The presence of baseline HBsAb-Specific B cells can predict HBsAg or HBeAg seroconversion of chronic hepatitis B on treatment

ABSTRACT

Indices for predicting HBsAg or HBeAg seroconversion in patients with chronic hepatitis B virus (HBV) infection during antiviral therapy remain elusive. We aimed to investigate if the presence of HBsAb-specific B cells at baseline can predict HBsAg or HBeAg seroconversion. In this study, 134 treatment-naive patients with chronic HBV were enrolled. A baseline HBsAb-specific B cell ELISpot assay was performed for all the patients that enrolled. Serum samples were collected at 12, 24, and 48 weeks for patients treated with Peg-IFN-α, or at 1 year, 3 years, and 5 years for patients treated with NAs. Laboratory testing of HBsAg, HBsAb, HBeAg, HBeAb, HBcAb, HBV DNA, ALT, and AST was done. We observed a significantly lower frequency of HBsAb-specific B cells in patients with chronic HBV than in healthy individuals . In the Peg-IFN-α-treated group, 41.2% of patients with baseline HBsAb-specific B cells achieved HBsAg seroconversion, while only 13.6% of patients without baseline HBsAb-specific B cells achieved HBsAg seroconversion (p = 0.006). By logistic regression analysis, patients with baseline HBsAb-specific B cells and HBsAg ≤ 1500 had higher HBsAg clearance at the end of treatment (p < 0.05). In the NA-treated group, 58.3% of patients with baseline HBsAb-specific B cells achieved HBeAg seroconversion, whereas only 30.0% of patients without baseline HBsAb-specific B cells achieved HBeAg seroconversion (p = 0.114). Our result revealed that baseline HBsAb-specific B cells by ELISpot assay might be a valuable predictive biomarker of HBsAg or HBeAg seroconversion in patients with chronic HBV on treatment.

KEYWORDS:

• HBsAb
• Chronic HBV
• HBsAg seroconversion
• Peg-IFN-α treatment
• ELIspot
• HBsAb-specific B cells

Introduction

Hepatitis B virus (HBV) infection is a worldwide epidemic. According to the World Health Organization, approximately 2 billion people worldwide have been infected with HBV, 240 million of whom have chronic infections. About 650,000 people die each year from liver failure, cirrhosis, and hepatocellular carcinoma (HCC) due to HBV infection. It causes 30% and 45% of the global cases of cirrhosis and HCC, respectively [1].

Although widespread HBV vaccination has reduced new infections, many people with chronic HBV infection still need effective treatment. Currently, the approved treatment regimens for chronic HBV include oral nucleotide DNA polymerase or reverse transcriptase inhibitors nucleoside/nucleotide analogues (NAs), and injectable immunomodulators (interferon-alpha, Peg-IFN-α). NAs is inexpensive and applicable to a wide range of patients. However, it requires long-term or even lifelong treatment that can result in drug-resistance mutations, which lead to treatment failure. Peg-IFN-α is less well-tolerated than NAs. It produces an antiviral response and inhibits HBV transcription and replication [2–4]. However, even in cases where the virus is suppressed after long-term therapy, patients still experience a viral relapse when therapy is discontinued. Long-term NA therapy with or without short-term IFN-α is the most common treatment option [5]. But challenges such as medication adherence, treatment cost, resistance mutations, and side effects still exist. Therefore, there is an urgent clinical need to explore an effective technique for monitoring therapy and predicting outcomes.

After HBV infection, B cells can produce a variety of HBV-specific antibodies against different epitopes of the HBV virus and non-infectious subviral particles (SVPs) that produced during HBV replication process [6]. Anti-HBs are antibodies against HBsAg. In most cases, only anti-HBs, which specifically recognize and bind HBsAg-associated epitopes, could offer significant protection [7]. Serological anti-HBs levels are currently the most common criterion for assessing the success of HBV vaccine immunization [8]. It has also been used as a marker for the clinical cure of HBV. However, due to being bound to the overabundant SVPs and the low clinical cure rates with current therapy, serologic anti-HB levels usually escape detection and are significantly limited in assessing the immune status of patients. Therefore, new biological indicators are needed to detect the specific immune response to the hepatitis B virus. Anti-HBs are theoretically produced by HBsAb-specific B cells that include antibody-secreting cells and memory B cells. Therefore, HBsAb-specific B cells may be a better indicator of anti-HBs production. We previously showed that the use of B-cell Enzyme Linked ImmunoSpot (ELISpot) was effective to enumerate the frequency of HBsAb-specific B cells in both healthy and HBV-infected individuals. Interferon treatment resulted in the dynamic frequency of HBsAb-specific memory B cell response among four HBV-infected individuals [9], suggesting that our established HBsAb-specific B-cell ELISpot might be useful to follow the clinical outcome of such immune interventions. Therefore, it is essential to further validate our observation by expanding the clinical samples using B cell ELISpot.

In our study, we aimed to investigate the characteristics of HBsAb-specific B cells in patients being treated with Peg-IFN-α or NAs using the ELISpot assay and correlate the treatment effect with the HBsAb-specific B cells in patients who completed treatment. We will evaluate the clinical value of peripheral blood HBsAb-specific B cells in guiding the optimal treatment and clinical cure of patients.

Materials and methods

Patients

We enrolled 156 patients with chronic hepatitis B in the study from the hepatitis clinic of Nanjing Gulou Hospital between March 2016 and March 2022. They were HBsAg positive for at least 6 months and had not received peg-IFN-α or NAs treatment before enrolment. Their baseline clinical features and peripheral blood samples were collected and recorded.

Patients with other types of hepatitis, cirrhosis, hepatocellular carcinoma, and other major diseases were excluded. They all signed an informed consent form. This study was approved by the Institutional Review Board of Nanjing Drum Tower Hospital.

The healthy controls involved in the research are all vaccinated by HepB vaccine and showed negative of anti-HBc. Health individuals always have been HBsAg negative. The exclusion criteria were as follows: (1) abnormal indicators of liver and kidney function; (2) presence of tumour; (4) other types of liver disease, such as hepatitis C, autoimmune hepatitis, etc. The characteristics of healthy controls were shown in Supplementary Table 1.

Study design

We collected the peripheral blood mononuclear cells (PBMCs) from all enrolled patients and performed a baseline HBsAb-specific B cell ELISpot assay. The serum samples were collected at baseline, 12, 24, and 48 weeks for patients on IFN-α treatment, or at 1 year, 3 years, and 5 years for patients on NAs treatment. During this follow-up period, we did a clinical evaluation and standard laboratory test for HBsAg, HBsAb, HBeAg, HBeAb, HBcAb, HBV DNA, ALT, and AST.

PBMC isolation

PBMCs were isolated from 15 mL of fresh heparinized peripheral blood by density gradient centrifugation using a SepMate™−15 (86415, Stemcell Technologies). We added blood diluted in phosphate-buffered saline (PBS) to Lymphoprep™ (07851, Stemcell Technologies) and centrifuged it at 2500 rpm for 15 min, and then collected the cell layer.

PBMC culture and B-cell stimulation

The fresh PBMCs obtained were washed with PBS, centrifuged at 1300 rpm for 5 min, and the supernatant was discarded. The bottom cells were suspended with an RPMI 1640 medium (C11875500BT, Gibco) that contained 10% penicillin–streptomycin-glutamine (10378016, Gibco) and 10% fetal bovine serum (35-081-CV, Corning® Fetal Bovine Serum). To effectively stimulate B cells, 1 μg/mL R848 (Mabtech) and 10 ng/mL of recombinant human IL-2 (Mabtech) were added. Cells were cultured in a 24-well plate for five days at 37°C, 5% CO₂.

B cell ELISpot assay

The detailed procedure of B cell ELISpot assay was previously reported and briefly described as following [9]. Sterile 96-well Multiscreen-IP filter plates with a PVDF membrane (MAIPSWU10, Millipore) were coated with either anti-human IgG (15 μg/mL, 3850-6-1000, Mabtech) or recombinant HBsAg (10 μg/mL, gifted by HuaBei Pharmacy and subtyped as adw2) overnight at 4°C. The anti-human IgG was used as positive control and wells with PBS were used as negative controls. Plates were washed with sterile PBS and blocked with an RPMI-1640 medium containing 2% penicillin–streptomycin-glutamine and 10% FBS for 2 h at 37°C, 5% CO₂. Serially diluted cells were added at 200 k or 400 k cells/well for HBsAg-coated wells. The culture plates were incubated for 18 hours at 37°C with 5% CO₂. Antibody-secreting cells were measured using the Human IgG ELISpot kit (3850-2H, Mabtech) after washing the cells with PBS and incubating them with biotin-labeled anti-IgG mAb and horseradish peroxidase (HRP)-conjugated streptavidin, according to the manufacturer’s protocol. An automated ELISpot image analyzer (Cellular Technology Limited, Hongkong, China) was used to analyze and count the spots. Spots numbers were converted into the number of spots per 10⁶ PBMCs.

HBV serologic biomarkers

Levels of serum HBV markers (HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb) were determined using the Architect-i2000 system (Abbott Laboratories). The quantitative determinations of biomarkers were considered positive according to the criteria set by the manufacturer. Serum HBV DNA was quantified by a real-time fluorescent quantitative polymerase chain reaction (RT–PCR, Aikang Biotechnology Co., Ltd) with a detection limit of 500 IU/mL. Serum ALT and AST levels were determined by commercial kits.

Ethical proof

The study was approved by the ethics committee of Nanjing Drum Tower hospitals (2008-022), and the process followed the ethical guidelines of the Declaration of Helsinki.

Statistical analyses

We reported continuous variables of normal distributions as means with standard deviations and continuous variables of skewed distributions as medians and interquartile ranges. We described categorical variables as counts and percentages, used independent t-tests to compare continuous variables with normal distribution, and Mann–Whitney U (non-normal distribution) were used to compare continuous variables between groups. The factors influencing “functional cure” were analyzed by binary logistic regression. The statistical analysis was conducted using IBM SPSS software, version 23.0 (IBM Corporation). A P value less than 0.05 (two-sided) was considered statistically significant. * indicates P  <  0.05, ** indicates P  <  0.01, *** indicates P  <  0.001, **** indicates P  <  0.0001, and ns indicates no significant difference.

Result

Baseline demographics and disease characteristics

We enrolled 156 treatment-naive patients with chronic HBV infection in the study between March 2016 and March 2022. Of these, some were excluded because of HCC or other malignancies (n = 3), follow-up data loss (n = 10), limited period of treatment (n = 8), or pregnancy (n = 1) (Supplementary Figure 1). Our study includes 134 CHB patients, including 16 treatment-naïve patients and 118 treated patients. Among 118 treated patients, 40 patients received NAs monotherapy. 78 patients received pegylated interferon-alfa (Peg-IFN) based therapy, including 29 patients were treated with Peg-IFN (180 μg/ stick/week) and 49 patients were treated with Peg-IFN combined with NAs. Combined with or without interferon, NAs regimen was 1 tablet/time, 1 time/day, and was taken daily during follow-up.

The overall demographic and baseline clinical characteristics of the study cohort are shown in Supplementary Table 2. The patients were grouped into those on Peg-IFN-α treatment, on NAs treatment, and on no treatment. As shown in Supplementary Table 2, the age was comparable between the three groups (median: 43.07 vs. 38.57 vs. 37.88 years old).

Patients with chronic HBV infection had fewer HBsAb-specific B cells

Using the established B-cell ELISpot assay [9], we observed a significantly lower frequency of HBsAb-secreting B cells in patients with chronic HBV compared with healthy individuals (4.16 vs. 44.78 cells per 10⁶ PBMCs, P  <  0.0001) (Figure 1(A and B)).

Figure 1. HBsAb-Specific B cells can be detected in peripheral blood of CHB patient (A) Representative B cell enzyme-linked immunosorbent spot (ELISpot) assay to detect IgG secreting or HBsAb secreting B cells from peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis B (CHB) and healthy controls (HC). (B) The frequency of HBsAb secreting B cells of PBMC from healthy controls versus CHB patients. (C) Patients were divided into four groups based on their circulating HBsAb levels: 0 mIU/mL (n = 47), 0–1 mIU/mL (n = 42), 1–10 mIU/mL (n = 33) and ≥10 mIU/mL (n = 12), the number of HBSAb-SFCs was compared. (D) Patients were divided into three groups based on their peripheral blood HBV-DNA levels: ≤500 IU/mL (n = 92), 500-10⁶IU/mL (n = 15) and ≥10⁶ IU/mL (n = 27), the number of HBSAb-SFCs was compared. (E) Patients were divided into three groups based on the level of HBeAg in peripheral blood: ≤1 SCO (n = 93), 1–100 SCO (n = 10) and ≥100 SCO (n = 31), the number of HBSAb-SFCs was compared. (F) Patients were divided into four groups based on their peripheral blood ALT levels: 0–20 U/L (n = 28), 21–40 U/L (n = 50), 41–80 U/L (n = 34) and ≥81 U/L (n = 22), the number of HBSAb-SFCs was compared. (F) Relationship between baseline HBsAg titres and the presence of baseline HBsAb-Specific B Cells was analysed. Mann-Whitney U (non-normal distribution) were used to compare continuous variables between groups. * p < 0.05, **** p < 0.0001.

Further, we stratified and analyzed the number of circulating HBsAb-specific B cells detected by ELISpot and the biochemical indices of the patients. The clinical characteristics of patients with or without baseline HBsAb-specific B cells was described as in Supplementary Table 3. First, we divided patients into four groups based on their circulating HBsAb levels: 0 mIU/mL, 0–1 mIU/mL, 1–10 mIU/mL, and ≥10 mIU/mL. We found that the number of HBsAb-specific B cells per 10⁶ PBMCs of patients was significantly less in the 0 mIU/mL group vs ≥10 mIU/mL group (adjusted p = 0.0455), ≤1 mIU/mL group vs ≥10 mIU/mL group (adjusted p = 0.0183) and 1–10 mIU/mL group vs ≥ 10 mIU/mL groups (adjusted p = 0.0455) (Figure 1(C)). The information of post hoc testing after Kruskal–Wallis test about the relationship between serum level of HBsAb and frequency of HBsAb-secreting B cells was shown as Supplementary Table 4. We divided the patients into three groups based on their serum level of HBV DNA levels: ≤500 IU/mL, 500-10⁶IU/mL, and ≥10⁶IU/mL. We found no significant difference in the number of HBsAb-specific B cells per 10⁶ PBMCs between the three groups (≤500 IU/mL group vs 500-10⁶ IU/mL group, adjusted p = 0.8813; ≤500 IU/mL group vs 10⁶ IU/mL group, adjusted p = 0.3865; 500-10⁶ IU/mL group vs ≥10⁶ IU/mL group, adjusted p = 0.4107) (Figure 1(D)). Further, we divided the patients into three groups based on the level of HBeAg in peripheral blood: ≤1 SCO, 1–100 SCO, and ≥100 SCO, and found no significant difference in the number of HBsAb-specific B cells per 10⁶ PBMCs among the three groups (≤10 SCO group vs 10-100 SCO group, adjusted p = 0.9979; ≤10 SCO group vs ≥100 SCO group, adjusted p = 0.3083; 10–100 SCO group vs ≥100 SCO group, adjusted p = 0.6814) (Figure 1(E)). We divided patients into four groups based on their peripheral blood ALT levels: 0–20 U/L, 21–40 U/L, 41–80 U/L, and ≥81 U/L, and revealed that there was no significant difference in the number of HBsAg specific B cells per 10⁶ PBMCs between the four groups (Figure 1(F)). Finally, relationship between baseline HBsAg titres and the presence of baseline HBsAb-Specific B cells was analysed (Figure 1(G)). The above results suggested that HBsAb-specific B cells in the peripheral blood of CHB patients were positively correlated with HBsAb in serum, but not with HBeAg, HBV-DNA, or ALT.

Baseline HBsAb-Specific B cells in patients on interferon therapy can predict HBsAg seroconversion

The patients enrolled in the study received different treatments, which had a significant impact on outcomes. During follow-up after retention of baseline characteristics, we divided the patients into three groups (IFN, NAs, and no treatment) for further analysis.

Patients who received IFN therapy were divided into the non-HBsAg seroconversion or HBsAg seroconversion groups after receiving the treatment for 48 weeks. There were 58 patients in the non-HBsAg seroconversion group, including 7 females and 51 males, with a mean age of 42.36 years. 20 patients in the HBsAg seroconversion group, including 2 females and 18 males, with a mean age of 43.97 years (Table 1). Further, we compared the biochemical and HBV-related virological indices of patients in the non-HBsAg seroconversion and HBsAg seroconversion groups before (0w) and at 12, 24, and 48 weeks after treatment. The ALT flare phenomenon was observed by comparing the ALT levels of the HBsAg seroconversion and the non-HBsAg seroconversion groups, both at 12 weeks (median: 76.3 U/L vs. 54.6 U/L, P = 0.02) and 24 weeks (median: 72.2 U/L vs. 40.7 U/L, P  =  0.049) (Table 1).

Table 1. Patient characteristics and follow-up data during Peg-IFN-αtherapy in 48 weeks.

	Patients, No./total No. (%)
Characteristic	non-HBsAg seroconversion Group				HBsAg seroconversion Group
Number	58 (74.36)				20 (25.64)
Sex
Female	7 (12.07)				2 (10.00)
Male	51 (87.93)				18 (90.00)
Age, median (IQR), y	43 (35–49)				44 (37–46)
Time point	0 w	12 w	24 w	48 w	0 w	12 w	24 w	48 w
ALT, median (IQR), U/L	31.10 (20.60–48.30)	54.60 (40.50–81.43)	40.70 (31.33–71.02)	29.70 (20.25–49.70)	27.60 (23.43–44.33)	76.30 (57.95–132.93)	72.20 (40.18–144.13)	36.90 (24.43–48.43)
AST, median (IQR), U/L	22.90 (19.90–30.90)	39.75 (31.13–51.00)	34.30 (26.05–54.40)	27.8 (20.08–34.75)	22.25 (20.18–28.85)	50.10 (35.93–112.78)	49.95 (31.00–87.35)	30.50 (25.18–38.23)
HBsAg, median (IQR), log10 IU/mL	2.85 (2.47–3.22)	2.84 (1.65–3.24)	2.53 (0.98–2.94)	2.33 (0.47–3.00)	1.17 (0.52–1.85)	−0.09 (−0.92–0.79)	−0.94 [(−1.64-(−0.36)]	−1.52 [(−1.85)-(−1.18)]
HBeAg status
Negative	43 (74.14)	45 (77.59)	46 (79.31)	46 (79.31)	18 (90.00)	18 (90.00)	20 (100.00)	20 (100.00)
Positive	15 (25.86)	13 (22.41)	12 (20.69)	12 (20.69)	2 (10.00)	2 (10.00)	0 (0.00)	0 (0.00)
HBV DNA, IU/mL
≤500	46 (79.31)	54 (93.10)	56 (96.55)	57 (98.28)	17 (85.00)	20 (100.00)	20 (100.00)	20 (100.00)
>500	12 (20.69)	4 (6.90)	2 (3.45)	1 (1.72)	3 (15.00)	0 (0.00)	0 (0.00)	0 (0.00)

NAs: nucleos(t)ide analogues; ALT: alanine transaminase; AST: aspartate transaminase; HBV: hepatitis B virus; HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B e antigen.

Furthermore, we divided the patients on IFN treatment into a positive group or a negative group based on their baseline existence of HBsAb-specific B cells. The analysis revealed that 41.2% (14 out of 34) of patients in the positive group achieved HBsAg seroconversion, while only 13.6% (6 out of 44) of patients in the negative group achieved HBsAg seroconversion (P  =  0.006) (Figure 2(A)). We followed up with the IFN-treated patients for only 48 weeks, possibly some of the patients with good responses had not yet achieved HBsAg seroconversion. Then we analyzed the reduction proportion of HBsAg reduction at 48 weeks and found that patients in the positive group had a lower reduction proportion than those in the negative group (median: −0.92 vs. −0.67, P  =  0.0055) (Figure 2(B)).

Figure 2. Existence of HBsAb specific B cells before treatment is an immunological indicator for predicting HBsAg seroconversion after interferon therapy. Patients under interferon treatment were divided into a possitive group or a negative group based on whether HBsAb-specific B cells could be detected by ELIspot at baseline. (A) The percentage of HBsAg seroconversion was compared between groups, (B) The reduction proportion of HBsAg loss was compared between groups. Mann-Whitney U (non-normal distribution) were used to compare continuous variables between groups. ** p < 0.01.

These results demonstrated that the existence of baseline HBsAb-specific B cells detected by ELISpot assay in IFN-treated patients was an immunological indicator that can be used to predict HBsAg seroconversion.

The presence of HBsAb-Specific B-cells before interferon therapy was an independent influencing factor for HBsAg seroconversion

The new switch study showed that nucleoside transcutaneous patients, with baseline HBsAg <1500 IU/mL, had higher HBsAg clearance at the end of treatment (p < 0.05). This result was confirmed by the previous OSST study [10]. We divided patients into advantaged (HBsAg ≤ 1500) and non-advantaged group (HBsAg > 1500) based on the existence of HBsAg in the patients before they received interferon therapy. The advantaged group had 61 patients, with 8 females and 53 males who had a mean age of 43.33 years old. The non-advantaged group had 20 patients, with 2 females and 18 males who had a mean age of 43.97 years old. The ALT, AST, HBsAg, HBeAg and HBV DNA levels of patients in both groups was detailed in Supplementary Table 5.

Further, we investigated the independent influences on HBsAg seroconversion in IFN-treated patients by logistic regression analysis. We found that the presence of HBsAb-specific B cells at baseline in the advantaged group was a positive predictor influencing HBsAg seroconversion (P  =  0.005). The HBeAg status (P  =  0.535), level of ALT (P  =  0.245), and AST (P  =  0.111) all had no significant effect on HBsAg seroconversion (Table 2).

Table 2. Univariate cox regression analysis of factors associated with HBsAg clearance in advanced CHB patients.

Univariate analysis	Advantaged patient (HBsAg ≤ 1500)
	OR (95%CI)	P value
Existence of HBsAb-SFCs	5.60 (1.68–18.70)	0.005
HBeAg status
Negative	1 [Reference]
Positive	0.59 (0.11–3.14)	0.535
ALT, U/L	1.02 (0.99–1.05)	0.245
AST, U/L	1.05 (0.99–1.12)	0.111

NAs: nucleos(t)ide analogues; ALT: alanine transaminase; AST: aspartate transaminase; HBV: hepatitis B virus; HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B e antigen

The existence of Baseline HBsAb-Specific B cells in patients on NAs therapy can predict HBeAg seroconversion

Though lifelong treatment with NAs is safe and effective in patients with chronic HBV infection, the overall reduction proportion of HBsAg loss remains low. To further validate the predicate effect of baseline HBsAg-specific B cells on HBeAg seroconversion in patients after NAs treatment, we divided the patients into the non-HBeAg seroconversion group or HBeAg seroconversion groups after 5 years of treatment. There were 19 patients in the non-HBeAg seroconversion group, with 5 females and 14 males who had a mean age of 37.11 years old. The HBeAg seroconversion group had 13 patients with 3 females and 10 males who had a mean age of 34.76 years (Table 3). We compared the biochemical and HBV-related virological indices of patients in the non-HBeAg seroconversion group and the HBeAg seroconversion group at 0 years, 1, 3 years, and 5 years after treatment with NAs therapy, and there were no significant differences (Table 3).

Table 3. Patient characteristics and Follow-up data during NAs therapy in 5 years.

	Patients, No./total No. (%)
Characteristic	non-HBeAg seroconversion Group				HBeAg seroconversion Group
Number	19 (59.38)				13 (40.63)
Sex
Female	5 (26.32)				3 (23.08)
Male	14 (73.68)				10 (76.92)
Age, median (IQR), y	37 (33–41)				35 (33–43)
Time point	0 year	1 year	3 year	5 year	0 year	1 year	3 year	5 year
ALT, median (IQR), U/L	46.90 (27.90–139.60)	27.80 (21.15–43.65)	23.20 (18.15–33.15)	22.10 (17.00–26.30)	95.70 (34.00–359.38)	28.80 (16.55–48.90)	20.30 (12.60–27.40)	19.95 (16.33–32.48)
AST, median (IQR), U/L	37.10 (23.60–79.50)	23.40 (20.60–36.00)	21.30 (16.75–30.05)	19.90 (17.10–22.90)	52.05 (25.98–138.78)	22.50 (20.05–34.15)	21.10 (18.40–23.60)	21.50 (17.95–32.93)
HBsAg, median (IQR), log10 IU/mL	4.43 (3.47–4.65)	3.93 (3.11–4.41)	3.89 (3.07–4.13)	3.76 (3.23–3.88)	3.30 (2.55–4.13)	3.45 (2.48–3.97)	2.92 (1.94–3.73)	3.32 (1.67–3.91)
HBeAg status
Negative	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	4 (30.77)	9 (69.23)	13 (100.00)
Positive	19 (100.00)	19 (100.00)	19 (100.00)	19 (100.00)	13 (100.00)	9 (69.23)	4 (30.77)	0 (0.00)
HBV DNA, IU/mL
≤500	2 (10.53)	15 (78.95)	18 (94.74)	19 (100.00)	2 (15.38)	12 (92.31)	13 (100.00)	13 (100.00)
>500	17 (89.47)	4 (21.05)	1 (5.26)	0 (0.00)	11 (84.62)	1 (7.69)	0 (0.00)	0 (0.00)

NAs: nucleos(t)ide analogues; ALT: alanine transaminase; AST: aspartate transaminase; HBV: hepatitis B virus; HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B e antigen

We divided the patients into positive group or negative group based on whether HBsAb-specific B cells could be detected by ELISpot at baseline in patients treated with NAs. The analysis revealed that 58.3% (7 out of 12) of patients in the positive group achieved HBeAg seroconversion, while only 30.0% (6 out of 20) of patients in the negative group achieved HBeAg seroconversion (Figure 3(A)). In addition, we counted the reduction proportion of HBeAg at 5 years after treatment and found that patients in the positive group had a significant HBeAg decline than those in the negative group, and there was a statistically significant difference (median: −0.96 vs. −0.81, P  =  0.0385) (Figure 3(B)).

Figure 3. Existence of HBsAb specific B cells before treatment is an immunological indicator for predicting HBeAg seroconversion after NAs therapy Patients under interferon treatment were divided into a possitive group or a negative group based on whether HBsAb-specific B cells could be detected by ELIspot at baseline. (A) The percentage of HBeAg seroconversion was compared between groups, (B) The reduction proportion of HBsAg loss was compared between groups. Mann-Whitney U (non-normal distribution) were used to compare continuous variables between groups. * p < 0.05.

The 5-year follow-up data of untreated patients was detailed in (Supplementary Table 6).

Discussion

We enrolled 156 treatment-naive patients with chronic HBV infection. Of these patients, some were excluded because of HCC or other malignancies (n  =  3), follow-up data loss (n  =  10), limited period of treatment (n  =  8), or pregnancy (n  =  1). Of the remaining patients, 78 were on Peg-IFN-α treatment, 40 were on NAs treatment, and 16 were not on treatment. The baseline HBsAb-specific B cells were evaluated by ELISpot. By expanding the study cohort and extending the follow-up time given the previous study [9], our current study further confirmed that baseline HBsAb-specific B cell was an important biomarker to predict HBsAg seroconversion among patients with interferon therapy.

NAs directly inhibit the reverse transcription of HBV polymerase, thereby suppressing viral replication. They are well-tolerated, significantly inhibit HBV replication, and have a high response rate after dosing [11]. However, NAs do not remove the viral transcriptional template, the covalently closed circular DNA molecule (cccDNA), from the nucleus of liver cells [12]. This commonly results in a rapid rebound of viral load after discontinuation of the drug because of viral re-replication. Interferon alpha (IFN-α) is a type I interferon and belongs to the alpha helix cytokine I family. Interferons made antiviral effects by acting directly on the HBV replication cycle. This includes inhibiting RNA and protein production and blocking the assembly of the nuclear capsid. Additionally, HBV replication can be indirectly inhibited by modulating cell-mediated immunity. Compared with NAs, IFN-α achieves higher rates of HBsAg and HBeAg clearance.

Adaptive immune responses are important for the control of HBV infection [13–15]. It is widely accepted that B cell responses was defective in unresolved chronic HBV infection. The role of HBsAb in the pathogenesis and clearance of the virus has often been overlooked. HBsAb can promote antiviral effects by influencing the following aspects of the immune responses: (i) HBsAb can mediate antibody-dependent cytotoxic effects and phagocytosis by binding specifically to antigens and activating intrinsic immunity [16]. (ii) HBsAb can bind free pathogen particles and achieve antibody neutralization. Studies suggested that HBsAb can inhibit HBV invasion into hepatocytes by blocking hepatocyte-expressed receptors [17,18], such as taurocholic acid co-transports peptides (NTCP). (iii) HBsAb can reduce the free virus and its substructure titre in vivo and activate cellular immunity. HBsAb in the serum and liver tissues can effectively reduce HBsAg concentration by neutralizing HBsAg thereby relieving its inhibition of CTL cells and activating cellular immunity to further limit HBV infection [8,19]. Because HBsAb binds to the overabundant SVPs that characterize HBV infection, it is quite usual to not detect it serologically.

We speculated that HBsAb secreting B cells could reflect the immune status of patients. Dysfunctional status of virus–specific B cells in chronic hepatitis B infection has been well characterized previously [20]. The presence of serum HBsAg affected function and phenotype of HBsAg-specific B cells that were unable to mature in vitro into Ab-secreting cells and displayed an increased expression of markers linked to hyperactivation (CD21^lo) and exhaustion (PD-1). Additionally, it is demonstrated that purified, differentiated HBsAg-specific B cells from patients with CHB had defective antibody production and an accumulation of CD21^– CD27^– atypical memory B cells (atMBC) [17]. The accumulation of atMBCs in HBsAg-specific B cells was resulted from the undetectable serum HBsAb. Thus those patients showed HBsAb secreting B cells revealed the functional status of humoral immune specific to HBV.

By analysing HBsAb-specific B cells at baseline, we tested the HBsAb secretion function of B cells, which could accurately reflect the strength of anti-viral humoral immunity in patients. First, the presence of HBsAg-specific B cells that secrete HBsAb directly by ELIspot assay demonstrates the presence of a large source of HBsAb in the patient. HBsAb is the only antibody known to have virus-neutralizing effect, and its presence is very important for seroconversion of HBsAg. Secondly, the presence of HBsAb-specific B cells is an indirect proof of the active immune state in the patient. Because in addition to the function of secreting antibodies, B cells also have antigen presentation, cytokine secretion and activation of downstream immune responses, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The presence of non-exhausted immune responses might have a higher probability of obtaining seroconversion of HBsAg. Our results revealed that effective B-cell responses was involved in and probably necessary for the clearance of HBV, and a defective B-cell response might account for failed treatment.

Our study has some limitations. Firstly, the number of patients in the cohort was limited and only a small number of them received interferon treatment. Secondly, we did the ELISpot test to analyse the baseline HBsAb-SFCs for all samples. However, for the follow-up timepoint, we only did laboratory testing for HBsAg, HBsAb, HBeAg, HBeAb, HBcAb, HBV DNA, ALT, and AST, but no B cell tests. It could have been more favourable to test the predictive performance of the index by collecting more B cell data. In addition, recent evidence suggested that ALT flare was an important indicator of HBsAg seroconversion during IFN therapy. However, our study only predicted the therapeutic effect according to the immunological status of patients before treatment, and did not involve the changes of various biochemical indicators during treatment. Therefore, whether peak ALT occurred during treatment was not included in the analysis.

In summary, our findings strongly implied that HBsAb secretion function of B cells is an important predictor of successful interferon treatment in patients with chronic HBV infection. Furthermore, the presence of baseline HBsAb-specific B cells is an important factor for HBsAg seroconversion that can be used as a prognostic marker in the future. Analysis of HBsAb responses in larger patient cohorts is warranted to better understand and manage chronic HBV infection.

Author’s contributions

All authors contributed to this study at different levels. All authors read and approved the final version. Study concept and design (Chao Wu, Yunxin Chen); acquisition of data (Shengxia Yin, Yawen Wan, R. Issa, Jiacheng Liu, Minxin Mao, Ming Li, Xiaoming Xu, Xin Tong, Chen Tian); statistical analysis and interpretation of data (Yao Zhang, Zhiyi Zhang, Yijia Zhu); drafting of the manuscript (Yawen Wan, Jiacheng Liu, Shengxia Yin); critical revision of the manuscript for important intellectual content (Jian Wang, Rui Huang, Qun Zhang, Jie Li, Chao Wu).

Data availability statement

The data that support the study findings are available upon reasonable request from the corresponding authors (Chao Wu).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Dr. Shengxia Yin wishes to acknowledge the support from the National Natural Science Fund (82002133) and Nanjing Medical Science and Technique Development Foundation (YKK20076). Dr. Jian Wang wishes to acknowledge the support from the Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (2021-LCYJ-PY-43) and Nanjing Medical Science and Technique Development Foundation (YKK21067). Dr. Jie Li wishes to acknowledge the support from the National Natural Science Fund (81970545 and 82170609), Natural Science Foundation of Shandong Province (Major Project) (ZR2020KH006) and Ji’nan Science and Technology Development Project (2020190790). Dr. Chen Tian wishes to acknowledge the support from the National Natural Science Fund (81902061). Dr. Qun Zhang wishes to acknowledge the support from Jiangsu Commission of Health (M2022001).