FormalPara Key Summary Points

Why carry out this study?

HBsAg clearance rate is extremely low with nucleoside (acid) analogues (NAs) monotherapy and most patients with CHB need lifelong medication.

The efficacy and optimization of PegIFNα-2b add-on therapy for HBeAg-negative patients with CHB were analyzed in this study.

What was learned from the study?

PegIFNα-2b add-on therapy could enhance HBsAg clearance for HBeAg-negative patients with CHB.

Low HBsAg level is a potential predictor of HBsAg clearance by PegIFNα-2b add-on therapy.

Prolonging PegIFN treatment duration after 48 weeks of PegIFN treatment may not improve the HBsAg clearance.

Introduction

Hepatitis B virus (HBV, a hepatotropic virus) can establish a persistent and chronic infection in humans through immune anergy. Currently, about 2 billion people worldwide suffer from HBV infection, and 3.5% of the global population (about 257 million individuals) is chronically infected [1, 2]. Chronic HBV infection is a major public health problem, leading to cirrhosis, liver failure, or hepatocellular carcinoma [3]. Active HBV infection includes hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients with chronic hepatitis B (CHB). Most HBeAg-negative patients with CHB have a long course, low spontaneous hepatitis B surface antigen (HBsAg) clearance rate, and low sustained response rate [4,5,6].

HBsAg clearance is the drug withdrawal indication and the goal of HBeAg-negative patients with CHB treatment [7]. However, HBsAg clearance rate is very low after long-term NAs therapy in this population and most of them need lifelong medication [8, 9]. Although nucleoside (acid) analogues (NAs) can suppress viral replication, reactivation is common when treatment is discontinued. For HBeAg-positive patients who achieved HBeAg seroconversion by NAs treatment, virological relapse was up to 71% within 1 year post NAs discontinuation. In addition, 90% of HBeAg-negative patients who maintained virological suppression on NAs therapy had a risk of relapse after consolidation therapy for at least a year or decreases [10,11,12,13,14]. Moreover, HBeAg-negative patients with CHB would suffer virological recurrence, liver function deterioration, severe hepatitis, and high incidence of liver cancer if they had drug withdrawal without obtaining HBsAg clearance [6, 15, 16]. Therefore, new treatment strategies are required to increase HBsAg clearance rate in HBeAg-negative patients with CHB.

Interferon alfa was the first antiviral drug for CHB treatment. Studies have reported that adding or switching to peginterferon could increase HBsAg clearance rate from 8.5% to 37.4% in NAs-treated patients with CHB [17,18,19,20]. The OSST trial and New Switch Study showed that patients with low HBsAg (< 1500 IU/mL) at baseline were more likely to achieve HBsAg clearance compared to those with HBsAg > 1500 IU/mL [19, 21]. Beyond previous studies, this study aimed to evaluate the efficacy and safety of peginterferon alfa-2b (PegIFNα-2b) treatment for HBeAg-negative patients with CHB who achieved HBsAg < 1500 IU/mL and HBV DNA < 100 IU/mL by NAs treatment.

Methods

Patients

NAs-treated patients with CHB in this study were enrolled from outpatient departments of Xiangya Hospital, Central South University during April 2018–October 2019. Patients paid for the drug by themselves in this real-world study and all enrolled patients chose to receive PegIFNα-2b treatment voluntarily with full knowledge and signed an informed consent. This study was in compliance with the Declaration of Helsinki and was approved by Xiangya Hospital Ethics Committee, Central South University.

Inclusion criteria:

  1. 1.

    Patients with CHB diagnosed according to the Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2019 Edition) in China [22].

  2. 2.

    NAs therapy ≥ 24 weeks and no interferon therapy within 6 months.

  3. 3.

    HBeAg-negative patients with HBsAg level < 1500 IU/mL and HBV DNA level < 100 IU/mL.

  4. 4.

    Aged 18–65 years old.

Exclusion criteria:

  1. 1.

    Patients with other viral hepatitis (hepatitis A, C, D, and E) or other liver diseases (such as autoimmune liver disease, Wilson’s disease, alcoholic liver disease, or drug hepatitis).

  2. 2.

    Decompensated cirrhosis or liver cancer.

  3. 3.

    HIV infection, lesions of important organs (such as lesions of the heart, brain, lung, kidney, or fundus), severe metabolic diseases, malignant tumors, severe mental diseases, etc.

  4. 4.

    Hyperthyroidism or hypothyroidism, combined with other autoimmune diseases.

  5. 5.

    Patients with interferon allergy, alcoholism, or drug addiction.

  6. 6.

    Patients receiving chemotherapy or immunosuppressive therapy.

Treatment Plan

In this non-randomized study, patients in the control group received continued NAs monotherapy. Patients in the PegIFNα-2b add-on treatment group received combination therapy of PegIFNα-2b (180 μg/week, subcutaneous injection) and NAs [entecavir (ETV), tenofovir (TDF), or tenofovir alafenamide (TAF)]. If patients took lamivudine (LAM), telbivudine (LdT), or adefovir (ADV) previously, they took ETV instead. If patients took ETV, TDF, or TAF previously, they continued previous NAs therapy.

All patients received PegIFNα-2b + NA or NA for at least 48 weeks. After finishing 48 weeks of treatment, patients chose to continue peginterferon therapy up to 72 weeks or discontinue peginterferon therapy voluntarily. If patients obtained HBsAg clearance at week 48, NA was stopped; otherwise, NA was continued.

Data Collection

  1. 1.

    Baseline information: gender, age, history of alcohol consumption, family history of hepatitis B, history of antiviral therapy, and history of interferon therapy.

  2. 2.

    Laboratory test: white blood cell (WBC), peripheral blood neutrophil (NEUT), platelet (PLT), hemoglobin (Hb), total bilirubin (TBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), HBV DNA quantification, HBsAg and HBeAg at baseline and at week 12, 24, 48, 72 during treatment.

  3. 3.

    Thyroid function, autoantibodies, alpha-fetoprotein (AFP), and abdominal ultrasound at baseline and during treatment.

The quantitative detection of HBV DNA was measured by high-sensitivity fluorescence quantitative real-time PCR with lower detection limit of 10 IU/mL. The quantitative detection of HBV markers (HBsAg, HBsAb, and HBeAg) were measured by Abbott Chemilescent Automatic Immunoanalyzer (ARCHTIECT i2000sr). The lower limit of HBsAg detection was 0.05 IU/mL, and HBsAb level > 10 IU/mL was defined as positive.

Evaluation of Therapeutic Efficacy

The primary endpoint was HBsAg clearance at week 48. The secondary endpoint was HBsAg serological conversion rate and HBV DNA levels at week 48, HBsAg clearance, and HBsAg serological conversion at week 12, 24 and 72. Complete response was defined as HBsAg < 0.05 IU/mL and non-complete response was defined as HBsAg ≥ 0.05 IU/mL.

Statistical Analysis

Data analysis of primary and secondary endpoints in this study was performed in the per-protocol (PP) population. Response rates were summarized by calculating percentage at week 48 and week 72. Data analysis for safety was performed in the safety population. Measurement data with normal distribution were expressed as mean ± standard deviation (SD). Measurement data with non-normal distribution were expressed by median [25th, 75th] (M[P25, P75]). Counting data were expressed in frequency (%). T test or non-parametric test was used for comparison of continuous variables between groups. Categorical variables were compared by chi-square test or Fisher exact test. All data were analyzed by SPSS 20.0 and graphpad prism 7.0. P < 0.05 was statistically significant.

Results

Patients and General Information

A total of 196 NAs-treated patients with CHB met the inclusion criteria, including three patients with loss to follow-up in the PegIFNα-2b add-on group (one because of hyperthyroidism, one because of continuous aminotransferase elevation, and one because of severe flu-like symptoms during the treatment), and 10 lost to follow-up in the NAs monotherapy group (Fig. 1). Therefore, 183 NAs-treated patients with CHB (108 in the PegIFNα-2b add-on group and 75 in the NAs monotherapy group) were enrolled in the study eventually. Baseline characteristics of gender, HBsAg level, HBV DNA, WBC, NEUT, Hb, TBIL, ALT, and AST were not statistically different between the two groups, whereas age and PLT level were (Table 1).

Fig. 1
figure 1

Flow chart of interferon therapy of 183 NAs-treated patients with CHB

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Table 1 Baseline characteristics of 183 patients with CHB treated with NAs or PegIFNα-2b combination therapy
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Virological Response

Ten patients were HBV DNA positive at baseline and all turned HBV DNA negative (HBV DNA < 10 IU/mL) after 48 weeks of PegIFNα-2b treatment. At the end of 48 weeks of treatment, 100% (108/108) and 97.33% (73/75) patients maintained HBV DNA suppression in the PegIFNα-2b add-on group and NAs monotherapy group, respectively. Three patients (4.0%, 3/75) had increased HBV DNA at week 48 by NAs monotherapy, but no significant difference compared with PegIFNα-2b add-on therapy (P = 0.067) (Table 2).

Table 2 Virological response at 48 weeks of treatment
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Serological Response

At week 48, HBsAg clearance (50.93%, 55/108) and HBsAg seroconversion (48.15%, 52/108) were achieved only in patients with PegIFNα-2b add-on therapy. Significantly more patients with PegIFNα-2b add-on therapy had low HBsAg level (0.05 ≤ HBsAg < 10 IU/mL) than those with NAs monotherapy (23.15% vs 4.0%, P = 0.000) (Table 3). In addition, HBsAg level showed a downward trend during PegIFNα-2b treatment. The percentage of patients with HBsAg < 0.05 IU/mL increased gradually, while those with HBsAg > 10 IU/mL decreased gradually during PegIFNα-2b add-on therapy (Fig. 2).

Table 3 Serological response at 48 weeks of treatment
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Fig. 2
figure 2

Different HBsAg levels during treatment. HBsAg level showed a downward trend during PegIFNα-2b treatment

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For 108 patients with PegIFNα-2b add-on therapy, HBsAg clearance and HBsAg seroconversion were increased over 48 weeks of treatment (Table 4). Patients with baseline HBsAg level < 100 IU/mL and HBsAg level 100–500 IU/mL achieved significantly higher HBsAg clearance than those with baseline HBsAg level 500–1500 at week 12, 24, and 48 (P = 0.001 vs P = 0.000 vs P = 0.042 respectively). Similarly, baseline HBsAg level 500–1500 IU/mL was associated with the lowest HBsAg seroconversion. In addition, at week 48, the HBsAg seroconversion was significantly different among patients with HBsAg level < 100 IU/mL, 100–500 IU/mL, and 500–1500 IU/mL (58.70% vs 54.84% vs 25.81%, P = 0.012) (Table 4).

Table 4 Serological response for PegIFNα-2b add-on therapy during the treatment according to baseline HBsAg level
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There were 95 patients with available data at week 72, including 47 HBsAg-positive patients and 48 HBsAg-negative patients at week 48. Both HBsAg clearance and HBsAg seroconversion were not significantly different between continued (18 patients) and discontinued (29 patients) PegIFNα-2b therapy at week 72 in 47 HBsAg-positive patients (16.67% vs 13.79%, P = 0.582 and 33.33% vs 20.69%, P = 0.493 respectively). In addition, 83.33% (5/6) and 95.24% (40/42) achieved HBsAg seroconversion for continued or discontinued PegIFNα-2b therapy respectively at week 72 in 48 HBsAg-negative patients (P = 0.336) (Table 5).

Table 5 Serological response for PegIFNα-2b add-on therapy at week 72 according to week 48 HBsAg status
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Safety

During the 48 weeks of treatment, more patients in PegIFNα-2b add-on therapy group experienced adverse events, such as transient influenza-like symptoms in the early stage (fever, fatigue, and muscle soreness), neutropenia (NEUT < 1.50 × 109/L), thrombocytopenia (PLT < 100 × 109/L), and elevated ALT or AST (Table 6). Three patients discontinued the treatment and were removed from the PegIFNα-2b add-on therapy group (one because of hyperthyroidism, one because of continuous elevation of transaminase, and one because of severe flu-like symptoms). One out of 111 PegIFNα-2b recipients developed ALT flare (ALT > 5 upper limit of normal). No NAs recipients discontinued treatment for safety reasons or experienced ALT flare. All side events were alleviated by timely treatment.

Table 6 Safety outcome of the study population
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Discussion

Most patients with CHB failed to withdraw the drug after achieving HBeAg clearance and HBV DNA inhibition by NAs therapy, because HBsAg clearance rate was extremely low by NAs monotherapy. PegIFN-based sequential/combination therapy is important to achieve optimal clinical cure. Previous study showed that 52.2% of HBeAg-negative patients with CHB with HBsAg < 10 IU/mL achieved clinical cure by PegIFN-based therapy [23]. This study focused on HBeAg-negative patients with CHB with low HBsAg level (< 1500 IU/mL) and low viral load (HBV DNA < 100 IU/mL) after NAs treatment, aiming to evaluate the efficacy and safety of PegIFNα-2b add-on therapy.

NAs and PegIFN can achieve virologic responses through different mechanisms [24,25,26]. After 48 weeks of add-on treatment of PegIFNα-2b, HBV DNA was undetectable in all patients. To our acknowledge, NAs are very effective in suppressing HBV DNA in patients with CHB. Previous studies showed that the virologic response of PegIFN and NAs combination therapy was as good as that of NAs monotherapy [27, 28]. Our study also showed that PegIFNα-2b add-on therapy was effective at maintaining viral suppression. No patients experienced viral rebound or HBV DNA increase during PegIFNα-2b add-on therapy.

HBsAg clearance represents clinical cure for HBeAg-negative patients with CHB. The OSST trial showed 22.2% HBsAg clearance among PegIFN alfa-2a-treated patients with HBeAg loss and HBsAg < 1500 IU/ml [19]. Yan et al. [25] showed 15% HBsAg clearance at week 48 among PegIFN add-on therapy patients with negative HBeAg. Wu et al. [20] showed 37.4% HBsAg clearance and 29.7% HBsAg seroconversion in PegIFNα-2a add-on therapy patients with HBsAg < 1500 IU/mL. Compared with those published studies [19, 20, 25], our study showed higher rates of HBsAg clearance (50.93%) and HBsAg seroconversion (48.15%) at week 48 following PegIFNα-2b add-on therapy. This better outcome might be attributed to the lower HBV DNA and HBsAg level at baseline in our study, since low HBsAg and HBV DNA levels were closely related to response to PegIFN add-on therapy and high HBsAg clearance rate [26, 28]. On the other hand, Chan et al. [29] reported that 50% of HBeAg-positive patients with CHB with baseline HBsAg < 500 IU/mL achieved HBsAg loss after 48 weeks of PegIFN treatment. In our study, we also showed that HBeAg-negative patients with CHB with low HBsAg (< 1500 IU/mL) and low HBV DNA (< 100 IU/mL) levels at enrollment had a good chance of achieving HBsAg clearance. In addition, we found that patients with HBsAg < 100 IU/mL had the highest rate of HBsAg clearance (up to 60.87% at week 48). Therefore, it would be valuable for clinical practice to identify them as the best candidates for PegIFN add-on therapy.

More interestingly, our results indicated that longer-term PegIFNα-2b add-on therapy could not improve HBsAg clearance or HBsAg seroconversion after 48 weeks of PegIFNα-2b add-on therapy. For HBeAg-negative or HBeAg-positive patients at week 48, HBsAg clearance and HBsAg seroconversion had no significant difference by continuing or discontinuing PegIFNα-2b therapy at week 72. Previous study showed that HBsAg clearance and seroconversion did not increase at week 72 compared with that at week 48 [25]. Hu et al. [21] also showed that the differences of HBsAg clearance were not statistically significant between week 48 and week 96, although HBsAg loss rates were improved from 14.4% to 20.7% by extending treatment from 48 to 96 weeks. Therefore, it would be unnecessary to prolong PegIFNα-2b treatment duration in clinical practice.

Generally, PegIFNα-2b add-on therapy was safe and well tolerated for NAs-treated patients with CHB. Most patients had transient flu-like symptoms, transaminase elevation (2–5 × ULN), and mild to moderate neutropenia, but could be mitigated by symptomatic treatment. Therefore, it is critical to regularly review and quickly treat adverse events during PegIFNα-2b application.

In summary, we analyzed the effect of PegIFNα-2b in HBeAg-negative patients with CHB with low HBsAg level through an observation study. We found that PegIFNα-2b could effectively inhibit HBV replication, reduce HBV antigen generation, and improve seroconversion. HBeAg-negative patients with CHB with low HBsAg level could achieve clinical cure and had drug withdrawal safely by PegIFNα-2b add-on therapy. Baseline HBsAg < 100 IU/mL predicted a better chance of HBsAg clearance. In addition, it would be unnecessary to prolong PegIFNα-2b treatment duration after 48 weeks of PegIFNα-2b add-on therapy. However, further study should be carried out to verify the results in the future.

This study has certain limitations: (1) This is a single-center real-world study, and the sample size of HBeAg-negative CHB population is not large; (2) The current study is still under way. Some patients had limited follow-up time, which did not involve the long-term response of HBsAg clearance after drug withdrawal. (3) Baseline age and PLT level between PegIFNα-2b + NAs therapy and NAs monotherapy were statistically different. (4) The treatment response of PegIFNα-2b is affected by HBV genotype. However, as a result of the continuous HBV DNA suppression (< 100 IU/mL) in the enrolled CHB population, the impact of HBV genotype on response was not explored.

Conclusions

HBeAg-negative patients with CHB with low HBsAg level could achieve high HBsAg clearance and seroconversion by PegIFNα-2b add-on therapy. Patients with low baseline HBsAg levels were more likely to obtain HBsAg clearance. PegIFNα-2b add-on therapy could be a good treatment option for NAs-treated patients with CHB with negative HBeAg, particularly for those with lower HBsAg level (< 100 IU/mL). This will have important implications for PegIFNα-2b treatment screening in HBeAg-negative patients with CHB. It would be unnecessary to prolong PegIFNα-2b treatment duration after fininshing 48 weeks PegIFNα-2b treatment in clinical practice.