Elsevier

Journal of Hepatology

Volume 76, Issue 1, January 2022, Pages 34-45
Journal of Hepatology

Research Article
Role of anti-HBs in functional cure of HBeAg+ chronic hepatitis B patients infected with HBV genotype A

https://doi.org/10.1016/j.jhep.2021.07.031Get rights and content

Highlights

  • HBsAg-IC peaks that coincided with either an ALT flare or a rise in ALT were detected before HBs-loss in 10/14 FC patients.

  • FcγRIIIa dimer binding was detected in 9/14 FC patients, independent of detection of overlapping HBsAg-IC/ALT peaks.

  • A reduction in HBsAg epitope occupancy was observed between 12 and 24 weeks of treatment in non-FC patients.

  • Convalescent sera recognised more HBsAg epitopes and neutralized HBV infection more potently than vaccinees' sera.

  • Neutralisation potency of sera increased up to 100 weeks after HBsAg loss in 4/5 FC patients examined.

Background & Aims

HBsAg-specific antibody responses are difficult to detect during chronic hepatitis B infection (CHB) and are often overlooked. The aim of this study was to examine whether anti-HBs may be involved in functional cure (FC) by profiling anti-HBs responses in patients with CHB using a panel of specific assays.

Methods

Longitudinal serum samples were obtained from 25 patients with CHB who were infected with HBV genotype A and were undergoing nucleos(t)ide analogue (NA) treatment: 14 achieved FC while 11 remained infected (non-FC). Anti-HBs immune complexes (HBsAg-IC), FcγRIIIa dimer binding, epitope specificity and neutralisation efficacy were measured.

Results

HBsAg-IC peaks were detected prior to HBsAg loss in 10/14 FC patients. These HBsAg-IC peaks overlapped with either an alanine aminotransferase (ALT) flare (8/10 patients), or a rise in ALT (2/10 patients). HBsAg-IC peaks were detected in 7/11 non-FC patients, but were not associated with an ALT flare. FCγRIIIa binding was detected in 9/14 FC patients, independent from detection of overlapping HBsAg-IC/ALT peaks. FC patients had stable HBsAg epitope occupancy across the study, whereas non-FC patients had a reduction in HBsAg epitope occupancy within the first 12–24 weeks of NA treatment. Convalescent sera from FC patients recognised more HBsAg epitopes and neutralised HBV infection more potently than anti-HBs derived from vaccinees. Neutralisation potency appeared to increase post-HBsAg loss in 4/5 FC patients examined.

Conclusions

Using these assays, we confirm that anti-HBs responses are present and fluctuate over time in this cohort of patients with HBeAg+ CHB, who were infected with HBV genotype A and treated with NAs. Key anti-HBs profiles associated with either FC or failure to achieve FC were also identified, suggesting a role for anti-HBs responses in FC.

Lay summary

Using a panel of assays to characterise hepatitis B surface antibody (anti-HBs) responses in a group of patients with chronic hepatitis B, we identified anti-HBs profiles associated with either functional cure, or failure to achieve functional cure. Functional cure was associated with immune complex peaks which overlapped with alanine aminotransferase flares. Conversely, in those who did not achieve functional cure, immune complex peaks were present, but were not associated with alanine aminotransferase flares, and a decline in anti-HBs diversity was observed early during treatment.

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.

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