Immunogenicity of 13-valent pneumococcal conjugate vaccine administered according to 4 different primary immunization schedules in infants: a randomized clinical trial.

IMPORTANCE: Immunization schedules with pneumococcal conjugate vaccine (PCV) differ among countries regarding the number of doses, age at vaccinations, and interval between doses. OBJECTIVE: To assess the optimal primary vaccination schedule by comparing immunogenicity of 13-valent PCV (PCV13) in 4 different immunization schedules. DESIGN, SETTING, AND PARTICIPANTS: An open-label, parallel-group, randomized clinical trial of healthy term infants in a general community in The Netherlands conducted between June 30, 2010, and January 25, 2011, with 99% follow-up until age 12 months. INTERVENTIONS: Infants (N = 400) were randomly assigned (1:1:1:1) to receive PCV13 either at ages 2, 4, and 6 months (2-4-6); at ages 3 and 5 months (3-5); at ages 2, 3, and 4 months (2-3-4); or at ages 2 and 4 months (2-4), with a booster dose at age 11.5 months. MAIN OUTCOMES AND MEASURES: Primary outcome measure was antibody geometric mean concentrations (GMCs) against PCV13-included serotypes 1 month after the booster dose measured by multiplex immunoassay. Secondary outcomes included GMCs measured 1 month after the primary series, at 8 months of age, and before the booster. RESULTS: The primary outcome, GMCs at 1 month after the booster dose, was not significantly different between schedules for 70 of 78 comparisons. The 2-4-6 schedule was superior to the 2-3-4 schedule for serotypes 18C (10.2 µg/mL [95% CI, 8.2-12.7] vs 6.5 µg/mL [95% CI, 5.4-7.8]) and 23F (10.9 µg/mL [95% CI, 9.0-13.3] vs 7.3 µg/mL [95% CI, 5.8-9.2]) and superior to the 2-4 schedule for serotypes 6B (8.5 µg/mL [95% CI, 7.1-10.2] vs 5.1 µg/mL [95% CI 3.8-6.7]), 18C (6.6 µg/mL [95% CI, 5.7-7.7]), and 23F (7.2 µg/mL [95% CI, 5.9-8.8]). For serotype 1, the 3-5 schedule (11.7 µg/mL [95% CI, 9.6-14.3]) was superior to the other schedules. Geometric mean concentrations for all 13 serotypes ranged between 1.6 and 19.9 µg/mL. Secondary outcomes demonstrated differences 1 month after the primary series. The 2-4-6 schedule was superior compared with the 3-5, 2-3-4, and 2-4 schedules for 3, 9, and 11 serotypes, respectively. Differences between schedules persisted until the booster dose. CONCLUSIONS AND RELEVANCE: The use of 4 different PCV13 immunization schedules in healthy term infants resulted in no statistically significant differences in antibody levels after the booster dose for almost all serotypes. The choice of PCV schedule will require a balance between the need for early protection and maintaining protection between the primary series and the booster. TRIAL REGISTRATION: trialregister.nl Identifier: NTR2316.

Changes in the composition of the pneumococcal population and in IPD incidence in The Netherlands after the implementation of the 7-valent pneumococcal conjugate vaccine.

The implementation of nationwide pneumococcal vaccination may lead to alterations in the pneumococcal population due to selective pressure induced by the vaccine. To monitor such changes, pneumococcal isolates causing invasive pneumococcal disease (IPD) before (2004-2005, n=1154) and after (2008-2009, n=1190) the implementation of the 7-valent pneumococcal vaccine (PCV7) in 2006 in the national immunization program (NIP) of The Netherlands were characterized by molecular typing using multiple-locus variable number tandem repeat analysis (MLVA) and capsular sequence typing (CST). The IPD incidence after the implementation of PCV7 in children <5 years of age declined, mainly due to an impressive reduction of cases caused by vaccine serotypes. In the age group of patients ≥5 years of age, the overall IPD incidence remained constant, but the IPD incidence due to vaccine serotypes declined in this age cohort as well, indicating herd immunity. IPD incidence of non-vaccine serotypes 1 and 22F isolates increased significantly and a shift in genetic background of the isolates belonging to these serotypes was observed. In general the composition of the pneumococcal population remained similar after the introduction of PCV7. Both before and after introduction of the vaccine several possible capsular switch events were noticed. We found 4 isolates from the pre-vaccination period in which the serotype 19F capsular locus had been horizontally transferred to a different genetic background. Remarkably, none of the 5 post-vaccination isolates in which we observed possible capsule switch belonged to the 19F serotype, possibly due to vaccine induced pressure. In the post-vaccine implementation period we found no evidence for capsular switch of a vaccine serotype to a non-vaccine serotype, indicating that capsular switch is not the main driving force for replacement. This study provides insights into the effects of nationwide vaccination on the pneumococcal population causing IPD.

Multiple-locus variable number tandem repeat analysis for Streptococcus pneumoniae: comparison with PFGE and MLST.

In the era of pneumococcal conjugate vaccines, surveillance of pneumococcal disease and carriage remains of utmost importance as important changes may occur in the population. To monitor these alterations reliable genotyping methods are required for large-scale applications. We introduced a high throughput multiple-locus variable number tandem repeat analysis (MLVA) and compared this method with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLVA described here is based on 8 BOX loci that are amplified in two multiplex PCRs. The labeled PCR products are sized on an automated DNA sequencer to accurately determine the number of tandem repeats. The composite of the number of repeats of the BOX loci makes up a numerical profile that is used for identification and clustering. In this study, MLVA was performed on 263 carriage isolates that were previously characterized by MLST and PFGE. MLVA, MLST and PFGE (cut-off of 80%) yielded 164, 120, and 87 types, respectively. The three typing methods had Simpson's diversity indices of 98.5% or higher. Congruence between MLST and MLVA was high. The Wallace of MLVA to MLST was 0.874, meaning that if two strains had the same MLVA type they had an 88% chance of having the same MLST type. Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%. For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup. Overall, MLVA is a promising genotyping method that is easy to perform and a relatively cheap alternative to PFGE and MLST. In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in The Netherlands before the introduction of the 7-valent conjugate vaccine.

Population structure of invasive Streptococcus pneumoniae in The Netherlands in the pre-vaccination era assessed by MLVA and capsular sequence typing.

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA).To assess the serotype of each isolate CST was used. Based on the determination of the partial sequence of the capsular wzh gene, this method assigns a capsular type of an isolate within a single PCR reaction using multiple primersets. The genetic background of pneumococcal isolates was assessed by MLVA. MLVA and CST were used to create a snapshot of the Dutch pneumococcal population causing invasive disease before the introduction of the 7-valent pneumococcal conjugate vaccine in The Netherlands in 2006. A total of 1154 clinical isolates collected and serotyped by the Netherlands Reference Laboratory for Bacterial Meningitis were included in the snapshot. The CST was successful in discriminating most serotypes present in our collection. MLVA demonstrated that isolates belonging to some serotypes had a relatively high genetic diversity whilst other serotypes had a very homogeneous genetic background. MLVA and CST appear to be valuable tools to determine the population structure of pneumococcal isolates and are useful in monitoring the effects of pneumococcal vaccination.

Optimization and application of a multiplex bead-based assay to quantify serotype-specific IgG against Streptococcus pneumoniae polysaccharides: response to the booster vaccine after immunization with the pneumococcal 7-valent conjugate vaccine.

We describe the optimization and application of a multiplex bead-based assay (Luminex) to quantify antibodies against polysaccharides of 13 pneumococcal serotypes. In the optimized multiplex immunoassay (MIA), intravenous immune globulin was introduced as an in-house reference serum, and nonspecific reacting antibodies were adsorbed with the commercial product pneumococcal C polysaccharides Multi. The antibody concentrations were assessed in 188 serum samples obtained pre- and post-booster vaccination at 11 months after administration of a primary series of the pneumococcal seven-valent conjugate vaccine (PCV-7) at 2, 3, and 4 months of age. The results of the MIA were compared with those of the ELISA for the serotypes included in the seven-valent conjugated polysaccharide vaccine and for a non-vaccine serotype, serotype 6A. The geometric mean concentrations of the antibodies determined by MIA were slightly higher than those determined by ELISA. The correlations between the assays were good, with R(2) values ranging from 0.84 to 0.91 for all serotypes except serotype 19F, for which R(2) was 0.70. The concentrations of antibody against serotype 6A increased after the administration of PCV-7 due to cross-reactivity with serotype 6B. The differences between the results obtained by ELISA and MIA suggest that the internationally established protective threshold of 0.35 microg/ml should be reevaluated for use in the MIA and may need to be amended separately for each serotype.