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. 2026 May 29;369:199758. doi: 10.1016/j.virusres.2026.199758

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

Sihang Zhang a,b,1, Xin Lai b,1, Haimei Dai b,1, Xiao Li b, Xingtong Li b, Fengwei Liu b, Taicheng Zhou b,⁎, Jia Wei a,b,⁎
PMCID: PMC13253202  PMID: 42217829

Highlights

  • •

    Large-scale cohort study (n = 955) in Yunnan, Southwest China, demonstrates that Patients with double-positive HBsAg/anti-HBs exhibit a dissociation phenomenon characterized by high viral load (56.8% >2 log IU/mL), an increased proportion of low HBsAg levels (< 100 IU/mL), and significant liver injury (elevated ALT/AST/AFP), indicating an elevated risk of disease progression.

  • •

    we demonstrate that the co-occurrence of the nt.T531C (I126T) and nt.A1762T/G1764A double mutations is strongly associated with the double-positive phenotype (p < 0.001), and this combination is independent of HBeAg status, representing a novel immune evasion strategy in viral evolution.

  • •

    The T531C mutation significantly upregulates HBsAg secretion and DNA replication in differentiated HepaRG cells, whereas in HepG2 cells it only enhances replication; the BCP double mutation suppresses antigen expression but maintains replication capacity, revealing the host-dependent modulation of mutational phenotypes.

  • •

    By achieving a balance between immune evasion and sustained replication, the triple mutation provides a molecular basis for clinical monitoring and therapeutic intervention targeting double-positive patients.

Keywords: Chronic hepatitis B, HBsAg/anti-HBs coexistence, Serological profile, HBV mutations, Nt. A1762T/G1764A

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

1. Introduction

Hepatitis B virus (HBV) infection remains a significant global public health issue, affecting approximately 257.5 million people positive for HBsAg worldwide and posing a considerable risk for severe liver diseases, including liver fibrosis, decompensated cirrhosis, Acute-on-chronic liver failure (ACLF) and hepatocellular carcinoma (HCC) (Hsu et al., 2023; Li et al., 2024; Polaris Observatory Collaborators, 2023; Wang et al., 2014). Typically, Hepatitis B surface antigen (HBsAg) is an essential serological marker indicating HBV infection, whereas antibodies against HBsAg (anti-HBs) suggest viral clearance and host immunity (Raimondo et al., 2019). However, the atypical coexistence of serum HBsAg and anti-HBs, known as double positivity (DP), challenges traditional serological patterns and indicates a complex host-virus interaction (Ali et al., 2024; Jiang et al., 2021; Wang et al., 2022). Recent epidemiological studies have demonstrated that this unusual serological profile occurs in approximately 2%-10% of chronic HBV infections and is associated with severe clinical outcomes, including progressive liver disease and an increased risk of HCC (Wang et al., 2022; Ding et al., 2015; Hou et al., 2020).

Simultaneous positivity for HBsAg and anti-HBs, hereafter referred to as double positivity (DP), represents an atypical serological pattern in chronic HBV infection.Multiple mechanisms have been proposed to explain the coexistence of HBsAg and anti-HBs, including viral genomic mutations, host immune responses, and genetic predisposition (Jiang et al., 2021; Wang et al., 2018). Notably, mutations within the HBV genome, particularly in the major hydrophilic region (MHR) of the S gene and basal core promoter/pre-core (BCP/PC) regions, have drawn significant attention due to their potential roles in immune escape, altered viral antigenicity, and increased viral replication (Kwon and Lee, 2011; Hee et al., 2025; Li et al., 2021; Locarnini and Zoulim, 2010; Tong and Revill, 20; Yan et al., 2019). Hepatitis B surface antigen (HBsAg) is composed of 226 amino acids encoded by the S gene (nt.155–835). The region encoding amino acids 124–147 constitutes the critical "a" determinant, mutations in which may lead to immune escape or diagnostic escape (Wang et al., 2010). Several key mutation sites within this region (Y134S, P142S, G145R) can impair the binding capacity of anti-HBs antibodies (Tarafdar et al., 2022). The nt. T813C/G mutation results in an F220C amino acid substitution in the S region, outside the “a” determinant, and may influence HBsAg structure, secretion, or antigenicity (Hou et al., 2020; Wang et al., 2021). Previous studies have reported that mutations at this position can affect the conformational structure of HBsAg and may contribute to immune escape, although the mechanism is less well-characterized than for "a" determinant mutation (Ding et al., 2015). Notably, the I126T/S/N amino acid substitutions (corresponding to nt. T531C/A/G in the S gene) occur within the “a” determinant and have been associated with altered HBsAg antigenicity and immune escape.As reported in Ying Yan et al. (Yan et al., 2022), the mutation I126T/S was identified as an immune escape variant in the S region. In addition, the research (Meldal et al., 2011) indicates that I126N was identified as an immune escape substitution. Furthermore, studies indicate that spatially distant amino acid variations on HBsAg (Wu et al., 2022) can cooperatively confer resistance to neutralizing antibodies, suggesting that the function of the "a" determinant may be influenced by other mutations. Compared to the single HBsAg-positive group, the double-positive group (HBsAg+/HBsAb+) exhibits a higher point mutation rate within the "a" determinant, with such mutations being more prominent in genotype C.

Research demonstrates that the emergence of HBsAg and anti-HBs double positivity is associated with the accumulation of amino acid variations, which are not confined to the "a" determinant; variations at other sites within the major hydrophilic region (MHR) (Song et al., 2025) may also contribute to the double-positive phenotype. Among these, the mutation at nt. T531C/A/G within the "a" determinant region of HBsAg and the double mutations at nt. A1762T/G1764A in the BCP/PC region are frequently detected in patients exhibiting the double-positive serological profile and have been associated with disease progression (Constantinescu et al., 2014; Dong et al., 2021; El-Mokhtar et al., 2021; Silva Souza et al., 2017; Wang et al., 2014). However, the detailed molecular mechanisms by which these specific mutations may cooperatively lead to the coexistence of HBsAg and anti-HBs, as well as their subsequent clinical implications, remain unclear.

In the present study, we aimed to elucidate the potential mechanisms underlying the serum coexistence of HBsAg and anti-HBs in chronic HBV-infected patients by focusing on the combined mutations at nt. T531C and nt. A1762T/G1764A of the HBV genome. Through clinical comparative analyses, sequencing, and in vitro functional assays, we investigated whether these combined mutations are associated with altered viral antigenicity, replication-related phenotypes, and the coexistence serological profile observed in these patients.

2. Materials and methods

2.1. Study design and subjects

This retrospective study enrolled chronic hepatitis B virus (HBV)-infected patients who visited a provincial hospital in Yunnan Province, China, between February and November 2017. According to established criteria and the method of Zhou TC et al. (Zhou et al., 2017), patients were divided into two groups: the double-positive (DP) group, defined by persistent HBsAg positivity for over 6 months with simultaneous HBsAg and anti-HBs positivity, and the single-positive (SP) group, defined by persistent HBsAg positivity for over 6 months with anti-HBs seronegativity. Exclusion criteria included coinfection with other hepatitis viruses (HAV, HCV, HDV, HEV), EBV, CMV, HIV, autoimmune liver diseases, inherited metabolic liver diseases, drug-induced or alcoholic liver injury, and nonalcoholic fatty liver disease. We systematically analyzed their serological profiles to ensure sufficient sample size in both groups for comparisons. In particular, we considered the potentially low proportion of DP cases in the patient population. Subject enrollment was not randomized; instead, all eligible DP patients were purposively included.

For all included patients, demographic information (age, sex) and laboratory data were collected, including quantitative HBV serological markers (HBsAg, anti-HBs, HBeAg, anti-HBe, HBcAb), HBV DNA viral load (IU/mL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and direct bilirubin (DBIL). All laboratory tests were performed in the clinical laboratory of the hospital using standard chemiluminescence immunoassays and biochemical analyzers.

For clarity, molecular analyses in this study consisted of two independent components: (i) mutation screening by PCR amplification and Sanger sequencing of clinical serum samples from 128 single-positive and 123 double-positive patients, and (ii) functional validation experiments using site-directed mutagenesis and plasmid transfection in HepG2 and HepaRG cells. These two experimental workflows were performed separately and addressed distinct research objectives.

2.2. HBV DNA extraction, PCR amplification, and sequencing

Serum samples from all subjects were stored at −80°C prior to analysis. Viral DNA was extracted using a commercial nucleic acid extraction kit, following the Ex-RNA/DNA Virus Nucleic Acid Extraction Kit (Tianlong, Suzhou) protocol. A 650-bp fragment encompassing partial reverse transcriptase (RT) domain and the S gene “a” determinant (including the nt.531 site) was amplified using seq-A primers (HBV-RT-456F, 5′-AAGGTATGTTGCCCGTTTG-3′; HBV-RT-1112R, 5′-TTGTAAGTTGGCGAGAAAGT-3′). In addition, the BCP/PC region (including the nt.1762/1764 sites) was amplified using seq-B primers (HBV-1604F, 5′-TCGCATGGAGACCACCGTGAAC-3′; HBV-2054R, 5′-GCTTGCCTGAGTGCCGTATG-3′) with Takara LaTaq DNA polymerase. The obtained sequences were aligned and compared with standard reference sequences obtained from GenBank: Genotype C (No. X04615) and Genotype B (No. D00329.1).

Amplified products were Sanger-sequenced (Kunming Shuoqing Biotechnology, China). Sequencing data were quality-checked; low-quality or ambiguous peaks and sequence ends (∼20 bp) were excluded from analysis. Sequences with rare or novel mutations were confirmed by chromatogram review. Multiple sequence alignments were performed using Clustal X, and genotype assignment was conducted both via the NCBI online HBV genotyping tool and by phylogenetic analysis using MEGA software, referencing all major HBV genotypes (A–I). Only samples with concordant results by both methods were included in downstream analyses. The quality of sequencing was analyzed using CodonCode aligner software (V. 3.7.1;CodonCode Corporation; http://www.codoncode.com/aligner) and all mutations were refined manually.

2.3. Plasmid construction, cell culture, and transfection

A 1.2-fold overlength HBV genome (genotype C) cloned into pcDNA3.1(+) (Y17-C1) was used as the wild-type template. Site-directed mutagenesis was performed to generate mutants carrying nt. T531C, nt. A1762T/G1764A, and the combined triple mutation using sequence-specific primers. All plasmids were verified by Sanger sequencing. High-quality plasmid DNA was prepared from DH5α E. coli using standard extraction kits and quantified by spectrophotometry to ensure sufficient concentration (>500 ng/μL) for transfection.

Human hepatoma HepG2 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO₂. HepaRG cells (Thermo Scientific) were maintained as undifferentiated, proliferating cultures in William's E medium supplemented with 10% fetal bovine serum, 2 mM GlutaMAX, 5 μg/mL human insulin, 50 μM hydrocortisone hemisuccinate, and 1% penicillin-streptomycin, at 37°C in 5% CO₂, with subculturing every 2–3 days at 80% confluence to prevent spontaneous differentiation. For functional assays, cells were seeded in 6-well plates and transfected at 70–80% confluence with wild-type or mutant HBV plasmids using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions. Undifferentiated HepaRG cells were used for transfection to bypass the receptor-mediated viral entry step, as HBV replicon plasmids are directly delivered into the cytoplasm (Macovei et al., 2010, Hofmann et al., 2023). Empty vector served as negative control. At 72 h post-transfection, culture supernatants were collected for downstream analyses.

2.4. Supernatant digestion and DNase I treatment

Culture supernatants were centrifuged at 3,000 rpm for 10 min at 4°C to remove cellular debris and then passed through a 0.22 μm membrane filter. DNase I (New England Biolabs, Nanjing, China) was added to the filtered supernatants at 5–10 U/mL, followed by incubation at 37°C for 15–30 min to degrade unprotected DNA. The reaction was terminated by adding EDTA to a final concentration of 2–5 mM and incubating at 65°C for 10 min to inactivate DNaseI. Subsequently, HBV DNA in the treated supernatants was quantified using the COBAS AmpliPrep/COBAS TaqMan 48 system, following the manufacturer's protocol for automated nucleic acid extraction and PCR amplification.

2.5. Quantification of HBV replication and antigen expression

Extracellular HBsAg and HBeAg levels in culture supernatants were measured using the Architect-i2000 system (Abbott Laboratories, USA) and HBs/eAg chemiluminescent microparticle immunoassay (CMIA) kits (Abbott Laboratories, Germany). Samples exceeding the upper detection limit were diluted. HBV DNA in supernatant analyzed using the COBAS AmpliPrep/COBAS TaqMan 48 analyzer. Alpha-fetoprotein (AFP) concentrations were determined by chemiluminescent microparticle immunoassay (CMIA), with results reported in ng/mL.

2.6. Association of different combinations of mutations with HBsAg and anti-HBs coexistence

We analyzed the association of the nt. T531C/A/G mutation in the "a" determinant and the high-frequency BCP/PC mutation nt. A1762T/G1764A with the double-positive (DP) group. The frequencies of individual mutations and combined mutation patterns were calculated and compared between patient groups, with statistical significance assessed using the chi-square test. This analysis was designed to delineate the influence of either the coexistence of HBsAg and anti-HBs or the presence of HBsAg alone on the mutation profiles. Notably, we also evaluated the impact of these mutations stratified by HBeAg-negative and HBeAg-positive status.

2.7. Statistical analysis

For multiple comparisons of mutation frequencies, false discovery rate (FDR) correction was performed using the Benjamini–Hochberg method.Continuous variables were presented as mean ± standard deviation (SD) or median (IQR), as appropriate. Group comparisons were conducted using the student’s t-test for normally distributed data or the Mann–Whitney U test for skewed data. Categorical variables were compared using the chi-squared test. Statistical significance was set at p < 0.05. Analyses were performed with SPSS 13.0 and GraphPad Prism 6.

3. Results

3.1. Comparison of demographic and clinical characteristics

In this retrospective study, we enrolled patients with chronic HBV infection who were hospitalized at a provincial hospital in Yunnan Province between February and November 2017. Participants were categorized into two groups based on hepatitis B surface antigen (HBsAg) and antibody (anti-HBs) serostatus: the HBsAg and anti-HBs double-positive group (DP group, n = 250; 26.2%) and the HBsAg-positive, anti-HBs -negative group (SP group, n = 705; 73.8%) (Fig. 1). There were no significant differences between the two groups in terms of age or sex (p > 0.05 for both comparisons, Table 1).

Fig. 1.

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The flowchart of patient selection.

Table 1.

Comparison of demographic and clinical characteristics between CHB with and without anti-HBs.

Characteristics Coexistence, n (%)
 P value
Yes (n = 250) No(n = 705)
Age 37.5 ± 11.9 37.4 ± 10.9 0.56
Gender
Male 151(60.4%) 389(55.2%) 0.15
Female 99(39.6%) 316(44.8%)
HBeAg
Positive 128(51.2%) 323(45.8%) 0.16
Negative 122(48.8%) 382(54.2%)
HBV-DNA (log IU/mL)
>2 142(56.8%) 257(36.5%) < 0.001
≤2 105(42%) 445(63.5%)
ALT (U/L)
> 40 92(36.8%) 171(24.5%) < 0.001
≤40 156(62.4%) 528(75.5%)
> 80 35(14.8%) 47(6.7%) 0.001
≤ 80 213(85.2%) 652(93.3%)
Genotypes
B 36(31.9%) 46(42.2%) 0.11
C 77(68.1%) 63(57.8%)
HBsAg(IU/mL)
>100 200(80.0%) 613(86.9%) 0.011
≤100 50(20.0%) 92(13.1%)
anti-HBs >10mIU/mL 245(98.0%) 0 < 0.001
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*ALT ≤ 40 U/L for males and ≤ 35 U/L for females, the study used the unified cut-off of > 40 U/L to define ALT abnormality

HBV DNA levels in both the DP and SP groups spanned the full spectrum from <100 IU/mL to 1×10⁸ IU/mL (Fig. 2A).The proportion of patients with HBsAg >100 IU/mL was significantly lower in the DP group than in the SP group (80.0% vs. 86.9%, P = 0.011). Anti-HBs seropositivity at a protective level was defined as anti-HBs ≥10 IU/L (Karime et al., 2024). In the present study, the proportion of individuals with anti-HBs ≥10 IU/L in the dual-positive group was 98% (Table 1). The proportion of individuals with HBV DNA >100 IU/mL was also significantly higher in the DP group than in the SP group (p ＜0.001, Table 1), indicating a higher level of viral activity in the DP group. No significant difference was found in the distribution of HBeAg status between the two groups (p = 0.16, Table 1).

Fig. 2.

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Comparison of HBV DNA and liver function clinical indices between single-positive and double-positive groups. DP was double-positive group (HBsAg+/anti-HBs+, n = 250), SP denotes single-positive group (HBsAg+/anti-HBs−, n = 705). (A) Serum HBV DNA levels were measured in chronic hepatitis B patients from the DP and SP groups respectively. A viral load below 100 IU/mL was defined as Undetectable HBV DNA. HBV DNA levels ranging from < 100 IU/mL to 108 IU/mL. The distribution differences of HBV DNA loads across different logarithmic magnitudes were compared between the two groups. (B-F) Blood samples from DP and SP patients were analyzed for liver function parameters including(B) alanine aminotransferase (ALT),The Mean ± SEM in DP group was 52.17 ± 4.150 while SP group was 36.73 ± 1.493. (C) aspartate aminotransferase (AST), The Mean ± SEM in DP group was 41.75 ± 2.505 while SP group was 33.82 ± 1.020. (D) total bilirubin (TBIL), The Mean ± SEM in DP group was 16.95 ± 0.543 while SP group was 15.88 ± 0.294. (E) represented the direct bilirubin (DBIL),The Mean ± SEM in DP group was 5.651 ± 0.305 while SP group was 4.792 ± 0.145. and (F) alpha-fetoprotein. The Mean ± SEM in DP group was 23.55 ± 9.958 while SP group was 6.014 ± 0.957. Intergroup comparisons were performed using Student's t-test. Intergroup comparisons were performed using Student's t-test. (**p < 0.01; *** p < 0.001; **** p < 0.0001).

ALT levels were significantly higher in the DP group than in the SP group (p < 0.0001, Fig. 2B). The prevalence of abnormal ALT also differed between the groups; the proportion of patients with ALT >40 U/L or ALT >80 U/L was higher in the DP group than in the SP group (p < 0.001; p = 0.001, Table 1), indicating a stronger hepatocellular injury in the DP group. In addition, AST, DBIL, and AFP levels were all significantly higher in the DP group than in the SP group (p < 0.001, p < 0.01, p < 0.01, Fig.s 2C, E, F). There was no significant difference in TBIL levels between the two groups.

3.2. HBV mutation characteristics and their association with double positivity

We randomly selected 251 chronic HBV-infected individuals for HBV sequence analysis, including 128 from the SP group and 123 from the DP group. Genotyping revealed only genotype B and C in our cohort, so all analyses were limited to these two genotypes (Table 1). No significant difference was found in the distribution of genotypes B and C between the two groups (p = 0.11).

Based on previously identified hotspot regions related to double positivity, we sequenced fragments covering the S region “a” determinant cluster (nt.519–nt.853, seq-A) and the BCP/PC region (nt.1655–nt.2014, seq-B). To minimize bias, only mutations detected in both groups were analyzed. For genotype C, 31 mutation sites were identified; for genotype B, 26 mutation sites were detected. Mutations with a frequency greater than 10% in either group were further analyzed: 12 sites for genotype C (Table 3) and 16 for genotype B (Table S1). In genotype B, nt. A519G and nt. A1762T mutations showed significant differences between the groups and were positively associated with the DP group (p < 0.05, OR>3). In genotype C, mutations at nt. T531C/A/G, nt. T813C/G, nt. A1762T, nt. G1764A, and the combined nt. A1762T/G1764A showed significant differences between the groups and were positively associated with the DP group (p < 0.05, OR>2.8, Table 2). There were no significant differences in the frequencies of nt. T531C/A/G, nt. T813C/G, nt. A1762T, nt. G1764A, or combined nt. A1762T/G1764A mutations between HBeAg-positive and -negative individuals in genotype C (p values were 0.601, 0.718, 0.536, 0.601, 0.359 sequentially, Table S2).

Table 3.

. Analysis of seq-A variations in single-positive (SP) and double-positive (DP) groups of genotypes B and C.

Genotype Mutation sites Amino acid substitutions DP SP total χ2 P values
C T531C/A/G * I126 T/N/S(S) 35.14% 14.75% 25.93% 7.23 0.007*
C G783A L209L(S) 32.43% 27.87% 30.37% 0.33 0.566
C T813G * F220C(S) 13.51% 1.64% 8.15% 4.81 0.028*
C C837T * N582N(P) 14.86% 3.28% 9.63% 5.16 0.023*
C C849A/T T586T(P) 10.81% 4.92% 8.15% – 0.614
B A519G * K122R(S) 17.65% 2.50% 9.46% – 0.033*
B A520G K122K(S) 5.88% 10.00% 8.11% – 0.418
B A529G * T125T(S) 38.24% 15.00% 25.68% 5.2 0.023*
B C837T N582N(P) 11.76% 2.50% 6.76% – 0.132
B A849C/T T586T(P) 2.94% 10.00% 6.76% – 0.289
B A853C K587K(P) 2.94% 10.00% 6.76% – 0.234
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Note: Genotype C: Double-positive (DP) group (n = 74), single-positive (SP) group (n = 61); Genotype B: DP group (n = 34), SP group (n = 40). (S): Amino acid substitutions in the S region; (P): Substitutions in the P region. Statistical analysis: Chi-square test; *p < 0.05 (significant); –Fisher’s exact test (SPSS).

Table 2.

Statistically significant mutation sites in single-positive and double-positive groups of genotypes B and C1.

Geno-type Mutation sites Mutation frequency
 Statistical analysis
DP SP total χ2 p values OR 95% CI
B nt. A519G 6(34) 1(40) 9.46% – 0.033* 8.36 (0.95, 73.34)
17.65% 2.50%
B nt. A1762T 10(34) 4(41) 18.67% 4.73 0.030* 3.85 (1.08, 13.70)
29.41% 9.76%
C nt. T531C/A/G 26 (74) 9 (61) 25.93% 7.23 0.007* 3.13 (1.33, 7.35)
35.14% 14.75%
C nt. T813C/G 10 (74) 1 (61) 8.15% 4.81 0.028* 9.38 (1.17, 75.46)
13.51% 1.64%
C nt. A1762T 33 (72) 14 (61) 35.34% 7.57 0.006* 2.84 (1.33, 6.05)
45.83% 22.95%
C nt. G1764A 34 (72) 13 (61) 35.34% 9.7 0.002* 3.3 (1.53, 7.12)
47.22% 21.31%
C nt. A1762T/G1764A 31 (72) 12 (61) 32.33% 8.25 0.004* 3.09 (1.41, 6.78)
43.06% 19.67%
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1

Frequency (total sample size in parentheses); * indicates p < 0.05 (statistically significant), – indicates Fisher's exact test was used based on SPSS analysis results.

Among these, the nt. T531C mutation located within the “a” determinant and the high-frequency BCP/PC mutations nt. A1762T/G1764A showed a strong association with the DP group. We evaluated both single and combined mutation patterns. Compared with the wild type, the frequencies of nt. T531C, nt. A1762T/G1764A, and their co-occurring mutations were significantly higher in the DP group than in the SP group (FDR-corrected p = 0.036 for each comparison), whereas no significant differences were observed among other mutation patterns (Fig. 3A).Interestingly, no significant differences in the frequencies of nt. T531C/A/G, nt. A1762T/G1764A, or their combined mutations were detected between HBeAg-positive and HBeAg-negative individuals (Fig. 3B), suggesting that these mutation patterns are independent of HBeAg status. Collectively, these findings indicate a strong association between the combined T531C and A1762T/G1764A mutations and the HBsAg/anti-HBs double-positive phenotype. We further performed a quasispecies-level analysis of these mutation sites in genotype C within the DP and SP groups, using data from our previous study (Zhou et al., 2017). The DP group (4 patients, 59 clones) exhibited significantly higher mutation frequencies for nt. T531C (35.6%), nt. A1762T (78%), nt. G1764A (78%), and the nt. A1762T/G1764A double mutation (78%) than the SP group (4 patients, 59 clones; all p < 0.05) (Fig. 3C). Moreover, the combined mutation pattern—defined as T531C co-occurring with the A1762T/G1764A double mutation—was significantly more prevalent in the DP group than in the SP group (p < 0.001). (Fig. 3C and Table S3). Notably, both the present cohort-level sequencing strategy (single PCR-based sequencing per individual) and previous quasispecies clone sequencing from a smaller cohort (PCR followed by cloning and sequencing of multiple clones) consistently demonstrate that the combined mutation pattern is strongly associated with the HBsAg/anti-HBs double-positive phenotype.

Fig. 3.

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Multilevel comparative analysis of HBV genotype C mutation patterns.

(A) Heatmaps showing FDR-adjusted P values derived from chi-square tests comparing four mutation patterns identified in patient samples: wild type (no mutations), nt. T531C, nt. A1762T/G1764A, and the combined nt. T531C with nt. A1762T/G1764A mutations. The upper-left triangular region (Y-axis vs. X-axis) represents intra-group and inter-group comparisons. Comparison between the single-positive (SP) and double-positive (DP) groups.(B) Comparison between the HBeAg-negative and HBeAg-positive groups. (C) Comparison of quasispecies mutation frequencies between the DP group (n = 4 patients, 59 clones) and the SP group (n = 4 patients, 59 clones) within genotype C. Data were derived from Zhou et al. (Zhou et al., 2017).

3.3. Functional analysis of nt. T531C and nt. A1762T/G1764A mutations

Given the high frequency and strong association of the nt. T531C and nt. A1762T/G1764A mutations with double positivity, we further explored their functional impact. First, we sequenced the 1.2-fold overlength HBV genome plasmid template (Y17-C1). This plasmid was then used as the template for constructing mutant plasmids carrying nt. T531C, nt. A1762T/G1764A, or the combined triple mutation(Fig. S2). Using site-directed mutagenesis, we generated four plasmid types: wild-type (A group), nt. T531C (B group), nt. A1762T/G1764A (C group), and a combination of nt. T531C with nt. A1762T/G1764A (D group); no other sites differed from Y17-C1. These plasmids, together with an empty vector (H group), were transfected into HepG2 cells for eukaryotic expression. At 72 h post-transfection, no obvious morphological differences were observed among the transfected cell groups.

In HepG2 cells, the T531C mutant (group B) did not significantly affect HBsAg secretion compared with the wild-type construct (group A), whereas the A1762T/G1764A mutant (group C) and the triple mutant (group D) significantly reduced HBsAg levels (****p < 0.0001; Fig. 4A). For HBeAg secretion, both group B and group C showed significantly lower levels than group A, while no significant difference was observed between the triple mutant and the wild type (Fig. 4B). In contrast, all mutant constructs exhibited significantly enhanced viral replication capacity, as reflected by increased levels of HBV DNA in the culture supernatant compared with group A (**p < 0.01; Fig. 4C).

Fig. 4.

Fig 4 dummy alt text

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Effects of mutations T531C and A1762T/G1764A on HBV replication and expression. We constructed four plasmid types: wild-type, nt. T531C, nt. A1762T/G1764A, and the combined mutation of nt. T531C with nt. A1762T/G1764A; no other sites differed from the Y17-C1 reference sequence. These plasmids, along with an empty vector control, were transfected into HepG2 and HepaRG cells for eukaryotic expression. (A-B) At 72 h post-transfection, we measured Secreted hepatitis B surface antigen (HBsAg), Hepatitis B e-antigen (HBeAg) levels across different groups, (C) HBV DNA load in cell culture supernatants. (**p < 0.01; *** p < 0.001; **** p < 0.0001).

In HepaRG cells, which display a more differentiated hepatocyte-like phenotype, the effects of these mutations differed substantially. The T531C mutant significantly increased HBsAg secretion relative to the wild type (**p < 0.01), whereas the A1762T/G1764A mutant and the triple mutant significantly decreased HBsAg levels (***p < 0.001 and ****p < 0.0001, respectively; Fig. 4A). HBeAg secretion was significantly reduced in all mutant groups compared with the wild type in this cell line (Fig. 4B). Regarding viral replication, group B showed significantly increased HBV DNA levels, whereas no significant differences were observed in group C or group D relative to group A (Fig. 4C).

4. Discussion

The coexistence of serum HBsAg and anti-HBs in chronic HBV infection is increasingly recognized as an atypical serological pattern associated with adverse clinical outcomes, such as severe liver fibrosis and an elevated risk of HCC (Wang et al., 2022; Afyon, 2016; Chu et al., 2002; Jin et al., 2019; Ding et al., 2016). However, the underlying molecular mechanisms facilitating this coexistence have not been fully elucidated. In this study, we found that specific HBV genomic mutations, particularly nt. T531C/A/G in the S gene “a” determinant and nt. A1762T/G1764A in the BCP/PC region, were associated with the coexistence phenotype (Fig. S1).

The significantly lower HBsAg levels observed in the double-positive (HBsAg+/anti-HBs+) group may reflect a distinct immunovirological state. This finding is consistent with a previous report (Thi Cam Huong, 2023) and suggests coexistence of anti-HBs with residual low-level HBsAg expression. Clinically, we observed higher HBV DNA levels and significantly elevated ALT, AST, DBIL, and AFP in patients exhibiting the double-positive serological profile, indicating enhanced viral replication and aggravated hepatic inflammatory responses in these individuals. These findings are consistent with previous reports linking the coexistence pattern with increased liver injury and progressive liver diseases (Hou et al., 2020). Our sequence analyses further identified a significant enrichment of the nt. T531C/A/G mutation in the S gene and the nt. A1762T/G1764A double mutation in the BCP/PC region among DP patients, particularly within genotype C, which is prevalent in Chinese populations (Wang et al., 2014; Changsri et al., 2024). Such mutations, especially in the MHR region, may alter the antigenicity of HBsAg, allowing escape from neutralizing antibodies and potentially facilitating coexistence with anti-HBs (Ding et al., 2016; Tong et al., 2013).

Interestingly, previous studies have indicated a close relationship between the nt. A1762T/G1764A mutations and HBeAg status, suggesting a role for these mutations in the modulation of HBeAg seroconversion and disease progression (Song et al., 2025; Chen, 2018). However, in our cohort, these mutations were specifically associated with the DP serological profile rather than HBeAg status. This discrepancy may reflect diverse viral-host interactions in different populations or varied impacts of combined mutations on viral antigenicity and replication. Further studies involving larger populations from various regions are required to clarify these differences and their clinical implications.

Functionally, our in vitro experiments revealed cell-type-specific effects of these mutations. In HepG2 cells, BCP double mutations (A1762T/G1764A) and the triple combination significantly suppressed HBsAg secretion, while T531C showed no significant effect compared to wild-type. For HBeAg, both T531C and BCP mutations significantly reduced levels in HepG2 cells, whereas the triple mutant showed no statistical difference from wild-type. Notably, all mutant constructs demonstrated enhanced viral DNA replication in HepG2 cells, suggesting increased viral fitness. In HepaRG cells, which exhibit a hepatocyte progenitor phenotype with preserved innate immune signaling and liver-specific transcription factor expression, the effects of these mutations differed substantially: T531C significantly upregulated HBsAg secretion and enhanced HBV DNA replication, while BCP mutations significantly suppressed both HBsAg and HBeAg production, with no significant effect on DNA replication. These differential effects highlight the importance of cellular context and hepatocyte differentiation status in modulating mutation phenotypes.This observation is partly consistent with previous studies (Wang et al., 2021; Hong et al., 2025). Mutations in the S region may alter HBsAg antigenicity and secretion without uniformly affecting other viral phenotypes. Conversely, dual mutations in the BCP downregulate HBeAg expression while permitting persistent viral replication (Lau et al., 2021; Elizalde et al., 2023; Elizalde et al., 2025). Collectively, these two classes of mutations illustrate HBV's evolutionary strategy to maintain chronic infection through genetic diversification under host immune pressure.

The nt. T531C mutation is located within the core region of the “a” determinant of the HBV S gene and results in an I126T amino acid substitution. This region represents a major neutralizing epitope of HBsAg. Previous studies have shown that mutations within the major hydrophilic region can reduce anti-HBs binding affinity and facilitate immune escape, thereby contributing to the coexistence of HBsAg and anti-HBs (Fu et al., 2017; De campos albuquerque et al., 2017).Mutations in the S region may alter HBsAg antigenicity and secretion without uniformly affecting other viral phenotypes. Consistent with this interpretation, the T531C mutation was significantly more frequent in the DP group than in the SP group.

The A1762T/G1764A double mutation is located in the BCP region of HBV and has been reported to enhance Pregenomic RNA (pgRNA) transcription and viral replication. (Li et al., 2024). In contrast, this mutation reduces precore mRNA transcription and suppresses HBeAg expression (Elizalde et al., 2025; Torres et al., 2019). Such coordinated regulation may facilitate viral persistence by maintaining replication capacity while reducing the production of highly immunogenic HBeAg. (Liu et al., 2020). In the present study, the BCP double mutation significantly reduced HBeAg secretion in both HepG2 and HepaRG cells. Interestingly, in HepG2 cells, the triple mutation partially restored HBeAg levels toward those of the wild type, suggesting that T531C may modulate the phenotypic effect of the BCP mutation in the context of the overlapping reading frame and regulatory structure of the HBV genome. In addition, because the BCP overlaps functionally with enhancer II, its effect on transcription may not be limited to precore/pgRNA regulation alone, and a broader influence on viral transcripts, including those related to HBsAg expression, cannot be excluded.

Moreover, T531C appeared in combination with the A1762T/G1764A BCP double mutation at a significantly higher frequency in the DP group, suggesting that cooperative effects between immune escape-associated and replication-related mutations may contribute to the development of the double-positive phenotype. In addition, the T813C/G mutation was also associated with the DP phenotype, further supporting the possibility that multiple mutations within or near the MHR may act together in immune evasion (Elizalde et al., 2025).

The coexistence pattern may reflect partial rather than complete immune escape, in which mutant HBsAg remains serologically detectable despite the presence of anti-HBs.This functional complementarity is fully demonstrated in the clinical characteristics of DP patients: high viral load (56.8% with DNA > 2 log IU/mL) and relatively low HBsAg (p = 0.011).These findings are consistent with the possibility that T531C contributes to altered antigenicity, while co-occurring A1762T/G1764A mutations may influence replication-related phenotypes and HBeAg downregulation. From an evolutionary perspective, the enrichment of the triple mutation pattern in the DP group may reflect viral adaptation under host immune pressure.

In this study, there was no significant difference in the median age of DP patients and SP patients, but liver injury markers were all elevated. it suggested that the triple mutant strain may have been selected at an earlier stage of infection and drive a faster disease progression. At the population level, A1762T/G1764A was more frequent than T531C, suggesting that these mutations may differ in their selective dynamics. Whether T531C accumulation requires additional immune selection remains to be determined.

Although anti-HBs is generally regarded as a marker of immune control, some DP patients still exhibited persistent liver inflammation. One possible explanation is that, under conditions of ongoing antigenemia, circulating HBsAg may bind to anti-HBs to form immune complexes (Xu et al., 2022).These immune complexes in the circulation may deposit in liver tissue, triggering complement activation and persistent inflammatory damage (Baidya et al., 2022). However, the pathogenic significance of such complexes in chronic hepatitis B remains uncertain, and this mechanism should be interpreted with caution. Current evidence indicates that HBV-specific T cell responses, particularly those directed against core-related antigens, play a central role in the immunopathogenesis of chronic hepatitis B.

Another most significant finding of this study is the pronounced cell-type dependency of triple combined mutation effects: in HepG2 cell transfection systems, both BCP double mutation and triple mutation combinations resulted in HBsAg secretion levels reduced by 50–70% compared to wild-type. T531C alone showed no significant effect in HepG2 cells. The T531C-associated phenotype observed in HepaRG cells may provide complementary information to the HepG2 model and may better reflect certain features of the DP clinical profile, including detectable but relatively higher HBsAg levels together with elevated HBV DNA. This suggests that reliance solely on traditional hepatoma cell lines (HepG2) may underestimate or misinterpret the true functions of certain mutations, particularly those involving host-virus interactions and immune regulatory mechanisms. Therefore, the contribution of HBsAg–anti-HBs immune complexes to liver injury in DP patients remains speculative and requires direct validation. Future studies using primary human hepatocytes, liver organoids, and humanized mouse models, together with direct assessment of immune complex deposition in liver tissue, will be needed to clarify this possibility.

Enhanced extracellular HBV DNA was observed for all mutant constructs in HepG2 cells, whereas in HepaRG cells this effect was significant only for T531C, indicating that the replication-related phenotype was cell-model dependent.

This study also found that triple mutation frequency was unrelated to HBeAg status, with no difference in HBeAg positivity between DP and SP groups. This enables the virus to maintain high replication regardless of HBeAg status (Chen et al., 2025), forming overlapping clinical phenotypes, suggesting that traditional clinical staging systems may underestimate disease risk in these patients.

Our study has certain limitations. The retrospective, cross-sectional nature of clinical data precludes definitive causal inference, and larger prospective cohorts are required to validate these findings longitudinally. Additionally, functional assays conducted in vitro may not fully recapitulate the complex host-virus immune dynamics in vivo. Further studies involving animal models or humanized mouse systems would be beneficial to confirm these molecular mechanisms. Additionally, hepatitis B core-related antigen (HBcrAg) testing was not performed in this study, which could have provided additional insights into viral replication status and cccDNA activity (Hepatitis B, 2026). Future studies incorporating HBcrAg measurements would enhance our understanding of the relationship between viral persistence and the double-positive serological pattern. An additional limitation is that our HepaRG transfection experiments were performed using undifferentiated, proliferating cells; future studies using differentiated HepaRG or primary human hepatocytes may further elucidate the impact of hepatocyte maturation status on mutation-associated phenotypes.

In conclusion, our findings underscore the critical role of combined HBV genomic mutations (nt. T531C and nt. A1762T/G1764A) in promoting the coexistence of serum HBsAg and anti-HBs through enhanced antigen production and altered antigenicity. These results provide valuable insights into the unique molecular mechanisms underlying this atypical serological pattern and emphasize the necessity for careful monitoring and targeted therapeutic interventions in affected individuals.

Author contributions

Sihang Zhang: Writing - review & editing, Writing - original draft, Visualization, Supervision, Formal analysis, Data curation. Xin Lai: Formal analysis, Data curation, Conceptualization. Haimei Dai: Methodology, Data curation. Xiao Li: Funding acquisition, Conceptualization. Xingtong Li: Resources, Funding acquisition. Fengwei Liu: Methodology. Taicheng Zhou: Writing - review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization. Jia Wei: Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Informed consent statement

Informed consent was obtained from all subjects involved in the study.

Datastatement

All relevant data are within the manuscript and its Supporting Information files, except for the raw clinical data, which cannot be shared publicly due to patient confidentiality. Requests for de-identified clinical data should be directed to the corresponding author.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements and Funding

This work was supported by grants from The Leading Talents in Medical Disciplines of Yunnan Province, China (No. D-2024002); The Reserve Talents Program for Middle-aged and Young Academic and Technical Leaders in Yunnan Province, China (No. 202205AC160023); The 14th Five-Year Plan Yunnan Provincial Key Clinical Specialty Project for Infectious Diseases, China (No. ZKF2025015); The Yunnan Provincial Major Science and Technology Special Project in Biomedicine, China (No. 202502AA310011); The Scientific Research Fund of the Yunnan Provincial Department of Education, China (No. 2025J0029); and The Medical Research Fund of Yunnan University, China (No. YDYXJJ2025-0004).

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2026.199758.

Contributor Information

Taicheng Zhou, Email: zhoutc@ynshhyy.com.

Jia Wei, Email: weijia19631225@163.com.

Appendix. Supplementary materials

mmc1.pptx (1MB, pptx)

Table S1. Analysis of seq-B variations in single-positive (SP) and double-positive (DP) groups of genotypes B and C

Table S2. Distribution of mutation sites in HBeAg-positive and HBeAg-negative groups of genotype C

Table S3 Comparison of quasispecies mutation frequencies within genotype C

mmc2.xlsx (15.7KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.pptx (1MB, pptx)

Table S1. Analysis of seq-B variations in single-positive (SP) and double-positive (DP) groups of genotypes B and C

Table S2. Distribution of mutation sites in HBeAg-positive and HBeAg-negative groups of genotype C

Table S3 Comparison of quasispecies mutation frequencies within genotype C

mmc2.xlsx (15.7KB, xlsx)

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