From the *Oxford Vaccine Group, NIHR Oxford Biomedical Research Centre and Department of Paediatrics, University of Oxford, Oxford, United Kingdom; †Specjalistyczny Zespol Opieki nad Matka i Dzeckiem, Poznan, Poland, Poznań University of Medical Sciences, Poznan, Poland; ‡GSK Biologicals, Rixensart, Belgium; and §Centre for Statistics in Medicine, University of Oxford, Oxford, United Kingdom.
Accepted for publication May 30, 2012.
This study was sponsored by GlaxoSmithKline Biologicals, Belgium. With the exception of the authors employed by GlaxoSmithKline Biologicals (DB, KP and NM), no authors have received direct payment from GlaxoSmithKline Biologicals. The sponsor funded the study and developed the study protocol in collaboration with AJP and MDS. Employees of the sponsor reviewed the manuscript before submission for publication. Funding from the NIHR Biomedical Research Centre Programme through the Oxford Partnership Comprehensive Biomedical Research Centre provides support to the Oxford Vaccine Group, including salary support for AK, MDS and TMJ. AJP is a Jenner Investigator and James Martin Senior Fellow. AK has received funding from the James Martin Vaccine Design Institute (James Martin Fellowship). AK, MDS and JW have received financial assistance from GlaxoSmithKline Biologicals to attend conferences. JW has received honoraria for giving lectures. AJP does not receive any personal financial support or payment from vaccine manufacturers but acts as a chief investigator for studies sponsored by vaccine manufacturers on behalf of Oxford University. DB, KP and NM are employees of GlaxoSmithKline Biologicals; DB and NM own stock options; DB is also an inventor of certain GlaxoSmithKline patents. All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author). The authors have no other funding or conflicts of interest to disclose.
Ethical approval was granted by Oxfordshire Research Ethics Committee B (Reference: 07/Q1605/33) in the United Kingdom, and the relevant Ethics committee in Poland.
Clinicaltrials.gov registration number: NCT00454987.
Address for correspondence: Ameneh Khatami, MBChB, Oxford Vaccine Group, Department of Paediatrics, CCVTM, Churchill Hospital, Oxford, OX3 7LJ, United Kingdom. E-mail: firstname.lastname@example.org.
Following introduction of Haemophilus influenzae type b (Hib) conjugate vaccines into routine infant immunization schedules in many countries in the 1990s, there was a rapid decline in the rates of invasive Hib disease. Data from 14 European countries demonstrate that between 1996 and 2006, the incidence of invasive Hib disease was 0.28 per 100,000 population, with >50% occurring in children less than 5 years of age.1 In England and Wales, the incidence of Hib in children aged <5 years declined from a peak of 28.3 per 100,000 in 1991 to 0.97 per 100,000 in 1998, after the introduction of Hib conjugate vaccines in 1992.2 Following a Hib vaccine booster campaign targeting children aged 6 months to 4 years in 2003 and the introduction of a routine combination Hib and serogroup C meningococcal (MenC) conjugate vaccine at 12 months of age, the incidence of invasive Hib disease fell further, and in 2007 notification rates in England and Wales were 0.17 per 100,000. In Poland, Hib vaccines were introduced into the routine schedule more recently in 2005 as 3 primary doses with a booster at around 18 months of age, and in 2007, the notification rate of invasive Hib disease was 0.05 per 100,000, more than 60% of which occurred in children aged less than 5 years.3
A dramatic decline in MenC disease was also seen following introduction of routine MenC conjugate vaccines and subsequent nationwide “catch-up” campaigns. The overall incidence of MenC disease in England and Wales fell from 1.85 per 100,000 in 1998–1999 to 0.02 per 100,000 in 2008–2009.4 In Poland, MenC conjugate vaccines are not included in the routine immunization schedule and, in 2007, the incidence of MenC disease was 0.36 per 100,000, representing about 35% of all reported cases of invasive meningococcal disease.3
However, waning of vaccine-induced antibody5,6 has been demonstrated for MenC and Hib conjugate vaccines, and in the absence of a booster vaccine, effectiveness wanes beyond 1 year4,6 after infant immunization. Consequently, a combination Hib-MenC tetanus-toxoid (TT) conjugate booster at 12 months was introduced in 2006 in the United Kingdom. This study aimed to assess the persistence of Hib and MenC antibody up to 4 years after Hib-MenC-TT booster vaccination. The persistence of antibody against pertussis antigens following primary and preschool booster immunizations was also assessed in a subgroup of participants (UK participants).
Study Design and Setting
One study visit was performed at 5 years of age in 9 centers in Poland and 1 center in the United Kingdom (Oxford) in 2010, for children previously enrolled in an open-label, randomized-controlled trial [t].
Participants and Vaccines
Four hundred seventy-eight children were enrolled in the original infant study [NCT00258700],7,8 randomized on a 3:1 basis to either the Hib-MenC-TT (immunized at 2, 3 and 4 months of age with combination diphtheria-tetanus-pertussis–inactivated poliovirus [DTaP-IPV; 3 pertussis antigens] and Hib-MenC-TT vaccines) or MenC-CRM group (immunized at 2, 3 and 4 months of age with DTaP/IPV-Hib [5 pertussis antigens] and monovalent MenC-CRM197–conjugated [MenC-CRM] vaccines). The vaccines received in the primary schedule were administered concomitantly, but in separate limbs at each visit (DTaP/IPV-Hib and DTaP-IPV vaccines administered in the right thigh, MenC-CRM and Hib-MenC-TT administered in the left thigh). Both groups received a Hib-MenC-TT booster at 12–15 months of age coadministered with a combined measles, mumps, rubella (MMR) vaccine, (Hib-MenC-TT administered into the right deltoid muscle, MMR administered subcutaneously in the left arm).
After completion of the original study, participants were invited to take part in a follow-on study [NCT00454987] with additional blood tests taken at approximately 2, 3½ and 5 years of age; the results from the first 2 visits have been previously reported.5 During this period, participants received nonstudy immunizations according to their national schedule: UK participants received booster doses of DTaP-IPV and MMR at 40–43 months of age; participants in Poland were given a DTP-combination booster vaccine and MMR in the second year of life.
The doses of antigens analyzed for immunogenicity results administered as part of the primary and booster vaccinations were as follows: 5 µg each of Hib and MenC polysaccharide, conjugated to 17.5 µg total of TT in the Hib-MenC-TT vaccine; 10 µg of MenC polysaccharide conjugated to 15 µg of CRM197 in the MenC-CRM vaccine; at least 40 IU of TT, 25 µg each of pertussis toxin (PT) and filamentous hemagglutinin (FHA) and 8 µg of pertactin (PRN) in the DTaP-IPV vaccine; and at least 40 IU of TT, 20 µg each of PT and FHA and 3 µg of PRN in the DTaP/IPV-Hib vaccine.
Eligible participants must have received all immunizations due in the original study, with no additional doses of Hib, MenC or (for UK participants) pertussis-containing vaccines. Additional eligibility criteria were good health, written informed consent from parents/guardians and no history of Hib, MenC or (for UK participants) pertussis disease.
All participants had up to 6mL of blood drawn at approximately 5 years of age. Serious adverse events considered to be related to vaccination were recorded.
IgG against polyribosylribitol phosphate (PRP) and the pertussis antigens (PT, FHA and PRN) were measured by enzyme-linked immunosorbent assay Anti-PRP concentrations of 0.15 and 1.0 µg/mL were used as correlates of short-term and long-term protection against Hib.9,10 The assay cutoff for IgG to the pertussis antigens was 5 EL.U/mL11,12; however, no universally accepted correlates of protection against disease have been demonstrated for the pertussis antibodies. Functional anti-MenC activity was determined by serum bactericidal assay using baby rabbit complement (rSBA-MenC). Titers were expressed as the reciprocal of the dilution resulting in 50% inhibition, and 1:8 and 1:128 were used as correlates of protection.13 An rSBA titer of ≥1:8 has been validated by postlicensure vaccine effectiveness studies in the United Kingdom as a more accurate correlate for short-term protection than the 1:128 threshold.4,14 The antibody response to diphtheria and tetanus were not measured in this follow-on study. All assays were performed at GlaxoSmithKline Biologicals’(Rixensart, Belgium) laboratories.
The sample size for the current study was determined by the number of participants available from the original study. Study objectives were to evaluate persistence of antibody against MenC and Hib in the 2 study groups, and persistence of antibody against the pertussis antigens in UK participants, by calculating the percentage achieving the assay thresholds defined earlier in the article. rSBA-MenC geometric mean titers (GMTs) and geometric mean concentrations (GMCs) for IgG against PRP and the pertussis antigens were calculated, with results below the threshold for detection being given a value of half of the threshold.
In an exploratory analysis, the 95% confidence interval (CI) of the difference between groups in the percentage achieving each endpoint was calculated with a CI not including 0, indicative of a statistically significant difference. Similarly, the 95% CI of the ratio of GMTs and GMCs between groups were calculated and those excluding 1 regarded as inferring a statistically significant difference.
Immune persistence data were based on the per-protocol cohort, which included participants who had received vaccines according to their randomization in the original study and who had assay results for at least 1 antigen. Antipertussis antibody persistence was only determined in UK participants because in Poland, children routinely receive a pertussis-containing vaccine in the second year of life.
Of the 478 children enrolled in the booster study, 268 children were enrolled at the “5-year-old” visit of this follow-on study (206 in Hib-MenC-TT group and 62 in MenC-CRM group). One Polish site did not participate (26 participants), 50 participants declined to participate, 120 participants were lost to follow-up and 14 children did not meet eligibility criteria. Median age at enrollment was 61 months (58–64 months). In total, 98.5% of participants were Caucasian, and 49.6% were female.
At 5 years of age, the percentage of children with rSBA-MenC titers ≥ 1:8 was 59.3% in the Hib-MenC-TT group compared with 44.8% in the MenC-CRM group (Table 1). Although this difference was not statistically significant (difference between groups 14.45% with 95% CI: −0.14 to 28.5), the children in the Hib-MenC-TT group had significantly higher rSBA-MenC GMTs (30.4 versus 11.3; ratio of GMCs 2.7 with 95% CI: 1.55 to 4.73). Persistence of Hib antibody was excellent in both groups with all children maintaining anti-PRP concentrations ≥ 0.15 µg/mL; however, anti-PRP IgG GMCs were higher in the Hib-MenC-TT primed children than in the DTaP-IPV-Hib primed children (3.82 versus 1.67) and this was statistically significant with the ratio of GMTs being 2.29, 95% CI: 1.59 to 3.28.
Higher anti-FHA and anti-PRN GMC values were observed in the DTaP-IPV primed group (FHA = 164.7 EL.U/mL and PRN= 102.8 EL.U/mL) compared with the DTaP-IPV-Hib primed group (FHA = 66.8 EL.U/mL and PRN = 23.4 EL.U/mL) based on nonoverlapping 95% CIs (Table 2), 2 years after the preschool booster of DTaP-IPV. No significant differences were noted between the groups with respect to anti-PT antibody levels. Tables 1 and 2 provide a summary of immunogenicity data for all tested antigens.
No serious adverse events assessed by the investigator as being possibly related to vaccination were reported in the 4 years following the 12-month Hib-MenC-TT booster.
This study is the first to report the persistence of anti-PRP antibody and rSBA-MenC up to 4 years following a Hib-MenC-TT booster administered at 12–15 months of age, and suggests that priming with Hib-MenC-TT vaccine induces better persistence of Hib and MenC antibody than does priming with MenC-CRM and DTaP-IPV-Hib vaccines. Use of the same (TT) carrier protein or the same polysaccharide formulation for priming and boosting could explain the enhanced persistence of functional MenC antibody in the Hib-MenC-TT group. Greater antibody response and improved persistence of immunity have been demonstrated with monovalent MenC-TT vaccines compared with MenC-CRM197 vaccines,15,16 and anti-PRP antibody responses to a CRM-conjugated Hib vaccine were enhanced by coadministration of an MenC-CRM vaccine, compared with coadministration of a Hepatitis B vaccine.17
Anti-PRP IgG GMCs and rSBA-MenC GMTs were more than 2-fold higher in the Hib-MenC-TT group compared with the MenC-CRM group. This is most likely due to the greater initial response to the Hib-MenC-TT booster in children primed with this vaccine7 and suggests that the strength of the booster response is a predictor of duration of antibody persistence, in line with evidence that the magnitude of immune response following primary immunizations is also associated with the duration of antibody persistence and response to boosters.18
In contrast to the rapid waning of rSBA-MenC titers, this study demonstrates that Hib antibody is sustained above the correlate of protection in all children, up to 4 years after the Hib-MenC-TT booster, with at least 62% having anti-PRP concentrations ≥1 μg/mL. Higher anti-PRP GMCs in the Hib-MenC-TT group, may partly be explained by separation of the Hib component from a pertussis-containing combination vaccine, which has been shown to lower anti-PRP responses6.
The introduction of conjugate vaccines has resulted in a dramatic decline in the incidence of invasive disease caused by Hib and MenC, largely due to reduced pathogen carriage.4,19,20 However, antibody against Hib and MenC wane beyond 1 year after infant immunizations, as does vaccine effectiveness.4,6 Although immune memory may contribute to immunity against these encapsulated organisms, active protection of individuals relies on sustained serum antibody levels. In this study, the MenC-CRM group (immunized with 3 doses of MenC-CRM vaccine at 2, 3 and 4 months, and a Hib-MenC-TT booster at 12 months) received immunizations that are closer to the current UK schedule (2 doses of monovalent MenC conjugate vaccine; usually a CRM197-conjugated vaccine, at 3 and 4 months of age, followed by a Hib-MenC-TT booster at 12 months); thus, the poor persistence of rSBA-MenC titers in this group is of particular concern for the United Kingdom. It is, however, important to note that the only MenC-CRM vaccine available in the United Kingdom currently is one supplied by Novartis Vaccines and Diagnostics (Menjugate; Liverpool, United Kingdom), whereas the vaccine used in this study was Meningitec, produced by Pfizer Vaccines (New York), although the immune response to these 2 CRM197-conjugated vaccines is likely to be similar.16
The persistence of antibody against the pertussis antigens remains difficult to interpret in the absence of valid correlates of protection. There is no consensus on the relative roles of the different antibody types in protection against disease, and the number of pertussis antigens included in vaccines varies according to manufacturer. Thus, although higher GMCs were seen in the DTaP-IPV primed group than the DTaP-IPV-Hib group for anti-FHA and anti-PRN, the clinical significance of these differences is unknown. There is some evidence that anti-PRN ≥7 EL.U/mL is correlated with protection from infection in individuals exposed to pertussis through household contact,21 with high anti-PT levels also playing a role. Existing epidemiologic data do not allow conclusions to be drawn regarding the effectiveness of either of these vaccines based on persistence of immune response, however, as for Hib and MenC, it appears that the nature of vaccines used for primary pertussis immunization influences the persistence of immune response to the booster vaccine.
The results of this study suggest that there is continued waning of antibody against MenC and to a lesser extent against Hib during early childhood, following a booster dose of Hib-MenC-TT at 12 months of age. The choice of vaccine for primary immunization and booster doses has a marked effect on the long-term duration of immunity and should be considered in planning immunization programs.
The authors acknowledge the contributions of the investigators, staff members and nurses who were involved in this study. The authors also thank the participants of this study and their family members. In addition, the authors thank Tracy Morrison for study coordination, Koen Maleux for the serological analysis, Archana M. Jastorff for clinical report writing and Wouter Houthoofd for publication coordination (all from GlaxoSmithKline Biologicals). DTaP-IPV (Infanrix-IPV), Hib-MenC-TT (Menitorix) and MMR (Priorix) vaccines are produced by GlaxoSmithKline Biologicals, Rixensart, Belgium. DTaP-IPV-Hib (Pediacel) is produced by Sanofi-Pasteur MSD, Lyon, France. MenC-CRM (Meningitec) is produced by Pfizer Vaccines, New York City, USA.
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meningococcal; serogroup C; vaccine; persistence; immunity; booster
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