tan KC, et al. IL-7 licenses activation of human liver intrasinusoidal mucosalassociated invariant T cells. J Immunol. 2013;190(7):3142–52. 92. Dias J, Hengst J, Parrot T, Leeansyah E, Lunemann S, Malone DFG, et al. Chronic hepatitis delta virus infection leads to functional impairment and severe loss of MAIT cells. J Hepatol. 2019;71:301–12. 93. Maini MK, Peppa D. NK cells: a double-edged sword in chronic hepatitis B virus infection. Front Immunol. 2013;4:57. 94. Thimme R, Dandri M. Dissecting the divergent effects of interferon-alpha on immune cells: time to rethink combination therapy in chronic hepatitis B? J Hepatol. 2013;58(2): 205–9. 95. Fisicaro P, Valdatta C, Boni C, Massari M, Mori C, Zerbini A, et al. Early kinetics of innate and adaptive immune responses during hepatitis B virus infection. Gut. BMJ Publishing Group Ltd and British Society of Gastroenterology. 2009;58(7):974–82. 96. Guy CS, Mulrooney-Cousins PM, Churchill ND, Michalak TI. Intrahepatic expression of genes affiliated with innate and adaptive immune responses immediately after invasion and during acute infection with woodchuck hepadnavirus. J Virol. 2008;82(17):8579–91. 97. Peppa D, Gill US, Reynolds G, Easom NJW, Pallett LJ, Schurich A, et al. Up-regulation of a death receptor renders antiviral T cells susceptible to NK cell-mediated deletion. J Exp Med. 2013;210(1):99–114. 98. Bertoletti A, Ferrari C. Adaptive immunity in HBV infection. J Hepatol. 2016;64(1 Suppl):S71–83. 99. Maini MK, Burton AR. Restoring, releasing or replacing adaptive immunity in chronic hepatitis B. Nat Rev Gastroenterol Hepatol. 2019;16(11):662–75. 100. 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(1):68–76. 101. 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(6):1386–96. 102. Sitia G, Isogawa M, Iannacone M, Campbell IL, Chisari FV, Guidotti LG. MMPs are required for recruitment of antigennonspecific mononuclear cells into the liver by CTLs. J Clin Invest. 2004;113(8):1158–67. 103. 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(3):486–500. 104. Sitia G, Iannacone M, Muller S, Bianchi ME, Guidotti LG. Treatment with HMGB1 inhibitors diminishes CTLinduced liver disease in HBV transgenic mice. J Leukoc Biol. 2006;81(1):100–7. 270 105. Iannacone M. Hepatic effector CD8+ T-cell dynamics. Cell Mol Immunol. 2014;12(3):269–72. 106. 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(5):1541–54. 107. 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 IFNgamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes. J Exp Med. 2001;194(12):1755–66. 108. 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(11):5707–19. 109. 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(8):1269–80. 110. Pallett LJ, Davies J, Colbeck EJ, Robertson F, Hansi N, Easom NJW, et al. IL-2(high) tissue-resident T cells in the human liver: sentinels for hepatotropic infection. J Exp Med. 2017;214(6):1567–80. 111. Alberti A, Diana S, Sculard GH, Eddleston AL, Williams R. Detection of a new antibody system reacting with Dane particles in hepatitis B virus infection. Br Med J. 1978;2(6144): 1056–8. 112. Corti D, Benigni F, Shouval D. Viral envelope-specific antibodies in chronic hepatitis B virus infection. Curr Opin Virol. 2018;30:48–57. 113. Salimzadeh L, Le Bert N, Dutertre C-A, Gill US, Newell EW, Frey C, et al. PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection. J Clin Invest. 2018;128(10):4573–87. 114. Le Bert N, Salimzadeh L, Gill US, Dutertre C-A, Facchetti F, Tan A, et al. Comparative characterization of B cells specific for HBV nucleocapsid and envelope proteins in patients with chronic hepatitis B. J Hepatol. 2020;72(1):34–44. 115. 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(8):4215–25. 116. Schuch A, Salimi Alizei E, Heim K, Wieland D, Kiraithe MM, Kemming J, et al. Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load. Gut. 2019;68(5):905–15. https://doi.org/10.1136/gutjnl-2018-316641. 117. 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(5):1835–45. 118. 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(3):167. 119. Fisicaro P, Barili V, Montanini B, Acerbi G, Ferracin M, Guerrieri F, et al. Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic hepatitis B. Nat Med. 2017;23(3):327–36. 120. Burton AR, Pallett LJ, McCoy LE, Suveizdyte K, Amin OE, Swadling L, et al. Circulating and intrahepatic antiviral B cells are defective in hepatitis B. J Clin Invest. 2018;128(10):4588–603. 121. Publicover J, Gaggar A, Nishimura S, Van Horn CM, Goodsell A, Muench MO, et al. Age-dependent hepatic lymphoid organi- A. Bertoletti and H. Huang 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. zation directs successful immunity to hepatitis B. J Clin Invest. 2013;123(9):3728–39. Cote PJ, Korba BE, Miller RH, Jacob JR, Baldwin BH, Hornbuckle WE, et al. Effects of age and viral determinants on chronicity as an outcome of experimental woodchuck hepatitis virus infection. Hepatology. 2000;31(1):190–200. Zhang Z, Wang C, Liu Z, Zou G, Li J, Lu M. Host genetic determinants of hepatitis B virus infection. Front Genet. Frontiers. 2019;10:696. Rivino L, Le Bert N, Gill US, Kunasegaran K, Cheng Y, Tan DZ, et al. Hepatitis B virus-specific T cells associate with viral control upon nucleos(t)ide-analogue therapy discontinuation. J Clin Invest. 2018;128(2):668–81. Chen Z, Diaz G, Pollicino T, Zhao H, Engle RE, Schuck P, et al. Role of humoral immunity against hepatitis B virus core antigen in the pathogenesis of acute liver failure. Proc Natl Acad Sci. 2018;115(48):E11369–78. Petersen J, Dandri M, Mier W, Lütgehetmann M, Volz T, von Weizsäcker F, et al. Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein. Nat Biotechnol. 2008;26(3):335–41. Urban S, Bartenschlager R, Kubitz R, Zoulim F. Strategies to inhibit entry of HBV and HDV into hepatocytes. Gastroenterology. 2014;147(1):48–64. 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(16):10460–6. 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(4953):361–4. Chisari FV. Cytotoxic T cells and viral hepatitis. J Clin Invest. 1997;99(7):1472–7. Gerlich WH. The enigma of concurrent hepatitis B surface antigen (HBsAg) and antibodies to HBsAg. Clin Infect Dis. 2007;44(9):1170–2. van der Molen RG, Sprengers D, Biesta PJ, Kusters JG, Janssen HLA. Favorable effect of adefovir on the number and functionality of myeloid dendritic cells of patients with chronic HBV. Hepatology. 2006;44(4):907–14. Op den Brouw ML, Binda RS, van Roosmalen MH, Protzer U, Janssen HLA, van der Molen RG, et al. Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus. Immunology. 2009;126(2):280–9. Woltman AM, Op den Brouw ML, Biesta PJ, Shi CC, Janssen HLA. Hepatitis B virus lacks immune activating capacity, but actively inhibits plasmacytoid dendritic cell function. PLoS One. 2011;6(1):e15324. Xu Y, Hu Y, Shi B, Zhang X, Wang J, Zhang Z, et al. HBsAg inhibits TLR9-mediated activation and IFN-α production in plasmacytoid dendritic cells. Mol Immunol. 2009;46(13):2640–6. Martinet J, Duchesne TD, Costa JB, Larrat S, Marlu A, Leroy V, et al. Altered functions of plasmacytoid dendritic cells and reduced cytolytic activity of natural killer cells in patients with chronic HBV infection. Gastroenterology. 2012;143(6): 1586–8. Gehring AJ, Haniffa M, Kennedy PT, Ho ZZ, Boni C, Shin A, et al. Mobilizing monocytes to cross-present circulating viral antigen in chronic infection. J Clin Invest. 2013;123(9):3766–76. Andrade BB, Santos CJN, Camargo LM, Souza-Neto SM, ReisFilho A, Clarêncio J, et al. Hepatitis B infection is associated with asymptomatic malaria in the Brazilian Amazon. Snounou G, editor. PLoS One. 2011;6(5):e19841. 16 Hepatitis B Virus 139. 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. 140. Michalak TI, Pasquinelli C, Guilhot S, Chisari FV. Hepatitis B virus persistence after recovery from acute viral hepatitis. J Clin Invest. 1994;94(2):907. 141. Pollicino T, Squadrito G, Cerenzia G, Cacciola I, Raffa G, Craxi A, et al. Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection. Gastroenterology. 2004;126(1):102–10. 142. Squadrito G, Spinella R, Raimondo G. The clinical significance of occult HBV infection. Ann Gastroenterol. 2014;27(1):15–9. 143. Seto W-K, Chan TSY, Hwang Y-Y, Wong DK-H, Fung J, Liu KS-H, et al. Hepatitis B reactivation in patients with previous hepatitis B virus exposure undergoing rituximab-containing chemotherapy for lymphoma: a prospective study. J Clin Oncol. 2014;32(33):3736–43. 144. Raimondo G, Locarnini S, Pollicino T, Levrero M, Zoulim F, Lok AS, et al. Update of the statements on biology and clinical impact of occult hepatitis B virus infection. J Hepatol. 2019;71(2):397–408. 145. Block TM, Gish R, Guo H, Mehta A, Cuconati A, Thomas London W, et al. Chronic hepatitis B: what should be the goal for new therapies? Antivir Res. 2013;98(1):27–34. 146. Liaw Y-F, Chu C-M. Hepatitis B virus infection. Lancet. 2009;373(9663):582–92. 147. Lok ASF, McMahon BJ. Chronic hepatitis B: update 2009. Hepatology. 2009;50(3):661–2. 148. Komatsu H, Inui A, Sogo T, Hiejima E, Tateno A, Klenerman P, et al. Cellular immunity in children with successful immunoprophylactic treatment for mother-to-child transmission of hepatitis B virus. BMC Infect Dis. 2010;10(1):103. 149. European Association for the Study of the Liver. Electronic Address: [email protected], European Association for the Study of the Liver. EASL 2017 clinical practice guidelines on the management of hepatitis B virus infection. J Hepatol. 2017;67:370–98. 150. 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(3):637–45. 151. Gill US, Pallett LJ, Kennedy PTF, Maini MK. Liver sampling: a vital window into HBV pathogenesis on the path to functional cure. Gut. 2018;67(4):767–75. 152. Zoulim F, Mason WS. Reasons to consider earlier treatment of chronic HBV infections. Gut. 2012;61(3):333–6. 153. Bertoletti A, Kennedy PT. The immune tolerant phase of chronic HBV infection: new perspectives on an old concept. Cell Mol Immunol. 2014;12(3):258–63. 154. Fitzsimons D, François G, Hall A, McMahon B, Meheus A, Zanetti A, et al. Long-term efficacy of hepatitis B vaccine, booster policy, and impact of hepatitis B virus mutants. Vaccine. 2005;23(32):4158–66. 155. Chen H-L, Lin L-H, Hu F-C, Lee J-T, Lin W-T, Yang Y-J, et al. Effects of maternal screening and universal immunization to prevent mother-to-infant transmission of HBV. Gastroenterology. 2012;142(4):773–81. 156. Chang MH, You S-L, Chen C-J, Liu C-J, Lai M-W, Wu T-C, et al. Long-term effects of hepatitis B immunization of infants in preventing liver cancer. Gastroenterology. 2016;151(3):472–80. 157. Ni YH, Huang LM, Chang MH, Yen C-J, Lu CY, You S-L, et al. Two decades of universal hepatitis B vaccination in Taiwan: impact and implication for future strategies. Gastroenterology. 2007;132(4):1287–93. 158. Lok AS-F. Hepatitis: long-term therapy of chronic hepatitis B reverses cirrhosis. Nat Rev Gastroenterol Hepatol. 2013;10(4):199–200. 271 159. Chen H-L, Lee C-N, Chang C-H, Ni YH, Shyu M-K, Chen S-M, et al. Efficacy of maternal tenofovir disoproxil fumarate in interrupting mother-to-infant transmission of hepatitis B virus. Hepatology. 2015;62(2):375–86. 160. Zoulim F, Locarnini S. Hepatitis B virus resistance to nucleos(t) ide analogues. Gastroenterology. 2009;137(5):1593–608. 161. 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(5):1611–20. 162. Fanning GC, Zoulim F, Hou J, Bertoletti A. Therapeutic strategies for hepatitis B virus infection: towards a cure. Nat Rev Drug Discov. 2019;18(11):827–44. 163. Allweiss L, Volz T, Giersch K, Kah J, Raffa G, Petersen J, et al. Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo. Gut. 2017;67:542–52. 164. Li D, He W, Liu X, Zheng S, Qi Y, Li H, et al. A potent human neutralizing antibody Fc-dependently reduces established HBV infections. elife. 2017;6:213. 165. Wedemeyer H, Schöneweis K, Bogomolov PO, Voronkova N, Chulanov V, Stepanova T, et al. GS-13-Final results of a multicenter, open-label phase 2 clinical trial (MYR203) to assess safety and efficacy of myrcludex B in cwith PEG-interferon Alpha 2a in patients with chronic HBV/HDV co-infection. J Hepatol. 2019;70(1):e81. 166. Lucifora J, Esser K, Protzer U. Ezetimibe blocks hepatitis B virus infection after virus uptake into hepatocytes. Antivir Res. 2013;97(2):195–7. 167. Shimura S, Watashi K, Fukano K, Peel M, Sluder A, Kawai F, et al. Cyclosporin derivatives inhibit hepatitis B virus entry without interfering with NTCP transporter activity. J Hepatol. 2017;66(4):685–92. 168. Long Q, Yan R, Hu J, Cai D, Mitra B, Kim ES, et al. The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation. Siddiqui A, editor. PLoS Pathog. 2017;13(12):e1006784. 169. Bloom K, Maepa MB, Ely A, Arbuthnot P. Gene therapy for chronic HBV-can we eliminate cccDNA? Genes. 2018;9(4):207. 170. Seeger C, Sohn JA. Targeting hepatitis B virus with CRISPR/ Cas9. Mol Ther Nucleic Acids. 2014;3(12):e216. 171. Sekiba K, Otsuka M, Ohno M, Yamagami M, Kishikawa T, Suzuki T, et al. Inhibition of HBV transcription from cccDNA with nitazoxanide by targeting the HBx-DDB1 interaction. Cell Mol Gastroenterol Hepatol. 2019;7(2):297–312. 172. Wooddell CI, Rozema DB, Hossbach M, John M, Hamilton HL, Chu Q, et al. Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection. Mol Ther. 2013;21(5):973–85. 173. Vaillant A. Nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis B and hepatitis D infection. Antivir Res. 2016;133:32–40. 174. Yuen M-F, Gane EJ, Kim DJ, Weilert F, Yuen Chan HL, Lalezari J, et al. Antiviral activity, safety, and pharmacokinetics of capsid assembly modulator NVR 3-778 in patients with chronic HBV infection. Gastroenterology. 2019;156(5):1392–7. 175. Bertoletti A, Le Bert N. Immunotherapy for chronic hepatitis B virus infection. Gut Liver. 2018;12(5):497–507. 176. 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(7):1655–65. 177. Ji C, Sastry KSR, Tiefenthaler G, Cano J, Tang T, Ho ZZ, et al. Targeted delivery of interferon-α to hepatitis B virusinfected cells using T-cell receptor-like antibodies. Hepatology. 2012;56(6):2027–38. 178. Menne S, Tumas DB, Liu KH, Thampi L, AlDeghaither D, Baldwin BH, et al. Sustained efficacy and seroconversion with the 272 179. 180. 181. 182. 183. 184. 185. 186. 187. A. Bertoletti and H. Huang toll-like receptor 7 agonist GS-9620 in the woodchuck model of chronic hepatitis B. J Hepatol. 2015;62(6):1237–45. Lanford RE, Guerra B, Chavez D, Giavedoni L, Hodara VL, Brasky KM, et al. GS-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees. Gastroenterology. 2013;144(7):1508–10. Gane EJ, Lim Y-S, Gordon SC, Visvanathan K, Sicard E, Fedorak RN, et al. The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection. J Hepatol. 2015;63(2):320–8. Jo J, Tan AT, Ussher JE, Sandalova E, Tang X-Z, Tan-Garcia A, et al. Toll-like receptor 8 agonist and bacteria trigger potent activation of innate immune cells in human liver. PLoS Pathog. 2014;10(6):e1004210. 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 HBV-specific CD8 T cells. PLoS Pathog. 2013;9(3):e1003208. Korolowizc KE, Li B, Huang X, Yon C, Rodrigo E, Corpuz M, et al. Liver-targeted toll-like receptor 7 agonist combined with entecavir promotes a functional cure in the woodchuck model of hepatitis B virus. Hepatol Commun. 2019;3(10):1296–310. Michel M-L, Deng Q, Mancini-Bourgine M. Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: perspectives and challenges. J Hepatol. 2011;54(6): 1286–96. Dembek C, Protzer U, Roggendorf M. Overcoming immune tolerance in chronic hepatitis B by therapeutic vaccination. Curr Opin Virol. 2018;30:58–67. 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(6):e1003391. Vandepapelière P, Lau GKK, Leroux-Roels G, Horsmans Y, Gane E, Tawandee T, et al. Therapeutic vaccination of chronic hepatitis B patients with virus suppression by antiviral therapy: 188. 189. 190. 191. 192. 193. 194. 195. 196. a randomized, controlled study of co-administration of HBsAg/ AS02 candidate vaccine and lamivudine. Vaccine. 2007;25(51): 8585–97. Gane E, Verdon DJ, Brooks AE, Gaggar A, Nguyen A-H, Subramanian GM, et al. Anti-PD-1 blockade with nivolumab with and without therapeutic vaccination for virally suppressed chronic hepatitis B: a pilot study. J Hepatol. 2019;71(5):900–7. Krebs K, Böttinger N, Huang LR, Chmielewski M, Arzberger S, Gasteiger G, et al. T cells expressing a chimeric antigen receptor that binds hepatitis B virus envelope proteins control virus replication in mice. Gastroenterology. 2013;145(2):456–65. Kah J, Koh S, Volz T, Ceccarello E, Allweiss L, Lütgehetmann M, et al. Lymphocytes transiently expressing virus-specific T cell receptors reduce hepatitis B virus infection. J Clin Invest. 2017;127(8):3177–88. Wisskirchen K, Kah J, Malo A, Asen T, Volz T, Allweiss L, et al. T cell receptor grafting allows virological control of hepatitis B virus infection. J Clin Invest. 2019;129(7):2932–45. Koh S, Kah J, Tham CYL, Yang N, Ceccarello E, Chia A, et al. Nonlytic lymphocytes engineered to express virus-specific T-cell receptors limit HBV infection by activating APOBEC3. Gastroenterology. 2018;155(1):180–6. 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;65(4):658–67. Dolman GE, Koffas A, Mason WS, Kennedy PT. Why, who and when to start treatment for chronic hepatitis B infection. Curr Opin Virol. 2018;30:39–47. Anderson RT, Lim SG, Mishra P, Josephson F, Donaldson E, Given B, et al. Challenges, considerations, and principles to guide trials of combination therapies for chronic hepatitis B virus. Gastroenterology. 2019;156(3):529–33. Revill PA, Chisari FV, Block JM, Dandri M, Gehring AJ, Guo H, et al. A global scientific strategy to cure hepatitis B. Lancet Gastroenterol Hepatol. 2019;4(7):545–58.