Complementary Presence of HBV-Specic Humoral and T-Cellular Immune Response Provide the Long-Lasting Immune Protection After Neonatal Immunization

Background: Hepatitis B vaccination is the most cost-effective way to prevent HBV infection. Currently, hepatitis B vaccine (HepB) ecacy was usually assessed by anti-HBs level, but there were little comprehensive analyses of humoral and cellular immune response to HepB in children after neonatal immunization. Methods: A total of 145 children with primary hepatitis B immunization history were involved in this study to evaluate the ecacy of HepB. Blood samples were obtained from 80 eligible children before one dose of HepB booster and 41 children post-booster. Children with anti-HBs at a low level (<10mIU/mL and [10,100) mIU/mL) were received one dose of HepB booster after informed consent. Subjects were be measured anti-HBs, HBsAg-specic T cell responses and frequency of B cell subsets before and after booster. Results: Among 80 subjects, 81.36% of children showed both T cell and anti-HBs responses positive at baseline. After one dose of booster, anti-HBs titer (P<0.0001), positive rate of HBsAg-specic T cell response (P=0.0036) and magnitude of SFCs (P=0.0003) increased signicantly. Comparing preexisting anti-HBs titer <10mIU/mL with anti-HBs titer [10,100) mIU/mL, anti-HBs response (P=0.0005) and HBsAg-specic T lymphocyte response (P<0.0001) increased signicantly. The change tendency of HBV specic humoral response is complementary to T cellular response with age. One This is a to spot-forming cells;


Introduction
Hepatitis B vaccination is the most cost-effective way to prevent acute or chronic HBV infection and reduce complications related to hepatitis B infection now. The Chinese government give priority to routine Hepatitis B vaccination for health care. In the late 1980s, the yeast-derived Hepatitis B vaccine (HepB) was imported by Chinese government. In 1992, HepB was introduced into routine immunization management, and in 2002 HepB was integrated into China's Expanded Program on Immunization (EPI) [1]. Since the announcement of Chinese HBV vaccination program in 1992, China had successfully transformed from HBV highly endemic country to a moderately endemic country. According to the serosurveys in China, from 1992 to 2014, the positive rate of HBsAg decreased 52% (from 9.8-4.7%) in the population, 97% (from 9.7-0.3%) in children < 5 years old and 92.4% (from 10.5-0.8%) in children < 15 years old respectively [2][3][4]. It is estimated that 80 million HBV infections and 20 million chronic HBV infections have been prevented since 1992 [5].
At present, the need for HepB booster in children after neonatal immunization is still controversial. On the one hand, many previous studies have identi ed no need for a booster dose to healthy children. The protection afforded by primary immunization with HepB can lasts 30 years [6,7] and only 0.7% of vaccinees had HBV breakthrough infection 5-20 years after neonatal HBV vaccination [8]. Immune memory for HepB exist persistently in children even with waning or undetectable concentrations of anti-HBs [9]. On the other hand, 25%-50% of HBV vaccinees have lost their immune memory to the HepB after 15 years old [10,11]. About 10.1% had no immune response to HepB booster after the initial vaccination [12]. These results recommended for at-risk youths who had primary immunization to receive HepB booster. Our previous study has identi ed protective antibody (anti-HBs) in children declined with age, from 93.7% in 1-year-old to 42.3% in 9-year-old [13,14]. Whether there is protective immune response in children without anti-HBs is little known, and the need for booster doses is in debate.
In this study, we aimed to carry out a comprehensive study to evaluate the protection acquired by primary immunization from the aspect of humoral and cellular immunity. Based on the analyses, we further assess HepB booster for children who have lost protective antibodies and pioneeringly investigate the e cacy of HepB booster in children with low anti-HBs at a level of [10,100)  (3) Any kind of vaccination in the past four weeks; (4) Any acute disease or anti-infective therapy in the past four weeks; (5) Fever history in the past one week (axillary temperature ≥ 38 ℃); (6) Blood transfusion history; (7) History of infectious diseases (hepatitis, AIDS, syphilis, gonorrhea, etc.); (8) The family history of HBV in three generations of lineal relatives; (9) Abnormal physical examination.
The owchart of inclusion of participants in this study was shown in Figure 1. In this study, 145 subjects aged 1-13 years ("0 year" means <12 months) were recruited through our program of our hospital's o cial website. Participants not accord with the criteria and those who refused to participate were excluded. Blood samples were obtained from 80 eligible children before a dose of hepatitis B vaccine booster (20μg of HBsAg, Huabei Pharmaceutical Co., Ltd) and 41 children one month after booster.
HBV seromarkers measure Blood sample was collected for HBV seromarkers testing by chemiluminescent microparticle immunoassay (CMIA) method by the Architect i system (Abbott Laboratories). The concentration of HBsAg was >0.05 IU/mL was regarded as positive. Anti-HBs-positive and seroprotection was regarded as anti-HBs titer ≥10 mIU/mL. Sample value/cut-off values (S/CO) were quantitative indicators of anti-HBe and anti-HBc. Anti-HBe S/CO ≤ 1.0 and anti-HBc S/CO ≥1.0 were de ned as positivity.
Interferon γ (IFN-γ)-secreting HBsAg-speci c T lymphocytes detection PBMCs were extracted using density gradient centrifugation. Enzyme-linked immunospot (ELISpot) assays were performed for quanti cation of HBsAg-speci c cytokine-secreting T cells by using the human IFN-γ ELISpot PLUS (ALP) (Mabtech, Sweden). IFN-γ pre-coated 96-well plates were preincubated with RPMI1640 (Gibco®, Invitrogen, USA) for half an hour at room temperature were then added in two replicates at 5×10 5 PBMCs/well in 200uL RPMI1640. PBMCs were stimulated with 10 g/ml of recombinant HBsAg (Bersee, China). Positive controls are wells containing PBMCs and RPMI1640 with anti-CD3 mAb (Mabtech, Sweden). Negative controls are wells without any stimulant. Incubations of plates were conducted for 48 hours at 37 degrees Celsius in 5 percent carbon dioxide and then developed for counting spots in ELISpot reader (Biosys, Germany). A positive result was regarded as a measurable response, 2 folds the negative control level as appropriate [15].

Analysis of frequency of B lymphocytes sub-populations
Peripheral blood was collected for use in ow cytometry to evaluate the phenotypes of immune B-cells. The following mAbs were used to de ne B-cell subsets: anti-CD19 (APC), anti-human CD24 (PE), anti-CD27 (BV450), anti-CD38 (PerCP-Cy5.5), and anti-IgD (BV510; all from BD Biosciences). Cells were then examined using a FACSCanto II ow cytometer (BD Biosciences) and raw data were analyzed using FACS Diva software. The gating strategy for de nition of the B-cell subsets is depicted in Supplementary  The data were analyzed and compared by SPSS (version 20.0). Graphs were made by GraphPad Prism (version 8.0). Continuous variables were compared by the Student t test. For categorical variables, comparisons were performed by the Chi-squared test or Fisher exact tests as appropriate. In addition, Spearman rank correlation was used to evaluate association between ELISpot results and the anti-HBs titer. P values smaller than 0.05 were assumed to be statistical difference.

Baseline information of participants
Demographic and characteristics of 80 subjects were presented in Table. All subjects had received 3-dose of neonatal HBV vaccination. We de ned groups on the basis of anti-HBs level, with anti-HBs<10mIU/mL group comprising 21 individuals, 10≤ anti-HBs<100 mIU/mL group comprising 30 children, 29 children in anti-HBs ≥100 mIU/mL group. We compared gender, age, pregnancy week, birth weight and disease history among each group. Compared to girls, boys were at a lower anti-HBs level (P=0.049). Preterm infants showed a higher rate of anti-HBs negative than normal baby (P=0.039).

HBsAg-speci c T cell responses are widespread in antibody-negative subjects
To make sure whether there is protective immune response in children without anti-HBs, we use ELISpot assay to measure HBsAg-speci c T cell responses. Distribution of positive and negative humoral and cellular immunity was shown in Higher levels of anti-HBs pre-booster achieved higher humoral response post-booster In order to explore the detailed changes in humoral response to Hepatitis B vaccine, we analyzed the anti-HBs level before and after booster in children ( Figure 3). After booster, anti-HBs titer signi cantly increased (P<0.0001). In this study, all individuals received a hepatitis B vaccine booster produced anti-HBs with titers over 100 mIU/mL. We further found that 56.25% and 100% of children with anti-HBs prebooster of [0, 10) mIU/mL and [10,100) mIU/mL produced anti-HBs with titers over 1000 mIU/mL respectively (P=0.0005). Higher levels of anti-HBs pre-booster achieved higher humoral response postbooster according to the increasing anti-HBs titers pre-booster with increasing post-booster.
The magnitude of HBsAg-speci c IFN-γ-producing T cells post-booster depended on the pre-booster anti-

HBs titer
We analyzed the HBsAg-speci c T lymphocyte responses pre-booster and post-booster ( Figure 4). ELISpot detection indicated that the positive rate (P=0.0036) and magnitude of SFCs (P=0.0003) increased signi cantly post-booster, while the magnitude of the HBsAg-speci c IFN-γ-producing T cells did not related to the anti-HBs titer after neonatal vaccination (P=0.1140). T lymphocyte responses postbooster showed the same change trend as humoral response. After booster, the quantity of HBsAgspeci c IFN-γ-producing T cells signi cantly increased (P=0.0004). Compared to children with low anti-HBs titer pre-booster, HBsAg-speci c T lymphocyte responses increased signi cantly post-booster in children with higher preexisting anti-HBs titer (P<0.0001). The intensity of T-cellular immunoreactivity post-booster also depended on the pre-booster anti-HBs titer.
A complementary presence between humoral and T-cellular response to HBV vaccine with age To explore the relation of humoral and T-cellular response with age, we analyzed the changes of anti-HBs level and HBsAg-speci c IFN-γ-producing T cells in children pre-and post-booster in different age groups ( Figure 5). Among the 41 vaccinees, post-booster anti-HBs titers increased signi cantly compared to prebooster in four age groups. 1-to 3-year-old group showed higher post-booster anti-HBs titer than 10-to 13-year-old group (P=0.031). Only 10-to 13-year-old group showed signi cantly difference in HBsAgspeci c IFN-γ-producing T cells pre-and post-booster (P=0.0172). At baseline, changes in anti-HBs titers are opposed to HBsAg-speci c T Cell responses in each group. After booster, children had the same trend. These results revealed that the change tendency of HBV speci c humoral response is complementary to T cellular response with age.

Change of immune B-cell subsets after booster in neonatal vaccines
We also analyzed the changes of immune B-cell subsets in participants pre-and post-booster

Discussion
The Chinese Centre for Disease Control and Prevention (CDC) reported that the coverage of three-dose vaccination before age 1 year was 83-99.53% between 2001 and 2017 [16]. According to our previous serosurvey, the proportion of seroprotection in 1-14 years old children was 46.03-72.29%, and the rate of anti-HBs < 10 mIU/ml in children ranged from 3.33-25.79% in all age groups [13]. The data were comparable with the CDC survey data of the HBV seroprevalence among different age groups in China [4].
Hepatitis B vaccine is one of the safest immunizations to prevent HBV infection and reduce the future risk of liver cancer [17]. In this study, all children had completed three-birth-dose HepB. We analyzed their immune response to HepB booster after neonatal immunization. We mainly focused on two questions: whether there is protective immune response in children without anti-HBs (titer < 10mIU/mL); whether or not need a dose of HepB booster in children.
This is the rst study to show that protective immunity from neonatal immunization in children persists long on account of the complementary presence of HBV-speci c humoral and T-cellular immune response. A detectable T-cell response to HBsAg (85.71%) was also found in anti-HBs negative children (< 10mIU/mL) when measured HBsAg-speci c INF-γ, which suggest that protection lasts long. In the study group of Wang RX et al, most anti-HBs negative vaccinees showed positivity in HBsAg speci c immune cells response [18]. Leuridan et al demonstrated the activation of immune cells in vaccinees based on cell proliferative response [19]. Long-lasting cellular immunity was also proved by detection of the secretion of cytokines by Th1 and Th2 lymphocytes after HBsAg stimulation [7]. These results con rmed T cell immunity persists long regardless of anti-HBs, which is consistent with our views. In addition, HBsAgspeci c T Cell responses showed a trend for an initial increase and then a decrease with age while anti-HBs titers were opposite. After birth, children can mount an immune response against pathogens, but adaptive immune responses are relatively weak and narrowly focused in neonates resulting in T cell hypo-responsiveness [20]. In younger children, HBV-speci c T cells are defective and not able to secrete enough IFN-γ but gradually improves with age [21]. Before and after HepB booster, the tendency of changes in anti-HBs titer is opposed to HBsAg-speci c T Cell response in each age group of children. In vaccine development, determining the balance between humoral and cellular response is the key challenge [22]. The complementary existence between protective antibody responses and T-cell responses is important to the persistence of protection from vaccine. So, there is no need to worry about the decline in anti-HBs in population. It is precisely because of this dynamic balance that screening for HBsAgspeci c T Cell immunity is not recommended for the general population. The routine screening for anti-HBs in vaccinees is su cient to evaluate the protection afforded by HepB.
One dose of HepB booster is e cient enough. All individuals who had received an HepB booster demonstrated protective anti-HBs and enhanced HBsAg-speci c T cell responses at 4 weeks post-booster. We found that humoral and T cellular immune response to Hepatitis B vaccine booster depended on the pre-booster anti-HBs titer. Only 56.25% of children with pre-booster anti-HBs < 10mIU/ml demonstrated anti-HBs ≥ 1000mIU/mL at 4 weeks post-vaccination. Those with pre-booster anti-HBs < 10mIU/ml were less likely to produce more anti-HBs compared to anti-HBs= [10,100) mIU/ml. Equally, the intensity of Tcellular immunoreactivity post-booster also depended on the pre-booster anti-HBs titer. After booster, the quantity of HBsAg-speci c IFN-γ-producing T cells in children with pre-booster anti-HBs= [10,100) mIU/ml signi cantly increased compared to anti-HBs < 10mIU/ml. This phenomenon has been observed in other previous studies [23]. Therefore, a series of 3-dose of HepB at 0, 1 and 6 months and the longterm immunity obtained by vaccine are of seminal importance.
Although immune memory for HepB exist persistently in children, HepB booster is recommended to children without anti-HBs in the endemic areas due to the increasing risk of exposure to HBV. Currently, although the available evidence does not provide a compelling basis for recommending a booster dose of Hepatitis B vaccine [6,24] and chronic HBV infection rarely occurs after primary immunization even when anti-HBs is lower than 10 mIU/mL [6,25]. However, according to our previous study, the prevalence of HBsAg and anti-HBc began to rise signi cantly in the 11-16-year-old children compared with 1-10-yearold children (from 0.46-1.40% and from 5.69-7.8% respectively) [14]. This may suggest that the risk of exposure to hepatitis B virus is increased in > 10 years old children, and more attention should be paid to this age group, and HepB booster should be conducted to reduce the risk of breakthrough infection. Some individuals with anti-HBs < 10mIU/ml who are at high risk of HBV exposure will require only one HepB booster to achieve protective anti-HBs. Hepatitis B vaccine has been continuously optimized since it was launched in 1986. The immunization strategy of HepB in China has been continuously improved and its safety has been con rmed [1]. Furthermore, serosurvey shows that the prevalence of HBV is signi cantly reduced [2][3][4]26], which is closely related to hepatitis B vaccination, which further proves that vaccination against hepatitis B is an economic and effective means to prevent and control hepatitis B [27].
In addition to T cellular immune response, B cells also participate in HBV vaccine response by generating a protective level of anti-HBs. Our results demonstrated that total B cells including some antibodysecreting cells decreased dramatically after booster. Furthermore, reductions in plasmablasts, memory B cells and unswitched memory B cells were observed in individuals, especially in children with pre-booster anti-HBs= [10,100) mIU/ml. After vaccination, there is rapid activation of circulating memory cells to terminally differentiate into low-a nity plasma or formation of germinal centers, which mediate further proliferation and selection for antigen binding later [28,29]. In this study, there was a decline in memory B cells, unswitched memory B cells and plasmablasts appearing in the peripheral blood post-booster. But subjects did show an increase in anti-HBs in the blood, so they must have produced antibody-secreting cells at some time before blood collection which may be high-a nity [30].
In conclusion, this study had comprehensively analyzed humoral and cellular immune response to Hepatitis B vaccine booster in children before and after neonatal immunization. Protection from primary HBV immunization persists long on account of the complementary presence of HBV-speci c humoral and T-cellular immune response. In addition, we demonstrated that one dose of HepB booster is e cient enough to produce protective anti-HBs and enhance HBsAg-speci c T cell responses. As the most economical and effective way, HepB booster immunization could be recommended to children without anti-HBs in the endemic areas to prevent HBV infection.