Research ArticleRole of anti-HBs in functional cure of HBeAg+ chronic hepatitis B patients infected with HBV genotype A
Graphical abstract
Introduction
Despite the success of the prophylactic hepatitis B vaccine in reducing transmission of HBV, an estimated 250 million people worldwide are still chronically infected.1 The currently approved antiviral and immune-modulating therapies can control viral replication, but complete clearance of the virus is uncommon,2 leaving patients at risk of severe hepatic complications including cirrhosis and hepatocellular carcinoma (HCC).3
A unique feature of HBV replication is the production of an enormous amount of non-infectious subviral particles (SVPs) which are comprised of the viral surface proteins (HBsAg) and are estimated to be over 10,000-fold more abundant than the infectious virions found circulating in the blood.4 SVPs are believed to render the humoral immune response ineffective in controlling infection by saturating plasma anti-HBs5 through the formation of immune complexes (HBsAg-IC), a feature that has made it difficult for researchers to detect the host's antibody response to HBsAg. Several studies have confirmed the presence of HBsAg-IC in chronic hepatitis B (CHB) using alternative detection methods[6], [7], [8], [9], [10] which have not been widely adopted, likely because of problems related to a standardised quantification method.
Functional cure (FC) of CHB is defined as clearance of HBsAg from the patient’s circulation with or without seroconversion to detectable anti-HBs, and is the endpoint of CHB treatment.11 FC can occur either spontaneously or during treatment but is very rare.11 There is strong evidence that alanine aminotransferase (ALT) flares may play an important role during FC, particularly in HBeAg-positive CHB (reviewed in12,13). Self-limiting, immune-mediated flares, described as ‘good’ flares, can occur during the treatment of non-cirrhotic patients and provide benefits including decreased viral DNA load, HBeAg seroconversion, and FC. These ALT flares can involve vigorous HLA-1 restricted, cytotoxic T lymphocyte (CTL)-mediated cytolysis of infected hepatocytes (reviewed in12), but the trigger for this CTL response, and the possible involvement of B cells and anti-HBs in ALT flares has not previously been reported.
Recently, our group reported that ALT flares were independently associated with FC in a cohort of patients with CHB infected with genotype A HBV, who were undergoing nucleos(t)ide analogue (NA) treatment.14 We also demonstrated that HBsAg clearance and the achievement of FC required the selection of serum antibodies targeting both loops of the ‘a’ determinant, described as an HBsAg clearance profile.15
In this study, a panel of assays was developed and utilised to further characterise the anti-HBs response in CHB. HBsAg-IC isolated from these samples was quantified, and the occupancy of HBsAg epitopes by anti-HBs was determined using a multiplex epitope mapping assay. The relative HBV neutralisation potency of unbound anti-HBs present in sera obtained from individuals who seroconverted was measured in parallel with HBsAg epitope specificity and FcγRIIIa dimer binding activity. Correlation of the resulting data with clinical observations and biochemical parameters revealed some previously unreported profiles which provide evidence that anti-HBs may contribute to CHB pathogenesis and clearance via multiple mechanisms.
Section snippets
Patient cohort and vaccinee recruitment
The serum samples analysed in this study were from a Gilead clinical trial of NA-treated patients (GS-US-174-0103; NCT00116805) followed-up for 196 weeks.14 All patients signed an informed consent form prior to screening and in accordance with local regulatory and ethics committee requirements. Experimental protocol in these trials was approved by Gilead Sciences and all local regulatory agencies. In this trial, 14 HBeAg+ CHB patients infected with genotype A HBV achieved functional cure and
Baseline characteristics
The FC and non-FC cohorts were closely matched with no significant differences in gender, age, BMI, treatment regimen, baseline ALT, aspartate aminotransferase or fibrosis scores. The FC cohort had significantly higher serum HBsAg (5.07 vs. 4.48 log10 IU/ml), HBeAg (3.59 vs. 2.52 log10 IU/ml) and HBV DNA levels (8.62 vs. 7.74 log10 IU/ml) than non-FC patients. There were no significant differences in baseline HBsAg-IC levels between the FC and non-FC patients (Table 1).
On-treatment HBsAg-IC peaks were associated with ALT flares in FC patients
Serum HBsAg-IC
Discussion
A possible role for anti-HBs in the pathogenesis and clearance of CHB has often been overlooked, as anti-HBs usually escapes detection by being bound to the overabundant SVPs that characterise HBV infection. By using a panel of complementary assays to profile free and bound anti-HBs before and after HBs loss we found that, in this study group, FC was associated with peaks in HBsAg-IC which preceded ALT flares as well as stable and broad anti-HBs diversity, potential induction of ADCC responses,
Financial support
Gilead Sciences funded and conducted the original clinical trial, including the collection and storage of serum samples. Gilead Sciences were not involved in the experimental design, analysis, interpretation, or the decision to submit for publication, but kindly provided clinical materials. This study is partly supported by grants from the National Health and Medical Research Council (NHMRC - GNT1127538) and from The Australian Centre for HIV and Hepatitis Virology Research (ACH2).
Authors’ contributions
HX, SL and NW designed the experiments; HX, RH, SS, DC, TH and NW performed the experiments; BW and MH contributed to the experimental design; HX, SL, RW, TS, AT and NW analysed the data; NW, HX, TS and SL wrote the manuscript. All authors critically reviewed the manuscript.
Data availability statement
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Conflict of interest
SL receives consulting fees from Aligos Therapeutics, Assembly Biosciences and Clear-B Therapeutics. SL is also involved in Institution for High Education Policy (IHep). AT receives research funding and consulting fees from Gilead Sciences, is also providing medical education to Gilead Sciences and is on the Clinical Advisory Board of Gilead Sciences. BDW and PMH receive grants from the National Health and Medical Research Council (NHMRC, GNT1145303). PR is supported by Virology Education to
Acknowledgements
We are grateful to Professor Stephan Urban at the University of Heidelberg, Germany for generously providing the HepG2-NTCP cells used in this study, to Professor Patrick Marcellin of the Viral Hepatitis Research Unit, Hospital Beaujon, Clichy, France for initial involvement in the clinical study, and to Anuj Gaggar, formerly of Gilead Sciences, Foster City, CA, USA for providing the serum samples from GS-US-174-0103 clinical trial used in this study.
References (35)
- et al.
The global burden of viral hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study 2013
Lancet (London, England)
(2016) - et al.
Hepatitis B surface antigen on subviral particles reduces the neutralizing effect of anti-HBs antibodies on hepatitis B viral particles in vitro
Virology
(2017) - et al.
Hepatitis B flares in chronic hepatitis B: pathogenesis, natural course, and management
J Hepatol
(2014) Acute flares in chronic hepatitis B: the natural and unnatural history of an immunologically mediated liver disease
Gastroenterology
(2001)- et al.
Results of a phase III clinical trial with an HBsAg-HBIG immunogenic complex therapeutic vaccine for chronic hepatitis B patients: experiences and findings
J Hepatol
(2013) - et al.
Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis
Gastroenterology
(1987) - et al.
Improved HIV-positive infant survival is correlated with high levels of HIV-specific ADCC activity in multiple cohorts
Cell Rep Med
(2021) - et al.
Hepatitis B reactivation associated with immune suppressive and biological modifier therapies: current concepts, management strategies, and future directions
Gastroenterology
(2017) - et al.
EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection
J Hepatol
(2017) - et al.
Present and future therapies of hepatitis B: from discovery to cure
Hepatology
(2015)
Hepatitis B virus morphogenesis
World J Gastroenterol
The serology of chronic hepatitis B infection revisited
J Clin Invest
Chronic liver disease: the detection and characterization of circulating immune complexes
Immunology
Experimental HBsAg/anti-HBs complex assay for prediction of HBeAg loss in chronic hepatitis B patients treated with pegylated interferon and adefovir
Antivir Ther
Analysis of viral proteins in circulating immune complexes from chronic carriers of hepatitis B virus
Clin Exp Immunol
Hepatitis B surface antigen circulating immune complexes (HBsAg-CICs) in patients with hepatitis B and asymptomatic HBsAg carriers
Clin Exp Immunol
Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance
Hepatology
Cited by (12)
Humoral immunity in hepatitis B virus infection: Rehabilitating the B in HBV
2022, JHEP ReportsCitation Excerpt :Following seroclearance of HBsAg, free HBsAb mostly appear in plasma as a hallmark of resolved CHB infection. While routine assays are not capable of detecting HBsAb-HBsAg immune complexes, PEG precipitation enabled the quantification of circulating immune complexes in a cohort of 25 patients with CHB, the kinetics of which seemed to correlate with ALT peaks and ultimate HBsAg loss.31,32 In addition, a proportion of chronic HBsAg carriers have concurrent free HBsAb in their plasma.33,34
Overview of the immunological mechanisms in hepatitis B virus reactivation: Implications for disease progression and management strategies
2024, World Journal of GastroenterologyEditorial: HBsAg seroclearance after pegylated interferon treatment—The beginning of the end. Authors' reply
2023, Alimentary Pharmacology and TherapeuticsThe Multiple Facets and Disorders of B Cell Functions in Hepatitis B Virus Infection
2023, Journal of Clinical Medicine
Author names in bold designate shared co-first authorship