Baseline data of enrolled IT children with CHB
This real-world study enrolled 48 IT children with CHB (male, n=28; female, n=20) and 32 cases completed consecutive antiviral therapy and other 16 cases did not receive antiviral treatment. There were no differences of the parameters between the treated and untreated IT children with the exception of the difference in liver inflammation grades (Table 1a). In treated group, twenty-eight cases (87.50%) were aged 1-7 years old. Twenty-six of them (81.25%) were perinatally infected with HBV or newborns who did not receive HBV vaccination in time (Table 1b). The majority of participants carried the HBV genotype C (62.50%, n=20) or B (15.63%, n=5). Twenty-eight patients underwent liver biopsy examination. Most of them showed mild degrees of hepatic inflammation (G1-G2) and/or fibrosis (S1-S2). Two of these 28 patients (7.14%) showed no hepatic inflammation, and 4 (14.29%) did not have hepatic fibrosis. The therapeutic regimens in this study were response guided, as we previously described. In brief, 19 of the 32 patients underwent combination therapy with IFN-a and NAs. The remaining 13 patients were initially treated with IFN-a monotherapy. After 6 months, 7 of those 13 patients showed significant viral suppression and continued to receive IFN-a monotherapy; the remaining 6 patients exhibited <2 log10 reductions in serum HBV DNA load at month 6, and began combination therapy with NAs. The therapeutic regimens and discontinuation standards for antiviral treatment are detailed in Fig. 1 and Supplementary Table 1. On contrast, there was not only without HBsAg loss, but also were without HBV DNA loss and HBeAg loss among the 16 IT children in untreated cases at the end of 36-month follow-up check (Table 1a).
Grouping of IT children according to HBsAg loss
Overall, of the 32 IT children on antiviral treatment in treated group, 84.38% (27/32) achieved full viral suppression, 50% (16/32) had HBeAg seroconversion, and 56.25% (18/32) had HBsAg seroconversion at the end of 36 months from the start of antiviral treatment (Supplementary Fig. 1). Using the criterion of serum HBsAg loss, there were 18 cured cases (56.25%) and 14 uncured cases (43.75%) (Table 1b). There were no significant differences in baseline HBV DNA loads, HBsAg quantity, serum ALT levels, grade of hepatic inflammation, or stage of liver fibrosis between the cured and uncured patients. In contrast, several baseline parameters differ between the cured and uncured cases, including age, counts of peripheral CD4+ T cells, CD8+ T cells, and B cells (Table 1b). Compared to the 18 cured cases that achieved sustained viral suppression and loss of both HBeAg and HBsAg in the peripheral blood, only 9 of 14 uncured cases (64.29%) achieved durable HBV DNA clearance, and only one of 14 cases had HBeAg seroconversion (Table 1b). The dynamic changes in serum HBV DNA, HBeAg and HBsAg loss or seroconversion, as well as the clinical parameters and potential factors associated with antiviral response were analyzed and compared between cured and uncured children as follows.
Faster viral suppression in cured children on antiviral therapy
In this study, antiviral therapy efficiently suppressed viral replication in both cured and uncured IT children. The cumulative rate of serum HBV DNA loss increased more rapidly in cured patients than in uncured patients over months 6 to 15 of antiviral treatment (Fig. 2a). At the same time, there was a rapid dynamic reduction in viral load and almost complete viral suppression in the cured IT cases at month 6 from baseline (Fig. 2b). In contrast, viral loads in uncured IT children decreased slowly, and three of five cases continued to have low levels of serum HBV DNA (1.3˟102-4.69˟103 IU/mL) at the end of 36 months of antiviral treatment. At 36 months, serum HBV DNA loss rates were 100% in the cured cases versus 64.29% in the uncured cases (p<0.0001) (Fig. 2a). Notably, the 18 cured patients with complete viral suppression did not experience viral rebound in this study. Therefore, the data showed that a sustained response of viral DNA, HBeAg, and HBsAg loss was achievable with antiviral treatment for IT children of the cured group. By contrast, there were still high level of serum HBV DNA in untreated children throughout the study period.
Significantly higher HBeAg loss and seroconversion in cured children
HBeAg loss and seroconversion often occur before HBsAg loss among CHB patients undergoing antiviral treatment. As shown in Fig. 3a, the cumulative rates of HBeAg loss were 11.11% (2/18), 61.11% (11/18), 83.33% (15/18), and 100% (18/18) in cured children compared with 0% (0/14),7.14% (1/14), 7.14% (1/14), and 7.14% (1/14) in uncured children at months 6, 12, 24, and 36 months from baseline, respectively. Accordingly, the cumulative rates of HBeAg seroconversion were 11.11% (2/18), 50% (9/18), 61.11% (11/18), and 83.33% (15/18) in cured children, respectively. These rates were significantly higher than those in uncured children, among whom only one individual showed HBeAg seroconversion (7.14%, p<0.0001) (Fig. 3b). As expected, HBeAg loss occurred significantly more rapidly in cured cases than in uncured cases (Fig. 3c), which was similar to what was seen with serum HBV DNA, as mentioned above. The appearance of anti-HBe also occurred sooner in cured versus uncured cases (Fig. 3d). Interestingly, 3 of the 18 cured children (P7, P10, and P14) achieved HBsAg loss but did not undergo HBeAg seroconversion, as shown in Supplementary Fig. 2a. The reason for this was still unknown. Overall, there were significantly higher rates of HBeAg loss and seroconversion in cured than in uncured children. In addition, sustained control of HBeAg loss was achieved in cured children.
Interestingly, HBsAg seroconversion occurred earlier than HBeAg seroconversion and HBV DNA loss in 2 cases (P1 and P8) (Supplementary Fig. 2b). Case P1 showed HBsAg seroconversion 7 months after the initiation of antiviral treatment. However, full virological suppression was not reached until the end of the 12th month, and HBeAg seroconversion was achieved only at 36 months. P8 reached HBsAg seroconversion at the 6-month mark. Notably, HBeAg seroconversion did not occur until month 9, when the anti-HBs titer reached ˃1000 IU/L; Serum HBV DNA was undetectable at month 12.
Close correlation of HBsAg loss with younger age and higher number of peripheral lymphocytes
HBsAg loss and/or seroconversion signify an optimal endpoint for CHB patients on antiviral treatment, reducing the risks of HCC and other hepatic events [19, 20]. In this real-world study, the cumulative rate of HBsAg loss in cured children was 16.67% (3/18), 61.11% (11/18), 83.33% (15/18) and 100% (18/18) at months 6, 12, 24 and 36, respectively (Fig. 4a). HBsAg seroconversion occurred in 11.11% (2/18), 50% (9/18), 83.33% (15/18), and 100% (18/18) of cured patients, respectively (Fig. 4b). Longitudinal analysis (Fig. 4c, d) showed rapid decreases in serum HBsAg load in the cured cases. 61.11% of the cases (11/18) reached HBsAg loss at 12 months, and 9 children had serum anti-HBs. At 36 months, 14 of 18 patients in the cured cases had high titers of serum anti-HBs (500-1000 IU/L). In contrast, serum HBsAg levels remained high among the uncured cases, although they showed a slow decrease over time with treatment (Fig. 4c, d).
To further investigate whether baseline age was an independent factor influencing the efficacy of antiviral therapy, we analyzed the association of age with HBsAg loss. The distribution of baseline age differed significantly between cured and uncured cases (p=0.002), as shown in Fig. 5a. Among the 18 cured patients, 9 were under 3 years old, 8 were aged between 3 and 6 years, and only one case was 6.4 years old (Supplementary Fig. 3a). Therefore, the cured group was significantly younger than the uncured group (9 cases aged 3-6 years old and 5 cases aged 6-13 years old) (Fig. 5a, Supplementary Table 1). In other words, 9 children under 3 years of age reached a functional cure in this study. Among the 19 patients aged 3-6.4 years old, nearly half (9/19) achieved clinical cure; however, 4 patients aged ≥7 years were not cured even with a 36-month period of antiviral treatment. Furthermore, the cumulative rate of cure gradually but significantly reduced with an increase in the baseline age at initiation of treatment (Supplementary Fig. 3b). Cox regression analyses also showed that the baseline age at the initiation of treatment (p=0.013) was closely correlated with HBsAg loss (Supplementary Table 2), corroborating the results of our previous study [18].
The absolute number and frequency of peripheral CD4+ T cells, CD8+ T cells, and B cells are typically higher in younger versus older children in Fig. 5, and peripheral neutrophils and lymphocytes exhibit a physiological curve, as shown in Supplementary Fig. 5. Interestingly, in our study, baseline lymphocyte counts differed significantly between cured and uncured patients (Fig. 5b, c, d). In line with the age distribution, CD4+ T (p=0.040), CD8+ T (p=0.008) and B cells (p=0.002) were significantly higher in the cured versus uncured cases (Table 1a). The higher counts of CD8+ T cells (p=0.013) and B cells (p=0.003) were significantly correlated to HBsAg loss rate (Supplementary Table 2); therefore, the role of higher lymphocyte counts in the cure of CHB in children with IT should be further investigated.
Analysis of hepatic pathology and HBsAg loss
Whether hepatic inflammation favors HBeAg seroconversion is an interesting issue to consider in this study, but it is difficult to draw any clear conclusions because the majority of IT children had mild or low degrees of active hepatic inflammation and fibrosis (Supplementary Fig. 4a, b). In brief, HBsAg loss was achieved in 61.54% (n=16) of 26 children with G1 or G2 inflammation. Only 2 patients with inflammation grade G0 were in the uncured cases. All patients had ALT levels lower than 60 IU/L. There were some cases of G0 and S0 inflammation and fibrosis, respectively, as shown in Table 1b and Supplementary Table 1. Inflammation grade did not significantly differ between the cured and uncured groups, possibly due to insufficient case numbers.
Safety evaluation
No serious adverse events were observed in any of the enrolled IT children receiving antiviral treatment; however, mild or moderate adverse events did occur. For example, fever (10/32) and vomiting (2/32) were the 2 most common adverse events during IFN-α treatment, and no other adverse events were reported. No viral breakthrough, rebound after treatment discontinuation, or drug resistance were observed during the 36-month follow up period.