Objective This study aims to investigate the impact of mutations within the major hydrophilic region (MHR) of the hepatitis B virus (HBV) S gene on the expression, secretion, and antigenicity of hepatitis B surface antigen (HBsAg), and evaluate the performance of commonly used commercial HBsAg detection systems in identifying these variants. Methods Seventeen high-frequency point mutations within the major hydrophilic region (MHR) of the HBV S gene were previously identified in individuals with occult HBV infection. These mutations were introduced into wild-type HBV sequences using site-directed mutagenesis to construct recombinant eukaryotic expression plasmids. Following sequence confirmation, the plasmids were transfected into Huh-7 cells. At 72 hours post-transfection, both culture supernatants and cell lysates were harvested. HBsAg levels were quantified using five commercial detection systems: Roche, Abbott, Autobio, CHIVD, and KHB. Results Compared with the wild-type strain, T126A (B) and M133I (B) variants presented significantly increased HBsAg levels (P0.05). Conversely, HBsAg expression was significantly reduced in K122E (B), C124R (B/C), M133T (B), C138R (B), T143S (B), D144G (B), and L162R (C) variants relative to the wild-type (P0.05). The L162R (C) variant produced no detectable HBsAg in either lysates or supernatants. The C124R (B/C) variant showed higher reactivity on the Abbott and KHB systems, whereas other systems failed to detect it effectively. In contrast, the C138R (B) and M133T (B) variants were detectable in lysates but undetectable in supernatants, suggesting impaired secretion. Among the evaluated systems, Abbott and KHB systems demonstrated comparatively higher sensitivity for variant detection. Conclusion Of the 17 MHR variants examined, only T126A (B) and M133I (B) enhanced HBsAg detectability. The remaining mutations impaired detection due to decreased expression, secretion defects, or altered antigenicity. Specifically, L162R (C) likely disrupts expression or antigenicity, C138R (B) and M133T (B) impair secretion, and C124R (B/C) alters antigenicity. Most other variants exhibited reduced detectability across systems. Continued refinement of immunoassays is needed to enhance the identification of HBsAg variants in clinical settings.
He et al. (Fri,) studied this question.