Diagnostics of Hepatitis B Virus: Standard of Care and Investigational

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Abbreviations

ALT

alanine aminotransferase

anti‐HBc

hepatitis core antibody

anti‐HBs

hepatitis surface antibody

BCP

basal core promoter

ccc

covalently closed circular

CHB

chronic hepatitis B

CTLA4

cytotoxic T lymphocyte antigen 4

dlDNA

double‐stranded linear/duplex‐linear DNA

HBcAg

hepatitis B core antigen

HBcrAg

hepatitis B core‐related antigen

HBeAg

hepatitis B e antigen

HBsAg

hepatitis B surface antigen

HBV

hepatitis B virus

HCC

hepatocellular carcinoma

IL

interleukin

mAb

monoclonal antibody

NA

nucleos(t)ide analogue

OBI

occult HBV infection

pgRNA

pregenomic RNA

Pol

polymerase

spDNA/RNA

spliced DNA/RNA

SVP

subviral particle

TLR7

toll‐like receptor 7

Hepatitis B virus (HBV) plays a significant role in global morbidity and mortality with around 257 million chronically infected people worldwide.1 In 2016, the World Health Organization released a global strategy for eliminating viral hepatitis as a major public health threat by 2030. Chronic hepatitis B (CHB) has become a key focus because it contributes to 50% of all viral hepatitis‐related deaths. Even though global testing, vaccination, and treatment are the keys to achieving elimination, this must be preceded by reliable and accurate diagnosis of infection through serological and virological testing.

A sterilizing cure, which involves eradication of detectable HBsAg and all HBV DNA including covalently closed circular (ccc) and integrated DNA, is the ultimate goal but remains elusive. A functional cure, defined by Lok and colleagues2 as sustained loss of hepatitis B surface antigen (HBsAg) with or without hepatitis B surface antibody seroconversion, is more feasible. Because there are currently multiple direct‐acting antivirals and immune modulators in the pipeline designed to promote this (see Table 1), biomarkers for detecting and monitoring intrahepatic activity and viral persistence will be crucial.

Table 1.

Potential Hepatitis B Virus Therapies in the Pipeline Including Direct‐Acting Antivirals and Immune Modulators.

Site of Action	Drug	Company	Status
Antiviral
Entry inhibitor	Myrcludex B	Hepatera/MYR GmbH	Phase 2
cccDNA inhibitor			Preclinical
RNA silencers	AROHBV	Arrowhead	Phase 1/2a
	ARB‐1467	Arbutus	Phase 2
	ARB‐1740	Arbutus	Preclinical
	ALN‐HBV	Alnylam	Phase 1/2
	GSK 3228836	GlaxoSmithKline/Ionis	Phase 1
Core protein/capsid inhibitors	AL‐3778	Alios/Johnson & Johnson	Phase 1/2
	ABI‐H0731	Assembly Bioscience	Phase 1
	BAY 41‐4109	AiCuris (Germany)	Phase 1
	GLS4	HEC	Phase 1
	JNJ‐56136379	Johnson & Johnson	Phase 1
	AB‐423	Arbutus	Preclinical
HBsAg release inhibitors	REP 2139	Replicor	Phase 2
	REP 2165	Replicor	Phase 2
Immune modulators
TLR7 agonist	GS‐9620	Gilead Sciences	Phase 2
Therapeutic vaccines	GS‐4774	Gilead Sciences	Complete phase 2
	ABX 203	ABIVAX (France)	Phase 2/3
	AIC649	AiCuris (Germany)	Phase 1
	FB‐02.2	Altimmune	Phase 1
	INO‐1800	Inovio	Phase 1
	TG1050	Transgene (France)	Phase 1
Small molecules*
RIG‐1 NOD2 activator*	SB 9200	SpringBank Pharmaceuticals	Phase 2
SMAC inhibitor*	Birinapant	TetraLogic	Terminated
Checkpoint inhibitors*	PD‐1/PD‐2 mAb	Merck Sharp and Dohme; Bristol‐Myers Squibb
	CTLA4 mAb

Abbreviations: CTLA4, cytotoxic T lymphocyte antigen 4; mAb, monoclonal antibody; NOD2, nucleotide‐binding oligomerization domain‐containing protein 2; PD‐1, Programmed cell death protein 1; RIG‐I, retinoic acid‐inducible gene I; SMAC, second mitochondria‐derived activator of caspases; TLR7, toll‐like receptor 7.

^*Adapted with permission from Gastroenterology & Hepatology.13 Copyright 2017, Millennium Medical Publishing.

HBV Biomarkers

Hepatitis B Surface Antigen

The presence of HBsAg is the prime biomarker for diagnosing acute or chronic infection (see Table 2 and Fig. 1). HBsAg is the surface protein that envelops infectious virions (Dane particles) and is produced in excess as noninfectious subviral particles (SVPs). HBsAg is produced from episomal cccDNA and integrated HBV DNA. Decline and ultimate loss of HBsAg is the current goal of therapy, and quantification of HBsAg during therapy is important in patient management. HBsAg levels may: (1) predict the phase of infection, with the lowest levels seen in HBeAg‐negative chronic infection; (2) predict HBsAg loss in HBeAg‐negative individuals; and (3) assist with response‐guided pegylated interferon therapy.3

Table 2.

Description of Virological and Serological Markers of Acute and Chronic HBV: Their Role and Use in Diagnosis.

Serum Biomarker	Description/Source	Role	Predictor/Outcome
HBsAg (essential)	Surface/envelope protein derived from cccDNA and/or integrated DNA	Required for infectivity, excessive quantities may inhibit immune response	Initial diagnosis of: acute HB—transient HBs antigenemia; CHB—persistent HBs antigenemia (>6 months) Loss/seroconversion results in functional cure, quantification is used to monitor therapeutic outcomes
HBV DNA (essential)	Found in infectious virions	Required for replication	Used to monitor therapy and assess risk to pregnant women to avoid perinatal transmission; HBV DNA >2000 IU/mL is a key predictor of HCC risk
HBeAg (essential)	Secreted nonstructural protein derived from precore	Tolerogenicity, immune regulation, persistence	Defines phase CHB; seroconversion is one of the main therapeutic endpoints; quantification is used to predict seroconversion and therapeutic response
HBcrAg (investigational)	Composed of viral HBcAg, HBeAg, and p22cr	Combines both structural and nonstructural proteins forming empty capsids and virions	Monitoring therapy and predictor of: treatment response; HBeAg seroconversion and HBV reactivation in occult infection during immunosuppression; and risk for HCC5
HBV pgRNA (investigational)	Derived directly from the TA of intrahepatic cccDNA/minichromosome	Transcriptional template for Pol protein production and HBV replication	Assesses intrahepatic cccDNA activity, used for monitoring current and novel therapies particularly when HBV DNA is suppressed, predictor of HBeAg seroconversion6
Anti‐HBc (optional)	Reliable marker of host response to HBV exposure	Immune response to HBcAg	Indication of HBV exposure, especially useful in diagnosis of occult infection
Anti‐HBs (optional)	Host response to hepatitis B vaccination and/or after decline/loss of HBsAg	Immune response to HBsAg	Presence confirms efficacy of vaccination or achievement of functional cure in CHB
dlDNA (future)	Genomic form of DNA	Involved in integration	May have the potential to reflect intrahepatic activity and in particular risk for HCC11
spDNA/RNA (investigational/future)	Truncated variants arising from spDNA/RNA during replication	May be involved in liver disease, including HCC, and impaired response to interferon	PCR assays for detection of spDNA/RNA may reflect increased risk for HCC10

Figure 1.

Biomarkers of chronic hepatitis B. Traditional (old) markers of CHB include HBsAg, HBV DNA, and HBeAg. Recent (new) experimental markers undergoing validation include HBV RNA and HBcrAg. Future markers include dlDNA. [Colour figure can be viewed at wileyonlinelibrary.com]

HBV DNA

HBV DNA in the peripheral component reflects the production of actively replicating virus within the hepatocytes. Quantification of serum HBV DNA is essential in staging the phase of CHB (Fig. 2) and also in monitoring effectiveness of nucleos(t)ide analogue (NA) therapy. Although suppressed HBV DNA to undetectable levels is ideal, it is currently achieved only through long‐term NA treatment, which does not target the intrahepatic cccDNA (minichromosome).

Figure 2.

Five phases of chronic hepatitis B infection in a natural history setting. Abbreviation: IL, interleukin. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3.

Geographic distribution of genotypes and subgenotypes. [Colour figure can be viewed at wileyonlinelibrary.com]

Hepatitis B e Antigen

Hepatitis B e antigen (HBeAg) is a nonstructural secreted protein that has tolerogenic properties and immune‐modulating activity, and plays an essential role in persistence. Its presence is used to distinguish the phase of CHB (Fig. 2). During the natural course of infection, seroconversion from HBeAg positivity to negativity often occurs. HBeAg may also be lost or reduced with the emergence of precore and/or basal core promoter (BCP) mutations. Regardless, HBeAg seroconversion is an established therapeutic endpoint associated with reduced morbidity and mortality provided HBV DNA is low or undetectable.4 Quantification of HBeAg is available clinically in many countries and is part of all HBV drug development roadmaps.

Hepatitis B Core Antigen and the Hepatitis B Core‐Related Antigen

The hepatitis B core‐related antigen (HBcrAg) assay is composed of antibodies that can detect three proteins that share a common 149‐amino acid sequence: hepatitis B core antigen (HBcAg), the structural nucleocapsid protein that cannot be easily measured directly; HBeAg; and a putative small core‐related protein, p22cr, found in DNA‐free virion‐like (empty) particles. HBcrAg is a potentially useful marker reflecting intrahepatic replication, particularly in NA‐suppressed and HBeAg‐seroconverted cases, but appears less robust for assessing cccDNA transcriptional activity (TA).5

HBV RNA

HBV pregenomic RNA (pgRNA) produced in virions is a key replicative intermediate that provides the template for protein translation of the polymerase (Pol) and genomic replication through reverse transcription. It is generally unaffected by NA therapy which targets the HBV DNA Pol. Hence, HBV RNA quantification is proving a useful investigational biomarker for monitoring therapy when HBV DNA is suppressed because its detection in serum6 may reflect intrahepatic TA.7

Hepatitis Core Antibody (Anti‐HBc)

Antibody to HBcAg, anti‐HBc, remains the most useful marker of past HBV exposure or infection. It becomes increasingly important in the diagnosis of occult HBV infection (OBI) where HBsAg is not detectable. Titers of anti‐HBc may be useful for assessing natural history, reactivation, and for predicting therapeutic response. It also indicates a lack of need for HBV vaccination.

Hepatitis Surface Antibody (Anti‐HBs)

Anti‐HBs develops as a response to prophylactic vaccination, or as a result of HBsAg seroconversion during the natural course of HBV infection or during therapy. Current diagnostic assays only detect free circulating anti‐HBs that are not complexed with the large quantities of HBsAg associated with virions or SVPs. Further studies are warranted to determine the true viral neutralizing capacity of these circulating anti‐HBs.

Genotype and HBV Mutants

There are currently 10 genotypes of HBV, A to J, that differ by 8% to 10% at the nucleotide level across the whole genome, and more than 40 subgenotypes (4% to 8% nucleotide divergence) within most genotypes (see Table 3). HBV genotype and subtype can influence disease progression and response to antiviral therapy,8, 9 as can the presence of mutations. BCP variants may increase the risk for hepatocellular carcinoma (HCC), and a deletion in preS may be a predictor of cirrhosis.9 Sequencing for drug resistance mutations may also be needed if viral loads persist and/or increase on therapy.

Table 3.

Characteristics of Hepatitis B Virus Genotypes.

Genotypes	Subtypes	Origin/Geographic Distribution	Clinical Notes	Predominant Route of Transmission	Mutations/Recombinations	Response to Antiviral Therapy
A	A1	Africa	More likely to lose HBsAg than C and D A1 is associated with aggressive liver disease	Parenteral or sexual	Higher frequency of BCP mutations than B and D	Responds better to interferon therapy than C and D
	A2	Europe/North America
	A3	Africa/Haiti
B	B1	Japan	Lower HBsAg than A and D More likely to lose HBsAg than C and D B1 is associated with fulminant hepatitis	Perinatal	93% of strains have recombination	Responds better to interferon therapy than C and D
	B2	China
	B3	Indonesia, China, Philippines
	B4	Vietnam, Cambodia, France
	B5	Eskimos, Inuits
C	C1	Thailand, Myanmar, Vietnam	High replication capacity can result in more severe disease Slower progression to HBeAg seroconversion Lower HBsAg levels than A and D	Perinatal	Higher frequency of BCP mutations than B and D
	C2	Japan, China, Korea
	C3	New Caledonia, Polynesia
	C4	Australian aborigines
	C5‐C12	Philippines, Indonesia
	C13‐C16	Indonesia
D	D1	Middle East, Central Asia	Early HBeAg seroconversion D3 often associated with OBI	Horizontal	High prevalence of precore mutations
	D2	Europe, Lebanon
	D3	Universal
	D4	Pacific Islands, Papua New Guinea, Arctic Denes, India
	D5	India
	D6	Tunisia, Nigeria
E	None	Africa—Western and Central		Horizontal
F	F1	Argentina, Costa Rica, El Salvador, Alaska		Horizontal
	F2	Nicaragua, Venezuela, Brazil
	F3	Venezuela, Colombia
	F4	Argentina
G	None	France, Germany, United States		Horizontal: sexual transmission in men who have sex with men	Two stop codons prevent HBeAg expression; requires another genotype, usually A, to establish CHB
H	None	Mexico, Japan, Nicaragua, United States	Often associated with OBI
I	I1	Vietnam, Laos, China		Perinatal	Recombinant of A, C, G
	I2	Vietnam, Laos, India
J (putative)		Japan/Borneo

Data are compiled from Honer et al. (2017),1 Kramvis (2014),8 and Sunbul (2014).9

Biomarkers Associated With Risk for Progression

Natural History of CHB

CHB is a prolonged, dynamic infection influenced by host responses and risk factors, immune selection of emerging quasispecies and mutants, and viral genotype. The five recognized phases of infection, summarized in Fig. 2, are defined by HBsAg status, HBV DNA load, HBeAg status, and alanine aminotransferase (ALT) level.

Future Perspectives

Double‐Stranded Linear/Duplex‐Linear DNA

Double‐stranded linear/duplex‐linear DNA (dlDNA) is the genomic form of the DNA that is involved in integration. Assays for detecting dlDNA in serum may have the potential to reflect intrahepatic activity and in particular risk for HCC. Zhao et al.10 demonstrated increasing dlDNA levels in patients with cirrhosis, HCC, and while receiving interferon and/or NA therapy.

Spliced DNA/RNA

Splicing of DNA and RNA during replication may result in truncated variants which may be involved in liver disease including HCC and impaired response to interferon. PCR assays for detection of spliced DNA/RNA (spDNA/RNA) may reflect increased risk for HCC.11

Empty Particles

HBV excretes excessive quantities of genome‐free empty particles that consist of enveloped nucleocapsids, that is, HBsAg and HBcAg.12 These particles are not affected by NA therapy, and hence may prove a useful biomarker for cccDNA TA.

Conclusion

Several biomarkers are available to accurately diagnose this complex and evolving viral infection in clinical practice. However, as new therapies emerge, developmental and future biomarkers will be crucial to monitor clinical and virological outcomes.