Elsevier

Journal of Hepatology

Volume 57, Issue 4, October 2012, Pages 720-729
Journal of Hepatology

Research Article
Influence of mutations in hepatitis B virus surface protein on viral antigenicity and phenotype in occult HBV strains from blood donors

https://doi.org/10.1016/j.jhep.2012.05.009Get rights and content

Background & Aims

This study aimed at investigating mutations in the hepatitis B surface protein (HBsAg) in occult hepatitis B virus (HBV) infection (OBI) and their influence on viral antigenicity and phenotype.

Methods

The characteristics of 61 carriers with OBI (OBI group), 153 HBsAg(+) carriers with serum HBsAg ⩽100 IU/ml (HBsAg-L group) and 54 carriers with serum HBsAg >100 IU/ml (HBsAg-H group) from 38,499 blood donors were investigated. Mutations in the major hydrophilic region (MHR) of the viral sequences were determined. Thirteen representative MHR mutations observed in OBI sequences were antigenically characterized with a panel of monoclonal antibodies (MAbs) and commercial HBsAg immunoassays and functionally characterized in HuH7 cells and hydrodynamically injected mice.

Results

Of 61 OBI sequences, 34 (55.7%) harbored MHR mutations, which was significantly higher than the frequency in either the HBsAg-L (34.0%, p = 0.003) or the HBsAg-H group (17.1%, p <0.001). Alterations in antigenicity induced by the 13 representative MHR mutations identified in the OBI group were assessed by reacting recombinant HBV mutants with 30 different MAbs targeting various epitopes. Four out of the 13 mutations (C124R, C124Y, K141E, and D144A) strongly decreased the analytical sensitivity of seven commercial HBsAg immunoassays, and 10 (G119R, C124Y, I126S, Q129R, S136P, C139R, T140I, K141E, D144A, and G145R) significantly impaired virion and/or S protein secretion in both HuH7 cells and mice.

Conclusions

MHR mutations alter antigenicity and impair virion secretion, both of which may contribute to HBsAg detection failure in individuals with OBI.

Introduction

Hepatitis B virus (HBV) persistently infects over 350 million people worldwide and leads to hepatitis, liver cirrhosis, and hepatocellular carcinoma. The hepatitis B surface protein (HBsAg) is an envelope glycoprotein that currently serves as primary target for diagnosis and immunoprophylaxis of HBV infection. The dominant epitopes of HBsAg, which are the targets of neutralizing B cell responses, reside in the “a” determinant (aa 124–147) within the major hydrophilic region (MHR). Amino acid substitutions in the MHR can cause reduced binding of anti-HBs antibodies, resulting in immune escape. The most common MHR mutation, G145R, was initially described in 1990 [1]. The emergence of single or multiple amino acid (aa) substitutions at this and other positions within the MHR has been observed in infants born to HBsAg(+) mothers who received the HBV vaccine with or without HBIG, in liver transplant recipients who received HBIG, in patients who experienced HBsAg loss after anti-HBV therapy, and in individuals with occult HBV infection (OBI) [2], [3], [4].

Occult HBV infection is an atypical form of HBV infection, which is defined as the long-term persistence of HBV DNA, despite undetectable HBsAg by regular immunoassays [5], [6]. Since its initial description in the late ‘70s, OBI has been a challenge in hepatitis B research, with its unique virological and clinical characteristics. Evidence from different geographical regions has demonstrated that OBI was distributed worldwide and was potentially a major source of HBV transmission by transfusion and organ transplantation [7]. Acute viral reactivation may occur in association with immunosuppressive therapies and/or immunodeficiency [8]. In addition, OBI virus maintains its pro-oncogenic properties [9]. High frequencies of MHR mutations have been observed in OBI strains of individuals from Europe and Africa [10], [11]. Generally, the modified proteins produced by MHR-mutated S genes, which are not well recognized by current HBsAg immunoassays, are considered a key cause of detection failure of HBsAg in OBI. However, the influence of MHR mutations on the characteristics of viral antigenicity and phenotype, which is important for uncovering the mechanism of OBI, has not been systematically investigated.

In this study, we investigated the molecular characteristics of OBI strains from a large cohort of blood donors. Representative MHR mutations were detected and were functionally characterized both in vitro and in vivo.

Section snippets

Specimens

From 2007 to 2010, 38,499 blood donations were collected by the Xiamen Blood Service (Xiamen, China). Among these specimens, 308 were HBsAg(+) and 38,191 were HBsAg(−) by ELISA (Murex-V3, Abbott-Murex, UK). The serum HBsAg levels of HBsAg(+) specimens were determined using CMIA (Architect HBsAg, Abbott Laboratories, USA). Of the 38,191 HBsAg(−) specimens, 70 yielded repeatable positive results by Cobas Taqscreen (Roche Inc., Switzerland) or nested PCR, as described elsewhere [4]. Anti-HBs and

MHR mutants in blood donors with OBI

The MHR (aa 110–160) of the viral S gene was successfully amplified and sequenced in 61 out of 70 OBI samples (OBI group) and in 207 out of 308 HBsAg(+) samples. These specimens were used for further molecular characterization. When HBsAg(+) individuals were divided into two groups according to their HBsAg level by a cut-off value of 100 IU/ml, which was widely recognized as a predictor of HBsAg seroclearance in CHB [14], [15], [16], 153 donors had a HBsAg level ⩽100 IU/ml (HBsAg-L group) and the

Discussion

In the present study, we observed a prevalence of OBI of 0.18% in a large cohort of blood donors in Xiamen (China), a highly HBV-endemic area with a documented HBsAg prevalence of 10.9% [17]. In viral sequences isolated from OBI samples, we observed a high MHR mutation frequency (55.7%), which was significantly higher than in the HBsAg(+) group. High mutation frequencies in the MHR have also been observed in OBI strains from Europe and Africa [10], [11]. Our analyses revealed that the MHR

Financial support

This work was supported by grants from the Excellent Youth Foundation of the Fujian Scientific Committee (Grant No. 2009J06020), the National Science Fund for Distinguished Young Scholars (30925030) and the National Scientific and Technological Major Project (2012ZX10002-005/2012ZX10004-503).

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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