The effects of booster vaccination on hepatitis B vaccine in anti-HBs negative infants of HBsAg-positive mothers after primary vaccination

Abstract

The purpose of this study was to investigate the changes in anti-HBs IgG levels after booster vaccinations in anti-HBs negative infants of HBsAg-positive mothers. After primary vaccination, the immunization effects of different dosages of booster vaccinations of hepatitis B vaccine (CHO) were compared.

A group of 472 newborns were vaccinated with three-dose hepatitis B vaccine at birth, 1 mo and 6 mo of age. Blood serum was collected within 6–12 mo after the third dose, and HBsAg, anti-HBs and anti-HBc levels were determined. Of this group, 101 infants who were both anti-HBs and HBsAg negative were revaccinated with 20 μg hepatitis B vaccine (CHO), and their antibody titers were monitored.

Among these 101 infants, the anti- HBs positive rates (defined as anti-HBs ≥ 100mIU/ml) differed after the first and the third dose (79% and 90%, respectively (p < 0.05), while differences in the corresponding geometric mean titers (GMTs) were not statistically significant (629 ± 3 mIU/ml and 572 ± 3 mIU/ml respectively, p < 0.05). The anti-HBs GMTs after booster vaccination were 10-fold larger than those before booster vaccination.

We conclude that a single booster dose is generally adequate for infants of HBsAg-positive mothers, whereas a further booster dose should be given for non-responders.

Keywords: :

• HepB (CHO)
• anti-HBs
• booster vaccination
• effect
• hepatitis B vaccine (HepB) made by recombinant deoxyribonucleic acid (DNA) techniques in Chinese ha
• mother-to-infant transmission

Introduction

Infection with hepatitis B virus (HBV) remains a worldwide public health problem, with more than 2 billion people infected with HBV, including 360 million people with chronic hepatitis. It is estimated that 600,000–1,200,000 chronic hepatitis patients will die from severe hepatitis related complications or hepatocelluar carcinoma annually.^1-4 Mother-to-infant transmission is the most important route of HBV transmission in Asian countries, followed by horizontal transmission, and 80–90% infants may become chronic HBV carriers.^5,6

Hepatitis B vaccination is an effective and economic measure against HBV infection.⁷ In China, mortality from HBV-associated diseases has been significantly reduced,^8,9 as has the prevalence of HBV infection. According to a national epidemiological survey, HBsAg carrier rates in people between 1 and 59 y old was 7.2% in 2006, and it is estimated that about 9.3 million Chinese in that age group years are chronic HBV carriers. Among children 0–4 y old, there are approximately 640,000 HBsAg carriers (1%).^10,11

Control of hepatitis B also prevents hepatitis D infection, as hepatitis D virus is a defective virus that only causes hepatitis in the presence of hepatitis B virus. There is also a high incidence of hepatitis A virus in China along with HBV, and patients super-infected by hepatitis A virus and HBV have a higher morbidity and mortality.¹² Control of hepatitis B decreases morbidity and mortality of patients super-infected by hepatitis A virus and HBV.

However, immunization fails in 5–15% of infants, with antibody titers falling under the protective level after hepatitis B vaccination.^13-15 Although some studies report that booster vaccinations are not necessary in a healthy population,^16,17 the risk of infection increases when antibody titers are under the protective level.^18,19 Furthermore, immunological memory responses decrease with age after primary immunization.^20-22 Consequently, the need for booster vaccination against Hepatitis B has received much attention,^23-25 but little is known about the effect of booster vaccination with HepB (CHO) in infants of HBsAg-positive mothers, who have a greater chance to be exposed to hepatitis B virus.

It is generally believed that chronic HBV carrying rates decrease with increasing age of HBV-infected children,¹⁶ and HBV-infected infants or young children are more likely to become chronic HBV carriers than older children.^26,27 Approximately 90% of HBV-infected newborns become chronic HBV carriers, as do infected infants (20–30%) and adults (1–10%). Furthermore, the post-dose-three antibody positive rate for booster vaccination is higher than the post-dose-one rate. ^[28] Vaccination with hepatitis B vaccine and HBV immune globulin (HBIG) within 12 h of birth, followed by two additional hepatitis B vaccinations decreases the chronic HBV carrying rate from 90% to 5–15%.^29-31 However, because current HBIG vaccination is on a “self-select and self-pay” basis and is costly, 100% coverage levels cannot be obtained. Therefore, in this study, infants over 1 y old were revaccinated with a three-dose vaccine and the results evaluated.

A previous study showed that the risk of HBV infection is inversely related to the titer after primary vaccination.¹⁴ In order to make sure that all high-risk infants in this study were adequately protected, a cutoff value of 100mIU/ml was used,^32-34 as recommended by Isolani.³³ From this examination of the booster immunization effect of HepB (CHO) in infants with anti-HBs < 100mIU/ml, we developed recommendations for a specific program for high-risk infant booster vaccination.

Results

Characteristics of study subjects

A total of 472 newborns of HBsAg-positive mothers received a course of three-dose vaccinations against HepB and were initially enrolled in screening. Among these 472 infants, four (0.9%) were positive to HBsAg, 362 (77%) were positive to anti-HBs, and 106 (22%) were both HBsAg negative and anti-HBs negative. Of the 106 HBsAg and anti-HBs negative infants, contact with five (4.8%) was lost during follow up, leaving 101 (95%) of these in the final study. The average age of the subjects before booster vaccination was 1.37 ± 0.31years, and the study included 53 males and 48 females.

Anti-HBs positive rates and GMTs after the first and third dose

In this group of infants of HBsAg-positive mothers, the post-dose-three anti-HBs positive rate of booster vaccination was about 11 percentage points higher than the post-dose-one rate (90% vs. 79%), and differences are statistically significant ( ײ = 37.9, p < 0.05, McNemar test). The corresponding GMTs were similar (572 ± 3 mIU/ml and 629 ± 3 mIU/ml, respectively). The anti-HBs positive rates are shown in Table 1.

Table 1. Anti-HBs positive rates and GMTs after the first and the third dose of booster vaccinations

Number with doses	Number	anti-HBs
		Number positive	Positive rate (%)	GMTs (x ± s, mIu/ml)
1st dose	101	77	76.2	629 ± 3
3rd dose	101	91	90.1	572 ± 3

In infants of HBsAg-positive mothers, the pre-dose-one GMTs, the post-dose-one anti-HBs GMTs and the post-dose-three anti-HBs GMTs were 30 ± 3. mIu/ml, 331 ± 5 mIU/ml and 432 ± 4 mIU/ml respectively. The post-dose-one anti-HBs GMTs were > 10-fold larger than the pre-dose-one GMTs, and the difference in anti-HBs GMTs between the pre-dose-one and post-dose-one is statistically significant (Z = -8.383,p < 0.05,Wicoxon Singed-Rank test). The post-dose-three anti-HBs GMTs were > 14-fold larger than the pre-dose-one GMTs, which is statistically significant (Z = -8.654, p < 0.05, Wicoxon Singed-Rank test). The post-dose-three anti-HBs GMTs were similar to the post-dose-one anti-HBs GMTs, and the difference in anti-HBs GMTs between post-dose-three and post-dose-one is not statistically significant (Z = -0.954, p < 0.05, Wicoxon Singed-Rank test).

Distribution of the pre- and post-booster HBV immune globulin vaccination

In infants of HBsAg-positive mothers, the pre-dose-one HBV immune globulin vaccination rate was 65% (306/472), and the difference in HBV immune globulin vaccination rate between positive anti-HBs and negative anti-HBs is not statistically significant ( ײ = 0.005,p < 0.05 Chi-square test).

The HBV immune globulin vaccination rate after booster vaccination was 66% (67/101), and the differences in rates of HBV immune globulin vaccination between positive anti-HBs and negative anti-HBs for both the post-dose-one and the post-dose-three are not statistically significant ( ײ = 0.002 or 0.639, p < 0.05, Chi-square test). The distribution of HBV immune globulin vaccination is shown in Table 2.

Table 2. Distribution on HBIG vaccinations before and after booster vaccinations

anti-HBs	HBIG vaccination
	Number	Number positive	Positive rate(%)
Before booster vaccinations
Negative	110	71	64.5
Positive	362	235	64.9
After one-dose booster vaccinations
Negative	24	16	66.7
Positive	77	51	66.2
After three-dose booster vaccinations
Negative	10	5	50
Positive	91	62	68.1

Discussion

In order to ensure that all high-risk infants were adequately protected, 100mIU/ml was used as the cutoff value for anti-HBs, as recommended by Isolani³³ for a good response to HepB. Although many studies consider a cutoff value of 10 mIU/ml anti-HBs to be protective against HBV infection, other studies have used 100mIU/ml.^32-34 Furthermore, the previous work showed that the risk of HBV infection was inversely the anti-HBs titer after primary vaccination, and that the incidence of HBV infection among children with 100 mIU/mL or higher anti-HBs titers after primary vaccination is about half of those children with initial titer lower than 100 mIU/mL after the primary vaccination. Infants who are born to HBsAg-positive mothers have a greater chance of HBV exposure than the general population of infants who are born to mothers not infected with HBV. In addition, infants are much more likely than adults to develop a chronic carrier state after HBV infection and develop grave consequences in the long run. HepB vaccination has been proven to be critical in protecting these infants from HBV infection.

In this study, we have demonstrated that a single booster dose is generally adequate for infants of HBsAg-positive mothers. About 80% of the infants were positive responders, and these levels of antibody positive rates have been shown to be effective at preventing infection.³⁵ This study also showed that the post-dose-one anti-HBs GMTs were increased 10-fold, compared with the pre-dose-one anti-HBs GMTs, which should give sufficient protection against infection and a strong immune memory, similar to the results reported by Wang.³⁶

This study also demonstrated that the immunization effects of booster vaccination with three doses of HepB (CHO) are superior to those of a single booster dose in anti-HBs-negative infants, whether or not the infants have a history of HBV immune globulin vaccination. Compared with a single booster dose, the anti-HBs positive rate is greatly increased after the third dose, similar to what has been previously reported.^28,37 This study gives the anti-HBs positive rates after booster vaccination lower than those previously reported.^28,37 Possible explanations for this result are as follows. Because the cutoff value for positive-responders in this study (100mIU/ml) is higher than the previously utilized cutoff value (10mIU/mL),^[28,37] the number of reported anti-HBs-positives is diminished, resulting in lower reported anti-HBs-positive rates.

This study is subject to some limitations. First, we were unable to collect blood samples from each subject after the second booster dose in order to detect antibody titer, so we were unable to analyze effects of the second booster dose. Second, because of the short observation time, the long-term effects of these immune boosters could not be evaluated.

Participants and Methods

Study participants

This research was performed in Changshan County, Kaihua County, Yuhuan County and Shangyu County in Zhejiang province, and a general hospital was selected in each county as a research site. Infants selected were born between 2008–2009, and had mothers who were chronic HBV carriers during pregnancy. The infants had received a course of three doses of 10 μg recombinant yeast dried hepatitis B vaccine (Shenzhen Kangtai Biological Products Co Ltd., Shenzhen, China) at birth and at 1 mo and 6 mo of age, but had not received further booster vaccinations. Three ml venous blood specimens were collected 6–12 mo after primary immunization, and HBsAg, anti-HBs and anti-HBc were determined for all specimens. Finally, infants who were both HBsAg negative and anti-HBs negative were selected as study subjects.

This study was approved by the institutional review board of the Zhejiang Center for Disease Control and Prevention (Hangzhou, China) and written informed consent was obtained from every child’s guardian. Specific inclusion and exclusion criteria were as follows:

Inclusion criteria

(1) Born between July 1, 2008 and December 31, 2009 and had received a course of three vaccinations against HepB in infancy at months 0, 1 and 6;

(2) Never received HepB booster vaccination;

(3) Permission from parents or guardians;

(4) Parental agreement to participate in the follow-up study and to have the infant’s blood sampled after vaccination.

Exclusion criteria

(1) HBsAg-positive;

(2) HBsAb-positive;

(3) Reluctant to participate in this project;

(4) Had experienced allergies or severe reaction to vaccination;

(5) Had known or foreseen immune dysfunction;

(6) Had received immune suppressive therapy (treated intravenously or orally with cortisone or with chemotherapy);

(7) Had high risk of compromised immunity;

(8) Had received any kind of vaccination during the previous four weeks;

(9) Had been vaccinated with any kind of observation drugs during the previous four weeks;

(10) Had any acute illness within the past 7 d;

(11) Had an infection that required treatment with antibacterial or antiviral therapy within the past 7 d;

(12) Had fever within the past 3 d (armpit temperature ≥ 38°C).

Vaccination and sampling

After acquiring informed consent from their guardians, a three ml blood sample from each subject was collected and tested. Booster vaccinations of 20 μg Hep B (CHO) (lot number: 200802A21 (01–05); NCPC Gene Tech Biotechnology Pharmaceutical Co Ltd, China) were administered by intramuscular injection in the upper arm deltoid, at 0, 1 and 3 mo. One month after the first and the third dose of booster vaccine injections, three ml blood samples from each subject were again collected and tested.

Lab testing

Sample processing

Frozen separated serum samples were sent to ADICON Clinical Laboratories, Inc. in Hangzhou, China for quantification of HBsAg, anti-HBs and anti-HBc by chemiluminescence immunoassay (CLIA). Samples with anti-HBs ≥ 1000 mIU/ml were diluted for further testing, whereas samples with antibody titer levels greater than 15000 mIU/ml were excluded from further analysis in order to eliminate large errors in the testing results.

Apparatus and reagents

An Architect-i2000 was used to perform the chemical luminescence immunoassay. The reagent lot number for the HBsAg tests was 70318HN00 (Abbott Laboratories, Chicago, US), with an S/n ≥ 0.05 considered to be positive. The reagent lot number for the anti-HBs tests was 75684M100 (Abbott Laboratories, Chicago, US), with an anti-HBs titer equal to or higher than 100 mIU/ml considered to be response positive, and an anti-HBs titer less than 100mIU/ml considered to be non-response (negative). The reagent lot number for the anti-HBc test was 72448M100 (Abbott Laboratories, Chicago, US), and an anti-HBc antibody level equal to or higher than 1 mIU/ml was defined as positive.

Data collation and analysis

A database EpiData3.2 (EpiData; Norway and Denmark) was established, and statistical analysis was performed using SPSS 18.0 and Excel 2003. The McNemar test was used for related samples enumeration data; the chi-square test for independent samples enumeration data; the Wicoxon Singed-Rank test for related samples measurement data; and a two-tailed probability in statistical tests, with an α of 0.05 considered to be significant.

Conclusions

A single booster dose is generally adequate for infants of HBsAg-positive mothers, whereas a further booster dose should be given for non-responders.Furthermore, active surveillance should be done in infants of HBsAg-positive mothers, and the infants' anti-HBs should be screened regularly.

Acknowledgments

We are grateful to the children and parents who volunteered to participate in the study and to the doctors in the Changshan, Kaihua, Yuhuan and Shangyu counties Center for Disease Control and Prevention. This study was supported by a grant from the scientific research fund of medical and health in Zhejiang province (No. 2009A035)

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.