Introduction

Chronic hepatitis B (CHB) affects approximately 260 million people worldwide and remains a major cause of liver cirrhosis and hepatocellular carcinoma (HCC) [1]. Long-term treatment with nucleot(z)ide analogues (NAs) effectively suppresses hepatitis B virus (HBV) replication and reduces the incidence of cirrhosis; however, reductions in HCC incidence have been reported only in limited regions, including Japan [2]. Because HBV persists in hepatocytes as covalently closed circular DNA (cccDNA), viral replication and antigen production can continue for prolonged periods, leading to persistent inflammation and fibrosis [3, 4]. Therefore, sustained and potent viral suppression is essential for improving long-term outcomes.

Entecavir (ETV), a first-line NA with potent antiviral activity and a high genetic barrier to resistance, has been shown to reduce the risk of cirrhosis progression and HCC development [5]. However, declines in hepatitis B surface antigen (HBsAg) levels are generally slow, and functional cure, defined as HBsAg loss, remains uncommon [6, 7]. Consequently, strategies to further reduce viral markers, including HBsAg and hepatitis B core-related antigen (HBcrAg), are needed.

Tenofovir alafenamide (TAF), a novel tenofovir prodrug, achieves effective intracellular delivery while maintaining low plasma tenofovir concentrations, thereby preserving antiviral efficacy and improving renal and bone safety [8, 9]. Although TAF is an attractive option for long-term CHB management, the clinical benefits of switching to TAF in patients who have achieved virological stability with long-term ETV therapy remain unclear.

In particular, data on long-term changes in virological markers, fibrosis markers such as Mac-2 binding protein glycosylation isomer (M2BPGi) and fibrosis-4 index (FIB4-index), and renal safety parameters, including estimated glomerular filtration rate (eGFR), serum phosphorus, urinary N-acetyl-β-D-glucosaminidase/creatinine (NAG/Cr), and urinary β2-microglobulin/creatinine (β2MG/Cr), are limited [10]. This issue is especially relevant in Japan, where HBV genotype C predominates and preservation of renal function is important in an aging population.

Therefore, we investigated the long-term efficacy and safety of switching from ETV to TAF in patients with CHB who had achieved stable viral suppression during long-term ETV treatment. We evaluated virological and biochemical markers, including HBcrAg, alanine aminotransferase (ALT), and M2BPGi, together with renal function and proximal tubular injury markers, to provide evidence for optimizing long-term treatment strategies.

Material and methods

Study population

Patients with CHB receiving ETV therapy at Kindai University Hospital were prospectively screened for participation. Only NA-naïve patients infected with HBV genotype C were included to maintain a homogeneous study population. Eligible patients were required to have documented HBsAg positivity for at least 6 months and to have achieved stable suppression of HBV DNA during ETV treatment. Participants should have received ETV therapy for at least 1 year and maintained HBV DNA levels < 2.1 log copies/ml. In addition, patients were required to have baseline HBsAg concentrations ≥ 800 IU/ml or between 80 and 800 IU/ml without a significant decline during the year preceding enrollment. The exclusion criteria were prior exposure to other antiviral agents, concomitant viral hepatitis, autoimmune liver disease, or alcohol-related liver injury. Patients with clinical evidence of HCC, decompensated cirrhosis, severe systemic illness, or impaired renal function were also excluded. Pregnant or breastfeeding women were not eligible for this study.

The study was approved by the Institutional Ethics Committee of Kindai University and was conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants.

Study design

This study was a single-center, prospective, observational study. A formal sample size estimation was not performed because the study was designed as a pilot analysis. Patients receiving ETV therapy were informed about the availability of TAF and were allowed to decide whether to continue their current treatment or transition to TAF. Patients who preferred to remain on ETV formed the continuation group, whereas those who elected to switch therapies were assigned to the TAF group. At enrollment, 32 patients continued ETV therapy and 48 switched to TAF. During the follow-up period, observation of three patients was discontinued owing to transfer to other institutions. Consequently, 31 patients in the ETV group and 46 patients in the TAF group were included in the final analysis (Fig. 1). ETV was administered orally at a dose of 0.5 mg once daily under fasting conditions, whereas TAF was administered at 25 mg once daily. The observation period extended to a maximum of 384 weeks after study entry.

Clinical assessments

Laboratory examinations were performed periodically during the follow-up period. Serum HBsAg, HB surface antibody, HB e antigen (HBeAg), and HB e antibody levels were measured by chemiluminescence immunoassay (CLIA) using the ARCHITECT kit (Abbott Japan, Tokyo, Japan). HBcrAg levels were measured by chemiluminescence enzyme immunoassay (CLEIA) using the Lumipulse HBcrAg kit (Fujirebio, Kanagawa, Japan). HBV-DNA levels were measured by real-time polymerase chain reaction as described previously (COBAS TaqMan HBV Test, version 2.0, Roche Diagnostics, Tokyo, Japan) [11]. HBV genotypes were identified by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against a type-specific epitope in the preS2 region (Immunology Research Institute, Tokyo, Japan) [12]. Liver biochemistry tests included alanine aminotransferase (ALT), platelet count, and serum phosphorus, and liver fibrosis was evaluated using M2BPGi. Renal function was assessed using eGFR, which was calculated according to the formula proposed by the Japanese Society of Nephrology. Additionally, markers of proximal tubular injury, including the urinary NAG/Cr ratio and urinary β2MG/Cr ratio, were evaluated.

Study endpoints

The primary endpoint of this study was the change in serum HBsAg concentration from baseline to week 384. Secondary outcomes included longitudinal changes in HBcrAg, ALT, M2BPGi, eGFR, serum phosphorus, and urinary tubular injury markers. These parameters were compared between the ETV continuation and TAF transition groups.

Statistical analysis

Continuous variables are presented as mean values with standard errors, whereas categorical variables are expressed as numbers and percentages. Between-group comparisons were performed using Student’s t-test for continuous variables and Fisher’s exact test for categorical variables. Longitudinal changes in clinical parameters were evaluated by comparing differences between baseline and the final observation point. All analyses were performed using SPSS Statistics for Windows, version 11.5 (SPSS Inc., Chicago, IL). Statistical significance was set at p < 0.05.

Results

Clinical characteristics

The baseline characteristics of the patients are summarized in Table 1. Overall, 31 and 46 patients infected with genotype C HBV were included in the ETV and TAF groups, respectively. The proportion of HBeAg-positive patients in the ETV and TAF groups was 10% (n = 3) and 9% (n = 4), respectively, with no significant difference between the two groups (p = 1.0). The mean HBsAg levels in the ETV group was 3.2 logIU/ml and in the TAF group 3.1 logIU/ml, with no significant difference between the two groups (p = 0.54). Furthermore, the mean reduction in HBsAg levels during the most recent year of ETV administration prior to the start of this study was −0.022 logIU/ml/year and −0.030 logIU/ml/year in the ETV and TAF groups, respectively, with no significant difference between the groups (p = 0.57). The ETV and TAF groups had HBcrAg levels and a mean eGFR of 3.7 logU/ml and 80, and 3.5 logU/ml and 78, with no significant difference between the two groups (p = 0.39, p = 0.43, respectively). The FIB-4 index was 1.54 in the ETV group and 2.05 in the TAF group, showing a significantly higher value in the TAF group (p = 0.02).

Virological and biological evaluations

The mean changes in serum HBsAg levels at week 384 were −0.575 ±0.151 log IU/ml in the ETV group and −0.531 ±0.160 log IU/ml in the TAF group, with no significant difference between the groups (p = 0.675) (Fig. 2). The mean changes in serum ALT levels were −1.588 ±2.265 U/l in the ETV group and −2.687 ±2.565 U/l in the TAF group, with no significant difference between the groups (p = 0.774) (Fig. 3). The mean changes in M2BPGi levels were −0.102 ±0.065 in the ETV group and 0.188 ±0.104 in the TAF group, with no significant difference between the groups (p = 0.105) (Fig. 4). The mean changes in the FIB-4 index were 0.412 ±0.101 in the ETV group and 0.373 ±0.191 in the TAF group, with no significant difference (p = 0.934) (Fig. 5).

Safety evaluation

A comparative analysis of renal function-related parameters revealed mean changes in eGFR of −9.125 ±1.562 in the ETV group and −7.026 ±2.242 in the TAF group, with no significant difference (p = 0.564) (Fig. 6). Furthermore, the mean changes in serum phosphorus levels were −0.100 ±0.118 and 0.043 ±0.119 in the ETV and TAF groups, respectively, with no significant difference between the groups (p = 0.562) (Fig. 7). Comparison of baseline and 384-week urinary NAG/Cr levels showed no significant differences in the ETV (0.040 ±0.005 vs. 0.047 ±0.004) and TAF groups (0.055 ±0.005 vs. 0.066 ±0.012) (p = 0.423 and 0.352, respectively; Fig. 8A). However, at 384 weeks, the urinary β2MG/Cr ratio was significantly elevated (1.149 ±0.184 vs. 2.512 ±0.353) in the ETV group (p = 0.001), but reduced in the TAF group (2.711 ±0.638 vs. 1.610 ±0.230) compared to that at baseline (p = 0.181) (Fig. 8B).

Discussion

This study examined the long-term clinical impact of transitioning antiviral therapy from ETV to TAF in patients with genotype C CHB who had already achieved sustained viral suppression. Over an extended follow-up period of up to 384 weeks, the magnitude of decline in HBsAg did not differ significantly between patients who continued ETV therapy and those who switched to TAF. These findings suggest that, in patients with stable virological control, replacing ETV with TAF does not substantially modify long-term antiviral efficacy as reflected by HBsAg kinetics.

The reduction in HBsAg observed during long-term NA therapy is generally gradual. In the present study, the mean decrease in HBsAg levels after 384 weeks was −0.575 log IU/ml in the ETV group and −0.531 log
IU/ml in the TAF group, indicating comparable antiviral outcomes between the two strategies. This observation is consistent with the biological characteristics of HBsAg production. Unlike serum HBV DNA, serum HBsAg levels reflect not only active viral replication but also transcriptional activity from covalently closed circular DNA and integrated viral sequences within hepatocytes. Consequently, once viral replication is effectively suppressed by long-term therapy, switching between potent NA may have limited influence on the rate of HBsAg decline.

Previously, Uchida et al. reported that the decline in HBsAg accelerated after patients switched from long-term ETV therapy to TAF [13]. Similarly, a multicenter cohort study by Ogawa et al. demonstrated a gradual decrease in HBsAg levels in patients who changed therapy from ETV to TAF [14]. These reports suggest that switching antiviral agents may enhance HBsAg reduction in certain clinical contexts. In contrast, our findings did not demonstrate a clear additional effect on HBsAg kinetics after the switch to TAF. One possible explanation is that the patients in the present study had already achieved prolonged viral suppression prior to treatment modification. Under such circumstances, differences in pharmacological mechanisms between NAs may have minimal impact on HBsAg production.

In addition to antiviral efficacy, long-term safety is an important consideration in CHB management because NA therapy is typically continued indefinitely. Renal safety is particularly important, as subtle changes in renal function may accumulate during prolonged treatment [15-17]. In our analysis, conventional markers of renal function, including eGFR and serum phosphorus levels, showed no significant differences between the two treatment groups throughout the observation period. These results suggest that ETV continuation and switching to TAF are associated with stable overall renal function during long-term therapy.

However, a difference in the markers of renal tubular injury was observed between the two treatment groups. The urinary β2MG/Cr ratio increased significantly over time in patients who remained on ETV, whereas no significant change was detected in those receiving TAF. The urinary β2MG/Cr ratio is considered a sensitive indicator of proximal tubular dysfunction, and its elevation may precede detectable declines in glomerular filtration, thereby providing early evidence of renal tubular stress. The absence of such an increase in the TAF group suggests that this drug may exert a lower burden on renal tubular cells during prolonged therapy. The favorable effect of TAF on tubular injury may be explained by its pharmacokinetic characteristics. TAF delivers tenofovir more efficiently to hepatocytes while maintaining substantially lower circulating tenofovir concentrations than earlier formulations. Reduced systemic exposure may decrease drug-related mitochondrial dysfunction and cellular stress in renal proximal tubular cells, thereby limiting cumulative tubular injury during long-term therapy. Although ETV is generally considered to have a favorable renal safety profile, our findings suggest that subtle tubular dysfunction may still accumulate during prolonged treatment and become detectable using sensitive biomarkers such as urinary β2MG. Accordingly, several clinical trials have demonstrated improved renal and bone safety profiles with TAF, and our long-term observational findings appear to support the persistence of this safety advantage during extended treatment. Notably, the differences observed in tubular biomarkers did not translate into measurable differences in global renal function parameters such as eGFR. Therefore, although switching from ETV to TAF may reduce tubular stress, the clinical relevance of this finding remains uncertain. Hence, longer follow-up periods and larger patient populations are required to determine whether these biomarker differences will translate into clinically meaningful renal outcomes.

Based on Japanese drug pricing, the estimated annual medication cost for the treatment of chronic hepatitis B (HBV) was approximately USD 372 for entecavir (ETV) and USD 1,987 for tenofovir alafenamide (TAF). Therefore, TAF was associated with an additional annual cost of approximately USD 1,615 compared with ETV. From a pharmacoeconomic perspective, ETV appears to be the more cost-effective treatment option because of its substantially lower acquisition cost. However, treatment selection should not be based solely on medication costs. TAF has demonstrated a favorable renal safety profile compared with some other antiviral agents and may provide additional clinical benefits in patients at increased risk of renal impairment. In such patients, the potential clinical benefits of TAF may justify its higher acquisition cost, although this would require confirmation in cost-effectiveness analyses.

This study also has some limitations that should be considered when interpreting the results. First, this was a single-center study with a relatively small sample size, which may have limited its statistical power and increased the possibility of selection bias. Second, the study only included Japanese patients infected with HBV genotype C; therefore, caution is warranted when extrapolating these findings to other ethnic groups or viral genotypes. Third, major clinical endpoints, such as HBsAg loss or HCC development, rarely occurred during the observation period, limiting evaluation of the impact of treatment strategy on long-term clinical outcomes. Despite these limitations, the present study provides valuable insights into the long-term outcomes of switching antiviral therapy in patients with stable CHB. The finding of a more stable urinary β2-microglobulin/creatinine ratio after switching to TAF, despite the small sample size, warrants further investigation in larger studies.

Conclusions

The long-term antiviral effects of TAF following switching from ETV appear to be similar to those of continued ETV therapy in patients with genotype C CHB. The overall renal function remained stable in both groups; however, the lack of significant increases in tubular injury markers among patients treated with TAF may suggest a more favorable renal safety profile during prolonged therapy compared to ETV. Therefore, larger, multicenter randomized studies are warranted to confirm these observations and determine whether switching therapy influences long-term clinical outcomes such as functional cure and prevention of HCC.

Disclosures

This research received no external funding.

This study was conducted in accordance with the principles of the 1964 Declaration of Helsinki and its later amendments. The study protocol was approved by the Institutional Review Board of Kindai University (approval number: 28-271). Written informed consent was obtained from all participants prior to enrollment.

The authors declare no conflict of interest.

References