Study design

A single-centre, parallel-group intervention study with two groups (PCV10 recipients and PCV13 recipients) was conducted in the Netherlands among infants eligible for the routine National Immunization Program (NIP): vaccinations at 2, 3, 4 and 11 months of age, or the 3+1 (primary plus booster) schedule (S1 Text). Through their parents, infants born in August 2011 were invited for the PCV13 group, and infants born in September-November 2011 were invited for the PCV10 group (Fig 1). Exclusion criteria were a serious and/or immunological disease that could interfere with the results of the study; previous administration of plasma products; presence of bleeding disorders, and prematurity (gestational age <37 weeks). No financial compensation for participants was provided. Infants in the PCV10 group received Synflorix (GlaxoSmithKline Biologicals s.a., Rixensart, Belgium) during regular well-baby clinic visits according to the NIP schedule. Infants in the PCV13 group received Prevenar-13 (Pfizer Limited, Sandwich, Hampshire, UK) during home visits by the study team according to the NIP schedule. Both groups received concomitantly DTaP-IPV-Hib-HepB (Infanrix hexa, GlaxoSmithKline Biologicals s.a., Rixensart, Belgium).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Enrollment and Study Participation.

PCV10 indicates 10-valent pneumococcal conjugate vaccine, Synflorix; PCV13 indicates 13-valent pneumococcal conjugate vaccine, Prevenar-13. * When the enrolment target was reached, further children were considered redundant, and their parents’ informed consent procedures were cancelled. ** Plasma cell and memory B-cell analysis published by van Westen et al.[27].

https://doi.org/10.1371/journal.pone.0144739.g001

For B-cell and immunogenicity assays, an 8-ml blood sample was collected by venipuncture and randomly allocated “pre-booster” for half of the children (group A) and “one week post-booster” for the other half (group B). From children of the PCV10 group, who gave the 8-ml post-booster sample, an additional pre-booster sample (300 µl) was collected by heel stick. In all subjects, to collect the one month post-booster blood (300 µl), which was the primary endpoint, the heel stick was used because it was easier for the children. This was an open-label study for parents and study staff, but immunogenicity analysis was performed blinded. An acknowledged national ethics committee approved the study (METC Noord Holland, NTR3069). All study-related activities were performed in accordance with European guidelines for Good Clinical Practice, which includes provisions of the Declaration of Helsinki, with written informed consent from the child’s parents or the guardian.

In the current article, we publish the quantitative IgG data for all 132 subjects, of all three time-points (pre-booster, one week post-booster, and one month post-booster) and all 13 serotypes. In addition, we publish the qualitative data, i.e., avidity and opsonophagocytosis of pneumococcal serotypes and IgG data of diphtheria, tetanus, pertussis and H. influenzae type b. Results on the B-cell assays were previously published, open access, by van Westen et al. [27] who also included IgG data for a subset of the children for 6 of the 13 serotypes, pre-booster and one week post-booster.

Humoral analyses

After blood collection, sera for serological analyses and plasma for opsonophagocytosis assays were separated and stored at -80°C. Pneumococcal serotype-specific IgG antibody concentrations against the 13 pneumococcal serotypes were measured using a fluorescent bead-based multiplex immunoassay (MIA), as described previously [28]. Avidity for all 13 serotypes was determined with a MIA using 0.5 M ammonium isothiocyanate (NH₄SCN) compared with phosphate buffered saline (PBS) [29]. Opsonophagocytosis activity (OPA) was determined for all 13 serotypes by multiplex opsonophagocytosis assay performed at UCL Institute of Child Health (London, UK) [30]. Antibody concentrations against DTaP-Hib antigens were determined by MIA, as described previously [31–33].

Statistical analyses

In this type of study, a 2- to 2.5-fold difference is considered to represent a true difference in immunogenicity [20, 34]. A minimum of 47 children per group were needed in order to provide 80% power to detect a 2-fold difference in IgG geometric mean concentrations (GMCs) between the two groups, with an assumed variance of the log-transformed concentration of 0.27 (based on serotype 6B, the serotype with the highest variance). The following formula was used n = 2k²σ² / δ². Since 66 children per group had already participated in the study for the B-cell assays [27], a 1.8-fold difference could be detected at one month post-booster the primary endpoint. For the other time-points, collected blood samples were fewer but sufficient to detect a 2- to 2.5-fold difference. Baseline characteristics including sex, age at vaccination and blood sampling, day-care attendance at 12 months of age, birth-weight, gestational age, presence of siblings < 5 year of age, duration of breast-feeding, and passive smoking were compared between the two groups, using a t-test for continuous variables and a chi-square test for categorical variables.

For each group, pneumococcal serotype-specific IgG GMCs, mean avidity indices (MAI), and OPA geometric mean titers (GMTs) with 95% confidence intervals (CI) were calculated. Percentages of vaccine recipients reaching an IgG threshold of 0.35 μg/ml for all time-points and 1.0 μg/ml post-booster were calculated. IgG GMCs were calculated diphtheria, tetanus, pertussis and H. influenzae type b antigens, and their internationally accepted thresholds for protection were used for seroprotection rate analysis. Differences in IgG GMCs, MIA, and OPA GMTs between groups were analyzed with a t-test, and differences in the percentage of subjects reaching a threshold were tested with a chi-square test or, when appropriate, a Fisher’s Exact test. Spearman correlations between IgG and OPA or avidity and OPA were calculated. For analyses between post-booster time-points (one week versus one month), a paired t-test was used.

Since the age at vaccination and blood sampling differed slightly between the groups, we performed secondary analyses in which we adjusted for age in days at vaccination and blood sampling using regression analysis.

All data were analyzed using SPSS 19. A p value of <0.05 was considered statistically significant.

Study population

In total, 132 infants were enrolled in the study, both groups (PCV10 recipients and PCV13 recipients) consisting of 66 infants (Fig 1). Baseline characteristics of the two groups are summarized in Table 1. Subjects of the PCV10 group, immunized during regular well-baby clinic visits, were slightly older at vaccination and blood sampling than children of the PCV13 group, who were vaccinated at home by the study team. This difference reflects the normal delays that occur when families plan clinic visits. Compared to the standard analyses described below, regression analyses for all time-points, with adjustment for age at vaccination and blood sampling (S1–S3 Tables), showed no change in the pattern of differences between the PCV13 group and the PCV10 group, although some differences were no longer statistically significant.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Characteristics of the participants.

https://doi.org/10.1371/journal.pone.0144739.t001

One month post-booster immunogenicity for the ten shared pneumococcal serotypes

One month post-booster, IgG GMCs were higher for serotypes 5, 6B, 9V, 14, 19F and 23F in the PCV13 group compared with the PCV10 group (Fig 2 and S1 Table). Avidity indices of IgG antibodies induced by PCV13 post-booster were higher for serotype 6B and 19F, but lower for 5 serotypes compared with the PCV10 group (Fig 2 and S2 Table). Nearly all participants reached the 0.35 μg/ml seroprotection threshold for all serotypes except for 19F in the PCV10 group (86%) (Table 2). The percentage of children with antibody concentrations >1.0 μg/ml was >90% for most serotypes, except in the PCV13 group for serotype 4 (89%) and in the PCV10 group for serotype 19F (73%).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Pneumococcal serotype-specific measurements.

IgG geometric mean concentrations (GMCs) pre-booster, one week post-booster and one month post-booster; Mean avidity indices of IgG antibodies pre-booster, one week post-booster, and one month post-booster; opsonophagocytosis geometric mean titers (GMTs) pre-booster and one week post-booster. *indicates significant differences (<0.05), higher in the PCV10 group than the PCV13 group ^#indicates significant differences (<0.05), higher in the PCV13 group than the PCV10 group **sample size for opsonophagocytosis assays: PCV10 group N = 22–27, PCV13 group N = 25–31

https://doi.org/10.1371/journal.pone.0144739.g002

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Seroprotection rate of the antibody concentrations against 13 pneumococcal serotypes for the PCV10 group and the PCV13 group with p-values for differences between the groups.

https://doi.org/10.1371/journal.pone.0144739.t002

Pre-booster immunogenicity for the ten shared pneumococcal serotypes

Compared with the PCV10 group, pre-booster IgG GMCs were lower in the PCV13 group for 5 of the 10 shared serotypes but higher for serotype 19F (Fig 2 and S1 Table). Avidity indices of the PCV13 group were lower for serotypes 5 and 14 but higher for 19F, compared with the PCV10 group (Fig 2). Functional OPA GMTs in the PCV13 group were lower for serotype 6B and 19F (Fig 2 and S3 Table). In contrast, the IgG GMC and avidity index for serotype 19F were higher for PCV13-vaccinated children compared with PCV10-vaccinated children. The percentage of children with antibody concentrations >0.35 μg/ml was above 75% for most serotypes (Table 2).

One week post-booster immunogenicity for the ten shared pneumococcal serotypes

Surprisingly, all one week post-booster IgG GMCs were significantly higher (p<0.002) than one month post-booster IgG GMCs (p values not shown), except for serotype 19F in the PCV10 group. Differences in IgG GMC and avidity data between the PCV13 and PCV10 groups were similar at one week and one month. However, in the one week post-booster groups, which included only half of the children of the PCV10 and PCV13 groups, the differences were in most cases not significant (Fig 2 and S1 and S2 Tables). OPA GMTs were significantly higher in the PCV13 group for serotypes 9V and 23F (Fig 2 and S3 Table). Spearman correlations were serotype-specific and found only found between IgG GMC and OPA; no correlation between avidity and OPA or IgG was found except for PCV10 serotype 1 (S5 Table and S1 Fig).

PCV13-specific serotypes 3, 6A and 19A

PCV13 induced IgG GMCs for serotype 3, 6A and 19A with pre-booster seroprotection rates ranging from 61% to 81% whereas the rates one month post-booster were 100% for antibody concentrations >0.35 μg/ml and 92–100% for antibody concentrations >1.0 μg/ml (Table 2 and S1 Table). PCV13 avidity indices these serotypes were comparable to other serotypes (Fig 2 and S2 Table), for all three time-points. In the PCV10-group IgG GMCs were slightly increased at one week and one month post-booster, compared with pre-booster, for serotypes 3, 6A and 19A (Fig 2, S1 Table), but avidity was not measured. One week post-booster in the PCV10 group, OPA GMTs had increased for serotypes 6A and 19A but were much lower than for the PCV13 group (S3 Table).

One month post-booster immune response of DTaP-Hib

Diphtheria- and pertussis-specific IgG GMCs and seroprotection rates induced by the concomitantly given DTaP-Hib vaccine did not differ between the groups (Table 3 and S4 Table). In both groups, all vaccine components showed a good booster response. However, Tetanus-specific IgG GMCs were significantly lower in the PCV13 group than the PCV10 group, whereas seroprotection rates were similar. Hib IgG GMCs were only significantly lower in the PCV13 group compared with the PCV10 group in the age-adjusted analyses.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 3. Seroprotection rate (%) for diphtheria, tetanus, pertussis, and Hib antigens for the PCV10 group and the PCV13 group with p-values for differences between the groups.

https://doi.org/10.1371/journal.pone.0144739.t003

Post-booster data

Post-booster, the IgG GMC levels for six shared serotypes were higher for the PCV13 group than for the PCV10 group. The PCV13 group also had higher functional OPA responses for two serotypes but higher avidity levels only for one serotype. The age at vaccination and blood sampling differed slightly between the groups, and the vaccination schedule varied more in the PCV10 group. However, the age differences between the groups did not appear to affect immune responses, as the age-adjusted analysis showed similar post-booster data for both products. Likewise, the results of the study of Spijkerman et al. showed that four different primary PCV13 vaccination schedules resulted in similar one month post-booster immunogenicity outcomes [29]. Differences found between our two groups could therefore be due to actual differences between the two PCVs, such as serotype concentrations, conjugates, and additives.

The most pronounced difference in immunological response between the two vaccines was found for serotype 19F, which showed a significantly higher IgG GMC and avidity index in the PCV13 group compared with PCV10 group. In our study, however, the serotype 19F-specific IgG GMC (2.09 μg/ml) induced by PCV10 one month post-booster was much lower than found in previous studies (4.23 to 9.72 μg/ml) [20, 21, 35–39]. Also, the related seroprotection rate (>0.35 μg/ml), which was 86% for serotype 19F for our PCV10 group, is lower than the 97–100% reported in other studies [20, 21, 35–39]. Seroprotection rates are based on internationally accepted ELISA IgG GMCs cut-off levels of >0.35 μg/ml for all serotypes [40]. Recently, Andrews et al. proposed serotype-specific ELISA thresholds for protection based on effectiveness data for PCV13 [41]. For serotype 19F, they determined a threshold of 1.17 μg/ml, which would translate to a serotype 19F-specific seroprotection rate of 70% for our PCV10 group and 100% for the PCV13 group one month post-booster. Moreover, our seroprotection rate was calculated using IgG GMCs determined in a MIA, which in general results in slightly higher levels than the WHO ELISA [28]. Serotype 19F IgG GMCs for our PCV13 group, analyzed in same assay run as for PCV10, indeed were relatively high (24.37 μg/ml), especially compared with data generated using the WHO ELISA. This finding indicates that the already low values for the PCV10 group might have been even lower if the WHO ELISA had been used. In spite of low serotype 19F IgG GMCs and avidity for the PCV10 group, OPA responses and OPA seroprotection rates were comparable to those in the PCV13 group, They were 88.9% and 100% for the PCV10 group and the PCV13 group, respectively, when using the 19F-specific seroprotection cut-off level of 430 [41]. This suggests a low correlation between IgG and OPA, which was indeed only 0.39 in the PCV10 group and 0.49 in the PCV13 group. The relatively high serotype 19F OPA response in the PCV10 group, despite the low IgG results could be explained by a higher IgM antibody response, since IgM is be effective at opsonizing bacteria [42, 43]. Moreover, the timing of the OPA samples in this study should be considered as we measured at one week post-booster, which could reflect the recall of the memory response, whereas if we would have measured at one month post-booster the time-point normally used for IgG antibodies, our measurements could have reflected a mixture of memory and newly generated antibodies.

Protection against disease by PCV10 seems adequate, SP carriage has remained low (1%) since its 2009 introduction in the Netherlands and no PCV10 serotype 19F vaccine failures have been observed [9, 44] despite low serotype 19F IgG GMCs and avidity.

Studies in Finland and Brazil have likewise found PCV10 to be 84–100% effective against IPD [45, 46].

For PCV13 serotype 19F, the current study showed an IgG GMC of 24.37 μg/ml, which is higher than in previous studies of both PCV7 (2.4–5.6 μg/ml)[20, 22, 23, 38, 47] and PCV13studies (6.6–11.06 μg/ml)[22, 23, 47] and could be related to the use of MIA instead of ELISA [28]. However, Grant et al. and Dagan et al. [22, 23], using ELISA, also reported higher 19F-specific responses for PCV13 than seen in previous PCV7 studies. Cross-reaction by serotype 19A could play a role [22, 48–50]. Overall, the reported higher serotype 19F-specific IgG GMCs, avidity, and OPA induced in our PCV13 group could result in better protection against pneumococcal disease than induced by PCV7, for which six vaccine failures in 5 years occurred in the Dutch population [44].

Van Westen et al. published the plasma and memory B-cells results of described trial for 6 serotypes, showing no clear correlation between IgG and plasma or memory B-cells. In our paper, we show a weak correlation between IgG GMCs and OPA and no correlation between avidity and OPA. Together with the fact that OPA measures actual killing of the bacteria and avidity index is a measure of antigen-antibody binding strength implies that for comparison of differences in vaccine-induced immunogenicity, IgG GMCs combined with OPA analysis should be performed.

Pre-booster data

Pre-booster seroprotection rates for pneumococcal vaccine serotypes remained high for both our groups, indicating that protection of children is adequate during the whole period between the primary series and the booster dose. Whether the 5 serotypes with lower IgG GMCs pre-booster in the PCV13 group, compared with PCV10, reflect a lower initial response after the primary series or a steeper decline cannot be concluded from data collected in this trial.

Interestingly, Spijkerman et al. showed that pre-booster antibody levels at 11 months of age were higher when the primary vaccinations started at an older age (three versus two months) or had longer intervals between doses (two versus one month) [29]. Children in our PCV13 group followed the NIP schedule for the primary vaccinations more strictly than the children in the PCV10 group, who in general had somewhat longer intervals between the doses. This might explain the differences between the groups in pre-booster GMCs at 11 months. However, the differences in the intervals are relatively small (at most 0.4 months at the third vaccination), and a more plausible explanation for the results is therefore the difference in vaccine composition between PCV13 and PCV10. Despite somewhat lower IgG GMCs for the PCV13 group, compared with the PCV10 group, PCV13 is still expected to be effective in the period between primary and booster vaccination. Its effectiveness has been shown in countries where it has been implemented, and even with herd protection induced by PCV7, a further decline of IPD caused by PCV7 serotypes is seen after introduction of PCV13 [51–53]. Also in other studies, comparable seroprotection rates for PCV13 at one month after the primary series have been shown [26, 29, 41].

Added value of serotype 3, 6A and 19A

Although serotypes 3, 6A and 19A are not present in PCV10, IgG GMCs against these serotypes could be detected in the PCV10 group, pre- as well as post-booster dose. For serotype 6A and 19A, a small but significant increase in IgG GMCs was found after the booster dose along with functional post-booster OPA (S1 Table). Both findings could be a result of carriage of 6A and 19A serotypes by 9–15% of the Dutch children at 11 months [4, 5, 9] or cross-reactivity between serotype 6B and 6A and between 19F and 19A antibodies [22, 48–50]. However, post-PCV10-booster, IgG GMCs for these serotypes were 10-30-fold lower than the IgG GMCs reached in PCV13-vaccinated children, and only 9 of 27 PCV10-vaccinated children showed serotype 19A-specific OPA titers comparable to the OPA titers measured in PCV13-vaccinated children, all others being lower. One week post-booster, 89% of the children of the PCV10 group and 100% of the PCV13 group reached the serotype 19A-specific OPA seroprotection threshold of 48 calculated by Andrews et al. as protective against serotype 19A [41]. In the study published by van Westen et al., the PCV10 group had no post-booster increase in plasma and memory B-cells for serotype 6A and 19A, possibly because the cross-reactive vaccine serotypes did not generate an optimal memory response for the non-vaccine serotypes [27]. Whether PCV10 induces protection against serotype 19A disease remains to be seen.

Influence on other NIP components

Serum IgG GMCs against the concomitantly given tetanus and H. influenzae type b (Hib) vaccine components were significantly lower in the PCV13 group compared with the PCV10 group. Enhancement of the antibody responses to Hib, diphtheria and tetanus vaccines by PCV10 vaccination have been reported [54] and could be related to the conjugate proteins used for this vaccine. The clinical relevance of these differences is unknown.

Strength and limitations of this study

The strength of our study is the head-to-head comparison within one trial of immunogenicity induced by PCV10 and PCV13, with all samples analyzed in the same lab using the same methods. An additional strength is that post-booster responses were evaluated at two short-term time-points (one week and one month after the booster), and multiple immunogenicity parameters, including functional activity, were evaluated (IgG GMC, avidity and opsonophagocytosis), generating a more complete immunological profile. We could therefore also determine if IgG or avidity is a better predictor of vaccine effectiveness. A study limitation is the small difference in age at vaccination and blood sampling between the two groups, due to different vaccination practices (well-baby clinic visits for the PCV10 group and home visits for the PCV13 group); however, the age-adjusted analyses showed no difference in immunogenicity.