With the optimization of hepatitis B antiviral treatment strategies, the number of patients achieving HBsAg seroclearance has increased [11, 12]. In turn, reducing HBsAg recurrence has become a major clinical issue of concern. Several clinical studies have shown that anti-HBs seroconversion and high levels of antibodies are strongly associated with recurrence after HBsAg seroclearance [6, 8, 13]. Our prospective study of 238 patients with HBsAg seroclearance following Peg-IFNα treatment with a median follow-up period of 160 weeks showed a cumulative HBsAg recurrence rate of 9.66% and the recurrence rate for those with anti-HBs ≥ 100 IU/L was significantly lower than for those with anti-HBs < 100 IU/L (4.3% vs. 21.1%, p < 0.001) [7]. Another meta-analysis that included a total of 43,924 patients showed that anti-HBs seroconversion was a protective factor against recurrence after HBsAg seroclearance [14]. The study also suggested that the incidence of HCC is much lower in patients with HBsAg seroclearance than in those without (RR = 0.41, P < 0.001). Current studies suggest that hepatitis B vaccination may increase anti-HBs levels which contributes to the reduction of the recurrence rate [9, 10, 15]. In a retrospective study of HBsAg seroclearance achieved with IFNα-based therapy, a significant difference in HBsAg recurrence rates was found among the vaccinated and anti-HBs ≥ 100 IU/L, vaccinated and anti-HBs < 100 IU/L, and unvaccinated individuals, at 7.7%, 58.5%, and 31.9% respectively (all p values were < 0.05) [15]. However, changes in immune function after hepatitis B vaccination in patients with HBsAg seroclearance have rarely been reported in the literature. Thus, we assembled a cohort of patients with HBsAg seroclearance after Peg-IFNα treatment, grouped them according to whether they received hepatitis B vaccination after HBsAg clearance, and investigated the changes in their antibody levels and immune function.
First, we analyzed the differences in antibody production between the groups with or without hepatitis B vaccination. Intra-group comparisons: Anti-HBs levels were significantly increased from baseline at both 12 and 24 weeks in the vaccinated group (all p values were < 0.05), and anti-HBs levels did not change significantly from baseline to these time points in the non-vaccinated group (all p values were > 0.05). Inter-group comparisons: The anti-HBs seroconversion rate reached 100% in the vaccinated group at 24 weeks but only 50% in the non-vaccinated group, the difference was statistically significant (p = 0.006). The proportions of anti-HBs ≥ 100 IU/L and ≥ 300 IU/L at 24 weeks in the vaccinated group was significantly higher than in the non-vaccinated group (all p values were < 0.05). Similar studies have also shown that the anti-HBs seroconversion rate after hepatitis B vaccination in individuals with HBsAg seroclearance after IFNα-based therapy can range from 81.8–100%, compared to only 0-36.4% in unvaccinated individuals [9, 10, 15]. Wu et al. showed that CHB patients treated with Peg-IFNα for 24 weeks had a significant increase in memory B cells and plasma cells relative to baseline [16]. In contrast, untreated CHB patients had difficulty producing anti-HBs even when given multiple doses of hepatitis B vaccine, which may be related to the severe dysfunction of B cells in patients with CHB [17, 18]. In vitro studies have shown that B cells from patients with HBsAg seroclearance can reverse B cell dysfunction and facilitate anti-HBs production through co-stimulation with HBsAg and other cytokines [19]. Therefore, in the present study, we selected patients with HbsAg clearance following Peg-IFNα treatment for hepatitis B vaccination. Our results showed that the rate of anti-HBs seroconversion and antibody levels significantly increased, indicating that vaccination is an effective clinical measure for reducing recurrence in patients with HBsAg seroclearance. Furthermore, no new adverse effects were seen with the increased use of hepatitis B vaccine.
To investigate the variability of immunity in individuals with HBsAg seroclearance with or without hepatitis B vaccination, we examined lymphocytes from peripheral blood samples. Compared to baseline, the vaccinated group had a significantly higher proportion of plasma cells at 12 weeks and higher proportions of total B cells and class-switched memory B cells at 24 weeks (all p values were < 0.05, Fig. 3A). In the vaccinated group, plasma anti-HBs titers and total IgG concentrations showed a significant increase from baseline to 24 weeks, suggesting that HBsAg seroclearance followed by vaccination results in significantly enhanced humoral immune function. Huang et al. also showed that total B cells and plasma cells were significantly higher in an anti-HBs ≥ 100 U/L group than in an anti-HBs < 100 U/L group in individuals with HBsAg seroclearance following Peg-IFNα treatment [8]. Other studies have found that the proportion of class-switched memory B cells increases after stimulation with exogenous antigens such as vaccines, and that they can further differentiate into plasma cells to produce antibodies [20–22]. The present study also found a significant elevation in the levels of class-switched memory B cells after vaccination. In addition to an elevated percentage of B cells, we found a significant increase in co-stimulatory molecules associated with cell activation. The results of the present study showed a significant increase in the proportion of CD80+ B cells at 12 weeks compared to baseline in the vaccinated group (p = 0.018). The expression of CD80, a co-stimulatory molecule on the surface of B cells, reflects the antigen presentation function of B cells. Studies on CD80 expression in the context of hepatitis B vaccination have not been reported; similar studies have been conducted in HIV-infected individuals. Rinaldi et al. found that trivalent influenza vaccination of HIV-infected patients resulted in a significant increase in CD80 expression on the surface of B cells [23]. Tfh cells are required for T cell-dependent B cell maturation and play a key role in memory B cell and plasma cell differentiation. As shown in Fig. 3B, the proportion of Tfh cells increased significantly from baseline to 12 weeks, consistent with the changes in plasma cells (p = 0.007). ICOS is an important ligand on the surface of Tfh cells that promotes their proliferation upon binding to its ligand on the surface of B cells [24]. The present study also observed a significant increase in the proportion of ICOS+ Tfh cells from baseline to 12 weeks in the vaccinated group (p = 0.001). In the non-vaccinated group, the changes in Tfh and ICOS+ Tfh cells were not significant. There are few reports on changes in Tfh cells and ICOS expression after hepatitis B vaccination in individuals with HBsAg seroclearance, with studies available only on changes during the Peg-IFNα treatment period. Zhang et al. reported that elevated Tfh cells at 48 weeks of Peg-IFNα treatment were significantly associated with decreased HBsAg [25]. Liu et al. also found a gradual increase in the proportion of ICOS+ Tfh cells in individuals with HBsAg seroclearance during the Peg-IFNα regimen, peaking at week 48, whereas the difference in individuals without HBsAg seroclearance was not significant [26]. Previous studies have also suggested that several vaccines, including the hepatitis B vaccine, cause elevated levels of Tfh cells in healthy adults [27–29]. In addition, the proportion of ICOS+ Tfh cells was lower in healthy adults who did not receive hepatitis B vaccination or had a weak response to it, suggesting that Tfh cells and ICOS are involved in humoral immunity and promote the production of anti-HBs antibodies [29]. However, we did not measure HBsAg-specific B cell and Tfh cell ratios, and the exact mechanism needs to be further investigated.
Among B cell subsets, Breg cells have a negative regulatory role. For example, their secretion of IL-10 inhibits B-cell activation and leads to reduced antibody production [30, 31]. The present study demonstrated that both types of Breg cells were significantly lower compared to baseline during treatment of patients in the vaccinated group, and that anti-HBs levels were significantly negatively correlated with the proportion of CD24+ CD38high Breg cells (all p values were < 0.05, Fig. 3A). The difference in Breg cells number was not significant in the non-vaccinated group. One study showed a significant decrease in Breg cells with prolonged Peg-IFNα regimens, similar to the findings of the present study [16]. However, some studies have also shown that Breg cells tend to increase and then decrease during IFNα treatment [32]. In contrast, Breg cells were significantly elevated in untreated CHB patients and were the primary source of elevated IL-10 [33]. There have not been reports on the changes in Breg cells in individuals with HBsAg seroclearance after Peg-IFNα treatment and hepatitis B vaccination. However, changes in Breg cells number following hepatitis B vaccination have been better studied in healthy adults, and it is generally believed that lower numbers of Breg cells are associated with higher anti-HBs antibody production. A study found that among healthy adults vaccinated against hepatitis B, the proportions of CD24+ CD27+ Breg and CD24+ CD38high Breg cells were significantly lower in the anti-HBs positive group than in the anti-HBs negative group [34]. In the present study, these two types of Breg cells were found to be significantly reduced in the vaccinated group during the course of treatment. In another study, the high-anti-HBs group not only had a lower percentage of CD24+ CD38high Breg cells but also lower levels of IL-10 production [35]. Therefore, the present study suggests that the concomitant use of Peg-IFNα and hepatitis B vaccination in individuals with HBsAg seroclearance may result in improved humoral immune function.
Th cells and their cytokines play regulatory roles in multiple stages of humoral immunity, including B cell development, differentiation, and antibody production [36]. We observed a significant positive correlation between anti-HBs antibodies and the proportion of Th2 cells at week 24 among the overall patient cohort (r = 0.456, p = 0.019). Among the patients in the vaccinated group, Th2/Th17 cells were significantly elevated at 12 and 24 weeks compared to baseline (all p values were < 0.05, Fig. 3B). In addition, the concentrations of the cytokines IL-2, IL-5, and IL-6 were significantly higher in the vaccinated group at 24 weeks compared to baseline (all p values were < 0.05). No statistically significant changes were observed in the non-vaccinated group. Thus, we hypothesized that Th cells may contribute to the production of anti-HBs. There have been few reports on the correlation between Th cells and HBsAg clearance, except for a study by Islam et al., who found that Peg-IFNα drove Th1/Th17 cell differentiation and that increased Th1/Th17 cells were associated with HBsAg seroclearance [37]. In addition, Doedée et al. reported that highly active Th2 cells were associated with higher anti-HBs antibody titers in healthy adults vaccinated against hepatitis B [38].
In conclusion, the present study suggests that hepatitis B vaccination in individuals with HBsAg seroclearance significantly enhances anti-HBs seroconversion rate and increases antibody levels. Therefore, we believe that concomitant hepatitis B vaccination to increase anti-HBs levels in the late stage of Peg-IFNα treatment may be an effective measure for preventing recurrence. The study also revealed a significant increase in plasma cells, Tfh cells, CD80 expression of B cells and ICOS expression in Tfh cells, and a significant decrease in Breg cells during the course of treatment. These results suggest that the treatment strategy of Peg-IFNα plus hepatitis B vaccination plays an excellent role in promoting the restoration of humoral immunity in chronic HBV-infected patients. However, due to the small sample size at our center and the fact that HBsAg-specific B and T cells have not been described, our conclusions must be further validated in future studies.