Hepatitis B surface antigen glycan isomer as a new potential biomarker in patients with hepatitis B virus infection

Abstract

It is estimated that over 254 million people worldwide are infected with HBV, causing roughly 1.1 million deaths annually¹ as HBV infection progresses to liver cirrhosis and HCC.^{2, 3} Nucleoside/nucleotide analogs and pegylated interferon are currently available antiviral regimens for managing chronic HBV infection. However, it remains difficult to eradicate the cccDNA of HBV in hepatocyte nuclei. Thus, serological biomarkers such as HBsAg, HBV DNA, and HBcrAg have been established to estimate HBV replication activity, predict therapeutic responses, and assess the risk of HCC development.^{4, 5}

Although the currently available quantitative HBsAg assay has been proven to correlate with serum HBV DNA and intrahepatic cccDNA levels,⁶ it cannot distinguish HBsAg in terms of HBV virions and noninfectious SVPs. Hepatitis B virus envelope proteins include three distinct types of HBsAg: S-HBsAg, M-HBsAg, and L-HBsAg. The HBV particles in a patient's blood include infectious Dane particles containing viral DNA and SVPs. The SVPs are composed mainly of S-HBsAg and are 1000 times more abundant than Dane particles, which are composed primarily of M-HBsAg protein. To accurately assess disease status, HBV virions must be closely monitored and clearly distinguished from SVPs.

Hepatitis B surface antigens are heavily glycosylated with N-glycans and O-glycans.^{7, 8} Whole-glycan structural analyses have revealed that the PreS2 domain on M-HBs, but not on L-HBs, contain a highly conserved O-glycosylated site in genotype C.^{9, 10} A recombinant monoclonal antibody against this HBsAgGi was then generated using an O-glycosylated PreS2 peptide. Unlike conventional HBsAg testing, which recognizes the entirety of viral particles, HBsAgGi specifically identifies infectious HBV particles containing M-HBsAg, that is, Dane particles containing DNA and RNA. Hepatitis B antigen glycan isomer testing is now commercially available and can be measured in patient serum with an enzyme-linked immunosorbent assay kit featuring a monoclonal antibody to O-glycosylated PreS2 on M-HBsAg. Evidence on the clinical utility of HBsAgGi is now growing.

Recent studies have described correlations of HBsAgGi with other HBV-associated markers along with HBsAgGi kinetics. We earlier reported that serum HBsAgGi had stronger correlations with serum HBV DNA than did total HBsAg after excluding patients under NA therapy (r = 0.4332 vs. 0.3927).¹¹ Murata et al. showed that serum HBsAgGi was significantly associated with HBcrAg at baseline (r = 0.452, p = 0.001) and after 48 weeks of NA therapy (r = 0.388, p = 0.007).¹² Similarly, Kozuka et al. showed that serum HBsAgGi was significantly associated with iTACT-HBcrAg at baseline (r = 0.56 p < 0.001) and after 48 weeks of NA therapy (r = 0.48, p < 0.001). On the other hand, their study result showed that iTACT-HBcrAg was not significantly associated with HCC development while HBsAgGi/HBsAg ratio was useful predictor of HCC development, as discussed below.¹³ Regarding the changes in HBsAgGi by NA treatment, multiple studies showed comparable results that serum levels significantly decreased from baseline to 48 weeks of NA therapy.^11-13 It has also been reported that the HBsAgGi antibody could immunoprecipitate HBV RNA-containing particles, thereby reflecting both HBV DNA and RNA virion levels.¹²

As chronic HBV infection has been associated with an increased risk of HCC, it is of clinical importance to predict HCC development on a patient-by-patient basis, especially in individuals with genotype C, which is a known independent risk factor for HCC.¹⁴ Low HBsAg (<3.0 log IU/mL) with high HBsAgGi (>3.0 log ng/mL) levels were associated with HCC history in a cross-sectional study (HR 5.00, p = 0.004).¹¹ Later, Kozuka et al. reported on the prognostic ability of HBsAgGi / HBsAg ratio (≥1.09) for HCC development (HR 10.099, p = 0.004) in a longitudinal multivariate analysis of a NA-treated cohort independent of iTACT-HBcrAg.¹³

In recent issue in this journal, Ikeda et al. have reported the clinical utility of HBsAgGi for predicting serum HBsAg clearance.¹⁵ As seroclearance is considered the ultimate goal of chronic hepatitis B treatment, its accurate estimation is essential when evaluating patient prognosis. In Ikeda et al.'s cohort, 22 of 232 patients achieved HBsAg seroclearance. Multivariate analysis revealed quantitative HBsAgGi level as an independent predictor of this end point. The adjusted HR indicated that individuals with low HBsAgGi (≤3.5 log ng/mL) had a seroclearance likelihood over fivefold greater than did those with high HBsAgGi. In support of this, Kaplan–Meier analysis indicated that the 10-year probability of HBsAg seroclearance was 21.0% and 3.0% in patients with low and high HBsAg levels, respectively (p < 0.001). Future elucidation of the underlying mechanisms linking low HBsAgGi level with HBsAg clearance is forthcoming.

The main limitations of the HBsAgGi assay are that only genotype C can currently be measured, and that its sensitivity is lower than those of established markers. However, HBsAgGi represents a possible new assay that can specifically measure M-HBsAg. Further testing in a larger number of samples is expected to lead to the validation and ultimate implementation of this promising marker.

The authors have no conflict of interest.

Approval of the research protocol by an institutional review board: N/A.

Informed consent: N/A.

Registry and the registration no. of the study/trial: N/A.

Animal studies: N/A.