Hepatitis B surface antigen levels during the natural history of chronic hepatitis B: A perspective on Asia
"In conclusion, this study demonstrates significant differences in the baseline serum HBsAg titres across the different phases of CHB infection. Quantitative HBsAg assays are non invasive, easy to perform and relatively inexpensive. Understanding the changing HBsAg titres throughout the natural history of CHB represent a step forward in further investigating the HBV viral life cycle and the influence of the host immune response. Baseline HBsAg quantification may help refine future treatment algorithms for both immune-modulator therapy and oral nucleos(t)ide analogue therapy. Larger prospective studies are now required to evaluate longitudinal changes in serum HBsAg, and evaluating their significance in predicting the ultimate goal of antiviral therapy, HBsAg seroconversion."
Jnl of Hepatology
Volume 52, Issue 4, Pages 508-513 (April 2010)
Tin Nguyen12, Alexander J.V. Thompson12, Scott Bowden1, Catherine Croagh2, Sally Bell2, Paul V. Desmond2, Miriam Levy3, Stephen A. Locarnini1
Received 30 August 2009; received in revised form 20 October 2009; accepted 22 October 2009. published online 17 February 2010.
Refers to article:
A new role for an old marker, HBsAg , 01 February 2010
Maurizia Rossana Brunetto
Journal of Hepatology
April 2010 (Vol. 52, Issue 4, Pages 475-477)
Background & Aims
Data from clinical trials suggest a potential role for on-treatment monitoring of serum HBsAg titres during interferon-alpha (pegIFN) therapy in predicting virological responses. However, baseline HBsAg titres during the natural history of chronic hepatitis B (CHB) have not been well-characterized. We aimed to define the serum HBsAg titres during the different phases of CHB in a cohort of Asian patients infected with either genotype B or C HBV.
Two-hundred and twenty patients were classified into immune-tolerant (IT), immune-clearance (IC), non/low-replicative (LR) or hepatitis B e antigen negative hepatitis (ENH) phases. Serum HBsAg was quantified using the ARCHITECT platform (Abbott Laboratories, Chicago, USA). Correlation of HBsAg titre with HBV DNA and serum ALT within each phase of infection was performed.
Median HBsAg titres were different between each phase of CHB (p=0.001): IT (4.53 log10IU/ml), IC (4.03 log10IU/ml), LR (2.86 log10IU/ml), and ENH (3.35 log10IU/ml). HBsAg titres were highest in the IT phase, and lowest in the LR phase. In general, median HBsAg titres were similar between genotypes B and C HBV. Serum HBsAg titres only correlated with HBV viral load in the IC phase. No correlation between the serum HBsAg level and ALT was observed.
This study demonstrated significant differences in median baseline serum HBsAg titres across the different phases of CHB. These results provide further insight into the HBV viral life cycle in the setting of the various phases of CHB. Baseline HBsAg quantification may help refine future treatment algorithms for both immune-modulator therapy and oral nucleos(t)ide analogue therapy.
The natural history of chronic hepatitis B (CHB) is typically regarded as consisting of four phases , ; immune-tolerant (IT), immune-clearance (IC), non/low-replicative (LR), and hepatitis B e antigen negative hepatitis (ENH). These phases have been classified by specific biochemical, serological and virological characteristics, including serum ALT levels, hepatitis B e antigen (HBeAg) serostatus, and hepatitis B virus DNA (HBV DNA) titre. It is important to note that these phases do not occur in all individuals, and do not always necessarily occur sequentially .
The understanding of the pathogenesis and natural history of CHB continues to evolve. This has been facilitated by the improved sensitivity of HBV DNA viral load assays, and the development of assays for the detection and measurement of HBV intrahepatic replicating forms of the virus such as covalently closed circular DNA (cccDNA) and other replicative intermediates , . Recently, sensitive and reliable assays have also been developed to quantify both serum hepatitis B surface antigen (HBsAg) and HBeAg.
HBsAg seroclearance represents the preferred endpoint of therapy for CHB, as it is believed to represent successful immunological control of active HBV replication. Recent data from clinical trials suggest a potential role for on-treatment monitoring of serum HBsAg and HBeAg titres during interferon-alpha (pegIFN) therapy in predicting virological responses , . However, baseline HBsAg titres have not been well-characterized in each phase of CHB, particularly in the IT and LR phases. The main objective of this study was to determine the serum HBsAg titres during the different phases of the natural history of CHB in a cohort of Asian patients infected with genotype B or C HBV.
A cross-sectional study was performed in patients with CHB from two tertiary hospitals (St. Vincent's Hospital Melbourne and Liverpool Hospital Sydney, Australia). Patients included from Liverpool hospital were a cohort of pregnant women. Only patients with genotype B or C HBV infection were included. All patients tested negative for markers of hepatitis C virus, hepatitis D virus, and human immunodeficiency virus (HIV). Markers for co-existent auto-immune or metabolic liver disease were negative.
Patient demographics, liver biochemistries, qualitative HBsAg and HBeAg status (by standard qualitative enzyme immunoassay), HBV DNA, and HBV genotype  were recorded. Biochemical and virological data were obtained from patient serum samples collected on the same day.
Patients were classified into a phase of CHB at the centers involved in the study after a follow-up period of 3-6months. The phase of CHB in each patient was determined by HBeAg/anti-HBe serostatus, and measurement of HBV DNA and serum ALT levels according to the recently published European Association for the Study of the Liver (EASL) clinical practice guidelines . The IT phase was defined as: HBeAg positive, high viral load, serum ALT <2X upper limit normal (ULN). The IC phase was defined as: HBeAg positive, elevated viral load, serum ALT >2X ULN. The LR phase was defined as; HBeAg negative, HBV DNA <2000IU/ml, normal serum ALT. The ENH phase was defined as: HBeAg negative, HBV DNA >2000IU/ml, serum ALT >2X ULN. For this study, the cut-off for a normal ALT was ∅30Units/L.
The study was conducted according to the guidelines of the Declaration of Helsinki, and was approved by the local institutional ethics research committee.
Quantitative serum HBsAg assay
Serum HBsAg was quantified using the ARCHITECT platform (Abbott Laboratories, Chicago, USA), as per the manufacturer's instructions. The ARCHITECT quantitative HBsAg assay is a chemiluminescent microparticle assay, internally calibrated using the World Health Organisation (WHO) standard for HBsAg . Quantitative HBsAg levels are reported in IU/ml, with a dynamic range of 0.05-250IU/ml. Given that most serum HBsAg titres are above this range, samples were initially tested at dilutions of 1 in 100 or 1 in 1000. ARCHITECT HBsAg Manual Diluent (Abbott Diagnostics) was used to dilute patient sera. This diluent contains recalcified human plasma that is non-reactive for HBsAg, HIV-1 RNA or HIV-1 Ag, anti-HIV-1/HIV-2, anti-HCV, or anti-HBs.
HBV DNA viral load
HBV DNA viral load testing was performed using the Versant HBV DNA 3.0 (Bayer Diagnostics, Emeryville, CA, USA), according to the manufacturer's instructions (dynamic range 3.5X102-1.8X107IU/ml). Samples with a viral load above the upper limit of the dynamic range were diluted by 103 to obtain a defined titre. ARCHITECT HBsAg Manual Diluent (Abbott Diagnostics) was used to dilute patient sera.
Genotyping (HBV genotypes A-H) was performed in 200/220 (91%) patients using methods as previously described . Genotyping was not possible in 20 patients in the LR phase due to insufficient HBV DNA. Briefly, HBV DNA was extracted from 200µl of patient serum using the QIAamp DNA MiniKit (QIAGEN, CA, USA) according to the manufacturer's instructions. Oligonucleotides were synthesised by Geneworks, Adelaide, Australia. For amplification of the polymerase gene the sense primer was 1877a (nt 1877-1996, CCT GCT GGT GGC TCC AGT TC) and the antisense primer 2996 (nt 2996-3014, GCG TCA GCA AAC ACT TGG C) was used. The amplified HBV envelope/surface gene was purified using PCR purification columns from MO BIO Laboratories Inc. and directly sequenced using Big Dye terminator Cycle sequencing Ready Reaction Kit Version 3.1 (Perkin-Elmer, Cetus Norwalk, CT). HBV consensus sequences were constructed using the DNA sequence analysis program Seqscape (Applied Biosystems, USA). HBV genotype was determined using a web-based program, SeqHepB (http://www.seqvirology.com/genome7/index.htm) . This program analyses HBV DNA to determine HBV genotype, to identify key mutations associated with antiviral resistance as well as other clinically important HBV variants by comparing the input sequence data with known HBV reference sequences.
Continuous and categorical variables were compared between groups, using the Mann-Whitney test and Kruskall-Wallis ANOVA for non-parametric continuous data, and χ2/Fisher's exact test for categorial data. Pearson's correlation coefficient (r) was used to describe the correlation between two variables. Statistical analysis was performed using GraphPad Software, San Diego California USA, www.graphpad.com.
Two-hundred and twenty treatment naïve patients were recruited. Patients from Liverpool Hospital, Sydney comprised a cohort of pregnant women (n=70). The classification of patients into respective phases of CHB were: IT (n=32), IC (n=55), LR (n=50), ENH (n=83).
The baseline patient characteristics are presented in Table 1. All patients were of Asian ethnicity and were infected with either genotype B or C HBV. There were overall more females in the study group (56%). HBeAg positive patients were younger than HBeAg negative patients (p<0.001). In HBeAg positive patients, there was no significant age difference between those in the immune-tolerant and immune-clearance phases. It should be noted that the participating hospitals did not include children or adolescents with CHB in this study. Patients in the ENH phase were older than those in the LR (p=0.02).
Population distribution of HBsAg titre
The distribution of serum HBsAg levels across the study population was evaluated (Fig. 1). HBsAg titres were different between each phase of CHB (p=0.001). The median HBsAg titres in each phase of CHB were: IT (4.53 log10IU/ml), IC (4.03 log10IU/ml), LR (2.86 log10IU/ml), and ENH (3.35 log10IU/ml), respectively. There was no difference in median HBsAg titres between pregnant (n=14) and non-pregnant (n=18) immune-tolerant cohorts (p=0.055). The median HBsAg in the LR and ENH were significantly lower than both HBeAg positive phases.
Correlation of serum HBsAg titres with serum ALT levels and HBV viral load
There was no observed statistical correlation between HBsAg titres with serum ALT levels in any phase of CHB. The correlation between serum HBsAg titres with serum HBV DNA in each phase of CHB is presented in Fig. 2A-D. There was a modest correlation observed in the IC phase (r=0.77, p=0.0001), but poor correlation between serum HBsAg and HBV DNA in either the IT (r=0.30, p=0.09), LR (r=0.22, p=0.11) or ENH (r=0.29, p=0.008).
The ratio of HBsAg (log10IU/ml) to HBV DNA (log10IU/ml) in each phase of CHB was also examined (Fig. 3). The HBsAg/HBV DNA ratio was significantly higher in the low-replicative phase compared to immune-tolerant, immune-clearance and HBeAg negative phases, respectively (1.05 vs 0.55, 0.55, 0.64. p<0.0001).
Serum HBsAg titres between genotypes B and C
The distribution of serum HBsAg titres in different phases of CHB was evaluated based on viral genotype (Fig. 4). In each viral genotype, median HBsAg titres were clearly different in each phase of CHB. The median serum HBsAg titre in the LR phase was lower in genotype B (2.24 log10IU/ml) compared to genotype C (3.34 log10IU/ml). However, it should be noted that viral genotype was not available in all patients in this phase as some patients had insufficient HBV DNA to allow for genotype determination by sequencing.
This study aimed to evaluate the baseline serum HBsAg titres in different phases of CHB infection. All patients were treatment naïve and chronically infected with either genotype B or C HBV, and were of Asian ethnicity. This study demonstrates that serum HBsAg titres differ between the four phases of CHB. This finding was also clearly demonstrated in an accompanying article by Jaroszewicz et al., which examined HBsAg levels in a predominantly European cohort (HBV genotypes A and D). In our article, HBsAg titres were higher in HBeAg positive compared to HBeAg negative CHB. The lowest HBsAg titres were evident in the low-replicative phase. The difference between median lowest and highest HBsAg titres in the different phases was less than 2 log10IU/ml, far smaller than that observed with changes in HBV DNA levels. Other than in the low-replicative phase, there was no difference in median HBsAg titres between genotypes B and C HBV. Genotyping was only performed in a subset of patients in the low-replicative phase due to insufficient HBV DNA.
Interest in quantitative HBsAg serology as a clinical biomarker has been based upon studies which showed a positive association with intrahepatic cccDNA levels ,  and HBV DNA , . HBV DNA quantification is currently the standard in selecting patients who are candidates for therapy, monitoring response to therapy, and detecting the emergence of drug resistance. Compared to HBV DNA, the assays for HBsAg quantification are less expensive, and are also fully automated with a high throughput capacity. However, the utility of HBsAg titres as a reliable surrogate for both cccDNA and HBV DNA remains unclear, as studies have also shown a poor correlation with HBV cccDNA , and only a positive correlation with HBV DNA in HBeAg positive CHB .
An understanding of HBsAg titre changes throughout HBV infection may provide some potentially useful insights into hepatitis B pathogenesis and viral life cycle. The mechanisms underlying HBsAg and viral replication during different phases of CHB are currently unclear. This current study observed a modest correlation of serum HBsAg with HBV DNA in the immune-clearance phase of CHB (r=0.77, p=0.0001). No correlation was observed in the IT, IC or ENH phases. Furthermore, the ratio of HBsAg to HBV DNA was significantly higher in the low-replicative phase compared to all other phases (1.05 vs 0.55, 0.55, 0.64. p<0.0001), a finding which is in accordance with previous studies . The apparent "disconnect" between HBsAg and HBV DNA at different phases may possibly be due to the processes of integrated viral envelope sequences. A second possible explanation is a different in the regulation of viral replication during different phases of infection, resulting in altered ratios of HBV virion to sub-viral HBsAg particles .
HBsAg synthesis during the HBV viral life cycle is complex, and typically occurs at the endoplasmic reticulum (Fig. 5). The envelope open reading frame (ORF) contains three in frame "start" codons which further divide it into preS1, preS2, and ORF-S domains. Envelope proteins are generated from two HBV mRNA transcripts, with subsequent translation resulting in small (ORF-S), medium (pres2+ORF-S) and large surface envelope proteins (preS1+preS2+ORF-S). These are also known as L, M and S surface proteins, respectively. Newly synthesised envelope protein interacts with mature HBV nucleocapsids at the endoplasmic reticulum prior to secretion from the hepatocyte. However, HBsAg production far exceeds that required for virion assembly, and excess surface envelope proteins are covalently linked by intermolecular disulphide bonds and secreted as non infectious filamentous or spherical sub-viral particles . These sub-viral particles may play a role in evading the host immune response , and may also co-exist with anti-HBs as part of circulating immune complexes . It is important to appreciate that whilst HBsAg quantification detects all three forms of systemic HBsAg (part of HBV virion, spherical, filamentous), differentiation between the relative proportions is currently not routine.
HBsAg may also be produced from HBV DNA integrated into the host genome. Although viral integration is an essential component of the life cycle of retroviruses such as HIV, it is not required for normal productive hepadnaviral infection. Rather, integration of HBV DNA occurs illegitimately through recombination mechanisms using host enzymes from double-stranded linear (DSL) HBV DNA (Fig. 5) , . In HBV infection, viral integration does seem to occur early in infection. Whilst HBV integration is believed to be a random event, a high preference for integration occurs at the DR1 and DR2 sequences on the HBV genome . Integrated sequences cannot provide a template for productive viral replication as a complete genome is not present . However, given that sequences of the S genes of the enhancer I elements are often present in integrated segments, HBsAg may be produced .
The role of quantitative HBsAg in predicting response to peginterferon therapy has been the focus of several recent studies , , , . In HBeAg negative patients treated with pegylated interferon, the decline in HBsAg titre at week 12 and 24 has been shown to be a useful predictor of achieving an undetectable viral load at 24weeks post therapy . The results of this current study may have implications for future treatment algorithms evaluating the on-treatment decline of HBsAg titres in both HBeAg positive and negative patients. For example, the median baseline serum HBsAg titre in the LR phase was 2.86 log10IU/ml. Future clinical trials could evaluate whether an on-treatment HBsAg titre decline to these levels is a predictor of durable suppression of viral replication.
A limitation of this study was its cross-sectional design, as it would have been useful to follow patients longitudinally through different phases of infection. However, such longitudinal follow-up is difficult given patients can remain in either the immune-tolerant or non/low-replicative phases for years, and patients in the immune-clearance and HBeAg negative hepatitis phases are potential treatment candidates.
In conclusion, this study demonstrates significant differences in the baseline serum HBsAg titres across the different phases of CHB infection. Quantitative HBsAg assays are non invasive, easy to perform and relatively inexpensive. Understanding the changing HBsAg titres throughout the natural history of CHB represent a step forward in further investigating the HBV viral life cycle and the influence of the host immune response. Baseline HBsAg quantification may help refine future treatment algorithms for both immune-modulator therapy and oral nucleos(t)ide analogue therapy. Larger prospective studies are now required to evaluate longitudinal changes in serum HBsAg, and evaluating their significance in predicting the ultimate goal of antiviral therapy, HBsAg seroconversion.