Breastfeeding enhances the antibody response to Hib and Pneumococcal serotype 6B and 14 after vaccination with conjugate vaccines

Vaccine 25 (2007) 1497–1502

Breastfeeding enhances the antibody response to Hib and Pneumococcal serotype 6B and 14 after vaccination with conjugate vaccines S.A. Silfverdal a,d,∗ , L. Ekholm a,c , L. Bodin b b

a Department of Paediatrics, Orebro ¨ ¨ University Hospital, Orebro, Sweden ¨ ¨ Clinical Research Centre, Biostatistics and Epidemiology Unit, Orebro University Hospital, Orebro, Sweden c Department of Children’s Health, Orebro ¨ ¨ County Council, Orebro, Sweden d Department of Mother’s and Children’s Health, Ostersund ¨ ¨ Hospital, Ostersund, Sweden

Received 19 May 2006; received in revised form 3 October 2006; accepted 12 October 2006 Available online 30 October 2006

Abstract Background: This study was performed in order to investigate the relationship between breastfeeding and the antibody response after vaccination with conjugate vaccines against Hib and pneumococcal diseases. Methods: This was an open non-randomised multi-centre study enrolling 101 healthy Swedish infants. PncCRM was administered concomitantly with DTaP/IPV/Hib at 3, 5, and 12 months at separate site. Duration of breastfeeding was calculated for days of almost exclusive as well as of total (any form of) breastfeeding. Results: At 13 months of age 6 out of 83 children did not reach 0.2 ␮g/ml against serotype 6B, and five of these were breastfed less than 90 days (Fisher’s Exact test, P = 0.011). Four children did not reach 1 ␮g/ml against Hib and all those were breastfed less than 90 days (Fisher’s Exact test, P = 0.008). One month after the second dose, at 6 months of age, children breastfed 90 days or more showed significantly higher GMC against serotype 14 (P = 0.003). Conclusion: This study indicates that children exclusively breastfed 90 days or more might get a better serological protection against Hib, and the pneumococcal serotypes 6B and 14 after vaccination, compared to children less breastfed. © 2006 Elsevier Ltd. All rights reserved. Keywords: Breastfeeding; Hib; Pneumococcus; Antibody response; Vaccination

1. Introduction Immunization is a very effective preventive strategy. The incidence of invasive Haemophilus influenza type b (Hib) disease has decreased rapidly after introduction of general Hib immunization [1]. However, an increase in invasive Hib disease has been reported in the UK in children 1–4 years old associated with waning immunity against Hib. Lack of a booster vaccination, loss of natural boosting by less Hib carriage and herd-immunity, as well as a change from a whole-cell Pertussis to an acellular Pertussis combo-vaccine ∗ Corresponding author at: Department of Paediatrics, Orebro ¨ University ¨ Hospital, SE-701 85 Orebro, Sweden. Tel.: +46 19 602 11 11; fax: +46 19 602 70 30. E-mail address: [email protected] (S.A. Silfverdal).

0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.10.025

have been suggested causes [2–4]. Besides these factors a low breastfeeding-rate in the UK population might be a neglected contributing factor [5]. Invasive pneumococcal disease (IPD) is a threat to infants and young children, especially in low-income countries, with a high mortality rate. In the U.S. this situation has rapidly changed after extensive immunization with a heptavalent pneumococcal conjugate vaccine against serotype 4, 6B, 9V, 14, 18C, 19F and 23F in childhood and a reduction of IPD is now documented not only in early ages but also in adults 20–39 years and >65 years [6,7]. This vaccine was given to infants at 2, 4, 6 and 12–15 months of age. In the Nordic countries and Italy the routine schedule for vaccinations in infancy against Diphtheria, Tetanus, Pertussis, Polio and Hib consists of 3-doses given at 3, 5, and 11–12 months of age. PCV7 given in this 3-dose schedule is as good

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as the 4-dose U.S. schedule except for a slightly lower protection against serotype 6B and 23F with the 3-dose schedule [8,9]. A protective effect by breastfeeding against morbidity, and especially against infections, is well established [10]. Breast-milk may have a direct and local effect on the mucosal membranes mainly through secretory-IgA, but also by a long lasting effect through priming of the infant’s immune system [10,11]. A protective effect by breastfeeding on invasive Hib disease is well documented [12–15]. In previous epidemiological studies, we have found a decreased risk for invasive Haemophilus influenza infection with 5% for each week of added exclusive breastfeeding [15]. The IgG2 antiHib response in children 18 months or older with invasive Hib disease is enhanced by breastfeeding with anti-Hib titres 2.5 times higher in children exclusively breastfed for 13 weeks or more than those breastfed less [16]. A stimulatory effect of breastfeeding on the antibody response to Hib after Hib immunization has been documented [17,18]. Formula fortified with an amount of nucleotides similar to human milk enhanced the Hib and diphtheria antibody response [19]. Immune-phenotypic differences between breast-fed and formula-fed infants indicate a greater maturity in development of the immune system of breastfed infants [20,21]. The aim of this study was to analyse the antibody concentrations to each of the Pneumococcal serotypes as well as to Hib in relation to the duration of breastfeeding. 2. Material and methods 2.1. Study-base This was an open non-randomised multi-centre study (PrevenarTM vaccine trial) enrolling 101 healthy Swedish ¨ infants at four sites (Malm¨o, Link¨oping, Ume˚a and Orebro). PncCRM was administered concomitantly, but at separate site, with DTaP/IPV/Hib at 3, 5, and 12 months. Antibody concentrations were determined at National Institute of Public Health, KTL, in Finland by EIA for anti-Pneumococcal IgG from serum samples taken at 3, 6, 12 and 13 months, and for anti-Hib IgG from serum samples at 6 and 13 months of age [8]. Duration of breastfeeding was based on parental interviews regularly during the study and measured in days of almost exclusive as well as days of any form of breastfeeding. Breastfeeding was analysed as recorded as well as with a pre-chosen dichotomisation at 90 days [15]. This breastfeeding study was a sub-protocol within the PrevenarTM vaccine trial with an estimated study size based on data from a similar immunogenicity study in Finland [8]. 2.2. Serological measures For analyses of serological protection and vaccine response against Hib, post-vaccination antibody concentrations of ≥0.15 ␮g/ml and ≥1.0 ␮g/ml were used, while for the pneumococcal serotypes the concentrations of ≥0.2 ␮g/ml,

≥0.35 ␮g/ml according the WHO recommendation, and ≥1.0 ␮g/ml were used [22]. 2.3. Statistics Geometrical means were calculated for the concentrations of antibodies using the statistical package SPSS-for Windows version 14.0 [23]. Because of skewed distributions we used non-parametric statistics in the basic analysis and transformations to reduce skewness in the parametric methods. Mann–Whitney’s U-test was used in analyses of antibody concentrations when two groups of children were compared. For two-group comparisons of the proportion above different protective levels Chi-square tests as well as Fisher’s exact test were employed. Regression analyses were done with logarithmic values of antibody titres with duration of breastfeeding as a continuous as well as a dichotomised explanatory factor. P-values less than 0.05 were chosen as statistically significant. 2.4. Ethics ¨ The Ethics committees of the County Councils in Orebro, Malm¨o, Link¨oping and Ume˚a approved the study.

3. Results One hundred and one (101) children were included in the vaccine trial. Two children did not complete the study, one because of serious adverse event and one at the request of the parents. Eighty-three were included in this study after exclusion for different reasons; two infants had no blood samples drawn, three infants did not follow the given time windows for blood sampling and 12 infants did not follow the schedule for vaccination. There were missing data on any breastfeeding for four infants. Twenty-six children were classified as exclusively breastfed less than 90 days, and 57 children more than 90 days. For any form of breastfeeding there were eight children less than 90 days and 71 children more than 90 days. For antibody assays there were 76 pre-vaccination serum samples available, 75 samples at 6 months, 83 at 12 months and 83 serum samples at 13 months, see Fig. 1. Before the first immunization, at 3 months of age, the children breastfed 30 days or more showed lower geometrical mean concentrations (GMC) of antibodies against serotype 6B, 9V and 19F than those children breastfed shorter than 1 month (data not shown). One month after the second dose, at 6 months of age, children breastfed 90 days or more showed significantly higher GMC against serotype 14 (Table 1). One month after the third dose, at 13 months of age, there was a tendency towards higher GMC of antibodies against Hib and five of the pneumococcal serotypes (4, 6B, 9V, 14 and 23F) in children breastfed 90 days or more (Table 1). At the same age 6 out of 83 children did not reach 0.2 ␮g/ml against serotype 6B and five of these were breastfed exclusively

0.2 16.9 12.3–22.2 9.6 3.9–22.6

0.4 5.1 3.3–7.3 3.9 1.8–6.3

Students’s t-test with log-transformed values. a

0.8 0.4–1.6 Hib

0.7 0.5–1.1

0.8

–

–

0.7 0.23 0.15–0.3 0.21 0.1–0.3 0.1 0.6 0.4–1.2 Serotype 23F

1.1 0.8–1.5

0.9 5.0 2.8–7.8 5.2 2.1–11.7 0.6 0.8 0.5–1.3 0.6 0.3–1.4 0.2 4.0 2.2–7.7 Serotype 19F

5.9 4.5–8.2

0.8 4.8 3.5–5.4 4.5 3.1–6.3 0.3 0.43 0.4–0.5 0.35 0.2–0.5 1.0 2.6 1.8–4.1 Serotype 18C

2.6 1.9–3.4

0.6 12.1 9.3–14.3 10.7 6.5–18.6 0.2 1.9 1.4–2.3 1.4 0.8–2.1 0.003 1.8 1.1–3.1 Serotype 14

4.6 3.3–6.2

0.4 7.2 5.7–8.8 6.2 4.6–9.0 0.5 0.8 0.6–0.9 0.7 0.4–1.0 1.0 3.2 2.3–4.5 Serotype 9V

3.2 2.5–4.3

0.1 6.2 4.9–9.2 3.4 1.1–8.0 0.1 0.26 0.2–0.3 0.18 0.1–0.3

0.9 0.3

0.8 0.30 0.2–0.4

0.3

P-valuea

Excl. bf ≥90 57 10.1 7.6–11.2 Excl. bf < 90 26 8.1 5.4–12.2

GMC post-dose 3 – at 13 months of age with 95% CI

P-valuea

Excl. bf ≥90 57 0.7 0.5–0.8 Excl. bf <90 26 0.7 0.5–1.0

GMC pre-dose 3 – at 12 months of age with 95% CI

P-valuea

0.28 0.2–0.5

The major limitation is the size of the study. The study was performed with a sub-protocol to a vaccine trial for investigation of reactivity and immunogenicity with a power calculation based on assumptions on immune response per se without including any assumption on breastfeeding exposure [15]. A posterior power calculation for the present sub-study showed a power between 5% and 67% to statistically verify differences in Hib and pneumococcal antibody concentrations between long and short breastfeeding exposure. Thus, the study was underpowered and in order to answer our questions more reliably the study size should be three times larger for the analysis of serotype 6B and 5–10 times larger for the other serotypes. With respect to the measures we claim that

Serotype 6B

4.1. Limitations

Excl. bf ≥90 53 4.7 3.6–5.4

Our understanding of the development of immunity in infancy and the role of breastfeeding is not fully understood. However, this study indicates that breastfeeding enhances the antibody response to some extent against Hib and the pneumococcal polysaccharides of serotype 6B and 14 after vaccinations with conjugate-vaccines.

GMC post-dose 2 – at 6 months of age with 95% CI

4. Discussion

Excl. bf <90 22 3.8 2.8–6.0

less than 90 days (P = 0.011; Fisher’s Exact test, 2-sided) (Table 2). At 13 months of age, two children of 83 did not reach an anti-Hib concentration of 0.15 ␮g/ml, and those were breastfed only 0–4 days; 4 of 95 children had an anti-Hib level of <1.0 ␮g/ml, and all those four children were breastfed less than 90 days (Fisher’s Exact test, P < 0.008). Regression models with breastfeeding as explanatory variable with sex and smoking as confounding variables did not revel any changes in the results. Pre-vaccination concentrations of antibodies were not correlated to post-vaccination concentrations. Weights at 3 months (first vaccination) or 13 months of age did not influence the results. Standard deviations for Hib and all pneumococcal serotypes, except serotype 9 (data not shown), were smaller in children breastfed 90 days or more compared to those less breastfed, thus, indicating a more homogenous distribution in children breastfed for a longer period.

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Exclusive breastfeeding days Number of samples Serotype 4

Fig. 1. Overview of the recruitment.

Table 1 Geometrical mean concentrations of antibodies against Hib and pneumococcal serotypes at post-dose 2, pre-dose 3 and post-dose 3 after vaccination with conjugated-vaccines in the 3-5-12 month vaccination schedule

S.A. Silfverdal et al. / Vaccine 25 (2007) 1497–1502

100 91a 100 100 100 81 93

57

28 12 39 75 9 32 11 38 8 27 65 15 27 8

P < 0.05. a

22

100 30 95 95 95 86 73 100 98 100 100 100 95 93

57 26

100 81 100 100 100 96 92 93 56 98 98 82 89 49

57 26 53 22

100 55 100 100 95 95 86 Serotype 4 Serotype 6B Serotype 9V Serotype 14 Serotype 18C Serotype 19F Serotype 23F

92 58 96 96 81 81 50

Ebf ≥90

100 64 98 100 94 96 87

Post-dose 3

92 77 100 96 96 85 88

57 26 53

96 23 85 92a 85 92 57 91 14 95 68 86 77 32

22 57

100 95 100 100 100 84 93 100 81 100 100 100 88 92

26

81 31 81 85 54 65 38 100 47 96 98 94 96 79

79 37 82 96 61 70 30

26 53

57

Ebf <90 Ebf ≥90

Post-dose 3

Ebf <90 Ebf ≥90

Pre-dose 3

Ebf <90 Ebf ≥90 Ebf < 90 Ebf ≥90

Post-dose 3

Ebf <90 Ebf ≥90

Several epidemiologic studies have demonstrated less Hib disease in breast-fed children [12–15]. Some vaccine studies, but not all, have documented a stimulatory effect on the immune response by breastfeeding. Such discrepancies might be explained by differences in measurement and classification of breastfeeding exposure, time between pre- and post-sampling, as well as by age at the immunization and post-immunization sampling. No difference in antibody levels against Hib at 7 months of age after vaccination with different conjugated Hib vaccines at 2, 4, and 6 months of age has been reported from three studies [24–26]. In contrast, Pabst et al. have documented higher anti-Hib concentrations at 7 and 12 months of age, and Greenberg et al. at 9–15 months of age, after vaccination with similar conjugated Hib vaccines using the same schedule as in the three studies mentioned above [17,18]. 4.3. Possible explanatory mechanisms

Ebf <90

Ebf <90

the breastfeeding data is valid and that recall bias is not a problem when data is collected prospectively. Duration of breastfeeding is often related to maternal smoking, in many societies also to educational level and social-economic situation. Parents in Sweden are supported by a paid maternal leave the first year why the financial reason to return to work is less likely. We have controlled for sex and smoking as well as weight at 3 and 13 months of age without changes in results. Larger studies might find significant associations but probably not of an important magnitude. 4.2. Findings in other studies

Ebf = exclusive breastfeeding in days Number of samples

Pre-dose 3

26

Ebf ≥90 Pre-dose 3

Ebf <90

Post-dose 2 Post-dose 2 Post-dose 2

Ebf ≥90

Proportion ≥1.00 ␮g/ml Proportion ≥0.35 ␮g/ml Proportion ≥0.2 ␮g/ml

Ebf <90

Ebf ≥90

S.A. Silfverdal et al. / Vaccine 25 (2007) 1497–1502 Table 2 Children vaccinated at 3-5-12 months of age with the proportion (%) of children above different protective levels at 6, 12 and 13 months of age in relation to duration of exclusive breastfeeding (Ebf) dichotomized at 90 days

1500

Both pneumococcal serotype 6B and Hib have weak antigenic properties compared to the other vaccine antigens in this study. Serotype 6B and Hib also cross-react with each other. Breastfeeding seems to influence the antibodymediated protection against both serotype 6B and Hib. For the other pneumococcal serotypes, except serotype 14, no significant influence by breastfeeding was found. An enhanced effect of breastfeeding might be a mere consequence from the fact that serotype 6B and Hib have weak antigenic properties and could not be sufficiently immunogenic on their own. If the antigens are very strongly immunogenic a slight enhancing effect by breastfeeding may not be visible. Their weak antigenic properties might be related to the similarities in antigenic structures and the tendency to cross-react with each other. The antibody response is dependent on many factors and the optimal post-vaccination antibody concentration is hard to predict. We do not know the real protective level and those we use are only surrogate measures [27,28]. Breastfeeding might be involved in the regulation and homeostasis of the immune response by enhancing weak antigens and modulate strong ones leading to less dispersion and higher degree of homogeneity. Before the first immunization, the children breastfed 30 days or more had lower antibody concentrations against

S.A. Silfverdal et al. / Vaccine 25 (2007) 1497–1502

serotype 6B, 9V and 19F than those children breastfed shorter than 1 month, probably because of less exposure of and mucosal encounter with bacteria through factors like oligosaccharides in breast milk that blocs adhesion to the mucosa. The maturation of the avidity and the development of B cell memory might be crucial for the understanding. After infection as well as after immunization it takes some weeks before the maturation of the avidity is optimal, and for the B cell memory to fully develop it takes at least 4 months [4,29,30]. According to this, studies on the antibody response too early in life but also too shortly after immunization, may hardly detect any influence by breastfeeding, and it may explain why some researcher [24–26] did not find any effect of breastfeeding on the anti-Hib response. This hypothesis is supported by our own findings of a more mature antibody response during invasive Hib disease in breastfed children as concern the isotype switch and the antibody concentrations in breastfed children [16]. Recent findings indicate that IL-7 in human milk plays an important role in the development of thymus [31]. IL-7 might be one factor in human milk that stimulates the avidity maturation and memory development leading to a long lasting immune-stimulatory effect [32]. In conclusion, this study indicates that children exclusively breastfed 90 days or more might get a better serological protection against Hib, and the pneumococcal serotypes 6B and 14 after vaccination, compared to children less breastfed. Due to the small sample-size we can neither confirm nor exclude that breastfeeding plays an important role in the immune response but the tendency in the results gives an indication that data on breastfeeding should be included in future research on vaccine response. Breastfeeding might be involved in the regulation and homeostasis of the immune response by enhancing weak antigens and modulate strong ones. Acknowledgments We wish to thank the children and their parents who have participated in this study, the personnel at the Prevenar study sites in Link¨oping (Lennart Nilssson, Lena Lindell, Christina Helander), Malm¨o (Carl-Erik Flodmark, Liselotte Andersson), Ume˚a (Leif Gothefors, Margareta B¨ackman), and ¨ Orebro (Laila Fj¨allryd), the personnel at National Institute of Public Health, KTL, in Helsinki, Finland (Teija Jaakola, ˚ Anne Holm, Helena K¨ayhty and Heidi Ahman), the Research ¨ Committee of Orebro County Council, and Wyeth Lederle ˚ Hanson, Gothenburg, Nordiska AB. Thanks to Prof Lars A for support with revising the manuscript. References ¨ Silfverdal SA, Hugosson S, Fredlund H, Bodin L, [1] Garpenholt O, Romanus V, et al. The impact of Haemophilus Influenzae type b vaccination in Sweden. Scand J Infect Dis 1996;28:165–9.

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