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Vaccine 27 (2009) 65306536

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Vaccine
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IgG responses after booster vaccination with different pertussis vaccines in Dutch children 4 years of age: Effect of vaccine antigen content
Lotte H. Hendrikx a,b, , Guy A.M. Berbers a , Reinier H. Veenhoven b , Elisabeth A.M. Sanders c , Anne-Marie Buisman a
a b c

Centre for infectious disease and control (Cib), National Institute for Public Health and the Environment, Bilthoven, The Netherlands Pediatric department, Spaarne Hospital Hoofddorp, Hoofddorp, The Netherlands Department of pediatric immunology and infectious diseases, University Medical Center Utrecht, Utrecht, The Netherlands

a r t i c l e

i n f o

a b s t r a c t
Since whooping cough is reemerging in the Netherlands from 1996 onwards, several changes in the national immunization program have been implemented regarding the pertussis vaccinations. The aim of this study is to investigate IgG responses in whole cell (wP) and acellular (aP) pertussis vaccine primed children following revaccination with different pertussis booster vaccines at 4 years of age. IgG levels to pertussis toxin (Pt), lamentous heamagglutin (FHA), pertactin (Prn) and mbriae type 2 and 3 (Fim2/3) and avidities of Pt and Prn antibodies were measured using a multiplex immunoassay. Before and after the booster we found signicantly higher IgG levels to Pt, FHA and Prn in aP compared to wP primed children. In all children a booster vaccination with a pertussis vaccine containing a high antigen dose (InfanrixTM ) induced higher IgG responses compared to a low antigen dose containing vaccine (TriaxisTM ). Avidities of Pt- and Prn-antibodies before and after booster vaccination were signicantly higher in aP than in wP primed children. This study shows that a booster vaccine with high pertussis antigen concentrations induces higher antibody levels than a low antigen containing vaccine. In children primed with the Dutch DTwP-IPVHib vaccine we suggest to administer a booster vaccine containing high pertussis antigens to optimize IgG responses. The pertussis vaccination history has to be taken into account in decisions on changes in pertussis vaccination policy. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 14 May 2009 Received in revised form 5 August 2009 Accepted 16 August 2009 Available online 1 September 2009 Keywords: Pertussis vaccines Antibodies Booster response

1. Introduction Since the introduction of the whole cell pertussis vaccination (wP) in the national immunization program (NIP) in the Netherlands in 1957, morbidity and mortality due to whooping cough has decreased. However, despite a high pertussis vaccination coverage in the Netherlands (>95%) as well as in other developed countries, whooping cough has reemerged worldwide [14]. In the Netherlands a three-yearly rise in the incidence of whooping cough has been observed since 1996 [46]. Several explanations have been proposed such as improved diagnostics, increased reporting, Bordetella pertussis strain variations and waning immunity [7,8]. To challenge the increasing incidence in the Netherlands, many changes regarding the pertussis vaccinations in the NIP have been implemented in the last decade. From 1996 to 2001 the pertussis incidence peaked in children 45 years of age concurrent with low

Corresponding author at: RIVM, LIS, Postbak 22, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands. Tel.: +31 30 2743944; fax: +31 30 274418. E-mail address: Lotte.Hendrikx@rivm.nl (L.H. Hendrikx). 0264-410X/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.08.052

serum antibody levels against pertussis antigens [9]. For this reason, the acellular booster vaccination was introduced in this age group in 2001. Later, in 2005, because of the considerable rate of systemic and local adverse effects of primary wP vaccination in infants, wP was replaced by the acellular pertussis (aP) vaccine. Nowadays, children receive DTaP-IPV-Hib combination vaccines at age 2, 3, 4 and 11 months and DTaP-IPV at 4 years of age. Following the implementation of the booster at 4 years of age, the peak incidence of whooping cough in children has shifted from 45 to 910 years of age. In addition, in time the pertussis incidence has also increased in adolescents and (young) adults [10]. For vulnerable as yet unvaccinated newborns, siblings and mothers seem to be the main sources of transmission [11,12]. To protect these infants, it is important to induce optimal and long lasting immunity in both children and adults. We studied the effect of the changes in the pertussis vaccines on immunogenicity against B. pertussis antigens in the last decade in the Netherlands. IgG responses elicited by different booster vaccines (with higher or lower pertussis antigen contents) at 4 years of age in children primed with either wP or aP vaccines were investigated in an observational cohort study (ISRCTN65428640).

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Table 1 Changes regarding pertussis vaccinations in the national immunization programme in the Netherlands since the start of the increasing incidence of whooping cough in 1996. Year End of 1997 1999 2001 2005 2006 2007 2008 2009 Schedule 3,4,5 and 11 months 2,3,4 and 11 months 2,3,4 and 11 months 4 years 2,3,4 and 11 months 4 years 2,3,4 and 11 months 4 years 2,3,4 and 11 months 4 years 2,3,4 and 11 months 4 years 2,3,4 and 11 months 4 years Vaccines DTwP-IPV + Hib DTwP-IPV + Hib DTwP-IPV + Hib DT-IPV + aP DTaP-IPV-Hib DTP + aP DTaP-IPV-Hib DTaP-IPV DTaP-IPV-Hib DTaP-IPV DTaP-IPV-Hib DTaP-IPV DTaP-IPV-Hib DTaP-IPV Change Dutch wP vaccine Potency 4 7 IU Adaptation schedule Introduction booster at 4 years old Monovalent aP (GSK) Infanrix-IPV-HibTM (GSK) Monovalent aP (GSK) PediacelTM (SP)/Infanrix- HexaTM (HepB) Triaxis-IPVTM (SP) PediacelTM (SP) Triaxis-IPVTM (SP)/Infanrix-IPVTM (GSK) PediacelTM (SP)/Infanrix-IPVTM (GSK) Triaxis-IPVTM (SP)/Infanrix-IPVTM (GSK) Infanrix-IPVTM (GSK) Infanrix-IPVTM (GSK)

Pertussis component of the vaccines are indicated in bold. Abbreviations D, diphtheria; wP, whole cell pertussis; aP, acellular pertussis; T, tetanus; IPV, inactivated polio virus; Hib, Heamophilus Inuenza type b.

Current IgG levels were compared with those measured in children 4 years of age in 1998 after boosting with different pertussis vaccines. The present data are important for future decisions on timing and implementation of pertussis vaccines with different compositions and concentrations of pertussis antigens, because serological immunity against whooping cough may depend on both timing of vaccinations and the choice of vaccines. 2. Subjects and methods 2.1. Vaccines All changes regarding pertussis vaccinations in the Dutch NIP in the last decade are shown in Table 1. All wP primed children had received DTwP-IPV-Hib (NVI, Bilthoven, the Netherlands) at age 2, 3, 4 and 11 months and all aP primed children had received DTaP-IPV-Hib (Infanrix-IPV-HibTM , GlaxoSmithKline Biologicals S.A., Rixensart, Belgium) at 2, 3, 4 and 11 months of age according to the Dutch NIP. The compositions of the booster vaccines studied are shown in Table 2. The ACV-SBTM and the InfanrixTM vaccine contained the same concentrations of pertussis toxin (Pt), lamentuous heamagglutin (FHA) and pertactin (Prn). 2.2. Study population The cohort of children 4 years of age as described in this study form a subset of a cross-sectional observational study (ISRCTN65428640) that investigates the immunity to B. pertussis following vaccination in children 39 years of age.

In 2007 we enrolled four groups of children previously immunised with DTwP-IPV-Hib at infant age: one group prior to booster vaccination (n = 61) and three groups postvaccination with different pertussis vaccines. One group consisted of 52 children 10 (1) days postvaccination with TriaxisTM , a group of 60 children 28 (2) days postvaccination with TriaxisTM and a group of 52 children 28 (2) days postvaccination with InfanrixTM . In 2008 comparable groups were enrolled, but now all had received DTaP-IPV-Hib vaccination at infant age: one group of 61 children pre-booster, a group of 53 children 10 (1) days postvaccination with InfanrixTM , a group of 41 children 28 (2) days postvaccination with InfanrixTM and a group of 16 children 28 (2) days postvaccination with TriaxisTM . In all children one venous blood sample (15 ml) was collected. In the two cohorts both sexes were equally distributed. IgG levels to the pertussis antigens measured in these children were compared with those previously measured in samples from children who participated in a study performed in the Netherlands in 1998. In this open, randomised, controlled trial several standalone pertussis vaccines were tested as a booster in children 4 years of age (TC1406, Dutch Trial Register). Children primed with DTwPIPV-Hib were included from which 43 received whole cell vaccine (DTwP-IPV, RIVM, Bilthoven, the Netherlands) and another 43 children acellular vaccine (ACV-SBTM , Smithkline Beecham Biologicals SA, Rixensart, Belgium) as a booster with DT-IPV simultaneously administered. Paired pre and post booster vaccination blood samples were collected from all these children according to the protocol. In the cohort both sexes were equally distributed. 2.3. Serological assays For measurement of IgG levels directed against the 5 B. pertussis vaccine antigens (pertussis toxin (Pt), lamentuous heamagglutin (FHA), pertactin (Prn) and combined mbriae type 2 and 3 (Fim2/3)) in plasmas of children included in 20072008, the multiplex immunoassay as previously described was used [13]. Fim 2/3 mix was obtained from Aventis Pasteur, Lyon, France. In the sera from children included in 1998 enzyme linked immunosorbent assays (ELISAs) had been performed at that time according to the U.S. Food and Drug Administration (FDA) ELISA originally described by Meade et al. [14] and with minor modications described by Van Gageldonk et al. [13]. Previous studies showed a good correlation with very similar absolute quantication of antibody levels between the multiplex immunoassay (MIA)

Table 2 Pertussis antigen components and concentrations of the different DTP-IPV vaccines used for booster vaccination in children 4 years of age. Triaxis-IPVTM is shortened to TriaxisTM and Infanrix-IPVTM is shortened to InfanrixTM . Vaccine (manufacturer) Pertussis antigen concentration ( g) Pt TriaxisTM (SP) InfanrixTM (GSK) ACV-SBTM (SB) DTwP-IPV (RIVM) Abbreviations: 2.5 25 25 0.16 FHA 5 25 25 2.6 Prn 3 8 8 n.d. Fim2/3 5 n.d.

g, microgram, n.d., not determined.

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and the FDA-ELISA [13,15,16]. At that time, Pt and FHA proteins were puried by the National Institute for Public Health and Environment (RIVM, Bilthoven, the Netherlands) and Prn was obtained from CHIRON (Siena, Italy). In both assays in-house reference and control sera were used. The in-house reference sera were calibrated against the FDA-human pertussis antiserum lot 3 and 4. The inhouse reference for the Fim 2/3 was calibrated against lot 3 that was arbitrarily set at 100 EU/ml. 2.4. Avidity assay

aP primed +28 days post TriaxisTM

629.1 (264.51496)

182.3 (99.0335.7)

44.7 (31.064.5)

aP primed +28 days post InfanrixTM

n = 16

2.7 (1.35.5)

Table 3 IgG levels against the pertussis vaccine antigens by group. GMC IgG levels (in bold) with 95% condence intervals are indicated as EU/ml.

The avidity of Pt- and Prn-antibodies was measured in the plasma of children included in 20072008 by using the multiplex immunoassay as previously described [13] with the following modications: duplicate plasma samples were diluted in assay buffer (3%BSA and 0.1%Tween-20 in PBS) to a nal concentration of 0.2 EU/ml for IgG-Pt and 0.05 EU/ml for IgG-Prn and were than allowed to bind for 45 min with Pt- or Prn-antigen that were covalently linked to carboxylated microspheres. Subsequently, 1.0 M or 1.5 M ammonium thiocyanate (NH4 SCN) was added to one row of plasmas and PBS was added to the corresponding row of the same plasmas for 10 min. After addition of R-PE conjugated anti-human IgG in PBS, the Pt- and Prn-IgG concentrations were measured in both corresponding rows of the same plasmas. The avidity index (AI) was expressed as a percentage of the remaining IgG levels in the presence of thiocyanate in comparison with the IgG levels measured after addition of PBS in which the avidity was set as 100%. 2.5. Statistical methods

192.3# (144.0256.7)

518.0# (429.4624.9)

1273.5# (932.51739) 1241.7 (963.11601) 186.7 (130.0268.1)

aP primed +10 days post InfanrixTM

n = 41

106.6 (80.6141.1)

564.8 (479.1665.7)

wP primed + 28 days post InfanrixTM

n = 53

60.7 (41.189.5)

194.7 (143.0265.1)

1.4 (1.11.6)

1.2# (1.01.4)

Signicant difference between pre-booster IgG levels in the wP and aP primed children. Signicant difference between wP and aP primed children in +28 days postvaccination with InfanrixTM IgG levels. Signicant difference between +28 days postvaccination with TriaxisTM IgG levels in the wP and aP primed children.

wP primed +28 days post TriaxisTM

IgG levels measured against the different pertussis vaccine antigens and AIs of Pt- and Prn-antibodies were presented in tables and gures in terms of respectively geometric mean concentrations (GMCs) and geometric mean avidity indexes (GMAIs). IgG levels and AIs in the different groups were compared with the MannWhitney U-test. A P-value <0.05 was considered statistically signicant. 3. Results 3.1. Pre-booster IgG levels after wP and aP priming IgG levels to the pertussis vaccine antigens for each child in the different groups are described in Table 3. In almost all wP and aP primed children low pre-booster IgG levels to the pertussis antigens were measured at 4 years of age. High IgG-Pt levels (>50 EU/mL) were measured only in one wP and two aP primed children. Despite low levels, wP primed children had a signicant 1.7-fold lower prebooster GMC of IgG-Pt and -FHA and a signicant 7.6-fold lower pre-booster GMC of IgG-Prn than aP primed children, whereas no signicant difference in pre-booster GMC of IgG-Fim2/3 was measured between the groups (Table 3). 3.2. Postvaccination IgG levels after wP and aP priming In wP primed children IgG-Pt levels were higher at day 10 and 28 after vaccination with TriaxisTM than pre-booster levels (Table 3). At day 28 postvaccination, children who had received InfanrixTM showed a signicant 3-fold higher GMC of IgG-Pt than those who received TriaxisTM . IgG-FHA and IgG-Prn levels had signicantly increased at day 10 and remained at the same high level at day 28 postvaccination following both TriaxisTM or InfanrixTM booster vaccination at 4 years of age. After vaccination with TriaxisTM , antibodies to Fim2/3 were signicantly higher at day 10 compared to day 28. Although InfanrixTM does not contain Fim2/3 antigens, a

n = 52

134.3 (100.1180.2) 131.7 (92.7187.1) 23.5* (18.429.9) 3.1 (2.34.3) Prn

123.1 (93.3162.5)

21.4 (13.833.2)

Postvaccination: vaccine and age group

wP primed +10 days post TriaxisTM

n = 60

16.1* (12.421.0)

aP primed

Prevaccination

wP primed

8.8 (5.813.3)

Fim 2/3

FHA

IgG

Pt

2.2 (1.62.9)

4.5 (3.36.0)

n = 61

1.4 (1.21.8)

n = 61

7.7* (6.09.8)

123.8 (71.3215.1)

n = 52

97.6 (70.4135.3)

10.6 (6.317.8)

50.4 (33.077.1)

7.0 (4.311.3)

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wP primed + 28 days post InfanrixTM

small, but signicant, increase in postvaccination IgG-Fim2/3 levels were measured at day 28 compared to pre-booster IgG-Fim 2/3 levels in wP primed children. In aP primed children, all IgG-Pt, -FHA and -Prn levels had signicantly increased at day 10 and remained at a similar high level until day 28 after booster vaccination with InfanrixTM (Table 3). Following TriaxisTM , IgG-Pt and -FHA levels were signicantly lower than after InfanrixTM at 28 days after vaccination, whereas IgG-Prn levels did not signicantly differ between TriaxisTM and InfanrixTM boostered children. Both pre- and postvaccination IgG-Fim2/3 levels were low in all aP primed children, irrespective of the type of booster vaccine received. Four weeks after booster vaccination with the same vaccine, GMCs to Pt, FHA and Prn were higher in aP compared to wP primed children (after InfanrixTM respectively a 2.4-, 2.2- and 6.4-fold and after TriaxisTM respectively a 2.3-, 1.5- and 5.5fold). All differences were signicant, except from GMCs to FHA after TriaxisTM vaccination that were non-signicant higher in aP than wP primed children (Table 3 and supplementary gure). Reverse cumulative distribution curves (RCDCs) illustrate the IgG-Pt, -FHA, -Prn and -Fim2/3 levels as measured in the prebooster and in the 28 days postvaccination groups of wP (Fig. 1A and B) and aP (Fig. 1C and D) primed children. Since no cut-off value is established for pertussis antibodies that indicates protection, an IgG-Pt level of 20 EU/ml was used as the arbitrary protective level [17]. Comparable numbers of the wP (90%) and aP primed children (85%) had pre-booster IgG-Pt levels below 20 EU/mL. Four weeks after vaccination with TriaxisTM , more than half (52%) of the wP and the majority (87%) of the aP primed children had IgG-Pt levels above 20 EU/ml (Fig. 1A and C), while after InfanrixTM this percentage was 77% in wP (Fig. 1B) and 97% in aP primed children (Fig. 1D). 3.3. Similar IgG responses in 1998 and 20072008 Since ACV-SBTM and InfanrixTM have the same pertussis antigen compositions (Table 2), postvaccination IgG levels measured in children in 1998 and 2008 can be compared. No signicant differences in postvaccination GMCs to Pt, FHA and Prn were measured in both groups (Tables 3 and 5). In 1998, 1 month after DTwP-IPV booster vaccination, IgG levels to Pt, FHA and Prn were signicantly lower than those measured after ACV-SBTM and also signicantly lower than the levels measured in 20072008 after TriaxisTM or InfanrixTM vaccination. Pre-booster, only small differences in IgG levels were measured; IgG levels to Pt were signicantly lower in children in 1998 than in children in 20072008 (GMC 1.9 and 4.5, respectively), whereas signicant higher IgG-Prn levels were measured in 1998 than in 20072008 (GMC 5.6 and 3.1, respectively). 3.4. Avidities of Pt- and Prn-antibodies after different booster vaccines The optimal ammonium thiocyanate (NH4 SCN) concentration was determined for 20 different samples at which differences in the relative avidity index (AI) were maximal. High and low relative AIs for Pt-antibodies were best distinguished at a NH4 SCN concentration of 1.0 M and for Prn-antibodies at 1.5 M (data not shown). After both booster vaccinations, the geometric mean avidity index (GMAI) of Pt- and Prn-antibodies increased from pre- to postvaccination respectively a signicant 2.3 and 2.0 -fold in wP primed and a signicant 1.3- and 1.1-fold in aP primed children (Table 4). No signicant differences in GMAIs of Pt- and Prn-antibodies between similar primed children vaccinated with TriaxisTM or InfanrixTM were measured at day 28 after vaccination. Pre- as well as post booster vaccination GMAIs of Pt- and Prn-antibodies were significantly higher in aP primed children than in wP primed children, irrespective of the booster vaccine. (Table 5)

aP primed +28 days post TriaxisTM

90.4 (83.497.9) n = 16 91.5# (86.297.3) n = 27 81.7 (76.587.3) n = 39 68.0 (60.975.8) n = 40

aP primed +28 days post InfanrixTM

aP primed +10 days post InfanrixTM

69.6 (62.178.0) n = 38 Prn 35.9 (29.144.3) n = 40 79.6* (75.184.3) n = 40 71.8 (65.379.0) n = 37 73.5 (66.980.8) n = 39

90.9 (87.694.3) n = 40
# *

88.0# (85.290.8) n = 23 Signicant difference in pre-booster AIs between wP and aP primed children. Signicant difference between wP and aP primed children in +28 days postvaccination with InfanrixTM IgG levels. Signicant difference in +28 days postvaccination with TriaxisTM AIs between wP and aP primed children.

Table 4 Avidity indexes of Pt- and Prn-antibodies by group. GMAI and 95% CI of the AIs are given in percentages.

wP primed +28 days post TriaxisTM

Postvaccination: vaccine and age group

wP primed +10 days post TriaxisTM

Pre-booster

GMAIs

Pt

28.3 (23.134.8) n = 40

wP primed

67.9* (60.875.9) n = 37

aP primed

53.2 (43.664.9) n = 38

63.8 (52.777.1) n = 40

86.4 (80.592.7) n = 16

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Fig. 1. Reverse cumulative distribution curves of the IgG levels against Pt (black), FHA (red), Prn (green) and Fim 2/3 (blue) measured pre-booster and at day 28 after booster vaccination: (a) wP + TriaxisTM , (b) wP + InfanrixTM and (c) aP + TriaxisTM (d) aP + InfanrixTM . The vertical line indicates the arbitrary level of protection for IgG-Pt (20 EU/mL). The x-axis is logarithmic.

4. Discussion In the Netherlands the switch from whole cell to acellular pertussis vaccines used for primary immunization took place in 2005. Hence, we had the unique opportunity to compare the effect of various acellular pertussis booster vaccines differing in antigen contents on the induction of IgG responses in wP and aP primed children at 4 years of age in an era with a high incidence of whooping cough. We found that following acellular pertussis vaccinations at infant age, the extent and avidity of the antibody responses to Pt, FHA and Prn were signicantly higher at 4 years of age in aP primed children than wP primed children. After booster vaccination with a 3-component, high antigen dose containing vaccine (InfanrixTM ), signicantly higher IgG responses were evoked compared to booster vaccination with a 5-component, lower antigen dose containing vaccine (TriaxisTM ) or following a whole cell vac-

cine with a very low dose of pertussis antigens. However, a strict antigen dose-response effect was lacking. 4.1. Pre-booster IgG responses Three years after wP and aP primary immunizations at 2, 3, 4 and 11 months of age, we observed low antibody levels to the pertussis vaccine antigens, suggesting a fast decay as previous studies already showed [1822]. Remarkably, despite low antibody levels in aP primed children relatively high avidity indexes of Pt- and Prn-antibodies were measured that seem to sustain even 3 years after primary immunization. In particular the pre-booster IgG-Prn response remained relatively high for aP primed children only. In individuals with low IgG levels, a high avidity index will help to distinguish between those who are primed for memory and those who are not [23]. In our study, some children showed very high (>50 EU/ml) pre-booster IgG-Pt levels, suggesting a recent infection

L.H. Hendrikx et al. / Vaccine 27 (2009) 65306536 Table 5 Pre and 28 days post booster IgG concentrations against the pertussis vaccine antigens for samples taken from children in the 1998 study. GMC IgG levels with 95% condence intervals are indicated as EU/ml. IgG Prevaccination Postvaccination: vaccine DTwP-IPV Pt FHA Prn 1.9 (1.52.3) 6.9 (5.58.7) 5.6 (4.47.1) 7.3 (4.212.8) 34.7 (26.046.4) 31.5 (23.243.0) ACV-SBTM 65.4 (43.997.3) 238.3 (163.0348.4) 143.3 (108.9188.8)

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large amount of Fim2/3 in the wP vaccine, through which wP vaccinated children are primed for Fim2/3, whereas aP (InfanrixTM ) primed children are not, since InfanrixTM contains no Fim2/3 at all. Lanzavecchia recently showed that a booster reaction could also induce an aspecic polyclonal stimulation of other antigens for which the individual is primed previously [34]. This immunological mechanism might explain the increase in IgG-Fim2/3 after InfanrixTM booster vaccination in wP primed children. Notably, in aP primed and TriaxisTM boostered children, a small, but nonsignicant increase in GMC IgG-Fim2/3 is noticed, reecting a weak primary immune response. 4.3. Limitations of the study Since we performed a cross-sectional study in 2007/2008, the exact course of the individual IgG response before and after vaccination could not be described. However, we were able to compare our data with the study performed in 1998 in which paired preand postvaccination blood samples were taken and measured with ELISA. In children immunized with the same pertussis vaccine some 10 years earlier, similar IgG levels were found despite the use of different assays and different antigen batches. The differences in time and assays make a real comparison very difcult. Despite this fact, some studies showed a good correlation between IgG levels measured with ELISA and with the multiplex immunoassay [13,15,16]. Therefore the higher antibody response measured after booster vaccination in the cross-sectional study seems to be induced by vaccination. In this study IgG levels were measured in children who had received the Dutch whole cell vaccine (DTwP-IPV-Hib). Since other wP vaccines have a different composition and concentration of pertussis antigens and possibly other immunogenicity proles, we can only describe here the effect of the Dutch wP vaccine on IgG levels in children. 4.4. Effect on protection After booster vaccination almost all aP primed children had IgG-Pt levels above the arbitrary protection level (20 EU/ml) [17], whereas in wP primed children only the high Pt containing booster vaccine InfanrixTM could elicit a notable serologic response. Hence, our study indicates that for the induction of an optimal protection against whooping cough, a booster vaccine with a high Pt dose must be administered in wP, and to a lesser extent in aP primed children. It is still under debate which components of the pertussis vaccine are protective and whether antigen-specic antibodies against them are reliable correlates of protection [35]. Low IgG-Pt levels are correlated with susceptibility to whooping cough [36,37] and antiPrn and -Fim2/3 levels are considered to be of protective value in Bordetella pertussis infection [38]. However, it is not clear to what extent immunity to single antigens will protect against whooping cough [39]. Plotkin recently mentioned that multiple components of the immune response reduce the risk of whooping cough [40]. As shown by Ausiello et al., after acellular vaccination, cellular immunity to Pt is still present when antibodies have decreased to undetectable levels [41]. Despite low IgG-levels, we measured relative high pre-booster AIs of Pt- and Prn-antibodies in aP primed compared to wP primed children, suggesting memory immunity. Thus, low antibody levels do not always correlate with poor immunity [42]. In conclusion, the pertussis antigen concentration in the vaccine is important for the induction of a notable antibody response. In view of the ongoing rises in the incidence of whooping cough in the Netherlands during the last decade, we advise to administer a high Pt containing vaccine as a booster in both Dutch wP and aP primed children. Antibody levels to pertussis toxin are considered

with B. pertussis. These data, combined with high IgG-Prn levels, emphasize that B. pertussis still circulates in the Dutch population [24]. However, about one third of the children had relatively high pre-booster IgG levels restricted only to FHA and no other pertussis antigens. This is probably due to cross reactive antibodies induced by proteins from other bacterial species like Haemophilus Inuenza and Mycoplasma pneumoniae [25,26]. Nonetheless, these outliers did not inuence the geometric means calculated. 4.2. Postvaccination IgG responses In children primed and boostered with the acellular pertussis vaccine InfanrixTM , the high IgG-Pt, -FHA and -Prn levels measured are consistent with other studies [2730]. The pertussis antigen concentration and composition in the vaccine used for primary and booster vaccination therefore seem important for the IgG response, as described previously by Podda et al. [31]. However, a clear cut antigen dose-response effect was lacking in our study, in agreement with the ndings of Knuf et al. [32]. We found postvaccination GMC levels of IgG-Prn to be identical in TriaxisTM and InfanrixTM boostered, wP primed children, despite a 2,5 times lower amount of Prn in TriaxisTM than in InfanrixTM . Remarkably, in aP primed children IgG-Prn antibodies had increased after booster vaccination to very high levels (GMC >1000 EU/ml). In addition, similar IgG-FHA booster responses were measured after both TriaxisTM and InfanrixTM in both wP and aP primed children, while again InfanrixTM contains a 5-fold more FHA antigen dose compared with TriaxisTM . This seems to indicate that after a certain dose the effect of higher vaccine antigen concentration no longer contributes to the extent of the IgG levels evoked. For the IgG-Pt response the optimal amount of Pt-antigen dose may not yet have been attained (Fig. 1). Since pertussis toxin in both whole cell and acellular pertussis vaccines is chemically modied and therefore less immunogenic, a relative large amount of Pt-antigen in the vaccine is needed to induce a proper (booster) response, compared to the amounts of FHA and Prn. In addition, during the production of wP, almost all pertussis toxin is being removed together with the culture medium [33]. Consequently, in wP primed children a weak IgG-Pt response was measured with a GMC of 7.3 EU/ml 1 month after booster vaccination with wP. After vaccination with TriaxisTM , the IgG-Pt response measured was also low due to the low amounts of Pt in this vaccine with a GMC of 21.4 EU/ml at day 28. These low IgG-Pt responses indicate poor immunological memory and appear to be linked to the primary vaccination. Remarkably, in wP, IgG-Fim2/3 levels were signicantly higher at day 28 after vaccination with InfanrixTM than pre-booster levels, although InfanrixTM does not contain Fim2/3. As previously shown by gel electrophoresis and monoclonal antibodies on a Western blot, the Fim2/3 preparation from Aventis Pasteur used is highly puried and does not contain any other pertussis vaccine antigens [24] [and unpublished data from the RIVM, the Netherlands]. In addition, we only measured a signicant higher IgG-Fim2/3 level after booster vaccination with InfanrixTM in wP primed and not in aP primed children. This could be explained by the relative

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L.H. Hendrikx et al. / Vaccine 27 (2009) 65306536 [18] Huang LM, Lee CY, Lin TY, Chen JM, Lee PI, Hsu CY. Responses to primary and a booster dose of acellular, component, and whole-cell pertussis vaccines initiated at 2 months of age. Vaccine 1996;14(9):91622. [19] Guiso N, Njamkepo E, Vie le Sage F, Zepp F, Meyer CU, Abitbol V, et al. Longterm humoral and cell-mediated immunity after acellular pertussis vaccination compares favourably with whole-cell vaccines 6 years after booster vaccination in the second year of life. Vaccine 2007;25(8):13907. [20] Versteegh FG, Mertens PL, de Melker HE, Roord JJ, Schellekens JF, Teunis PF. Agespecic long-term course of IgG antibodies to pertussis toxin after symptomatic infection with Bordetella pertussis. Epidemiol Infect 2005;133(4):73748. [21] Giuliano M, Mastrantonio P, Giammanco A, Piscitelli A, Salmaso S, Wassilak SG. 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to be protective against whooping cough and seem dependent on the concentration of pertussis toxin in the booster dose. However, multiple components of Bordetella pertussis are considered important for protection against whooping cough. Long-term pertussis antibody responses to the pertussis vaccine antigens and cellular immunity need to be further investigated in wP and aP primed children of different ages. Acknowledgements We would like to thank all children who participated in the MEMORY study, Kemal zturk, Pieter van Gageldonk and Rutger Schepp (Cib, RIVM, Bilthoven) for technical assistance and all research staff from the Spaarne hospital Hoofddorp and the RIVM in Bilthoven who were involved in this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2009.08.052. References
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