Hepatitis B Surface Antigen Loss: Too Little, Too Late and the Challenge for the Future

Abstract

See “Factors associated with rates of HBsAg seroclearance in adults with chronic HBV infection: a systematic review and meta-analysis,” by Yeo YH, Ho HJ, Yang H-I, et al, on page 635. See “Factors associated with rates of HBsAg seroclearance in adults with chronic HBV infection: a systematic review and meta-analysis,” by Yeo YH, Ho HJ, Yang H-I, et al, on page 635. Detectable hepatitis B surface antigen (HBsAg) is the serological hallmark of persistent hepatitis B virus (HBV) infection and disease. Loss of HBsAg signifies a favorable outcome of the natural history, particularly if HBsAg loss occurs before the accrual of significant liver disease. Loss of HBsAg has been defined, for reference, as a functional “cure.” However, HBsAg is infrequently cleared in those with chronic hepatitis B. In the current issue of Gastroenterology, Yeo et al1Yeo Y.H. Ho H.J. Yang H.-I. et al.Factors associated with rates of HBsAg seroclearance in adults with chronic HBV infection: a systematic review and meta-analysis.Gastroenterology. 2019; 156: 635-646Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar have performed a systematic review and meta-analysis of HBsAg clearance rates and predictors of clearance. Of 42,588 patients, 3194 cleared HBsAg. The pooled annual rate of HBsAg seroclearance was 1.02% (95% confidence interval, 0.79–1.27). Favorable factors for HBsAg loss included hepatitis B e antigen (HBeAg) negativity, a lower quantitative HBsAg level, and lower HBV DNA concentrations at baseline. Numerically, genotype A had the highest HBsAg seroclearance rates. No marked regional differences were noted, but seroclearance rates were numerically higher in community versus health service-based cohorts. Treatment had little effect, although higher HBsAg seroclearance rates were observed in interferon-treated patients than nucleos(t)ide analogue (NUC) recipients. The authors’ detailed analysis extracts a comprehensive body of data and is the last word on the subject. Although there was substantial statistical heterogeneity in the pooled cohorts, the data verify that HBsAg seroclearance rates are low in both untreated and treated patients. It should not come as a surprise that HBsAg loss is a rare event, based on prior data.2Liu J. Yang H.I. Lee M.H. et al.Spontaneous seroclearance of hepatitis B seromarkers and subsequent risk of hepatocellular carcinoma.Gut. 2014; 63: 1648-1657Crossref PubMed Scopus (124) Google Scholar, 3Lim T.H. Gane E. Moyes C. et al.HBsAg loss in a New Zealand community study with 28-year follow-up: rates, predictors and long-term outcomes.Hepatol Int. 2016; 10: 829-837Crossref PubMed Scopus (16) Google Scholar Although the cumulative incidence rate increased to 17.9% at 15 years (95% confidence interval, 6.18–23.24), the confidence intervals are wide. The inclusion of treated patients may be somewhat problematic, because untreated patients are likely to have low baseline levels of HBV DNA, whereas treated patients have more active disease. The fact that those with decreasing HBsAg levels are more likely to become undetectable for HBsAg is something of a self-fulfilling prophecy. What are the implications and how can this substantive analysis be leveraged? HBsAg loss is regarded as the ideal end point of therapy; however, although considered a functional cure of chronic hepatitis B, it is recognized that HBV viral genomes endure in the liver even if HBsAg is undetectable. What then is the value of HBsAg loss? It is important to emphasize that the clinical outcomes are likely to be more favorable if HBsAg loss occurs at a young age. In a study in New Zealand, spontaneous HBsAg loss occurred in 33% of patients after 12,702 person-years of follow-up (1.14 per 100 person-years); liver stiffness measurements were lower if HBsAg loss occurred before the age of 50 years.3Lim T.H. Gane E. Moyes C. et al.HBsAg loss in a New Zealand community study with 28-year follow-up: rates, predictors and long-term outcomes.Hepatol Int. 2016; 10: 829-837Crossref PubMed Scopus (16) Google Scholar Conversely HBsAg loss, after the onset of significant hepatic fibrosis, does not obviate the risk of hepatocellular carcinoma.4Simonetti J. Bulkow L. McMahon B.J. et al.Clearance of hepatitis B surface antigen and risk of hepatocellular carcinoma in a cohort chronically infected with hepatitis B virus.Hepatology. 2010; 51: 1531-1537Crossref PubMed Scopus (197) Google Scholar In the REVEAL-HBV studies the incidence of hepatocellular carcinoma was 93.7 in 100,000 person-years in those who achieved HBsAg clearance compared with 106.2 in 100,000 person-years in those who did not.2Liu J. Yang H.I. Lee M.H. et al.Spontaneous seroclearance of hepatitis B seromarkers and subsequent risk of hepatocellular carcinoma.Gut. 2014; 63: 1648-1657Crossref PubMed Scopus (124) Google Scholar The independent effect of undetectable or low HBV DNA and HBsAg clearance on hepatocellular carcinoma risk can be difficult to tease out, but there may be added value to HBsAg loss in NUC-treated patients.5Yip T.C. Wong G.L. Chan H.L. et al.HBsAg seroclearance further reduces hepatocellular carcinoma risk after complete viral suppression with nucleos(t)ide analogues.J Hepatol. 2018 Oct 28; ([Epub ahead of print])Abstract Full Text Full Text PDF Scopus (118) Google Scholar Given the low rates of HBsAg loss with treatment, what therapeutic interventions may enhance HBsAg seroclearance? Several strategies are being used, including cessation of NUCs. In a recent controlled trial of cessation of tenofovir, 19% of patients achieved HBsAg loss after 144 weeks follow-up.6Berg T. Simon K.G. Mauss S. et al.Long-term response after stopping tenofovir disoproxil fumarate in non-cirrhotic HBeAg-negative patients - FINITE study.J Hepatol. 2017; 67: 918-924Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar Withdrawal often results in viral relapse, however, and such a strategy will only be applicable in patients without severe liver disease.7Bourliere M. Rabiega P. Ganne-Carrie N. et al.Effect on HBs antigen clearance of addition of pegylated interferon alfa-2a to nucleos(t)ide analogue therapy versus nucleos(t)ide analogue therapy alone in patients with HBe antigen-negative chronic hepatitis B and sustained undetectable plasma hepatitis B virus DNA: a randomised, controlled, open-label trial.Lancet Gastroenterol Hepatol. 2017; 2: 177-188Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 8Van Hees S. Bourgeois S. Van Vlierberghe H. et al.Stopping nucleos(t)ide analogue treatment in Caucasian hepatitis B patients after HBeAg seroconversion is associated with high relapse rates and fatal outcomes.Aliment Pharmacol Ther. 2018; 47: 1170-1180Crossref PubMed Scopus (40) Google Scholar, 9van Bommel F. Berg T. Stopping long-term treatment with nucleos(t)ide analogues is a favourable option for selected patients with HBeAg-negative chronic hepatitis B.Liver Int. 2018; 38: 90-96Crossref PubMed Scopus (27) Google Scholar Quantitative HBsAg may be valuable in selecting patients who could benefit and who may require retreatment.10Jeng W.J. Chen Y.C. Chien R.N. et al.Incidence and predictors of hepatitis B surface antigen seroclearance after cessation of nucleos(t)ide analogue therapy in hepatitis B e antigen-negative chronic hepatitis B.Hepatology. 2018; 68: 425-434Crossref PubMed Scopus (164) Google Scholar, 11Liaw Y.F. Jeng W.J. Chang M.L. HBsAg Kinetics in retreatment decision for off-therapy hepatitis B flare in HBeAg-negative patients.Gastroenterology. 2018; 154: 2280-2281Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Pegylated interferon treatment may also enhance the risk of HBsAg loss, as highlighted in the analysis. Both switch to and add-on strategies combining interferon and NUCs have been assessed.12Qiu K. Liu B. Li S.Y. et al.Systematic review with meta-analysis: combination treatment of regimens based on pegylated interferon for chronic hepatitis B focusing on hepatitis B surface antigen clearance.Aliment Pharmacol Ther. 2018; 47: 1340-1348Crossref PubMed Scopus (38) Google Scholar Again, a low quantitative HBsAg and HBV DNA is favorable, and an early response seems to determine the rates of HBsAg loss. Newer markers including quantitative hepatitis B core-related antigen and HBV pregenomic (pgRNA) could be used to predict the sequence of HBeAg and HBsAg loss during treatment.13Martinot-Peignoux M. Lapalus M. Maylin S. et al.Baseline HBsAg and HBcrAg titres allow peginterferon-based ‘precision medicine' in HBeAg-negative chronic hepatitis B patients.J Viral Hepat. 2016; 23: 905-911Crossref PubMed Scopus (44) Google Scholar, 14Wang B. Carey I. Bruce M. et al.HBsAg and HBcrAg as predictors of HBeAg seroconversion in HBeAg-positive patients treated with nucleos(t)ide analogues.J Viral Hepat. 2018; 25: 886-893Crossref PubMed Scopus (33) Google Scholar If HBsAg seroclearance is associated with improved prognosis, what are the mechanistic considerations we need to understand to improve rates of loss? HBV envelope variability may play a role, because more frequent mutations in the major hydrophilic region have been observed in those who have lost HBsAg (such mutations may have a key role in immune recognition and antigenicity).15Eschlimann M. Malve B. Velay A. et al.The variability of hepatitis B envelope is associated with HBs antigen persistence in either chronic or acute HBV genotype A infection.J Clin Virol. 2017; 94: 115-122Crossref PubMed Scopus (5) Google Scholar Patients with detectable basal core promoter/precore variants and higher viral diversity showed a lower probability of HBsAg loss during NUC therapy.16Bayliss J. Yuen L. Rosenberg G. et al.Deep sequencing shows that HBV basal core promoter and precore variants reduce the likelihood of HBsAg loss following tenofovir disoproxil fumarate therapy in HBeAg-positive chronic hepatitis B.Gut. 2016; 66: 2013-2023Crossref PubMed Scopus (41) Google Scholar Increased natural killer cell functionality and HBV-specific T-cell responsiveness have been shown in patients experiencing HBsAg seroclearance after stopping NUC therapy.17Zimmer C.L. Rinker F. Honer Zu Siederdissen C. et al.Increased NK cell function after cessation of long-term nucleos(t)ide analogue treatment in chronic hepatitis B is associated with liver damage and HBsAg loss.J Infect Dis. 2018; 217: 1656-1666Crossref PubMed Scopus (43) Google Scholar, 18Rinker F. Zimmer C.L. Honer Zu Siederdissen C. et al.Hepatitis B virus-specific T cell responses after stopping nucleos(t)ide analogue therapy in HBeAg-negative chronic hepatitis B.J Hepatol. 2018; 69: 584-593Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Finally, what is the source of the enduring HBsAg, especially in patients without a detectable viral load? It is apparent that HBsAg can be transcribed from covalently closed circular DNA and integrated viral genomes (Figure 1). Loss of HBeAg is usually associated with a 3–4 log10 decrease in HBV DNA, whereas HBsAg concentrations are reduced by only 1 log10. In HBeAg-negative patients, the relatively smaller reduction of S gene messenger RNA than of pgRNA (suggesting transcription from integrated sequences) could explain why HBsAg remains high.19Larsson S.B. Malmstrom S. Hannoun C. et al.Mechanisms downstream of reverse transcription reduce serum levels of HBV DNA but not of HBsAg in chronic hepatitis B virus infection.Virol J. 2015; 12: 213Crossref PubMed Scopus (13) Google Scholar HBsAg expression has been found to correlate negatively with methylation of HBV DNA in HBsAg transgenic mice. Interestingly, loss of HBsAg expression ameliorates cell stress and liver integrity.20Zhang Y. Mao R. Yan R. et al.Transcription of hepatitis B virus covalently closed circular DNA Is regulated by CpG methylation during chronic infection.PLoS One. 2014; 9: e110442Crossref PubMed Scopus (64) Google Scholar, 21Graumann F. Churin Y. Tschuschner A. et al.Genomic methylation inhibits expression of hepatitis B virus envelope protein in transgenic mice: a non-infectious mouse model to study silencing of HBV surface antigen genes.PLoS One. 2015; 10: e0146099Crossref PubMed Scopus (10) Google Scholar Integrated viral genomes and integrant-derived RNAs are abundant in malignant liver tissue, suggesting that integrant RNAs are a significant source of HBV envelope protein independent of viral replication, explaining why treatment with interferon or NUCs fail to achieve a loss of HBsAg. RNA interference-based treatment of chronically infected patients and chimpanzees reveals that integrated HBV DNA is a major source of HBsAg, particularly in HBeAg-negative patients.22Freitas N. Lukash T. Gunewardena S. et al.Relative Abundance of integrant-derived viral RNAs in infected tissues harvested from chronic hepatitis B virus carriers.J Virol. 2018; 92Crossref PubMed Google Scholar, 23Wooddell C.I. Yuen M.F. Chan H.L. et al.RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.Sci Transl Med. 2017; 9Crossref PubMed Scopus (273) Google Scholar This “underrecognized” source of HBsAg, hiding in plain sight, has therapeutic implications. A deeper understanding will be required to identify differences in the composition and source of subviral particles of HBsAg derived from covalently closed circular DNA versus integrated HBV DNA.24Peiffer K.H. Kuhnhenn L. Jiang B. et al.Divergent preS Sequences in virion-associated hepatitis B virus genomes and subviral HBV surface antigen particles from HBV e antigen-negative patients.J Infect Dis. 2018; 218: 114-123Crossref PubMed Scopus (32) Google Scholar The estimated HBsAg loss reported in this meta-analysis by Yeo et al,1Yeo Y.H. Ho H.J. Yang H.-I. et al.Factors associated with rates of HBsAg seroclearance in adults with chronic HBV infection: a systematic review and meta-analysis.Gastroenterology. 2019; 156: 635-646Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar and the results reported in trials of pegylated interferon and NUCs, or after NUC cessation, provide a benchmark against which future clinical trials on functional cure will be designed. We are entering a new era, where the objective of achieving higher rates of HBsAg loss at an early stage of disease is the goal. High HBsAg antigen loads imprint on the host immune response. New therapeutic strategies and innovative technologies, including enhanced infected cell loss, gene editing, and targeted antiviral drugs, are required to decrease the continuing cache of HBsAg emanating from a hitherto relatively inaccessible store. Several potential strategies exist to achieve higher rates of HBsAg loss with treatment of a finite duration, which suggest several editorialized projections of where the field may go.•Covalently closed circular DNA is not eliminated by treatment with NUCs, which have less effect on HBV RNA and HBsAg, despite inhibiting HBV replication.•Novel capsid assembly modulators deepen inhibition of HBV replication by disrupting crucial early and late states of the HBV life cycle; these agents cause a decrease in HBV DNA and HBV RNA, but have a limited effect on HBsAg after 28 days and will require a longer duration of therapy to reduce HBsAg.•Sequential combinatorial therapy ideally should cause a precipitous decrease in HBsAg that, when followed by an immunomodulatory therapy, leads to sustained HBsAg loss.•Indeed, GalNac small interfering RNA knockdown in transgenic and transduced mice of HBsAg followed by an adjuvant therapeutic vaccine provides an elegantly choreographed proof of concept.25Michler T. Kosinska A. Grimm D. et al.Suppression of hepatitis B Virus antigens by RNA interference enables therapeutic vaccination to achieve immune control in high-titer virus carriers.Hepatology. 2018; 68Google Scholar These data require further clinical experimentation in humans with chronic hepatitis B.•Nucleic acid polymers, which block assembly and release of subviral particles, followed by pegylated interferon result in marked HBsAg reduction (and anti-HBs development), but require further validation and safety testing.26Bazinet M. Pantea V. Placinta G. et al.Establishment of high rates of functional control and reversal of fibrosis following treatment of HBeAg negative chronic HBV infection with REP 2139-Mg / REP 2165-Mg, Tenofovir disoproxil fumarate and pegylated interferon alpha-2a.Hepatology. 2018; 68: 234APubMed Google Scholar•Several immunomodulatory strategies including RIG-1 agonism or oral Toll-like receptor agonists or check point inhibition could trigger effective innate and adaptive immune responses after HBsAg reduction.•These strategies will need tailoring to the immunologic phenotype in patients with high and lower levels of HBV replication, to adjudge their effectiveness in different populations and at different stages of disease. Factors Associated With Rates of HBsAg Seroclearance in Adults With Chronic HBV Infection: A Systematic Review and Meta-analysisGastroenterologyVol. 156Issue 3PreviewSeroclearance of hepatitis B surface antigen (HBsAg) is a marker for clearance of chronic hepatitis B virus (HBV) infection, but reported annual incidence rates of HBsAg seroclearance vary. We performed a systematic review and meta-analysis to provide more precise estimates of HBsAg seroclearance rates among subgroups and populations. Full-Text PDF