Immune Memory and Immune Response in Children From Bulgaria 515 Years After Primary Hepatitis B Vaccination

Teoharov, Pavel MD, PhD*; Kevorkyan, Ani MD; Petrova, Nedyalka MD, PhD; Baltadzhiev, Ivan MD§; Van Damme, Pierre MD, PhD

Pediatric Infectious Disease Journal: January 2013 - Volume 32 - Issue 1 - p 51–53
doi: 10.1097/INF.0b013e31826f354e
Vaccine Reports

Background: Bulgaria adopted the World Health Organization recommendation of routine universal infant vaccination against hepatitis B in 1991. Nevertheless, only a few studies evaluated the protection after the vaccination against hepatitis B, especially in children. The objective of this study was to investigate the duration of protection against hepatitis B in children aged 5–15 years after primary immunization, by measuring the immune and anamnestic immune response and possible breakthrough infections.

Methods: A total of 141 children (aged 5–17 years) were recruited randomly and divided into 3 groups, approximately 5 years (group 1), 10 years (group 2) and 15 years (group 3) after primary immunization with a recombinant hepatitis B vaccine; they were tested for hepatitis B markers: hepatitis B surface antigen anti-hepatitis core antibody and antibodies to hepatitis B surface antigen (anti-HB). A booster dose of vaccine was administered to 23 children with titers of anti-HBs antibodies below the threshold considered to be protective (<10 mIU/mL). Anti-HBs concentrations and geometric mean concentration (GMC) were determined before and 21–28 days after the booster vaccination.

Results: Protective anti-HBs antibodies were detected in 95 of 141 (67.4 %) tested children, with a GMC of 63.57 mIU/mL. The seroprotection rate and GMC by groups was respectively: 84.6% and GMC of 76.05 mIU/mL in group 1; 55.8% and GMC of 58.1 mIU/mL in group 2; and 61.1% and GMC of 50.33 mIU/mL in group 3. Hepatitis B surface antigen and anti-hepatitis core antibody were found in 1 of the 141 subjects (0.7%). Of the remaining 140 children, 95 had anti-HBs ≥10 mIU/mL, and anti-hepatitis core antibodies were not detected. A booster dose of hepatitis B vaccine was administered to 23 of 45 (51%) children with anti-HBs <10 mIU/mL. Anamnestic immune response was shown in 100% of the children: the GMC was 337.38 mIU/mL and protective antibodies ranged between 15 and 955 mIU/mL.

Conclusion: The study demonstrates the presence of immune memory and protection 5–15 years after the initial course of newborn immunization with recombinant vaccines against hepatitis B.

From the * Department of Virology, National Centre of Infectious and Parasitic Diseases, Sofia, Bulgaria; Departments of Hygiene, Ecology and Epidemiology and Social Medicine and Public Health and § Infectious Diseases, Parasitology and Tropical Medicine, Medical University, Plovdiv, Bulgaria; and Faculty of Medicine and Health Science, Centre for the Evaluation of Vaccination, Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.

Accepted for publication August 14, 2012.

This study was supported by Medical University of Plovdiv grant no. 9/2009. PT has received speakers fees for presentations on vaccines from GlaxoSmithKline, Rixensart, Belgium. He has received no personal remuneration for this article. PVD acts as chief and principal investigator for vaccine trials conducted on behalf of the University of Antwerp, for which the university obtains research grants from vaccine manufacturers; speaker’s fees for presentations on vaccines are paid directly to an educational fund held by the University of Antwerp. PVD receives no personal remuneration for this work. The authors have no other conflicts of interest or funding to disclose.

Address for correspondence: Pavel Teoharov, MD, PhD, Department of Virology, National Centre of Infectious and Parasitic Diseases, 1233 bl. Stoletov 44a, Sofia, Bulgaria. E-mail:

Article Outline

Bulgaria is one of the first countries that adopted the World Health Organization recommendation for routine universal infant vaccination against hepatitis B in 1991.1 Since then, only 1 study has evaluated the protection of Bulgarian infants immediately after the hepatitis B immunization. So far, no studies on the duration of protection induced by hepatitis B immunization programs in the country have been conducted.2 Currently available vaccines are safe and highly immunogenic but data show a decline of antibody titers over time.3–5

There is evidence that immune memory persists up to 20 years after primary immunization but studies in immunized infants suggest that with increasing age some lose the ability to show immune memory.6–8

The objective of this study was to evaluate the duration of the vaccine-induced protection against hepatitis B virus 5–15 years after a 3-dose course in Bulgarian children, vaccinated in the first year of life, by measuring immune and anamnestic immune response and screening for the presence of hepatitis B markers.

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A total of 141 children (70 boys and 71 girls) between the ages of 5 and 17 years were randomly recruited in the Central-South region of Bulgaria. All were born after 1992 and were documented to have received 3 doses of recombinant infant hepatitis B vaccines, starting at birth according to a 0, 1 and 6-month schedule. Since 1992, 2 hepatitis B vaccines were used in Bulgaria (Engerix B, SmithKline Beecham, Rixensart, Belgium, and Euvax B, Aventis Pasteur Korea, Seoul, South Korea). Engerix B (SmithKline Beecham) was used from 1992 until 2011 with a break in procurement from 1999 to 2003, when Euvax B (Aventis Pasteur Korea) was used. The study was performed as part of the research project “Changes in the dynamics of the epidemic process of viral hepatitis in the Central South region of Bulgaria” and was funded and approved by the Medical University of Plovdiv, Plovdiv, Bulgaria.

Serum samples were collected through outpatient medical centers with the assistance of pediatricians and general practitioners (children of 3 age groups) and 3 schools (children from groups 2 and 3) from the region.

Three groups of children were recruited: group 1 (n = 53), 2 (n = 52) and 3 (n = 36), approximately 5, 10 and 15 years after the hepatitis B immunization, respectively. To collect data (demographic, family contact with hepatitis B surface antigen [HBsAg] carrier, immunization status) questionnaires and documentary methods were used.

After obtaining informed consent, all children underwent blood tests for HBsAg, anti-hepatitis core antibody (HBc) and antibodies to hepatitis B surface antigen (anti-HB). Children with anti-HBs titers >10 mIU/mL were considered to be immune. Those with anti-HBs levels <10 mIU/mL were offered a booster dose of infant recombinant hepatitis B vaccine (Engerix B). The second serum sample was obtained 21–28 days after the administration of the booster dose and the response was defined as an anti-HBs rising to a level >10 mIU/mL.

Serologic specimens from participants were tested for the presence of HBsAg and anti-HBc antibodies by commercial enzyme immunoassay kits (ETI-MAK-4, ETI-AB- COREK PLUS, DiaSorin, Vercelli, Italy). To determine the concentration of anti-HBs in serum, commercial kits based on the immunoenzymatic method were used (Monolisa anti-HBs Plus, Bio-Rad, Marnes-la-Coquette, France). Five calibrators: C0 (0 mIU/mL), C1 (10 mIU/mL), C2 (100 mIU/mL), C3 (400 mIU/mL), C4 (1000 mIU/mL) were used for a quantitative determination of anti-HBs antibodies. Quantitative anti-HBs levels were presented as geometric mean concentrations (GMCs).

Data were analyzed using the following statistical analyses: descriptive statistics, nonparametric analysis χ2 (Pearson χ2 test), Fisher Exact test and Student t test. To detect the relation between age and antibody titer, a correlation test with the Pearson coefficient was used. Quantitative parameter “age” is presented as mean ± standard deviation; P < 0.05 was considered statistically significant. Data were analyzed by using statistical software SPSS version 11 (SPSS Inc., Chicago, IL).

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Of 141 children in this study, 7 (5%) had a close family contact with HBsAg-positive relatives; only 1 child (0.7%) from an HBsAg-positive mother was positive for HBsAg and anti-HBc, after testing of all sera. Protective anti-HBs antibodies were detected in 95 of the 140 children (67.9%) with a GMC of 63.6 mIU/mL whereas 45 children (32.1%) had anti-HBs levels<10 mIU/mL. Most of the children with undetectable anti-HBs levels (40/45) had anti-HBs levels between 5 and 9 mIU/mL (Table 1). The nonparametric analysis shows that the age and interval since primary vaccination was determinant for the anti-HBs titers below the protective level (χ2 = 10.93, P < 0.01); weak negative correlation was found between age and anti-HBs levels (r = −0.13). There were no significant differences in seroprotection rates by gender: 71% in boys and 64.8% in girls (P > 0.05).

A booster dose of infant recombinant hepatitis B vaccine (Engerix B/GlaxoSmithKline Biologicals, Rixensart, Belgium) was administered to 23 of the 45 (51%) children with a titer <10 mIU/mL. The parents of the remaining 22 children declined participation. All 23 (100%, 95% confidence interval: 88.05–100%) children responded to the booster dose; the anti-HBs GMCs by study group were: 250.1 mIU/mL for group 1 (n = 3), 371.6 mIU/mL for group 2 (n = 11) and 331.3 mIU/mL for group 3 (n = 9).

The GMCs of these 23 children increased from 4.28 mIU/mL prebooster to 337.38 mIU/mL postbooster (78.5-fold). Most of the children had anti-HBs titers between 200 and 1000 mIU/mL and only 1 had a marginal response reaching levels between 10 and 20 mIU/mL (Fig. 1).

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In this study, persistence of vaccine-induced anti-HBs was evaluated in 5- to 17-year-old children, who had received 3 doses of recombinant hepatitis B vaccine, administered in the first year of life. Follow-up studies of children vaccinated against hepatitis B at birth have demonstrated approximately similar or more rapid decline in anti-HBs levels at 15 years of age, compared with our observations. Anti-HBs rates above 10 mIU/mL have been reported to decrease significantly with increasing age and timing since primary immunization—possibly reflecting waning anti-HBs levels over time.6–8

Bulgaria is an intermediate endemicity country for hepatitis B virus infection with an average HBsAg seroprevalence rate of 3.9% in the general population (4.2% in the Central-South region of Bulgaria).9 In this study, 7 children (5%) had a close family contact with HBsAg-positive relatives but only 1 case (0.7%) showed a breakthrough infection; all remaining 140 children were found to be negative for HBsAg and anti-HBc. A booster response was determined if the anti-HBs titer >10 mIU/mL was reached in those with undetectable anti-HBs.3,6

In this study, the immune memory was evaluated at a longer interval (5–15 years) in children immunized at birth. The presence of immune memory response was determined in children with anti-HBs titers <10 mIU/mL by measuring the anti-HBs response after administration of a booster dose.

Booster immune response was found in all children and antibody titer rose with a 78.5-fold increase in GMC. No clear trend to a higher anamnestic response was seen in those immunized more recently. Previous studies on immune memory, which persists beyond anti-HBs disappearance, demonstrated in children a 92–100% anamnestic immune response after booster dose offered 5–15 years after primary schedule.6,10,11 Our data are in line with this and support the statement of the European Consensus Group on Hepatitis B Immunity, as well as the Viral Hepatitis Prevention Board, the Centers for Disease Control and Prevention and the World Health Organization recommendation that, as far as no contrary evidence emerges from longer term follow-up of hepatitis B vaccinees, there is evidence to date not to introduce a booster dose in universal hepatitis B immunization programs.12–15

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The authors thank GlaxoSmithKline, Sofia, Bulgaria, for the provided hepatitis B vaccines.

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hepatitis B vaccine; immune response; immune memory; 5–15 year follow-up

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