Department of Public Health, Taipei Medical University, Taipei City, Taiwan

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1 Page 1 of 30 1 TITLE PAGE Title: Chronic hepatitis B infection in adolescents who had received primary infantile vaccination. Authors and affiliations Tzu-Wei Wu 1,*, Hans Hsienhong Lin 2,3*, Li-Yu Wang 1,4 * The first two authors contributed equally to this work. 1 Department of Medicine, Mackay Medical College, New Taipei City, Taiwan 2 Department of Gastroenterology, Tzu Chi Hospital Taipei Branch, New Taipei City, Taiwan 3 Department of Internal Medicine, School of Medicine, Tzu Chi University, Hualien, Taiwan 4 Department of Public Health, Taipei Medical University, Taipei City, Taiwan Keywords: immunity, vertical transmission, hepatitis B vaccination, long-term efficacy, population This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an Accepted Article, doi: /hep.25988

2 Page 2 of 30 2 FOOTNOTE PAGE Contact Information Dr. Li-Yu Wang, Department of Medicine, Mackay Medical College, New Taipei City, Taiwan. No. 46, Sec. 3, Jhong-Jheng Rd., San-Jhih District, New Taipei City, Taiwan. yannbo@mmc.edu.tw; Tel. no: ext 1213; Fax: List of Abbreviation anti-hbs, antibodies to hepatitis B surface antigen; anti-hbc, antibodies to hepatitis B core antigen; HB, hepatitis B; HBIG, hepatitis B immunoglobin; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus. Financial Support This research was supported by grants from the National Science Council, Taiwan (NSC B MY3), and grants from Mackay Medical College, Taiwan (981B03 & 991A01). Conflicts of interest The authors disclose no conflicts.

3 Page 3 of 30 3 ABSTRACT Hepatitis B virus (HBV) infection is a global health issue. Universal infantile hepatitis B (HB) vaccination is very efficacious. However, HBV infections among those immunized subjects have been reported. The long-term efficacy of postnatal passive-active HB vaccination in high-risk subjects is not well explored. A total of 8733 senior high school students who were born after July 1987 were assayed for hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-hbs). The overall HBsAg and anti-hbs positive rates were 1.9% and 48.3%, respectively. The HBsAg positive rate was 15% in HB immunoglobulin (HBIG) recipients (adjusted odds ratio [OR]: 15.63; 95% confidence interval [CI]: ). Among students who didn t receive HBIG, there was a significantly negative association between HB vaccination dosage and HBsAg positive rate (p for trend=0.011). Adjusted ORs for those who received 4, 3 and 1-to-2 doses were 1.00, 1.52 (95% CI: ) and 2.85 (95% CI: ) respectively. Among HBIG recipients, HBsAg positive rate was significantly higher in subjects with maternal hepatitis B e antigen (HBeAg) positivity and who received HBIG off-schedule. A booster dose of HB vaccination was administered to 1974 HBsAg- and anti-hbs-negative subjects. A prebooster and a postbooster blood samples were drown for anti-hbs quantification. The proportions of postbooster anti-hbs titer <10 miu/ml was 27.9%. Subjects with prebooster anti-hbs titers of miu/ml had significantly higher postbooster anti-hbs titers than those with prebooster anti-hbs titers of <1.0 miu/ml (p<0.0001). Conclusion: Having maternal HBeAg positivity is the most important determinant for HBsAg positivity in adolescent who had received postnatal passive-active HB vaccination 15 years before. A significant proportion of complete vaccinees may have lost their immunological memories against HBsAg.

4 Page 5 of 30 5 Hepatitis B virus (HBV) infection is a global issue, affecting two billion people in the world, with 360 million chronic carriers of hepatitis B surface antigen (HBsAg). 1 The sequalae of chronic HBV infection, including hepatic failure, liver cirrhosis and hepatocellular carcinoma, shorten lives and impose great economic burdens on society. Taiwan has been an endemic area of HBV infection, with an HBV infection rate of 95% and a 15-20% HBsAg carrier rate in the general population. 2 Vertical transmission is the main cause of persistent HBV infection in Taiwan, 3 fortunately, it can be blocked by passive-active vaccination after birth. 4-6 To control HBV infection, a hepatitis B (HB) vaccination program was launched in Taiwan in 1984, starting with newborns of highly infectious mothers, and expanded to all newborns in The remarkable effectiveness of universal infantile HB vaccination program (UIHBVP) is well documented In Taiwan, the HBV infection and carrier rates of children born after the program has declined dramatically The incidence of infantile fulminant hepatitis 13 and childhood hepatocellular carcinoma has also declined However, the efficacy of the postnatal passive-active HB vaccination is not 100%. HBsAg chronic infections in immunized infants and children has been reported in Taiwan and other countries Immunoprophylaxis failure occurred in a few high-risk infants even though HB immunoglobin (HBIG) were administered immediately after delivery. 4,17,18,22,23 The HBsAg positive rates in high-risk infants aged 0.5-to-2 years were 2-9% after postnatal passive-active HB vaccination Several studies had shown that the protective effects of HB vaccination can last for at least 10 years However, the long-term protective effects of HB vaccination in high-risk subjects are not well explored. Additionally, it seems that the risks of HBV infection are

5 Page 6 of 30 6 increased dramatically during adolescence. The cumulative 10-year risk of HBV infection in postnatal passive-active HB vaccinated subjects was less than 15%. 24,25 On the contrary, more than 30% of these high-risk subjects were anti-hbc positive at 15 years of age. 26 Here, we report our recent study on HBV chronic infection among senior high school students who were born after the implementation of UIHBVP. We found that more than 15% of vaccinated students born by highly infectious mothers became HBsAg carriers. We also found that approximate one sixth of complete vaccinees may have no or low immunological memories against HBsAg.

6 Page 7 of 30 7 Subjects and Methods The universal infantile HB vaccination program (UIHBVP) In July 1984, the Government of Taiwan initiated a HB mass vaccination program aiming primarily at interrupting mother-to-infant vertical transmission. High-risk infants were inoculated freely with a single dose of 145 IU of HBIG (Abbott Laboratories, Cutter, or Green Cross, Taiwan) within 24 hours after delivery. A 5 µg of a plasma-derived HBV vaccine (HavacB; Pasteur-Merieux, Lyon, France, or its equivalent derivative, Lifeguard hepatitis B vaccine, Hsin-Chu, Taiwan) was given to the infants at the first week of age. Two additional doses of HB vaccine were given at ages of 5 and 9 weeks. A booster dose of HB vaccination was given at 12 months of age. 7 Recombinant HB vaccine was not available in Taiwan until July After 1 November 1992, the plasma-derived vaccine was replaced by recombinant yeast vaccines H-B-Vax II (Merck Sharp & Dohame, Rahway, NJ) and Engerix-B (SmithKline Beecham, Rixensart, Belgium). 10 In July 1986, this program was extended to all neonates, then to preschool children in July 1987, to primary-school children in 1988, to middle-school children in 1989, and to adults in Since 1991, the vaccination log-sheet of first grade children were checked and unvaccinated or incompletely vaccinated children were given catch-up HB vaccination. 10 Study area Hualien County is located in the east Taiwan. It has an area of 4629 km 2 and a population of 336,733 citizens in Jan Approximately 90% of the land area of Hualien County is constituted by mountains in which the Central Mountain Chain lays at the west and the East Coast Mountains lay at the east. The population density for Hualien County and the whole Taiwan area were 72.8 and persons per km 2, respectively. 29 Hualien has one city, nine rural and three mountainous townships. Fifty-six percentages of the total population of

7 Page 8 of 30 8 Hualien County resided in Hualien City and Jian Township in which both are located on the northern Hualien. The prevalence rate of HBsAg in subjects born before Year 1978 was 17.0%, 11 which was similar to that of the whole islands. 2 There are 7 public and 6 private senior high schools in Hualien County and the total number of freshmen is approximately 4 thousands annually. Study subjects There were two parts in the present study, including HBV seroepidemiology and HB revaccination study. Procedures of study subject recruitment of the seroepidemiological study were similar to our previous study. 12 In brief, an invitation letter coupling with a short questionnaire and informed consent form was delivered to freshmen of seven public senior high schools during October 2003 to May Consenting students were asked to provide their vaccination record and assayed for serum HBsAg and antibodies to HBsAg (anti-hbs). Students with seronegativity for both anti-hbs and HBsAg were further invited to participate the HB revaccination study. The study protocols were similar to our previous study with little modification. 30 In brief, a booster dose of 20 µg HB vaccine (Engerix-B, GlaxoSmithKline Biologicals, Rixensart, Belgium) was administered to each participant. One prebooster and one postbooster blood samples were obtained at the date and 4-to-6 weeks after, respectively, the booster dose for anti-hbs quantification. All students were tested for serum antibody against HBV core antigen (anti-hbc). The study protocols were approved by the Institutional Review Board of Tzu Chi Medical Center and Taipei Medical University. Records of primary infantile HB vaccination Since 1983, the Department of Health of Taiwan implemented vaccination registration. A yellow vaccination log-sheet was provided to each woman who gave birth recently. The

8 Page 9 of 30 9 health institutions were obligated to register the dates and associated information on the yellow log-sheet. In the study, consenting students were asked to provide a copy of their vaccination record. To increase the completeness of the vaccination data, we also reviewed and abstracted HB vaccination records from 13 local health stations and major health care institutions in Hualien County. For those who had two sources of HB vaccination data, the copies that were provided by the study subjects were used as the primary source. Records of maternal HBeAg status for HBIG recipients were also reviewed and abstracted. Definitions of attributes of and responses to HB vaccination All the study subjects were born between Jul 1987 and Jul During this period, only high risk infants who were born by mothers with HBeAg positivity or reciprocal serum titers of HBsAg higher than 2560 were administered a single dose of HBIG and the recommended dates of HB vaccination were the 1 st, 5 th and 9 th weeks after delivery. 30 In the present study, infantile and early childhood HB vaccination was defined as those administered before 3 years of age. Completion of HB vaccination was defined as subjects received at least 3 doses of HB vaccine before 12 months of age. On-schedule HB vaccination was defined as followed: (1) the first dose was administered at the first week after delivery; (2) the second dose was administered at 4-to-6 weeks of age; and (3) the third dose was administered at 8-to-10 weeks of age. The response rate to a booster dose of HB vaccine was defined as the proportion of booster recipients whose postbooster titer 10 miu/ml. 30,31 The overall anti-hbs seropositive rate, designated as PR T, after a booster dose of HB vaccine was approximated by the following formula: PR T =PR 1 +(1-PR 1 ) PR 2 Where PR 1 is the anti-hbs seropositive rate before booster and PR 2 is the response rate

9 Page 10 of in the booster recipients. Serologic testing HBsAg and anti-hbs serostatus in blood samples obtained for the seroepidemiological study were detected by microparticle enzyme immunoassays (MEIA) with commercial kits AxSYN HBsAg (V2) (Abbott Diagnostics, North Chicago, Illinois A) and anti-hbs (Abbott Diagnostics), respectively. Anti-HBc serostatus was also detected by MEIA with commercial kit AxSYN CORE (Abbott Diagnostics). Serum anti-hbs titers in pre- and post-booster blood samples were assayed by a quantitative method with commercial kit AxSYN AUSAB (Abbott Diagnostics). The detection limit of this quantitative method was 1.0 miu/ml. Statistical Analysis Pearson's Chi 2 -test and Fisher's exact test were used to assess whether significant difference exist in the positive rate of HBsAg among students with different attributes. A p-value of 0.05 or less was regarded as statistically significant. Multivariate logistic regression models were used to estimate the adjusted odds ratio (aor) and the 95% confidence interval (95% CI) of being HBsAg-positivity for potential determinants.

10 Page 11 of Results A total of 8813 (83%) freshmen were recruited. Eighty students who were born before July 1987 or didn t have blood drown were excluded, leaving a total of 8733 individuals in the seroepidemiological study. The mean age with standard deviation at enrollment was 15.6 (0.4) years, 52.8% were male. The seropositive rate of HBsAg and anti-hbs were 1.9% and 48.3%, respectively. Histories of infantile HB vaccination were not available for 819 (9.4%) subjects. Approximately 87% of subjects had official documentation of receiving 3 doses of HB vaccination before 3 years old. Three hundred and eighty-one (4.8%) subjects had documented HBIG injection. Records of maternal HBeAg status of HBIG recipients were left blank in 211 (55.4 %) subjects. Among 7914 subjects had official documentation of HB vaccination, 6804 of them had data from two different sources. The consistency in the dates of HB vaccination was 94.5%. To assess the effects of HB vaccination on HBV chronic infection, we restricted analyses in 7914 subjects had official documentation of HB vaccination. Table 1 showed that there was a significantly negative association between HB vaccination dosage and HBsAg positive rate (p for trend=0.0085). As compared with those who received 4 doses of HB vaccination, adjusted ORs of HBsAg positivity were 1.40 (95% CI: ) and 2.38 (95% CI: ) for those who received 3 and 1-to-2 doses, respectively. The HBsAg positive rate was significantly higher in HBIG recipients than that of subjects without HBIG injection (1.3% vs 15.4%; adjusted OR=15.63, ). Table 2 showed that among 7537 students who didn t receive HBIG, there was a significantly negative association between HB vaccination dosage and HBsAg positive rate

11 Page 12 of (p for trend=0.011). As compared with those who received 4 doses of HB vaccination, adjusted-ors were 1.52 (95% CI: ) and 2.85 (95% CI: ) for those who received 3 and 1-to-2 doses, respectively. Among 377 HBIG recipients, no significant association was observed. Table 3 showed the HBsAg positive rates and timing of HBIG injection and maternal HBeAg serostatus among 373 passive-active HB vaccination recipients. The HBsAg positive rates was non-significantly lower for subjects received HBIG injection at the date of birth than that of the next date after delivery (14.6% vs 19.4%, one-sided p=0.14). Among 162 subjects with documented maternal HBeAg status, the HBsAg positive rate was significantly higher for HBeAg sero-positivity than that of sero-negativity (17.8% vs 11.1%, one sided p=0.014). Among 135 students with maternal HBeAg sero-positivity, 101 and 31 of them received HBIG injection at the date of birth and the next day after delivery, respectively. The HBsAg positive rate was significantly lower for those who dosing at the date of birth than that of dosing at the next day (14.9% vs 29.0%, one sided p=0.032). Among 4364 HBsAg- and anti-hbs-negative students, 2057 of them were recruited in the HB revaccination study. There was no significant difference in the HB vaccination histories between responders and no-responders. Twenty-two participants were anti-hbc positive and another 35 participants with prebooster anti-hbs titers 10 miu/ml were excluded. Additionally, 26 participants who failed to provide postbooster blood sample were further excluded, leaving a total of 1974 participants in the revaccination study. The proportions of postbooster anti-hbs titers <10, and 100 miu/ml were 27.9, 29.7 and 42.4%, respectively (Table 4). There was a significant association between pre- and post-booster anti-hbs titers (p<0.0001). Among 1252 booster recipients who had

12 Page 13 of prebooster anti-hbs titers of <1.0 miu/ml, the postbooster anti-hbs titers <10, and 100 miu/ml were 40.6, 31.6 and 27.8%, respectively. The corresponding figures in 722 booster recipients who had prebooster anti-hbs titers of miu/ml were 5.8, 26.6 and 67.6%, respectively. The anti-hbs seropositive rate before booster was 48.3% [PR 1 ] and the response rate in the booster recipients was 72% [PR 2 ]. The overall anti-hbs seropositive rate after a booster dose of HB vaccine was estimated to be 84.3%.

13 Page 14 of Discussion In the present study, we found that the HBsAg seropositive rate in high-risk subjects who received HBIG injection 15 years ago was as high as 15%. The HBsAg seropositive rates were much higher in subjects with maternal HBeAg-positivity and received HBIG injection off-schedule. To the best of our knowledge, there was no research that reported the cumulative risk of 10 years or more of HBV chronic infection in subjects who received HBIG injection during infancy. It had been suggested that mother-to-infant transmission was the primary route of HBV infection in Taiwan. 3 Approximately 90% of infants of HBeAg positive mothers were persistently positive for HBsAg. 4,5 Although postnatal passive-active HB immunization were proven to be very efficacious, nonetheless, persistent HBV infection in immunized subjects were observed in Taiwan and other countries Moreover, a prospective study enrolled 665 high-risk newborns who were immunized with HBIG within 24 hours after delivery and received subsequently 3 doses of HB vaccination. 19 Sixteen of the cohort members were positive for HBsAg within 1 hour after delivery and remained HBsAg positive at 6 months of age. 19 These evidences indicated that immunoprophylaxis failure occurred in a few high-risk infants even though HBIG were administered immediately after delivery. In Taiwan, only high-risk infants whom were born by women with HBeAg positivity or high levels of HBsAg were eligible for HBIG injection. It is reasonable to hypothesize that the observed high HBsAg seropositive rate in HBIG recipients in the present study was associated with high maternal HBV infectiousness. It was recommended that passive-active HB vaccination should be administered as soon as possible after birth. 1 In the present study, around 25% of all HBIG recipients and 22% of HBeAg positive recipients did not received HBIG at the date of birth. Statistically, those who

14 Page 15 of did not receive HBIG injection at the date of delivery had higher HBsAg seropositive rate. Consequently, off-schedule HBIG injection could possibly contribute to the observed significantly higher HBsAg positive rate in the present study. HBV transmission during early childhood is another major determinant of countries with HBsAg hyperendemicity. 1 Herd immunity induces by mass or national-wide vaccination program is well documented The HBsAg seropositive rates in newborns with maternal HBeAg-positivity and had received passive-active HB immunization were 4~6% at age of 1 year. 4,16,17 Among infants who were born by women with low or unknown levels of HBsAg, the HBsAg positive rate at 15 months of age were 2.5% and 5.5% for subject who had received HB vaccines on- and off-schedule, respectively. 16 A more recent population study of passive-active HB immunized infants reported that 15 (4.2%) of 357 subjects with maternal HBeAg-positivity were HBsAg positive at 2 years of age. 18 Among infants who were born by HBeAg negative carriers and non-carriers, the corresponding figures were 0.8% and 0.2%, respectively. 18 These evidences suggested that HB vaccination program significantly decreased the risk of HBV infection during infancy and early childhood in the low- and high-risk populations in Taiwan. All subjects in the present study were born after the implementation of UIHBVP, therefore, the attributable risk for HBV infection during infancy and early childhood in HBsAg chronic infection should not be too high. HBV reactivation or infection in later life span, especially in those who have low or no immunological memories against HBsAg, is the major determinants of HBV chronic infection in low HBsAg endemic countries 1 and should be taken into consideration. HBV reactivation in transplant recipients is well documented A long-term prospective study of HB vaccination and risk of HBV infection in infants born by HBeAg positive mother showed that the natural infection rate of HBV before 3 years of age was 12%. 17 Additionally, during the 5-year follow-up period, none of anti-hbc positive infants became HBsAg positive. 17 In

15 Page 16 of another prospective study of 805 infants who were un-infected and anti-hbs positive by 1 year of age, 52 of them became HBV infected at aged of 1-to-5 years. Additional 61 subjects became HBV infected at aged of 5-to-10 years. The cumulative 10-year risk of HBV infection was approximately 15%. 24 Additionally, among 113 HBV-infected subjects, 4 became chronic infected. 24 In a more recent study, the anti-hbc and HBsAg positive rates were 12% and 0%, respectively, in children aged 5-to-10 years. 25 These evidences indicated that even in high-risk children, the cumulative 10-year risk of HBV reactivation or infection is moderate and the risk of becoming a HBV carrier is low. These findings were supported by the evidenced that the protective effects of HB vaccination can last for at least 10 years. 27,28 However, whether protective effects of HB vaccination can persist for much longer in high-risk subjects as well as the risk of becoming a HBV carrier in subjects who contracted HBV infection after 10 years of age are both far from investigation. In the present study, the overall anti-hbs seropositive rate after a booster dose of HB vaccine was estimated to be 84.3%. It was suggested that an anti-hbs titer of 10 miu/ml is necessary for the prevention of HBV infection. 38 Therefore, about one sixth of subjects who had received infantile HB vaccination may have no or low immunological memories against HBsAg at 15 years of age and subsequently had elevated risk of HBV chronic infection when exposure to HBV. A recent study assayed for HBV markers in a cohort of 78 adolescents with maternal HBeAg positivity and had developed protective levels of anti-hbs during infancy. The anti-hbc positive rate of the cohort member was 33.3% at 15 years of age. 26 One child became HBsAg positive at the age 7-to-15 years old. 26 On the contrary, the cumulative risks of HBV infection in high-risk subjects younger than 10 years of age were less than 15%. 24,25 However, whether the risks of HBV infection or reactivation in high-risk subjects increase significantly at 10 years or more after infantile HB vaccination need further investigation.

16 Page 17 of In the present work, we found a significant proportion of adolescents who were born by high-risk mothers and received postnatal passive-active HB immunization were HBsAg positive. It seems that HBV infection or reactivation in adolescents with low or no immunological memories against HBsAg and mother-to-infant HBV transmission were the primary determinants. HBsAg chronic carriers were at significantly higher risks of end-stage liver diseases. 39,40 Our findings indicated that more efforts should be devoted to further reduce HBV transmission. Booster HB vaccination in subjects aged 15 years or more should be considered, especially in subjects born by HBsAg positive mothers or had a high-risk of HBV exposure. Additionally, it is now routine practice to use antiretroviral therapy in pregnant women with human immunodeficiency virus infection to prevent mother-to-infant transmission. 41,42 Preliminary studies have shown that lamivudine 43,44 and telbivudine 45 uses during pregnancy may reduce vertical transmission of HBV. The routine use of anti-hbv treatment during pregnancy for those with high viral load awaits further study to confirm efficacy and safety. There were concerns needed to be addressed. Undetermined accuracy of the HB vaccination histories could result in information bias. Before the implementation of National Health Insurance Program in March 1995 in Taiwan, the main provider of primary health care was the local health station. Hualien County is an area with limited health care resources. It was reasonable to hypothesize that most of the infants received vaccination in nearby local health stations and their vaccination data were recorded immediately in the yellow log-sheet by well-trained health professionals. Our estimates were comparable with previous researches. The population-based study, which selected a random sample of 1500 children born during September 1986 to August 1987 from the whole Taiwan area, found that 87.2% received 3 doses of HB vaccination. 46 In the present study, 4.8% of study subjects had documentation of HBIG injection, which also similar to previous

17 Page 18 of researches. 14,18 More importantly, among subjects who had vaccination data from two different sources, the consistency in dates of HB vaccination was as high as 94.5%. We abstracted records of HB revaccination without knowing subject s HBsAg serostatus. Consequently, information bias should be minimal and non-differential in the present study. Additionally, less than half of the HBsAg- and anti-hbs-negative students were recruited in the HB revaccination study and less than 45% of the HBIG recipients had known maternal HBeAg status. We found that there was no significant difference in the HB vaccination histories between responders and no-responders of the HB revaccination study (HB vaccination 3 dosages: 82.8% vs 84.4%; complete rate: 75.3% vs 77.3%) and between HBIG recipients with known and unknown maternal HBeAg status (HB vaccination 3 dosages: 97.5% vs 98.1%; complete rate: 89.8% vs 90.7%). It is therefore believed that bias associated with non-response was minimal in the HB revaccination study. In conclusion, having a high-risk mother is one of the most important determinants for HBsAg positivity at adolescence. A significant proportion of adolescents who had received primary infantile HB vaccination may have lost their immunological memories against HBsAg. Routine use of anti-hbv treatment during pregnancy might be an option, if safety and efficacy are proven by further large-scale studies. Booster HB vaccination in subjects aged 15 years or more should be considered, especially in subjects born by HBsAg positive mothers or had high-risk of HBV exposure. Acknowledges We thank to the staffs of the health stations in Hualien County and the Center of Disease Control, Department of Health, Taiwan, for their valuable contribution.

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24 Page 25 of 30 1 Table 1. HBsAg positive rates in 7914 adolescents with documentation of HB vaccination histories. HBsAg+ Crude OR Adjusted OR Total N (%) OR 95% CI aor a 95% CI HB vaccination during infancy and early childhood b Dosage 4 doses (1.8) # 3 doses (2.3) 1.29 ( ) 1.40 ( ) 1~2 doses (2.8) 1.64 ( ) 2.38 * ( ) Completion c Yes (1.9) No (2.0) 1.09 ( ) 1.29 ( ) On-schedule d Yes (2.0) No (1.8) 0.89 ( ) 1.05 ( ) HBIG injection ND (1.2) Yes (15.4) * ( ) * ( ) a Defined as HB vaccination administered before 3 years of age. b In addition to histories of HB vaccination, ORs were also adjusted for age and gender. c Defined as 6 those who received 3 doses of HB vaccination before 12 months of age.

25 Page 26 of 30 2 d 0-7 days, 4-6 weeks, and 8-10 weeks after birth for the 1 st, 2 nd, and 3 rd dose, respectively. # test for trend, p=0.0085; * p<0.05 OR, adjusted odds ratio; CI, confidence interval; HB, hepatitis B; HBIG, hepatitis B immunoglobin; HBsAg, hepatitis B surface antigen; ND, no documentation.

26 Page 27 of 30 3 Table 2. HBsAg chronic infection and HB vaccination histories in 7914 with documentation of HB vaccination histories, stratified by HBIG. Without HBIG With HBIG HBsAg+ HBsAg+ Total N (%) aor a 95% CI Total N (%) aor a 95% CI HB vaccination during infancy and early childhood b Dosage 4 doses (1.0) 1.00 # (15.2) doses (1.6) 1.52 ( ) (17.5) 1.21 ( ) 1~2 doses (2.9) 2.85 * ( ) 4 0 (0.0) - Completion c Yes (1.1) (15.8) 1.00 No (1.8) 1.59 ( ) 28 3 (10.7) 0.64 ( )

27 Page 28 of 30 4 On-schedule d Yes (1.0) (17.2) 1.00 No (1.3) 1.30 ( ) (13.5) 0.76 ( ) a In addition to histories of HB vaccination, ORs were also adjusted for age and gender. b Defined as HB vaccination administered before 3 years of age. c Defined as those who received 3 doses of HB vaccination before 12 months of age. d 0-7 days, 4-6 weeks, and 8-10 weeks after birth for the 1 st, 2 nd, and 3 rd dose, respectively. aor, adjusted odds ratio; CI, confidence interval; HB, hepatitis B; HBIG, hepatitis B immunoglobin; HBsAg, hepatitis B surface antigen; ND, no documentation. #test for trend, p=0.011; * p<0.05

28 Page 29 of 30 5 Table 3. HBsAg positive rates in 373 adolescents who had received postnatal passive-active HB vaccination. HBsAg+ Total No. (%) P Date of HBIG dosing 0.60 Date of birth (14.6) The next date (19.4) After the next date 6 1 (16.7) Maternal HBeAg status 0.60 ND (14.7) Negative 27 3 (11.1) + Positive (17.8) + HBIG at date of birth (14.9) * HBIG at the next date 31 9 (29.0) * HBIG at the other 3 0 (0.0) dates + * Exact one-sided p= Exact one-sided p= HBeAg, hepatitis B e antigen; HBIG, hepatitis B immune globulin; HBsAg, hepatitis B surface antigen; ND, no documentation.

29 Page 30 of 30 6 Table 4. Relationship between pre- and post-booster anti-hbs titers Post-booster anti-hbs levels Pre-booster anti-hbs levels (miu/ml) (miu/ml) n < All < < ~