Clinical significance and mutation analysis of HBsAg and Anti-HBs coexistence in Chronic Hepatitis B

The coexistence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) represents an atypical serological profile in chronic hepatitis B (CHB). The clinical significance and molecular characteristics of this double-positive phenomenon remain unclear. This study characterized the clinical and virological features of CHB patients with coexisting HBsAg and anti-HBs and assessed associations between them and key HBV mutations, specifically nt. T531C and nt. A1762T/G1764A double nucleotide substitution.

A retrospective analysis was performed on 955 chronic HBV-infected patients treated at a provincial hospital in Yunnan, China, between February 2017 and November 2017. Patients were categorized as double-positive group (HBsAg+/anti-HBs+, n = 250) and single-positive group (HBsAg+/anti-HBs-, n = 705) based on serological profiles. Demographic, clinical, and biochemical data were collected. HBV DNA from patient sera was analyzed by PCR amplification and Sanger sequencing to evaluate mutation patterns. Functional effects of key mutations were assessed by transfection of wild-type and mutant HBV plasmids into HepG2 and HepaRG cells.

DP patients exhibited higher HBV DNA levels (56.8% vs 36.5% with DNA >2 log IU/mL, p < 0.001) and elevated ALT, AST, DBIL, and AFP (all p < 0.01), indicating enhanced viral replication and liver inflammation. Combined mutations at nt. T531C(resulting in the I126T in the S region) and nt. A1762T/G1764A were significantly enriched in DP patients (p < 0.001).The proportion of patients with HBsAg >100 IU/mL was significantly lower in the DP group than in the SP group(p < 0.05). Functional assays revealed cell-type-specific effects: In HepG2 cells, BCP double and triple mutations significantly suppressed HBsAg secretion (p < 0.0001), while T531C and BCP double mutations reduced HBeAg levels; Notably, all mutant constructs demonstrated enhanced HBV DNA levels (p < 0.01). In HepaRG cells, T531C significantly upregulated HBsAg secretion (p < 0.01) and HBV DNA levels (p < 0.01), whereas BCP mutations suppressed HBsAg (p < 0.001) and showed no effect on extracellular HBV DNA levels. These mutations were independent of HBeAg status.

The coexistence of HBsAg and anti-HBs in chronic hepatitis B is associated with enhanced viral replicative activity, increased liver inflammation, and a distinct mutation pattern characterized by the co-occurrence of nt. T531C and nt. A1762T/G1764A substitutions. T531C was associated with increased extracellular HBV DNA in a cell-type-dependent manner and showed variable effects on HBsAg secretion, suggesting a potential role in immune escape and viral persistence.