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

Abstract

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.

Figure 1

Alignment of amino acid sequences of HBV isolates in Indonesian blood donors with frequently-reported variants associated with failure of diagnostic assays. Three amino acid substitutions were identified in 7 HBV isolates in blood donors: Pattern 1, T123A, in one isolate; Pattern 2, M133L, in one isolate; Pattern 3, T143M, in five isolates. HBV DNA isolated from the remaining 18 samples showed wild type (wt) sequences with no amino acid substitution. Consensus of each of the three single mutation patterns and wt were aligned with five known variants frequently associated with problems in diagnostic assays and/or escape to vaccine/HBIg therapy: T123N, M133I, M133T, M133V, and T143L, together with M54923 sequence (genotype B/adw) retrieved from GenBank as a reference.

Figure 2

Comparison of tertiary structure prediction. Tertiary structure prediction of M54923 (reference sequence), T123A, M133L, and T143M mutants. The 'a' determinant is shown in blue, yellow, magenta, and green, respectively, while residues of importance are labelled with the side chains shown.