Hepatitis B virus (HBV)

1.2.1 Genomic structure and genotypes

Hepatitis B virus (HBV) is an enveloped DNA virus with a diameter of 42 nm (Dane particle) and classified taxonomically in hepadnaviridae. The viral genome of HBV is a relaxed circular partially double-stranded DNA with a length of 3.2 kb (24). Negative strand of HBV genomic DNA is in full length and covalently linked with viral

polymerase on 5’-end, while positive strand is shorter with a capped oligoribonucleotide on 5’-end. After viral entry, the genomic structure is converted into

covalently-closed-circular DNA (cccDNA) in the nucleus of hepatocytes. The viral genome contains 4 open reading frames (ORF) which are highly overlapped: C-ORF,

coding for capsid core protein (HBcAg) and HBV e antigen (HBeAg); P-ORF, coding for HBV polymerase protein; S-ORF, coding for 3 HBV envelope proteins: large surface Ag (pre-S1 or LHBsAg), middle surface Ag (pre-S2 or MHBsAg), and small surface Ag (S, HBsAg, or SHBsAg).

The 4 major RNA transcripts are denominated by their lengths (25). 3.5 kb mRNA includes pregenomic RNA and precore mRNA. The former is responsible for the synthesis of HBcAg and polymerase protein, and serves as the template of for synthesis of DNA negative strand. 2.4 kb mRNA encodes pre-S1 envelope protein and 2.1 kb mRNA is the template for synthesis of pre-S2 envelope protein and HBsAg. 0.7 kb mRNA encodes HBx protein. All the transcripts share the same sequences at 3’-end. At least 8 genotypes, denominated from A to H, have been identified and classified

according to an intergroup divergence of more than 8% in the complete nucleotide sequence (26).

1.2.2 Viral proteins

HBcAg is a structural protein with molecular weight of 21 kDa. Dimers of HBcAg comprise the viral nucleocapsids, which encapsidate pregenomic RNA and viral polymerase protein.

HBeAg (precore protein) shares the same ORF with HBcAg with extra 5’ sequence, which encodes a signal peptide that direct the newly synthesized precore protein to secretory pathways (27). After cleavage of the N-terminus, the molecular weight of HBeAg decreases from 22 kDa to 17 kDa, smaller than HBcAg. In contrast to HBcAg, which is particulate, HBeAg is non-particulate and secretory. Studies illustrated that HBeAg is dispensable for the replication and infection of HBV (28, 29). However, researches in terms of viral immunology revealed a protective role of HBeAg for HBV

to evade host immunity (30, 31).

HBV polymerase (90 kDa) consists of 3 functional domains: terminal protein (TP) domain, reverse transcriptase (RT) domain, and RNase H domain (RH) (32). TP domain is responsible for protein-priming step of viral replication, while RH domain possesses an enzyme function for digesting the RNA template after reverse transcription by RT domain.

The molecular weights of pre-S1, pre-S2, and S envelope proteins are 39, 33, and 25 kDa, respectively. These proteins share common C-terminal sequences. After

synthesized and modified with glycosylation, these proteins are secreted as 22-nm subviral particles, which contain no nucleocapsid of HBV, whereas the 42-nm Dane particles, which are infectious, comprise nucleocapsid. In patients’ serum, Dane particles are outnumbered by the non-infectious subviral particles (10⁴ to 10⁶ folds).

HBx protein has a molecular weight of 17 kDa. HBx protein is inessential for HBV replication but can enhance viral replication (33). Nonetheless, it has been demonstrated that HBx protein can interact with several cellular proteins and regulate their

physiological functions, which leads to onsets of hepatocellular carcinoma (HCC) (34).

1.2.3 Replication and life cycle

Hepatocyte is the major tropism of HBV. The putative receptor of HBV has recently been identified and further studies are required to confirm this discovery (35, 36). After the nucleocapsid is transported to the nucleus, the partially double-stranded DNA is converted to cccDNA by cellular DNA polymerase. The episomal cccDNA serves as the template for viral transcripts. After transcription and translation, the pregenomic RNA is bound by viral polymerase and subsequently encapsidated into nucleocapsid, where replication of viral DNA takes place. After reverse transcription by polymerase,

the newly formed negative strand DNA becomes the template for synthesis of positive strand. The mature nucleocapsid containing the partially double-stranded DNA genome is transported back to the nucleus to replenish the pools of cccDNA or enveloped by pre-S1, pre-S2 and HBsAg at a ratio of 1:1:4 followed by secretion.

1.2.4 HBV infection

Patients who are serologically positive for HBsAg for more than 6 months are diagnosed as chronic hepatitis B (CHB) infection. More than 350 millions of people worldwide are suffering from chronic hepatitis B infection. HBV infection in adulthood usually becomes asymptomatic and self-limited (90-95%), whereas most neonatal infection via vertical transmission and infection during childhood develop into chronic infection (more than 90%) (37). Additionally, acute HBV infection in persons under immunosuppression conditions is more likely to turn into chronic infection (38).

1.2.5 Immune responses in HBV infection

Persistent infection of hepatitis B virus (HBV) is a common cause of liver cirrhosis and hepatocellular carcinoma worldwide and elucidating the mechanisms of the immunological interaction between HBV and host is the key step toward resolving the diseases (39-41). In acute HBV infection, viral clearance requires specific CD8 T cells which are induced vigorously after exposure to viral antigens and equipped with fully activated antiviral functions, including both cytolytic and non-cytolytic effects that lead to self-limited infection. In contrast, clinical observations of chronic carriers reveal functional defects of HBV-specific CD8 T cells in terms of both quantity and quality. In line with other chronic viral infections, the levels of viremia of chronic HBV patients are inversely correlated to the frequencies of HBV-specific CD8 T cells and their

production of cytokines such as IFN-γ which control the viral replication (23). There is a correlation between the expression of PD-1 of these T cells and the viremia levels in patients and blocking PD-1:PD-L1 pathway improve the function of specific CD8 T cells in vitro (42). Nonetheless, the investigation of the direct effect of HBV burdens on T cell dysfunction is limited by the diversities of genetic background of patients, viral genotypes, and time of exposure to antigens. It is also difficult to address this issue due to the lack of small animal models of natural chronic HBV infection.

1.2.6 Animal models

Since the natural hosts of HBV are human, chimpanzee, and tupaia, proper animal models with available immunological reagents are absent (43, 44). Several substitutive animal models were developed and used for the research of HBV. Alternative

hepadnaviruses that possess infectivity toward hepatocytes of ducks (duck hepatitis B virus, DHBV) and of woodchucks (woodchuck hepatitis virus, WHV) have been used in virological and immunological researches (45, 46). Nevertheless, these viruses differ from HBV and manipulation of experiments of these alternative animals is quite

difficult. Immunodeficient mice baring human hepatocytes as xenografts provide mouse model for virological researches and drug screening. However, the lack of immune system and the laborious experimental procedures render this model limited (47).

HBV-transgenic mice, with HBV genome integrated chromosomally as, were firstly introduced as a mouse model of chronic carrier for the studies of anti-HBV immunity in chronic infection (48, 49). It has a limitation that HBV-specific CD8 T cells are absent in these mice due to central tolerance. As a result, HBV-specific CD8 T cells have to be generated by other donors and adoptively transferred into the transgenic recipients.

Hydrodynamic injection of plasmids containing terminally redundant HBV genome lead

to chronic infection in part of injected mice (30 to 40%) depending on mouse strains (50). Liver damages are observed in injected mice with elevated serum alanine

aminotransferase (ALT). Infection of adenoviral vectors carrying the full-length HBV genome can bypass the viral entry step but can only establish transient expression of HBV. A recent report showed that low dose of adenoviral vectors can achieve long-term HBV replication and protein expression in mouse hepatocytes (51). However, it is difficult to manipulate viral loads in these models due to a relatively narrower window to establish persistent infection.

Using adeno-associated viral (AAV) vectors containing two split genomes of HBV (AAV2/8-5’-HBV-SD and AAV2/8-3’-HBV-SA), we establish persistent viral

replication and expression after recombination of two HBV half genomes in the hepatocytes of immunocompetent mice (52). The persistent rate is 100% and

independent on mouse strains, as the presence of HBV virions and proteins are detected in the sera and the livers of 4 different strains. The profiles of antibody responses are similar to those observed in chronic carriers, while HBs- but not HBc-specific IFN-γ production of CD8 T cells are detected both in the liver and, to a less extent, the spleen.

The frequencies of HBs-reacting CD8 T cells peak at 2 weeks post-infection (p.i.) decrease gradually during persistent infection. The cellular immunity against HBV peaks at week 2 p.i. but diminishes during the persistent expression of virus, indicating a tolerant phenotype to HBV in the immunocompetent hosts. Mice infected with AAV/HBV show hyporesponsiveness to HBV vaccination, but the immunity against ovalbumin (OVA) induced by immunization is comparable to uninfected mice, suggesting the immune tolerance is HBV specific.

There are several advantages of this AAV-mediated mouse model of chronic HBV infection. First, transduction of AAV/HBV shows poor induction of innate immune

responses in immunocompetent hosts (53, 54), which recapitulates HBV infection (55, 56). Second, the AAV DNA genomes are converted into concatamer, an episomal form of extrachromosomal DNA (57), which resembles cccDNA of HBV. Third, the

recombinant AAV vectors is defective and no viral genes of AAV are expressed in transduced cells (58). Most importantly, viral loads of HBV in infected hosts can be controlled by adjusting AAV/HBV titers used for infection.