Introduction
Hepatitis B virus (HBV) infection results in more than 0.6 million deaths, and 350 million chronic HBV infections are reported every year. Patients with chronic hepatitis B (CHB) can suffer from liver cirrhosis and hepatocellular carcinoma, despite having a healthy life style, and require periodic monitoring and proper treatment [
1,
2]. Several antiviral agents have been developed to effectively inhibit the replication of HBV and to reduce the level of HBV DNA in the blood [
3]. Although the characteristics of HBV patients treated with nucleos(t)ide analogues (NA) that cause HBsAg seroclearance are not known, the cumulative incidence of HBsAg seroclearance is known to increase with a decrease in the baseline HBsAg titer before the initiation of treatment [
4,
5]. However the HBsAg titer tests are expensive, and only one test per year is covered by medical insurance in Korea. Therefore, it is necessary to develop highly accurate and inexpensive predictive biomarkers.
Usually, the qualification of HB core antibody (anti-HBc IgG) is always checked during initial screening of CHB. It is also a surrogate indicator of HBV-specific activation of adaptive immune response [
6]. Previously published studies have reported that the level of anti-HBc IgG is highly correlated with serum ALT levels [
7,
8], and is also associated with HBsAg seroclearance in NA-naïve CHB patients who naturally exhibit seroconversion of HBeAg [
4]. However, the level of anti-HBc IgG needs to be validated in patients with NA-induced HBeAg seroclearance. Although it is difficult to quantify anti-HBc IgG levels, qualification can be checked easily and is also less expensive. In this study, we investigated the suitability of using the indirect ratio of anti-HBc IgG as a predictor of HBsAg seroclearance in patients with NA-induced HBeAg seroclearance.
Discussion
Conventionally, the goal of therapy in chronic hepatitis B patients is viral suppression [
9,
11,
12]. This outcome ameliorates hepatic inflammation as well as fibrosis, and reduces the occurrence of HCC [
13,
14]. To achieve this goal, antiviral agents aimed at suppressing the replication of HBV DNA have been developed. HBsAg seroclearance with sustained HBV DNA suppression is regarded as the optimal treatment endpoint, called ‘functional cure’ [
9]. However, the capacity of current NA treatments to achieve functional cure is limited [
15], primarily due to the ability of HBV to survive in the form of cccDNA in infected hepatocytes [
13]. In our data, HBsAg seroclearance was observed in only 16% of the patients despite the presence of HBeAg seroclearance. Furthermore, not much is known about the predictors of HBsAg seroclearance before the initiation of NA treatment. In previous studies, the cumulative incidence of HBsAg seroclearance was higher when baseline HBsAg titer was lower before treatment [
4]. As HBsAg titer test is costly, the development of accurate predictive biomarkers of functional cure is necessary.
In multivariate analysis, age serum ALT and anti-HBc IgG were predictors of HBsAg seroclearance. Our data suggest that old age (≥ 50 years), elevated ALT (≥ 40 IU/L) at the time of NA induced HBeAg seroclearance, and low anti-HBc IgG level (< 11 RLU) prior to antiviral therapy have high probability of NA induced HBsAg seroclearance. In previous study, age ≥ 60 years old, and serum ALT ≥ 45 IU/L were predictors of spontaneous HBsAg seroclearance in HBeAg-sereonegative individuals [
4]. However, gender is not a predictor in our study unlike previous study.
Qualitative anti-HBc IgG assay is used to determine the presence of current or past HBV infections in an individual [
16]. In a recent study [
4], anti-HBc IgG levels have been shown to be associated with HBsAg and HBV DNA in NA-naïve CHB patients who showed natural seroconversion of HBeAg. Additionally, anti-HBc IgG levels were also higher in HBeAg seropositive patients when compared with those seen in HBeAg seroclearance patients, who showed undetectable HBV DNA and HBsAg seroclearance.
Although baseline anti-HBc IgG levels were checked by qualification methods, multivariate analysis (Table
4) shows the odds ratio of 2.213, when the anti-HBc IgG level is less than 11 RLU as a predictor of HBsAg seroclearance among patients with NA-induced HBeAg seroclearance. The HBV core antigen (HBcAg) can stimulate humoral and cellular immune responses [
17], and CHB patients show late, transient, or narrowly focused T-cell responses [
18]. Therefore, anti-HBc IgG serves as a surrogate indicator for both the HBV-specific adaptive immune activation and the intrahepatic viral antigen load [
4]. In our study, the percentage of HBsAg seroclearance was relatively high at 68.8%, when anti-HBc IgG levels were less than 11 RLU, and no HBsAg seroclearance was observed when the anti-HBc IgG levels exceeded 16 RLU (Table
3). Therefore, the low anti-HBc IgG levels could serve as a predictor of HBsAg seroclearance in CHB patients treated with NA.
Entecavir treatment showed significantly higher HBsAg seroclearance when compared with that of tenofovir (
p = 0.031, Table
1), possibly due to the difference between treatment durations of entecavir and tenofovir (79 months versus 34 months). In Korea, entecavir and tenofovir were introduced in 2007 and 2012, respectively. Therefore, in the absence of randomized, controlled study, we could not compare the effectivity for HBsAg seroclearance of these two drug treatments [
19].
Our study also has some limitations. First, it relies on qualitative methods instead of quantitative methods. We were only able to test the baseline anti-HBc IgG levels in CHB patients. We believe that studying the changes in anti-HBc IgG levels during treatment could reveal interesting observations. We did not analyze HBsAg titer. The HBsAg quantification is also an important contributing factor for functional cure. However, it was not covered by medical insurance system at the time of patients’ enrollment in Korea. Second, Korea has a high prevalence of the alcohol-related liver disease. Because it is a retrospective study, a selection bias can occur that does not exclude patients with the absence of alcohol history in the medical record. Third, at the time when HBeAg loss or seroconversion occurred, there were no patients with HBeAg loss and HBsAg loss simultaneously. HBsAg test is not checked regularly in real clinical practice, but at the time of HBeAg loss in HBeAg positive patients, and once a year in HBeAg negative patients with undetectable HBV DNA. Unfortunately, the HBsAg test was done arbitrarily in intervals in quite a few patients who take oral NA due to retrospective studies. Fourth, if a serum anti-HBc IgG test had been performed at the time of NA-induced HBeAg seroclearance, it would have been a better study. However, almost all patients performed serum anti-HBc IgG test prior to antiviral therapy. Lastly, this study was based on data obtained from a single center. Therefore, these results need to be validated in multiple cohorts.
In conclusion, even if current anti-HBc IgG qualification test is the indirect method, anti-HBc level < 11 RLU might be associated with HBsAg seroclearance in patients with NA-induced HBeAg seroclearance. We hope that our results can help physicians in predicting HBsAg seroclearance in patients with NA-induced HBeAg seroclearance as like naturally developed HBsAg seroclearance. Our results need to be validated using a new quantitative anti-HBc IgG technique. Additionally, combining the model on quantitative HBsAg, anti-HBc IgG, HBV DNA, and host factors (age and ALT) may predict functional cure.
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