Clinical Implications of the Molecular Biology of Hepatitis B Virus
Introduction
Growing options exist for prevention, early detection, and therapeutic intervention of Hepatitis B Virus (HBV) infections.
Universal recombinant vaccine use nearing its third decade in the U.S.
Clinically apparent liver diseases (cirrhosis, hepatocellular carcinoma (HCC)) arise after decades of chronic infection.
These diseases result from chronic necrotic inflammation mediated by the immune response to HBV antigens.
Association between liver diseases and virological/host biomarkers is increasingly apparent, guiding early detection and therapeutic intervention.
Eight medications for chronic hepatitis B management are FDA-approved.
Advances in HBV biology and immunopathology enable identification of new antiviral strategies and drugs for improved efficacy and potential functional cures.
Molecular biology of the virus explains these advances.
HBV Infection
HBV infection can be transient (resolution in six months) or chronic.
Transient infection is mostly asymptomatic but can manifest as acute hepatitis or, rarely, fulminant hepatitis.
Chronic infection involves virus persistence, often without apparent clinical disease; however, cirrhosis and/or HCC may develop in as many as a third of chronically infected individuals.
Despite vaccine availability and therapeutic options, chronic HBV infection remains a significant public health problem.
More than 270 million people are chronically infected worldwide, and without intervention, as many as 90 million may die from liver diseases.
The CDC estimates 1.4 million chronically infected people in the U.S., but the actual number may be above 2 million as of 2000, considering demography and HBsAg prevalence in high-risk ethnic groups.
The HBV Life Cycle and Targets of Antiviral Medications
HBV is a member of the Hepadnaviridae family.
It contains a partially double-stranded, relaxed circular DNA (rcDNA) genome of approximately 3.2 kb.
Unlike mammalian DNA viruses, HBV replicates its genomic DNA by reverse transcription of its pregenomic RNA (pgRNA); thus HBV is a retrovirus with a DNA genome.
HBV genomic DNA integration into host chromosomes is not obligatory.
Upon hepatocyte entry, viral genomic rcDNAs are transported into nuclei and converted into episomal covalently closed circular DNA (cccDNA), serving as the template for viral RNA transcription.
HBV infection of hepatocytes is initiated by binding to a low-affinity heparan sulfate proteoglycan receptor via the S domain of envelope proteins.
This is followed by a more specific and higher affinity binding of the N-terminal myristoylated peptide in the pre-S1 domain of L protein in the virion envelope to sodium taurocholate cotransporting polypeptide (NTCP), the bona fide receptor of HBV, on the hepatocyte plasma membrane.
HBV virions are then internalized into hepatocytes via endocytosis, and nucleocapsids are subsequently released into the cytoplasm.
They are transported by dynein motor complex to nuclear pore complex, where viral genomic rcDNA is released into the nucleus for cccDNA synthesis.
Molecular mechanisms involved in the conversion of virion-derived genomic rcDNA into nuclear cccDNA are just beginning to be understood.
To form cccDNA from viral rcDNA, the viral DNA polymerase, covalently linked to the 5′ terminus of minus-strand DNA, and the RNA oligomer linked to the 5′ end of plus-strand DNA must be removed.
Recent studies indicate that the ends of rcDNA may be processed by the host cellular tyrosol-DNA phosphodiesterase 2 (TDP2) and/or flap endonuclease 1 (FEN 1) [18, 19].
For cccDNA synthesis to occur, the incomplete plus strand of rcDNA needs to be filled in.
Genetic screening in HBV infection of NTCP-expressing HepG2 cells found that cellular DNA polymerase and, to a lesser extent, DNA polymerase , are required for de novo cccDNA synthesis.
DNA polymerases and play a role in translesion DNA synthesis and the nonhomologous end joining (NHEJ) DNA repair pathway, respectively.
A recent study also showed that both cellular DNA ligase I and DNA ligase III are required for cccDNA synthesis, presumably by ligation of both strands of viral DNA [21].
Viral Replication Cycle
HBV virion binds to sodium taurocholate cotransporting polypeptide (NTCP) receptor and enters hepatocytes by endocytosis.
Viral nucleocapsids are delivered into the cytoplasm, and viral genomic DNA is transported into the nucleus after the removal of the DNA polymerase covalently linked to the minus strand of viral DNA.
Covalently closed circular DNA (cccDNA) is generated by repairing the deproteinized viral DNA and serves as the transcription template to produce viral RNAs.
In the cytoplasm, viral pregenomic RNA and DNA polymerase are encapsidated to form nucleocapsid where HBV DNA synthesis occurs.
“Mature” relaxed circular DNA (rcDNA)‐containing nucleocapsids are assembled into virion secreted from hepatocytes.
Progeny rcDNA in the cytoplasmic nucleocapsids can be shuttled back into the nucleus to form more cccDNA.
Empty capsids and RNA‐containing capsids can also be enveloped and secreted out of cells as genome‐free virions or RNA‐containing virions.
Subviral particles are secreted via the ER/Golgi complex pathway, while virions and virion‐like particles are secreted via multivesicle bodies (MVBs).
*Forms of HBV DNA: HBV DNA forms in virions and intracellular HBV DNA replication intermediates can be revealed by Southern blot hybridization.
HBV rcDNA in virions are heterogeneous in length.
HBV DNA in intracellular nucleocapsids represents all forms of HBV DNA replication intermediates, including incomplete single‐stranded DNA, full‐length single‐stranded DNA (ss), partially double‐stranded DNA, mature rcDNA (RC) and complete double‐stranded linear DNA (DSL).
Intracellular protein‐free HBV DNA species, including deproteinized rcDNA and cccDNA, are extracted by Hirt method and separated by agarose gel electrophoresis.
In the nucleus, the cccDNA is assembled into a minichromosome and serves as the template for transcription of viral mRNAs.
The pgRNA, which is longer than full-length genomic DNA and contains terminally redundant sequences, is translated to produce both the core protein and viral DNA polymerase (Pol).
The Pol protein binds to the ε stem-loop structure within the 5′‐terminal region of pgRNA to prime viral DNA synthesis and initiate nucleocapsid assembly.
Inside the nucleocapsid, viral DNA polymerase converts the pgRNA first to a single-stranded DNA and then to rcDNA.
The rcDNA-containing mature nucleocapsids can either acquire an envelope and be secreted out of cells as infectious virions or deliver the rcDNA into the nucleus to amplify the cccDNA pool, the sole transcription template to support HBV replication.
Selectively blocking any of the essential steps of the HBV life cycle should ultimately stop its replication.
Currently available antiviral drugs for hepatitis B are either interferon alpha, which inhibits multiple steps of HBV replication, or nucleos(t)ide analogs that inhibit viral DNA polymerase by chain termination.
Inhibitors of other steps of the viral life cycle and drugs to activate host antiviral immune responses against HBV are currently under preclinical or clinical development.
Biological Function and Clinical Significance of Viral Proteins
The HBV genome contains four open reading frames (ORFs).
Because of its overlapping coding regions, the virus actually specifies a total of seven proteins from these ORFs, including DNA polymerase, core protein, and a secreted viral protein HBeAg, three envelope proteins (surface antigens), and the “X” protein (HBx).
The biological functions of these proteins in viral replication and pathogenesis have been studied over the past decades, and the clinical significance of the related discoveries in the management of HBV infection is summarized
DNA Polymerase
HBV DNA polymerase (Pol) is a 90 kDa multifunctional protein that consists of four structural domains: terminal protein (TP), spacer, reverse transcriptase (RT), and RNase H domain in the order from the N‐ to the C‐terminus.
Although the two C‐terminal proximal domains are phylogenetically homologous to the reverse transcriptases of retroviruses, the two N‐terminal proximal domains share no homology with any known proteins.
Genetic studies have revealed that while the spacer domain is dispensable for HBV DNA replication, the TP domain is essential and contains a tyrosine (Tyr63) residue to which the first nucleotide of minus‐strand DNA is covalently attached during the priming reaction of DNA synthesis.
Besides catalyzing the reverse transcriptional synthesis of viral DNA and degrading the pgRNA template, HBV DNA polymerase also binds to the epsilon structure at the 5′‐terminal region of pgRNA to initiate nucleocapsid assembly and prime minus‐strand DNA synthesis.
There are now six FDA‐approved nucleoside/nucleotide analog inhibitors of the enzyme for the treatment of chronic hepatitis B, and a few more are in clinical trials.
Therapy with DNA polymerase inhibitors has been associated with a rapid and multi‐log decline of viremia.
Presumably, due to the lack of a direct effect on cccDNA, DNA polymerase inhibitor‐based therapy rarely cures chronic HBV infection.
Long‐term treatment is required for sustained suppression of virus replication, which frequently leads to the emergence of drug‐resistant variants and failure of antiviral therapy.
Drug‐resistant HBV variants are distinctively selected by the different inhibitors, and the mutant viruses selected by one class of the DNA polymerase inhibitors are usually sensitive to others.
For example, lamivudine‐resistant HBV can be inhibited by adefovir and entecavir, although lamivudine‐resistant virus is then predisposed to becoming entecavir‐resistant.
In addition to reverse transcriptase activity, the RNase H activity of HBV DNA polymerase catalyzes the degradation of pgRNA templates during minus‐strand DNA synthesis and generates RNA primer for plus‐strand DNA synthesis.
A small molecular inhibitor of HBV RNase H has now been demonstrated to inhibit HBV replication in vivo in a mice model.
Iron protoporphyrin (heme) and several related analogs selectively disrupted the formation of RT‐epsilon RNA complex in vitro by binding to the TP domain of DNA polymerase.
A cellular molecular chaperone complex consisting of heat shock protein 90 (Hsp90) and its cofactors (Hsp70, Hsp40, Hop, and p23) are required for the proper folding and the interaction between DNA polymerase and epsilon RNA, which is essential for nucleocapsid assembly and priming of minus‐strand DNA synthesis.
Hsp90 inhibitors had been shown to inhibit HBV replication in cultured cells, and their antiviral effects are worthy of further evaluation in animal models.
Lack of structural information of the enzymes has prevented detailed structure–function analyses and rational design of antivirals against HBV.
Core Protein
Core protein or core antigen (HBcAg) is a small polypeptide of 183 or 185 amino acid residues and contains two structural domains connected by a linker region of 9 amino acid residues.
The N‐terminal 140 amino acids form the capsid assembly domain, which is sufficient to assemble into empty capsids.
The C‐terminal 34 amino acid residues specify the C‐terminal domain (CTD) containing multiple arginine‐rich motifs and seven conserved serines or threonines that can be dynamically phosphorylated and dephosphorylated during the viral life cycle.
Studies with duck hepatitis B virus (DHBV) show that modification of the capsid protein by phosphorylation and dephosphorylation plays an indispensable role in pgRNA encapsidation, viral DNA replication, and virion particle morphogenesis.
Core protein is hyperphosphorylated in free dimers and empty capsids but hypophosphorylated in pgRNA‐ and DNA‐containing nucleocapsids.
The CTD of core protein is hyperphosphorylated in empty virions but unphosphorylated in complete HBV virions.
Core protein phosphorylation regulates capsid stability/uncoating and delivery of viral rcDNA into the nucleus for cccDNA synthesis.
Capsid assembly is driven by hydrophobic interaction between the inter‐dimer interfaces of core protein dimers.
Binding of small molecule core protein allosteric modulators (CpAMs) to a hydrophobic pocket, designated as the HAP pocket, at the dimer–dimer interface accelerates the assembly kinetics and misdirects their assembly to form noncapsid polymers of core proteins or morphologically “normal” empty capsids, and thus precludes viral DNA replication.
Several CpAMs have been demonstrated to potently inhibit HBV replication in animal models and in human clinical trials.
Antibodies to capsid protein (anti‐HBc) are associated with previous or current infection with HBV and are not “protective.”
High levels of IgM anti‐HBc are associated with acute infection. IgG anti‐HBc persists throughout the chronic infection, and IgM anti‐HBc may become detectable again during exacerbation of chronic hepatitis B.
Anti‐core antibodies are present in the serum of people who have had acute, transient infections or chronic infections, they are used by blood collection agencies to screen donors for exposure to HBV infection.
In the United States, anti‐HBc‐positive blood is not used for transfusion or preparation of blood products, and donors are informed of their positive test results.
HBV e‐antigen (HBeAg)
HBeAg is translated from precore mRNA, originally, as a 25 kDa precore protein, which is sequentially processed into p22 and p17 by proteolytic cleavage of N‐terminal signal peptide and C‐terminal domain in the endoplasmic reticulum (ER) and secreted as a 17 kDa soluble protein.
Detection of HBeAg in the circulation is a marker of a high level of viral replication.
Loss of HBeAg and appearance of antibodies to HBeAg (anti‐HBe) is called HBeAg seroconversion, which is associated with viral clearance in acute infection and usually with a reduction in levels of viremia in persistent infections.
HBeAg seroconversion is considered to be a beneficial milestone in antiviral therapy.
Many chronic HBV carriers who appear to lose HBeAg have done so because of mutations in the precore region, and these HBeAg mutant viruses may be more pathogenic than are the wild type.
Loss of detectable HBeAg in individuals who have moderate to high levels of circulating viral DNA is evidence for emergence of a mutant virus with lesions within the precore or basal core promoter region, which may represent a more pathologically aggressive virus.
HBeAg is not required for infection in cultured cells or in animals; however, since all known hepadnaviruses specify an HBeAg protein, it is reasonable to assume that HBeAg plays a critical function in some aspect of the viral life cycle.
HBeAg might play a role in immunosuppression and induction of immune tolerance
Maternal HBeAg predisposes hepatic macrophages by upregulation of inhibitory ligand PD‐L1 and altered polarization upon restimulation with HBeAg, which impairs CD8+ T cell responses to HBV in her offspring and facilitates HBV persistence.
Envelope Proteins
HBV encodes three envelope glycoproteins called large (L, LHBs), middle (M, MHBs), and small (S, SHBs, or more commonly abbreviated as HBsAg) surface proteins.
While LHBs is translated from 2.4 kb mRNA, MHBs and SHBs are translated from 2.1 kb mRNA by using different starting codons.
Virions contain approximately 100 copies of HBsAg for every 5 MHBs and 1 LHBs proteins.
The viral envelope proteins are essential for the assembly, secretion, and infectivity of HBV virions.
The N‐terminal portion of pre‐S1 domain of LHBs mediates the binding of HBV virions to NTCP receptor and initiates infection.
HBV‐infected cells usually secrete 100–1000 times as many subviral particles with spherical or filamentous shapes made mostly of SHBs (and less MHBs).
The biological significance of those subviral particles is unclear and has been suspected to play a role in inducing the exhaustion of host antiviral adaptive immune response.
The association between accumulation of LHBs in hepatocytes, in the absence of other viral proteins, and malignant transformation, was reported nearly three decades ago in a transgenic mice model
LHBs and mutant LHBs polypeptides can be detected, in the absence of other viral proteins, within sections of HCC tissues, providing more circumstantial evidence for a role of aberrant expression of HBV envelope polypeptides and hepatocarcinogenesis.
Retention of LHBs proteins (including pre‐S2 mutant LHBs) in the ER activates ER stress pathways that predispose the cell to induction of oxidative stress, DNA damage/genomic instability, and ultimately cell death.
The LHBs‐driven hepatocyte death, liver inflammation, and regeneration would place large numbers of hepatocytes at risk for acquisition of transforming mutations and development of HCC.
X protein (HBx)
HBV X is a 17 kDa, 155 amino acid protein.
HBx is not essential for WHV DNA replication in cultured cells but is required for its infectivity in vivo.
HBx is not required for cccDNA synthesis after de novo infection but is essential for viral RNA transcription.
The cccDNA minichromosome is transcriptionally silenced by the binding of host polypeptide, such as the cellular structural maintenance of chromosomes 5/6 (SMC5/6) complex, including Smc5, Smc6, Nse1, Nse2, Nse3, and Nse4 proteins.
By interacting with DNA‐damage binding protein 1 (DDB1), HBx protein recruits DDB1‐Cullin4 E3 ubiquitin ligase complexes to degrade SMC5/6 complex, thereby relieving its restriction of cccDNA transcription.
As a key regulator of viral infection, the stability of HBx is regulated by poly‐ubiquitination‐mediated proteasomal degradation and E3 ligase HDM2‐mediated Neddylation that stabilizes HBx.
The roles of the HBx protein in HCC development and regulation of cell death and proliferation have been suggested by many studies, but the molecular mechanism remains controversial.
HBV Genomic DNA and Its Replication Intermediates: Biological Function and Clinical Significance
The structure of HBV virion DNA is presented in Figure 64.1. The intracellular replicative forms of HBV DNA are illustrated in Figure 64.2b–d.
Although not a functional replication intermediate, HBV DNA that has integrated into the cellular chromosome may play a role in HCC development and, more recently, was used as a genetic marker to study the fate of virally infected hepatocytes during the progression of HBV infection
Virion DNA and Viral Load
Virion DNA is conventionally detected by serum analysis and reported in laboratory assessments of people chronically infected, and referred to as “viral load.”
Approximately 15–30% of all people with chronic HBV infection eventually develop HCC.
An elevated serum HBV DNA level (10,000 copies per mL) is significantly correlated with risk of HCC, independently of HBeAg, serum alanine aminotransferase level, and liver cirrhosis.
Sustained suppression of HBV replication with antivirals has been consistently shown to greatly reduce the likelihood of cirrhosis and HCC.
Covalently Closed Circular DNA (cccDNA)
cccDNA is the first viral DNA replication intermediate detected in the cell following infection of hepatocytes by the virus.
In addition to being formed from the input viral capsid rcDNA during the initial infection, cccDNA can be produced by an intracellular pathway.
Newly synthesized cytoplasmic core DNA can be shunted into the nuclei and converted into cccDNA molecules.
The pool size of cccDNA in the nuclei of infected cells is regulated by viral and perhaps also cellular mechanisms.
The general consensus is that cccDNA is the most stable form of viral replication intermediate and extremely resistant to DNA polymerase inhibitor‐based antiviral therapy and host immunological response.
It is the source of rebound viremia following discontinuation of therapy.
It is also the source of viremia in people who had presumptively “resolved” infections but are experiencing “reactivation,” which has been observed following immunosuppression and/or chemotherapy.
Elimination of cccDNA is thought to be an important therapeutic goal, since doing so would presumably eliminate all sources of recurrence.
HBV RNA
cccDNA in the nucleus is the template for all viral RNAs:
3.5 kb precore mRNA (pre‐C)
pregenomic (pg) RNAs
2.4 kb mRNA for large (L, LHBs) envelope protein
2.1 kb mRNA for middle (M, MHBs) and small surface (S, SHBs) proteins
0.7 kb mRNA for the X protein.
Transcription of viral mRNA depends upon the host cellular RNA polymerase II and liver‐enriched transcription factors.
A helioxanthin analog can inhibit HBV replication by selectively blocking viral pgRNA transcription.
The relative transcription efficiency (RNA made per cccDNA molecule) of pgRNA, but not mRNAs for envelope proteins, has been reported to be reduced in the livers of HBeAg‐negative individuals, suggesting a role for host antiviral immune response in regulation of cccDNA‐based transcription.
Cytoplasmic Forms of HBV DNA
HBV replication occurs in the cytoplasm where the Pol protein binds to pgRNA and recruits HBcAg to form nucleocapsid.
HBV DNA is produced within the nucleocapsids (or core particles) by synthesis of the minus-strand DNA from pgRNA and then the positive-strand DNA from the newly synthesized minus-strand DNA.
The nucleocapsid is matured as rcDNA is formed and assembles with envelope proteins in the ER to form virions and is secreted from hepatocytes.
Therefore, all reverse transcription intermediates of HBV DNA replication, which include incomplete and full-length minus-strand DNA, partially double-stranded and mature forms of rcDNA and double-stranded linear (dsl)DNA, can be found in the cytoplasmic nucleocapsids.
DNA polymerase inhibitors inhibit both strand DNA synthesis, and thus all forms of capsid-associated DNA are reduced or even eliminated in treated cells.
Integration of HBV DNA into Host Cellular Chromosomes
HBV DNA integration into host chromosomes is not required for viral replication, it does occur randomly in infected hepatocytes.
Integration can be detected as early as a few days post infection, and the frequency of integration increases in infected livers with the duration of viral infection
The most likely precursor of integrated viral DNA is dslDNA, a replication product of in situ priming of plus-strand DNA [99].
Integrated HBV DNA is able to be transcribed into functional mRNAs for envelope proteins, but not 3.5 kb precore mRNA and pgRNA, and thus would be unable to support the production of infectious virus.
A significant amount of the SHBs in the circulation of people chronically infected with HBV, particularly HBeAg‐negative patients, is produced from mRNA transcribed from integrated HBV DNA [100].
HBV DNA integration could activate cellular proto‐oncogenes by an insertion activation mechanism and thus contribute to the development of HCC.
In human HCC, HBV DNA integration is largely random and has only been seen in a few cases, close to important cell growth regulatory genes.
Viral DNA integration may play a role in HCC carcinogenesis by promoting instability of chromosomal DNA in virally infected cells, influencing the expression levels of cellular genes, or by specifying the expression of viral gene products, such as X or the envelope polypeptides.
Integrated viral DNA has been used as a genetic marker for individual hepatocyte lineages to determine the fate of virally infected hepatocytes during transient and chronic infections.
Despite the low inflammation, HBV-infected hepatocyte clonal expansion and possibly, hepatocarcinogenesis could be occurring in patients with early-stage chronic HBV infection .
Genome-free Virions and RNA-containing Virions
Empty capsids and RNA‐containing capsids can also be enveloped and secreted as virion‐like particles, designated as genome‐free (GF) virions and RNA‐containing virions, respectively [104].
GF virions are usually more abundant than rcDNA-containing or complete virions, but RNA-containing virions are less abundant than complete virions [105].
The RNA species in RNA-containing virions can be either pgRNA or spliced viral RNA [106, 107].
Production of complete virions can be inhibited by viral DNA polymerase inhibitors, production of GF virions and RNA-containing virions cannot be suppressed by DNA polymerase inhibitors.
Quantification of plasma HBV GF virions or viral RNA may reflect the cccDNA load and transcription activity under nucleos(t)ide analog therapy, and undetectable GF virions or viral RNA may indicate low load/activity of HBV reservoir and thus an indication to stop nucleos(t)ide analog therapy [105, 109].
Antiviral Therapy of Chronic Hepatitis B From The Molecular Perspective
Available Medications to Manage Chronic Hepatitis B
Among the eight US FDA-approved medications for the treatment of chronic hepatitis B, two are the injected regular and pegylated alpha-interferons (IFN-α) and six are orally available nucleotide or nucleoside analogs.
All the nucleot(s)ide analogs ([[Lamivudine]], [[Adefovir Dipivoxil]], [[Entecavir]], [[Telbivudine]], [[Tenofovir Disoproxil Fumarate]], and [[Tenofovir Alafenamide]]) are viral DNA polymerase inhibitors and therefore directly inhibit viral DNA synthesis in the cytoplasmic nucleocapids,
The therapeutic efficacy of IFN-α is due to its modulation of host antiviral immune response [110].
IFN-α reduces viral RNA transcription and pgRNA encapsidation and promotes the decay of nucleocapsids [28–30].
Treatment of HBV-infected primary human hepatocytes with IFN-α noncytolytically reduces the amount of cccDNA by inducing the expression of APOBEC3A and APOBEC3B. These APOBEC3 proteins can be recruited to cccDNA to deaminate cytosines in the negative strand of cccDNA and promote its decay [111].
Who Should Be Treated, When Should They Be Treated, and For How Long?
Current therapeutic interventions have generally been reserved for those who have what is termed “active hepatitis”: higher levels of serum HBV DNA and some evidence of “active” liver disease, as manifested histologically or by elevated levels of liver-derived enzymes in serum [2, 114].
Since there is evidence that as many as a third of those with chronic hepatitis B and normal serum alanine aminotransferase (ALT) levels may still have fibrotic or even cirrhotic livers [115], treatment of all chronically infected individuals may be indicated.
Severe liver disease and cancer associated with HBV usually, but not by any means always, comes after many years of infection.
“Beneficial responses to therapy have generally been defined in virological and biochemical terms”
Virological response is characterized by the loss of HBeAg (in HBeAg‐positive patients) and reductions of serum HBV DNA to undetectability (by PCR, meaning less than 102–103 genomes per mL).
Biochemical and histological responses are normalization of serum ALT levels and improvement in liver disease scores, respectively [1].
Therapeutic objectives are thus measured by the ability to achieve what has been called “sustained virological and biochemical” responses, (SVR, SBR, respectively) [2].
Off-therapy, SVR is rare in HBeAg-negative patients [122]. Therefore, for HBeAg-positive patients, DNA polymerase inhibitor can be withdrawn six months after HBeAg seroconversion, but for HBeAg-negative patients, the general practice is to suppress HBV replication as long as possible.
Emergence of Drug-Resistant Viruses
The current virological goals or “twin pillars” of oral antiviral therapy are potent, long-term viral suppression and avoidance of resistance [123].
Emergence of HBV variants resistant to the currently available HBV DNA polymerase inhibitors remains one of the greatest threats to their effective use [124, 125].
Based on the assumption that reverse transcription of HBV DNA polymerase has an error rate of per base per replication cycle [126], Perelson and Ribeiro [127] propose that approximately 0.1 base changes are made per genome replication.
Circulating virus concentration is usually in the range of – per mL, assuming a half‐life of one day for circulating HBV virions, and approximately HBV virions are produced and cleared every day.
For example, HBeAg-positive people with high viral loads and HBeAg-negative people with low viral loads that differ by orders of magnitude develop viral mutants resistant to polymerase inhibitors at approximately the same rate [122].
New Antiviral Drugs on the Horizon
Extensive efforts are currently underway to develop novel antivirals that target other viral or host functions to achieve a functional cure of chronic hepatitis B.
Antiviral efficacy of compounds that inhibit virus entry, nucelocapsid assembly, or induce viral RNA decay have been demonstrated in animal models in vivo [131, 132].
Myrcludex B, a synthetic N‐acylated pre‐S1 lipopeptide that inhibits HBV infection of hepatocytes [133], and multiple HBV CpAMs that disrupt HBV capsid assembly and disassembly [134, 135] are currently in clinical trials for treatment of chronic hepatitis B [132, 136].
Small interfering RNA (siRNA) targeting HBV mRNA has also been shown to efficiently reduce virus load and HBsAg in chimpanzees [100].
Restoration of a functional host antiviral immune response is essential to achieve a functional cure for chronic hepatitis B [4, 17, 137].
Reconstitution of antiviral immune response via adoptive transfer of engineered HBV-specific T cells, such as T cells expressing chimeric antigen receptor (CAR-T) against HBV, activation of exhausted T cell function in chronic HBV carriers by T cell checkpoint blockade, and therapies with the agonists of pattern recognition receptors (PPRs), particularly TLR7 and TLR8, have been explored to induce an effective immune control of chronic HBV infection in preclinical and clinical studies.
HBV Variants and Their Clinical Significance
Genotypes
Genotypes are defined as having a sequence divergence of greater than 8% in the entire genome.
There are currently 10 different genotypes, designated A–J, and they are not evenly distributed throughout the world [143, 144].
Genotype A is associated with a greater chance of spontaneous resolution, and genotype C tends to be less responsive to interferon and has a more aggressive clinical course, being associated with HCC more often than genotype B, for example [145].
Mutations in Envelope Genes
The “a” antigenic determinant, is created by two “loops” of amino acids 120–163 within the SHBs (HBsAg) polypeptide [96–98].
The serotypes of HBV are defined by the three epitopes and denoted as serotype “adw,” “adr,” “ayw,” “ayr,” etc
Point mutations leading to amino acid substitutions in “a” determinant have been associated with escape from neutralizing antibody [146].
It has been reported that the frequency of pre-S mutant viruses in circulation increases as viral titer declines [148].
Increased pathogenic potential of envelope protein with mutations in the pre-S2 region is highlighted by transgenic mouse studies, in which it has been found that by 2 years of age, a majority of homozygous male mice develop hepatocellular neoplasias, including HCC [149].
HBsAg Mutations in DNA Polymerase Inhibitor-Resistant Viruses
Given the complete overlap of the coding regions of HBsAg and the N-terminal two-thirds of the DNA polymerase RT domain [150] (Figure 64.4), it is not surprising that the sequence changes associated with antiviral resistance in the polymerase usually lead to changes in the surface antigen [151, 152].
Given the complete overlap of the coding regions of HBsAg and the N-terminal two-thirds of the DNA polymerase RT domain [150] (Figure 64.4), it is not surprising that the sequence changes associated with antiviral resistance in the polymerase usually lead to changes in the surface antigen
There is also a report showing that some of the lamivudine-treated patients apparently cleared HBsAg, based on commercial immunoassay, but remain with detectable circulating HBV DNA. The failure to detect HBsAg in these patients is due to the selection of a sP120A mutation that reduces anti-HBs binding
Mutations in Core and Precore Genes
Loss of HBeAg is generally considered a favorable serological sign.
Loss of detectable HBeAg, under conditions where viral DNA levels (viral load) remains high, is often associated with emergence of HBV variants containing mutations in precore and/or basal core promoter regions
One of the most common precore mutations is G1896A, which changes tryptophan codon (UGG) to a termination codon (UAG) [155].
Acute infection with HBeAg-negative HBV has been associated with severe chronic liver diseases and fulminant hepatitis [156, 157]. Mutations affecting capsid protein sequences have also been reported [111].
Core Promoter Mutations
The “basal” core promoter (BCP), with its associated enhancer, controls transcription of the precore and pg RNA.
A pair of mutations in this region, A1762T and G1764A, may be associated with HBeAg-negative viremia and have been reported to associate with more severe liver diseases and especially with cirrhosis and the development of HCC [159–161].
Overall, perhaps as many as 50% of the HBV chronic carriers who develop HCC are viremic with basal core mutants. the BCP mutation may merely be a phenomenon of the duration of infection, as is the development of HCC.
The HBV Vaccine
Administration of HBsAg subviral particles to people and to animals elicits production of antibodies specific for the “a” and “b” epitopes.
Antibodies to the “a” epitope appear to be sufficient, when present in adequate amounts, to prevent the establishment of chronic HBV infection [165, 166].
The original HBV vaccine was purified from inactivated preparations of HBV subviral particles from the circulation of people chronically infected with the virus [167].
Chronic HBV infection, which is the cause of the largest proportion of HCCs in the world, is 90% preventable with proper use of the hepatitis B vaccine [169].
Envelope Gene Mutations and The Vaccine
The recombinant vaccines that are in current use elicit antibodies against only, or mostly, the “a” epitope.
Vaccine escape mutants that have mutated amino acid sequences within the “a” epitope can and do occur, and the possibility that this will become a problem over time remains [165, 173].
In one report, the incidence of HBsAg mutants in infants born from HBV carrier mothers and treated with vaccine and HBIg was 4% [178].
Waning Immunity to The Vaccine
There is some evidence that people who were vaccinated with recombinant HBV vaccine at birth or in infancy might not have protective levels of antibody to HBV when they reach adulthood [179].
In 2017, a new, two-dose vaccine called Heplisav for immunization against hepatitis