Effects of pre-S mutations of hepatitis B virus on hepatitis B surface antigen expression

Abstract

Backgrounds & Aims: For patients with chronic hepatitis B virus (HBV) infection, the use of quantitative HBV surface antigen (qHBsAg) level as a biomarker for monitoring viral replication and optimizing treatment strategies has been highlighted recently. However, whether mutations in pre-S region may have any effect on circulating qHBsAg levels remains uncertain. The aim of this study is to determine whether intracellular and extracellular qHBsAg levels differed between wild-type HBV and HBVs with naturally-occurring mutations in the pre-S region.Methods: Plasmids expressing pre-S1 mutation (1-8 amino acid [aa] deletion) or pre-S2 mutation (3-25 aa deletion) were constructed. At 72 h post-transfection into Huh-7 cells, qHBsAg levels were analyzed in supernatant and cell lysate using electrochemiluminescence immunoassay analyzer.Results: Extracellular qHBsAg levels were on average 3.87-fold higher in Huh7 cells transfected with the pre-S1 Δ1-8 aa mutant than in cells transfected with wild-type HBV. In contrast, those in cells transfected with the pre-S2 Δ3-25 aa mutant were slightly lower than in cells transfected with wild-type HBV (on average 0.92-fold lower, but without statistical significance). Conversely, compared to cells transfected with wild-type HBV, intracellular qHBsAg levels were on average 0.57-fold lower in cells transfected with the pre-S1 Δ1-8 aa mutant, whereas those were on average 1.60-fold greater in cells transfected with the pre-S2 Δ3-25 aa mutant. Consistent with these results, immunofluorescence staining of cellular HBsAg showed that cells transfected with the pre-S1 Δ1-8 aa mutant were stained to a lesser degree and that cells transfected with the pre-S2 Δ3-25 aa mutant were more densely stained. Co-transfections of wild-type HBV with deletion mutants at the following ratios (either the pre-S1 Δ1-8 aa mutant or the pre-S2 Δ3-25 aa mutant : wild type = 0:10, 1:9, 3:7, 5:5, 7:3, 9:1, and 10:0) were performed to mimic the milieu of quasispecies. As the ratios of either the pre-S1 Δ1-8 aa mutant or the pre-S2 Δ3-25 aa mutant : wild type increased from 0:10 to 10:0, relative extracellular qHBsAg levels increased accordingly from 1.0 to 3.85 in the pre-S1 Δ1-8 aa mutant co-transfection models, whereas those decreased from 1.0 to 0.88 in the pre-S2 Δ3-25 aa mutant co-transfection models.Conclusion: Pre-S deletion mutants exhibit different phenotypes with respect to HBsAg expression and the effects seem to be dependent on the precise location of the deletion sites. Thus, the use of qHBsAg level as a biomarker for diagnosis and prognostification should be approached more cautiously, considering the emergence of pre-S deletion mutants.