**3. Contributions of hepatitis B surface, core and e antigens to the pathogenesis of chronic infection**

#### **3.1. Hepatitis B surface antigen (HBsAg)**

More than 90% of these children became HBV carriers, characterized by the persistence of virus or virus antigens in their blood for years to decades. These children were a high risk for the development of chronic liver disease (CLD), which progressed from hepatitis, to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) [4]. Fortunately, newborns in many countries receive the HBV vaccine at birth, which helps to prevent mother-to-infant transmission as well as protect from exposure later in life. Among unvaccinated adults engaging in unprotected sex, roughly 5–10% become carriers, and these individuals are also at high risk for the development of CLD and HCC. Although estimates vary, there are ~300–350 million carriers of HBV worldwide [5]. HCC is the sixth most prevalent cancer worldwide, with about 600,000 newly diagnosed cases annually, and the second leading cause of cancer deaths [6]. Interferon, and in more recent years, powerful nucleoside analogs, have successfully treated patients with chronic hepatitis B, but presently there is no cure [7, 8]. HCC is curable by surgical resection, but this is often accompanied by relapse. Dozens of drugs, alone or in combination, have been evaluated in clinical trials for patients with advanced HCC, but only the multi-kinase inhibitors, Sorafenib and regorafenib, and the immune checkpoint inhibitor nivolumab, have been useful in modestly extending the lifespan of such patients [9, 10]. Given that the carrier state and CLD are the major risk factors for HCC [11], there is strong rationale to better understand

the host-virus relationship that contributes to the pathogenesis of chronic infection.

A hallmark in the pathogenesis of HBV infection is its' variability. Among acutely infected adults, up to 65% develop a subclinical infection characterized only by the appearance of one or more viral antibodies in the blood, while another 25% develop acute resolving infection, which may or may not include a bout of hepatitis. The remaining 10% of patients develop chronic infection (i.e., the persistence of virus and virus antigens in the blood for more than 6 months). In chimpanzees [12] and woodchucks [13], acute infections are characterized by the nearly complete clearance of virus from the blood and liver followed by seroconversion from surface antigen to corresponding antibody. In this case, virus is mostly cleared by non-cytolytic cytokines (e.g., interferon gamma [IFNɣ] and tumor necrosis factor alpha [TNFα]) prior to the appearance of T and other inflammatory cells in the liver, suggesting that most virus clearance occurs prior to the development of acute hepatitis. Further work showed that CD4<sup>+</sup>

T cells, natural killer (NK) cells, Fas, various IFNs and corresponding receptors, and

T cells to facilitate virus elimination in acute infection

the TNF receptor 1 participate in virus clearance, suggesting redundant pathways inhibit HBV replication in the liver [14]. The subsequent contribution of a T cell response appears to clear virus infected cells by cytolytic mechanisms involving Fas and granzymes. In this context,

[14]. When this happens in acute, resolving infection, the T cell response to HBV is vigorous, polyclonal and multi-specific, while among those who go on to develop chronic infection, adaptive immunity is relatively weak and narrowly focused, suggesting that clearance of HBV is T cell dependent. When T cell responses are not adequate, CLD may develop and progress to cirrhosis and HCC. However, CLD may spontaneously resolve at any of these stages. While the origin of this variability is not completely characterized, it is clear that the ability of the

host to mount adaptive immune responses is a key element to limiting virus spread.

**2. Variations in pathogenesis**

T cells are required to prime CD8<sup>+</sup>

and CD8+

14 Liver Cancer

CD4+

HBV is a small virus consisting of only four open reading frames (ORF) [15]. One ORF encodes a family of envelope polypeptides (**Figure 1**). The major envelope polypeptide, HBsAg, triggers neutralizing antibody which is central to virus clearance after acute exposure and is the major component of the HBV vaccine [2]. HBsAg polypeptides are transmembrane proteins and glycoproteins that are on the envelope of virus particles, and are also secreted as small, spherical and variably long filamentous subviral particles that lack the virus nucleocapsid and HBV DNA. It is thought that these subviral particles, which are produced at concentrations several logs above that of infectious virus particles, absorb neutralizing antibody and trigger immunological tolerance, both of which promote virus persistence in the blood. Moreover, in patients with CLD, there does not seem to be any correlation between intrahepatic HBsAg expression patterns and inflammatory infiltrates [16, 17], nor have HBsAg specific T cell clones been isolated from such patients [18]. In addition, T cell sensitization to HBsAg in acute and chronic HBV infection is usually undetectable [19],

**Figure 1.** Genetic organization of HBV showing the ORFs (in color). The positions of enhancer 1 (EN1) and 2 (EN2) are also shown. The direct repeat 1 (DR1) and 2 (DR2) sequences at the ends of the long and short DNA strands are also indicted. The pregenomic RNA (3.5 kb) is greater than genome length, while the 2.1 and 2.4 kb subgenomic mRNAs encode surface antigen polypeptides, and the 0.7 kb mRNA encodes the X protein. Reproduced from [20] with permission.

so while HBsAg clearance occurs in acute, resolving infections, it is not clear that it is an immunological target in established infections.

#### **3.2. Hepatitis B core antigen (HBcAg)**

The second ORF, or core gene, encodes the hepatitis B core antigen (HBcAg) or nucleocapsid protein that polymerizes as an icosahedron around the virus replication complex, the latter of which consists of the virus nucleic acid and HBV encoded polymerase [20]. The fact that the pregenomic RNA and the reverse transcribed viral DNA product are sequestered within a nucleocapsid means that they are not readily detected by pattern recognition receptors, (e.g., toll-like receptors, retinoic acid inducible gene 1 [RIG-1], and mitochondrial anti-viral signaling [MAVS]) that trigger innate immunity [21]. Moreover, innate immune responses do not develop in the liver of acutely infected chimpanzees [22], suggesting that HBV replication and spread may be conducted in "stealth" mode with virus nucleocapsids upon infection and again during virus replication. If so, then this may explain why up to 70% of acutely infected adults who become carriers do not develop CLD. However, carriers who develop CLD also have intrahepatic core antigen, suggesting that HBcAg may be an important immunological target in CLD [23]. Alternatively, patients with acute, resolving hepatitis show a vigorous peripheral blood mononuclear cell response to HBcAg that is temporally associated with the clearance of HBsAg, while in patients with chronic infection, T cell responsiveness to HBcAg is relatively weak, providing an opportunity for HBV to spread in the liver and establish a chronic infection [19].

double stranded viral DNA is made fully double stranded by the endogenous DNAp activity [20] (**Figure 2**). The HBV genome then appears as a supercoiled mini-chromosome in the nuclei of infected cells, and this acts as a template for the transcription of subgenomic RNAs and a greater than genome length pre-genomic RNA. The latter then migrates into the cytoplasm, where it is packaged with the virus polymerase into nascent ("immature") core (or nucleocapsid) particles, where the pregenomic RNA is reverse transcribed into minus strand DNA, with the latter then being used as a template for partial plus strand synthesis just prior to the budding and secretion of progeny virus (**Figure 2**). Some immature core particles are recycled into the nucleus to replenish the pool of covalently closed circular (ccc) HBV DNA. Although the HBV polymerase triggers antibody responses [31], there is no evidence that immune responses against the polymerase directly impact pathogenesis or virus persistence. However, HBV polymerase inhibits RIG-1 and nuclear factor kappa B (NF-ĸB) induction of IFNβ, suggesting that the polymerase could block innate signaling [32, 33], thereby contributing to virus persistence.

**Figure 2.** General scheme of HBV replication. See the text for additional details. Reproduced from [20] with permission.

Pathogenesis of Hepatitis B Virus Associated Chronic Liver Disease

http://dx.doi.org/10.5772/intechopen.79746

17

**4. Relationship between persistent virus replication, integration of** 

There is evidence to suggest that persistent, high levels of HBV replication correlate with the progression of CLD to HCC [34]. However, independent work showed an elevated risk for HCC among patients with CLD but low virus titers [35, 36]. Other observations have shown

stage of liver disease in carriers [37, 38]. In addition, it is controversial as to whether long term nucleoside analog therapy resulted in a decreased risk for the development of HCC [6, 35]. Given that HBV is not directly cytopathic [39], that carriers with high levels of HBV DNA in serum are often asymptomatic, and that the pathogenesis of CLD is immune mediated [17,

copies/ml) and histological grade or

**HBV DNA, and the risk for the development of HCC**

no correlation between HBV DNA levels in serum (>10<sup>5</sup>

#### **3.3. Hepatitis B e antigen (HBeAg)**

A proteolytic fragment of HBcAg, known as HBeAg, is secreted into the circulation and serves as a surrogate marker of virus replication. Seroconversion from HBeAg to anti-HBe is usually accompanied by a significant decrease in virus replication in both the liver and blood and resolution of CLD [24]. The detection of HBcAg specific cytotoxic T lymphocytes (CTL) is associated with the clearance of virus replication, often a transient exacerbation of CLD, and seroconversion to anti-HBe during the natural history of infection [24], suggesting that HBcAg is an important virus target in CLD. HBcAg specific T cells have been detected in the peripheral blood and liver [18, 25] of patients with CLD, suggesting that HBcAg is an immunological target in chronic hepatitis B. Interestingly, HBeAg in serum may attenuate immune responses against virus infected liver, because some patients who develop mutations in HBV that no longer express HBeAg, continue to support high levels of virus replication and ongoing, CLD [26, 27]. In fact, HBeAg appears to be a T cell tolerogen that down-regulates immune responses against HBcAg [28]. HBeAg may also stimulate the appearance of regulatory dendritic cells, which would also suppress virus specific immunity and promote virus persistence [29] by up-regulating the expression of suppressor of cytokine signaling 2 (SOCS2), which in turn represses IFN signaling, thereby blunting innate anti-viral responses and promoting virus persistence [30]. Thus, HBeAg polypeptides, like subviral HBsAg particles, promote chronicity by acting as tolerogens.

#### **3.4. Hepatitis B polymerase**

The HBV encoded polymerase, encoded by a third ORF, has DNA dependent and RNA dependent DNA polymerase (DNAp) activities, and RNase H activity. Upon infection, the partially

so while HBsAg clearance occurs in acute, resolving infections, it is not clear that it is an

The second ORF, or core gene, encodes the hepatitis B core antigen (HBcAg) or nucleocapsid protein that polymerizes as an icosahedron around the virus replication complex, the latter of which consists of the virus nucleic acid and HBV encoded polymerase [20]. The fact that the pregenomic RNA and the reverse transcribed viral DNA product are sequestered within a nucleocapsid means that they are not readily detected by pattern recognition receptors, (e.g., toll-like receptors, retinoic acid inducible gene 1 [RIG-1], and mitochondrial anti-viral signaling [MAVS]) that trigger innate immunity [21]. Moreover, innate immune responses do not develop in the liver of acutely infected chimpanzees [22], suggesting that HBV replication and spread may be conducted in "stealth" mode with virus nucleocapsids upon infection and again during virus replication. If so, then this may explain why up to 70% of acutely infected adults who become carriers do not develop CLD. However, carriers who develop CLD also have intrahepatic core antigen, suggesting that HBcAg may be an important immunological target in CLD [23]. Alternatively, patients with acute, resolving hepatitis show a vigorous peripheral blood mononuclear cell response to HBcAg that is temporally associated with the clearance of HBsAg, while in patients with chronic infection, T cell responsiveness to HBcAg is relatively weak, providing an opportunity for HBV to spread in the liver and establish a chronic infection [19].

A proteolytic fragment of HBcAg, known as HBeAg, is secreted into the circulation and serves as a surrogate marker of virus replication. Seroconversion from HBeAg to anti-HBe is usually accompanied by a significant decrease in virus replication in both the liver and blood and resolution of CLD [24]. The detection of HBcAg specific cytotoxic T lymphocytes (CTL) is associated with the clearance of virus replication, often a transient exacerbation of CLD, and seroconversion to anti-HBe during the natural history of infection [24], suggesting that HBcAg is an important virus target in CLD. HBcAg specific T cells have been detected in the peripheral blood and liver [18, 25] of patients with CLD, suggesting that HBcAg is an immunological target in chronic hepatitis B. Interestingly, HBeAg in serum may attenuate immune responses against virus infected liver, because some patients who develop mutations in HBV that no longer express HBeAg, continue to support high levels of virus replication and ongoing, CLD [26, 27]. In fact, HBeAg appears to be a T cell tolerogen that down-regulates immune responses against HBcAg [28]. HBeAg may also stimulate the appearance of regulatory dendritic cells, which would also suppress virus specific immunity and promote virus persistence [29] by up-regulating the expression of suppressor of cytokine signaling 2 (SOCS2), which in turn represses IFN signaling, thereby blunting innate anti-viral responses and promoting virus persistence [30]. Thus, HBeAg

polypeptides, like subviral HBsAg particles, promote chronicity by acting as tolerogens.

The HBV encoded polymerase, encoded by a third ORF, has DNA dependent and RNA dependent DNA polymerase (DNAp) activities, and RNase H activity. Upon infection, the partially

immunological target in established infections.

**3.2. Hepatitis B core antigen (HBcAg)**

16 Liver Cancer

**3.3. Hepatitis B e antigen (HBeAg)**

**3.4. Hepatitis B polymerase**

**Figure 2.** General scheme of HBV replication. See the text for additional details. Reproduced from [20] with permission.

double stranded viral DNA is made fully double stranded by the endogenous DNAp activity [20] (**Figure 2**). The HBV genome then appears as a supercoiled mini-chromosome in the nuclei of infected cells, and this acts as a template for the transcription of subgenomic RNAs and a greater than genome length pre-genomic RNA. The latter then migrates into the cytoplasm, where it is packaged with the virus polymerase into nascent ("immature") core (or nucleocapsid) particles, where the pregenomic RNA is reverse transcribed into minus strand DNA, with the latter then being used as a template for partial plus strand synthesis just prior to the budding and secretion of progeny virus (**Figure 2**). Some immature core particles are recycled into the nucleus to replenish the pool of covalently closed circular (ccc) HBV DNA. Although the HBV polymerase triggers antibody responses [31], there is no evidence that immune responses against the polymerase directly impact pathogenesis or virus persistence. However, HBV polymerase inhibits RIG-1 and nuclear factor kappa B (NF-ĸB) induction of IFNβ, suggesting that the polymerase could block innate signaling [32, 33], thereby contributing to virus persistence.
