Prediction of conformational changes by single mutation in the hepatitis B virus surface antigen (HBsAg) identified in HBsAg-negative blood donors.

BACKGROUND: Selection of hepatitis B virus (HBV) by host immunity has been suggested to give rise to variants with amino acid substitutions at or around the 'a' determinant of the surface antigen (HBsAg), the main target of antibody neutralization and diagnostic assays. However, there have never been successful attempts to provide evidence for this hypothesis, partly because the 3 D structure of HBsAg molecules has not been determined. Tertiary structure prediction of HBsAg solely from its primary amino acid sequence may reveal the molecular energetic of the mutated proteins. We carried out this preliminary study to analyze the predicted HBsAg conformation changes of HBV variants isolated from Indonesian blood donors undetectable by HBsAg assays and its significance, compared to other previously-reported variants that were associated with diagnostic failure. RESULTS: Three HBV variants (T123A, M133L and T143M) and a wild type sequence were analyzed together with frequently emerged variants T123N, M133I, M133T, M133V, and T143L. Based on the Jameson-Wolf algorithm for calculating antigenic index, the first two amino acid substitutions resulted in slight changes in the antigenicity of the 'a' determinant, while all four of the comparative variants showed relatively more significant changes. In the pattern T143M, changes in antigenic index were more significant, both in its coverage and magnitude, even when compared to variant T143L. These data were also partially supported by the tertiary structure prediction, in which the pattern T143M showed larger shift in the HBsAg second loop structure compared to the others. CONCLUSIONS: Single amino acid substitutions within or near the 'a' determinant of HBsAg may alter antigenicity properties of variant HBsAg, which can be shown by both its antigenic index and predicted 3 D conformation. Findings in this study emphasize the significance of variant T143M, the prevalent isolate with highest degree of antigenicity changes found in Indonesian blood donors. This highlights the importance of evaluating the effects of protein structure alterations on the sensitivity of screening methods being used in detection of ongoing HBV infection, as well as the use of vaccines and immunoglobulin therapy in contributing to the selection of HBV variants.

Chronic hepatitis B in pregnant women: is hepatitis B surface antigen quantification useful for viral load prediction?

BACKGROUND: New cases of hepatitis B virus (HBV) infection continue to occur worldwide. Most of these are due to mother-to-child transmission (MTCT), with maternal viraemia as the most important contributing factor. The hepatitis B surface antigen (HBsAg) level, which correlates positively with viral load, has been used for treatment monitoring in chronic hepatitis B. This study evaluated the usefulness of quantitative HBsAg for viral load prediction in HBsAg-positive pregnant women. METHODS: A total of 943 pregnant women in Makassar, Indonesia, were screened for HBsAg. Sixty-four women were HBsAg-positive and investigated. HBsAg level and hepatitis B e antigen (HBeAg)/hepatitis B e antibody (anti-HBe) status were determined serologically. Viral load was measured by real-time PCR. HBV DNA was sequenced and analysed for identification of genotype and basal core promoter (BCP)/precore (PC) mutations. RESULTS: Of 64 subjects, 12 (18.8%) were HBeAg-positive and 52 (81.3%) were HBeAg-negative. HBsAg and HBV DNA levels were significantly higher in the HBeAg-positive group (p<0.001). HBsAg and HBV DNA levels were positively correlated in the HBeAg-positive group (r = 0.659; p=0.02), but not in the HBeAg-negative group (r=0.194; p=0.168). Low HBsAg levels (<3.0 log10 IU/ml) corresponded with HBV DNA levels<6.0 log10 IU/ml (r=0.404; p=0.001), a recognized threshold for MTCT. Genotype C was more prevalent than genotype B, but not associated with HBsAg level, viral load, or HBeAg status. Two-thirds of HBeAg-negative subjects with high HBV DNA levels harboured BCP (A1762T/G1764A) and/or PC (G1896A) variants. CONCLUSIONS: HBsAg levels provide a good viral load predictor in HBeAg-positive but not HBeAg-negative pregnant women. The HBeAg-negative group had a frequent occurrence of BCP/PC variants, which may have contributed to the lack of correlation observed. Samples with a low HBsAg level, which is associated with a low risk of MTCT, do not require HBV DNA measurement.

HBsAg, HBeAg and HBV DNA level changes and precore/basal core promoter mutations in the natural history of chronic hepatitis B in Indonesian patients.

INTRODUCTION: Chronic hepatitis B (CHB) is a state of complex interactions between the hepatitis B virus (HBV) and host. We studied the changes in hepatitis B surface antigen (HBsAg), hepatitis B 'e' antigen (HBeAg) and HBV DNA levels, considering the implications of HBV genotype, basal core promoter (BCP) A1762T/G1764A and precore G1896A mutations in CHB. METHODS: One hundred fifty-two treatment-naïve CHB patients were classified into immune-tolerant (IT), immune-clearance (IC), low/non-replicative (LR) and 'e'-negative hepatitis B (ENH) phases, based on HBeAg status, HBV DNA and ALT levels. HBV DNA was detected and quantified by polymerase chain reaction, then analyzed by sequencing. HBsAg and HBeAg levels were measured serologically. RESULTS: HBsAg and HBV DNA levels varied between CHB phases, with HBsAg highest in IT and lowest in LR, and HBV DNA high in IT and IC, and lowest in LR. Both markers increased in ENH. Correlation between HBsAg and HBV DNA was significant in IT and IC, modest in ENH, but missing in LR. HBeAg and HBV DNA levels were dissociated in HBeAg-positive patients. Genotypes B and C were similarly distributed, with precore mutations higher in HBeAg-negative patients and BCP mutations comparable in all phases. Temporal association between HBeAg seroconversion and an increase of BCP/precore mutations was observed. CONCLUSION: HBsAg and HBV DNA levels were high and correlated in early CHB phases and dissociated after HBeAg seroconversion, indicating different controls affecting HBV replication and HBsAg production. Selection of BCP/precore mutants may affect disease course and explain the HBeAg-HBV DNA dissociation, a precaution for clinical application of quantitative HBeAg.

Occult hepatitis B in blood donors in Indonesia: altered antigenicity of the hepatitis B virus surface protein.

BACKGROUND AND AIMS: Occult hepatitis B virus infection (OBI) poses a challenge to the safety of blood donation. The prevalence of OBI is not well documented in Indonesia, although this information in such an endemic country is needed. This study was aimed to evaluate the prevalence of occult hepatitis B in blood donors from two cities of Indonesia, and to study the genetic variation and its effect on the predicted antigenicity of HBsAg. METHODS: Serum samples of 309 regular blood donors negative for HBsAg were tested for anti-HBs and anti-HBc. Hepatitis B virus (HBV) DNA isolated from anti-HBc-positive samples were analyzed by polymerase chain reaction, cloned and sequenced. Antigenic properties of identified HBsAg mutants were predicted by calculation of the antigenic index. RESULTS: Of the 309 HBsAg-negative samples, anti-HBc was positive in 134 (43.4%) and HBV DNA was detected in 25 (8.1%). Seven of the viremic samples had nucleotide substitutions (A521G, A551T, C582T, and A562G) in the S gene, causing amino acid mutations (T123A, M133L, and T143M) in the 'a' determinant of HBsAg that resulted in changes in the predicted antigenicity. CONCLUSIONS: OBI was detected in blood donors' samples in Indonesia. Anti-HBc was shown to be a better screening parameter than HBsAg, however, it might result in the loss of donors particularly in endemic countries. HBsAg detection failure in this study might be due to mutations altering the protein antigenicity and/or the low-level carriage of HBV.