Immune memory to hepatitis B persists in children aged 7–8 years, who were vaccinated in infancy with 4 doses of hexavalent DTPa-HBV-IPV/Hib (Infanrix™ hexa) vaccine

Abstract

Protection against hepatitis B disease relies on either protective serum antibodies or on the ability of the immune system to mount an anamnestic response when confronted with the hepatitis B virus (HBV). This open multicenter study (EUDRACT: 2010-022538-10) measured antibodies to HBV surface antigen (anti-HBs) in 7–8-year-old children who had received 4 doses of hexavalent diphtheria-tetanus-acellular pertussis-HBV-inactivated poliovirus-Hemophilus influenzae type b conjugate vaccine (DTPa-HBV-IPV/Hib: Infanrix™ hexa; GlaxoSmithKline Vaccines) in the first 2 years of life through routine vaccine services in Germany. The ability of these children to mount an anamnestic response to a challenge dose of monovalent HBV vaccine (Engerix™ B Kinder; GlaxoSmithKline Vaccines) was also assessed.

Before the challenge dose, 78.5% of children had anti-HBs levels ≥6.2 mIU/mL (seropositive) and 72.2% had anti-HBs levels ≥10 mIU/mL (seroprotected). Post-challenge, 98.9% had anti-HBs levels ≥10 mIU/mL and 95.8% had anti-HBs ≥100 mIU/mL. An anamnestic response to the challenge was observed in 96.6% of all subjects. The challenge dose was well tolerated, with a reactogenicity and safety profile consistent with published data. DTPa-HBV-IPV/Hib induces long-lasting immune memory to HBV that appears very similar to that induced by monovalent HBV vaccines. Protection against hepatitis B may be conferred through immune memory in subjects who responded to primary vaccination, even when they subsequently lose detectable levels of circulating anti-HBs antibodies.

Introduction

Protection against hepatitis B disease relies on either circulating protective serum antibodies or on the ability of the immune system to mount an anamnestic response when confronted with the hepatitis B virus (HBV). Studies conducted after primary vaccination of infants with monovalent recombinant HBV vaccines show persisting immune memory responses up to 20 y after vaccination.^1,2 Based on such data, consensus groups, including the World Health Organization (WHO), the European Union Consensus Group and the Viral Hepatitis Prevention Board do not recommend administration of booster doses after primary vaccination in immunologically competent individuals.^3,4,5

The HBV antigen may be administered as a monovalent vaccine, such as Engerix™ B (GlaxoSmithKline Vaccines), or in multivalent combination vaccines that facilitate inclusion and uptake in infant vaccination schedules. The combined hexavalent diphtheria-tetanus-pertussis-HBV-inactivated poliomyelitis and Hemophilus influenzae type b conjugate vaccine (DTPa-HBV-IPV/Hib, Infanrix™ hexa; GlaxoSmithKline Vaccines) has been licensed for the primary vaccination of infants and as a booster dose in the second year of life since 2000. The immunogenicity and tolerability of DTPa-HBV-IPV/Hib has been extensively documented in clinical trials, and through 12 y of post-marketing experience.^6,7 Combining the hepatitis B surface antigen (HBsAg) with other vaccines routinely administered in infancy promotes higher coverage than can be achieved using monovalent vaccines, or through vaccination programmes targeting adolescents and at-risk groups.^8,9,10 HBV vaccination is important even in countries of low endemicity, where a proportion of chronic disease cases arise through perinatal or early childhood infections.¹¹

Many of the long-term follow up studies of HBV immunity have been performed in cohorts vaccinated in infancy with monovalent HBV vaccines. However, it is important to establish that protection against hepatitis B after vaccination with combination vaccines will also be long lasting.

In Germany, the routine infant vaccination schedule, including the HBV vaccine, is given at 2, 3, and 4 mo of age and a booster dose is given before 18 mo of age. Here we report antibody persistence in healthy 7–8 y old children who received 4 doses of DTPa-HBV-IPV/Hib in the first 2 years of life through routine vaccine services in Germany. The ability of the children to mount an anamnestic response to a challenge dose of HBV was also assessed.

This study is the second of a series of 4 studies evaluating anti-HBs persistence and immune memory after infant vaccination with DTPa-HBV-IPV/Hib in routine clinical practice. The studies will evaluate progressively older cohorts in Germany, as they become available. Evaluation of the 5-y-old cohort study was reported previously.¹²

Results

Of the 300 subjects who were enrolled and vaccinated in this phase IV, open-label study, 3 were eliminated from all of the statistical analyses—one subject was found to have received primary vaccination with a vaccine other than Infanrix™ hexa, one subject had missing laboratory results, and one subject provided duplicate blood samples after the challenge dose. Thirteen subjects were eliminated from the according-to-protocol (ATP) immunogenicity analysis because they violated protocol-specified inclusion or exclusion criteria (n = 10)—had not complied with the blood sampling schedule (n = 2) or had missing serological data (n = 1). The ATP immunogenicity cohort consisted of 284 subjects with a mean age of 7.5 y (standard deviation [SD] 0.5), of which 48.9% (139) were female. All but 2 subjects (99.3%) were Caucasian.

In this study 78.5% (223/284) of 7–8 y olds had persisting antibodies to HBV surface antigen (anti-HBs) levels ≥6.2 mIU/mL (seropositive) and 72.2% (205/284) had anti-HBs levels ≥10 mIU/mL (seroprotected). One month after the challenge dose, 98.9% (259/262) of children had anti-HBs ≥10 mIU/mL and 95.8% (251/262) had anti-HBs levels ≥100 mIU/mL (Table 1).

Table 1. Antibody persistence and response to challenge (ATP cohort for immunogenicity).

Sub-group	Timing	N	≥6.2 mIU/mL		≥10 mIU/mL		≥100 mIU/mL		GMC mIU/mL
			n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	(95% CI)
<6.2mLU/mL	Pre	61	0	0.0 (0.0–5.9)	0	0.0 (0.0–5.9)	0	0.0 (0.0–5.9)	3.1 (3.1–3.1)
	Post	58	55	94.8 (85.6–98.9)	55	94.8 (85.6–98.9)	48	82.8 (70.6–91.4)	458.4 (278.1–755.7)
≥6.2–10mLU/mL	Pre	18	18	100 (81.5–100)	0	0.0 (0.0–18.5)	0	0.0 (0.0–18.5)	7.8 (7.2–8.4)
	Post	15	15	100 (78.2–100)	15	100 (78.2–100)	15	100 (78.2–100)	1873.1 (960.3–3653.5)
≥10mLU/mL	Pre	205	205	100 (98.2–100)	205	100 (98.2–100)	87	42.4 (35.6–49.5)	86.9 (72.5–104.2)
	Post	189	189	100 (98.1–100)	189	100 (98.1–100)	188	99.5 (97.1–100)	10692.4 (8607.5–13282.4)
Overall	Pre	284	223	78.5 (73.3–83.2)	205	72.2 (66.6–77.3)	87	30.6 (25.3–36.4)	36.5 (29.5–45.0)
	Post	262	259	98.9 (96.7–99.8)	259	98.9 (96.7–99.8)	251	95.8 (92.6–97.9)	4819.1 (3749.7–6193.6)

N, number of subjects with available results; n (%), number (percentage) of subjects with concentration within the specified range. 95% CI, 95% confidence interval; GMC, geometric mean concentration calculated on all subjects; Pre, pre challenge dose time-point; Post, post challenge dose time-point

An anamnestic response to the HBV vaccine challenge (defined as a 4-fold or greater increase in the post-challenge anti-HBs antibody concentration in subjects who were seropositive prior to the challenge dose—or a post-challenge anti-HBs antibody concentration ≥10 mIU/mL in initially seronegative subjects) was observed in 96.6% (253/262) of subjects overall (Table 2). The anti-HBs antibody geometric mean antibody concentration (GMC) increased by 132-fold compared with pre-challenge concentrations.

Table 2. Anamnestic response to the HBV challenge dose based on the pre-challenge serostatus (ATP cohort for immunogenicity).

Pre-vaccination status	N	Anamnestic response
		n	% (95% CI)
<6.2 mIU/mL	58	55	94.8 (85.6; 98.9)
6.2 – <10 mIU/mL	15	15	100 (78.2; 100)
≥10 mIU/mL	189	183	96.8 (93.2; 98.8)
Total	262	253	96.6 (93.6; 98.4)

N, number of subjects with both pre- and post-vaccination results available. n (%), number (percentage) of responders. 95% CI, exact 95% confidence interval. Anamnestic response, ≥4-fold increase in the post-challenge anti-HBs antibody concentration in subjects who were seropositive prior to the challenge dose; or a post-challenge anti-HBs antibody concentration ≥10 mIU/mL in initially seronegative subjects.

Sixty-one children had undetectable pre-challenge anti-HBs antibodies (<6.2 mIU/mL; i.e., seronegative, Table 1). Of the 58 who had post-challenge results, 55 (94.8%) reached the 10 mIU/mL cut off after the challenge dose (an anamnestic response). Eighteen children had anti-HBs 6.2- <10 mIU/mL) before the challenge dose. Of the 15 subjects with post-challenge results, all recorded anti-HBs concentrations ≥10 mIU/mL after the challenge dose.

About 50% of the variation in response variable (post-challenge dose results) is explained by the pre-challenge dose results (Fig. 1, R² 0.5093).

Figure 1. Anti-HBs antibody concentrations post challenge as a function of pre-challenge concentrations, with regression line (ATP cohort for imunogenicity). Regression equation and R² is given by: y = 2.4142+0.8113x; R2 0.5093. Where, y = post challenge dose (log); x = pre challenge dose (log); R² = proportion of variation in post challenge dose (log) that is predictable from pre challenge dose (log). Note: The number of subjects considered for the analysis is 262. Twenty-two out of 284 subjects in the ATP cohort were excluded due to missing results (“IR,” “QNS’).

Reactogenicity and safety of the challenge dose

Within 4 d (Day 0–3) of vaccination with monovalent HBV, pain was reported by 30.6% (95% confidence interval [CI]: 25.4%; 36.2%) of subjects, redness by 28.3% (95% CI: 23.2%; 33.8%), and swelling by 16.5% (95% CI: 12.5%; 21.2%) of subjects. Fatigue, gastrointestinal symptoms, headache, and fever were reported by 12.5% (95% CI: 8.9%; 16.8%), 9.1% (95% CI: 6.1%; 13.0%), 12.1% (95% CI: 8.6%; 16.4%), and 4.7% (95% CI: 2.6%; 7.8%) of subjects, respectively. Solicited symptoms of grade 3 intensity were reported by 1.7% of subjects or fewer. One unsolicited adverse event (injection site hematoma) reported within 31 d after vaccination was considered by the investigator to be related to vaccination. Three serious adverse events reported in 2 children occurred during the study—one subject had an allergic reaction 15 d after vaccination, one subject experienced Lyme disease and facial palsy that began 39 d after vaccination. None of the serious adverse events were considered by the investigator to be vaccine related, and all resolved.

Discussion

Primary and booster vaccination of infants with DTPa-HBV-IPV/Hib in routine practice induced sustained immune memory to HBV until 7–8 y of age. More than 70% of children retained anti-HBs antibody concentrations ≥10 mIU/mL at 7–8 y of age, i.e., more than 6 and a half years after their fourth dose of DTPa-HBV-IPV/Hib. The majority of subjects with undetectable anti-HBs antibody concentrations at the time of the challenge dose showed evidence of immune memory, with many-fold increases in anti-HBs antibody concentrations observed after the challenge dose. This is consistent with other studies undertaken in populations of low HBV endemicity where immune memory has been shown to persist in individuals primed with monovalent HBV or other recombinant vaccines in whom circulating antibody has been lost.^13,14,15 We observed only a moderate correlation between pre- and post-challenge anti-HBs concentrations, implying that the magnitude of the post-challenge response was not directly associated with the level of persisting anti-HBs antibodies prior to challenge. Furthermore, an anamnestic response was demonstrated in a similar proportion of subjects regardless of their seroprotection status before the challenge (94.8% and 100% with pre-challenge anti-HBs concentrations <6.2 and 6.2- <10 mIU/mL, respectively compared with 96.8% in those with pre-challenge concentrations ≥10 mIU/mL). These observations bring additional evidence to the notion that immune memory to hepatitis B is not driven only by the presence of persisting antibodies, but by persisting memory cells capable of responding rapidly to antigen exposure^16,17

This study builds on a growing body of evidence demonstrating prolonged persistence of immune memory to HBV after primary vaccination with DTPa-HBV-IPV/Hib (summarized for countries, like Germany with a low HBV endemicity in Table 3). In clinical trials in Germany, in which children were vaccinated with 4 doses of DTPa-HBV-IPV/Hib in infancy, between 85.3% and 91.0% had persisting anti-HBs ≥10 mIU/mL at 4–6 y of age.^12,18,19 This compares with anti-HBs ≥10 mIU/mL in 92.9% of 4–6 y old vaccinated with 4 doses of Engerix™ B.¹⁸ An anamnestic response to the challenge dose was observed in 95.7% to 96.8% of DTPa-HBV-IPV/Hib-primed children.^12,19 In 7- to 9-y-old children vaccinated with 4 doses of DTPa-HBV-IPV/Hib in infancy, anti-HBs concentrations ≥10 mIU/mL persisted in 78.0% of subjects and an anamnestic response was observed in 98.9%.¹⁹ These results are consistent with those of the present analysis.

Table 3. Prolonged persistence of immune memory to HBV after primary vaccination with either HBV or DTPa-HBV-IPV/Hib.

Infant priming	Study	Age at challenge (years)	N ATP Persistence cohort	Pre-challenge Anti-HBs > 10mIU/mL	N ATP immunogenicity cohort	Post-challenge Anti-HBs > 100mIU/mL	Anamnestic response	GMC fold-increase (post/pre)	Ref.
DTPa-HBV-IPV/Hib	DTPA-HBV-IPV-112	4–5	293	85.3%	286	95.8%	96.8%	103	12
DTPa-HBV-IPV/Hib	DTPA-HBV-IPV-111	4–6	198	86.4%	188	92.0%	95.7%	83	19
DTPa-HBV-IPV/Hib	DTPA-HBV-IPV-096	4–6	89	91.0%	0	N/A	N/A	N/A	18
DTPa-HBV-IPV + HBV	DTPA-HBV-IPV-096	4–6	28#	92.9%	0	N/A	N/A	N/A	18
HBV	HBV-315	7–8	282	83.3%	280	98.2%	99.6%	233	20
DTPa-HBV-IPV/Hib	DTPA-HBV-IPV-110	7–9	193	77.2%	187	93.6%	98.9%	110	19
HBV	DTPA-HBV-IPV-110	7–9	11	81.8%	9	100.0%	100.0%	216	19
DTPa-HBV-IPV/Hib	DTPA-HBV-IPV-113	7–8	287	72.1%	262	95.8%	96.6%	132	Current paper
HBV	HBV-318	12–13	286	78.3%	284	93.7%	97.2%	161	20

N, subjects with available results. # total cohort excluding 7 subjects without booster dose. Anamnestic response, ≥4-fold increase in the post-challenge anti-HBs antibody concentration in subjects who were seropositive prior to the challenge dose; or a post-challenge anti-HBs antibody concentration ≥10 mIU/mL in initially seronegative subjects.

Our results are also consistent with findings of a study in a similar population vaccinated with monovalent HBV vaccine.²⁰ In 7- to 8-y-old German children vaccinated in infancy with 3 doses of monovalent HBV through routine vaccination services, 83.3% had persisting anti-HBs ≥10 mIU/mL. Of the 21/280 children with undetectable anti-HBs prior to the challenge dose, all demonstrated an anamnestic response to challenge.²⁰ These studies indicate that the persistence of antibodies and immune memory is similar after vaccination with monovalent HBV or DTPa-HBV-IPV/Hib.

The challenge dose was well tolerated, with a reactogenicity and safety profile in line with published data on monovalent HBV.²¹

Although limited by the absence of a control group vaccinated with monovalent HBV vaccine, this study provides evidence of the long-term immunogenicity of DTPa-HBV-IPV/Hib in subjects vaccinated in a routine clinical practice setting.

DTPa-HBV-IPV/Hib induces long-lasting immune memory to HBV that appears to be very similar to that induced by monovalent HBV vaccines.^18,20 Protection against HBV infection may be conferred through immune memory in subjects who responded to primary vaccination, even when they subsequently lose detectable levels of circulating anti-HBs antibodies.

Methods

This open study was conducted in 12 centers in Germany between 26 April 2011 and 28 September 2011 (EUDRACT: 2010-022538-10). The protocol was approved by the Landesärztekammer Baden-Württemberg Ethics committee. The study was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki. Written informed consent was obtained from subject’s parent/legally acceptable representative prior to enrolment.

Participants were healthy with documented evidence of previous vaccination with 4 consecutive doses of Infanrix™ hexa as part of routine vaccination in Germany—3 doses of primary vaccination received by 9 mo of age and one booster dose received between 11 and 18 mo of age. Children who had received HBV vaccine at birth or any HBV vaccine after the fourth dose administered in the second year of life, and those with a history of HBV infection, were excluded. Children were also excluded if they were immunosuppressed from any cause, including chronic (>14 d) receipt of oral immunosuppressant medication.

All eligible subjects received a single challenge dose of monovalent HBV vaccine (Engerix™ B Kinder; GlaxoSmithKline Vaccines) administered intramuscularly into the deltoid region of the non-dominant arm.

A blood sample was taken before and one month after the challenge dose. Anti-HBs antibodies were measured using an FDA approved and EU marketed Chemiluminescence Immunoassay (CLIA) (Centaur™) with a cut-off of 6.2 mIU/mL defining seropositivity. Previous HBV infection, as evidenced by testing seropositive for antibodies against hepatitis B core antigen (anti-HBc), was determined in all children at the post-challenge blood sampling time-point. No samples were found to be anti-HBc positive.

Reactogenicity was assessed using diary cards that were provided to participants at the first study visit. Parents of children were asked to record any solicited local and general adverse events that occurred during 4-d (Day 0–3) follow-up after the challenge dose. The occurrence of solicited local (pain, redness, and swelling) and general (fatigue, gastrointestinal symptoms, headache, and fever) adverse events that occurred during 4-d (Day 0–3) follow-up after the challenge dose was recorded. Grade 3 (severe) symptoms were defined as redness or swelling ≥50 mm diameter—temperature >39.0 °C—and as preventing normal activity for all other symptoms. All other adverse events were recorded for 31 d after vaccination. Serious adverse events were recorded over the entire study period.

Statistical analyses

The primary objective of the study was to assess the percentage of subjects who achieved anti-HBs antibody concentrations ≥100 mIU/mL after the challenge dose. A sample size of 270 evaluable subjects provided 86% power for the lower limit of the 95% CI for the percentage of subjects with anti-HBs antibody concentrations ≥100 mIU/mL one month post- challenge to be >90%, if the true percentage of responders was 95%.

The Total Vaccinated Cohort included all subjects who received the HBV challenge dose. The ATP Immunogenicity Cohort included all eligible subjects who received the challenge dose, who met eligibility criteria, complied with protocol-defined procedures and who had post-challenge blood sampling results available.

Glossary

Abbreviations:

anti-HBs

antibodies to HBV surface antigen

ATP

according-to-protocol

CI

confidence interval

CLIA

Chemi Luminescence Immuno Assay

DTPa-HBV-IPV/Hib

diphtheria-tetanus-acellular pertussis-HBV-inactivated poliovirus-Haemophilus influenzae type b conjugate vaccine

GMC

geometric mean concentration

HBsAg

hepatitis B surface antigen

HBV

hepatitis B virus

SD

standard deviation

WHO

World Health Organization

10.4161/hv.28480