Direct Comparison of Immunogenicity Induced by 10- or 13-Valent Pneumococcal Conjugate Vaccine around the 11-Month Booster in Dutch Infants

Background & Aims Since 2009/10, a 10- and a 13-valent pneumococcal conjugate vaccine (PCV) are available, but only the 10-valent vaccine is now being used for the children in the Netherlands. As the vaccines differ in number of serotypes, antigen concentration, and carrier proteins this study was designed to directly compare quantity and quality of the antibody responses induced by PCV10 and PCV13 before and after the 11-month booster. Methods Dutch infants (n = 132) were immunized with either PCV10 or PCV13 and DTaP-IPV-Hib-HepB at the age of 2, 3, 4 and 11 months. Blood samples were collected pre-booster and post-booster at one week and one month post-booster for quantitative and qualitative immunogenicity against 13 pneumococcal serotypes, as well as quantitative immunogenicity against diphtheria, tetanus, pertussis and Haemophilus influenzae type b. We compared immunogenicity induced by PCV13 and PCV10 for their ten shared serotypes. Results One month post-booster, pneumococcal serotype-specific IgG geometric mean concentrations (GMCs) for the PCV13 group were higher compared with the PCV10 group for six serotypes, although avidity was lower. Serotype 19F showed the most distinct difference in IgG and, in contrast to other serotypes, its avidity was higher in the PCV13 group. One week post-booster, opsonophagocytosis for serotype 19F did not differ significantly between the PCV10- and the PCV13 group. Conclusion Both PCV10 and PCV13 were immunogenic and induced a booster response. Compared to the PCV10 group, the PCV13 group showed higher levels for serotype 19F GMCs and avidity, pre- as well as post-booster, although opsonophagocytosis did not differ significantly between groups. In our study, avidity is not correlated to opsonophagocytotic activity (OPA) and correlations between IgG and OPA differ per serotype. Therefore, besides assays to determine IgG GMCs, assays to detect opsonophagocytotic activity, i.e., the actual killing of the pneumococcus, are important for PCV evaluation. How differences between the two vaccines relate to long-term protection requires further investigation. Trial Registration www.trialregister.nl NTR3069

A controlled randomized intervention trial with 2 groups (see figure 2 and table 1).
o Divided in group 1a and 1b; 33 infants per group o Divided in group 2a and 2b; 33 infants per group Group 1 and 2 are split in sub groups in order to reduce the burden of the 8 ml blood samples. Randomization will be done within group 1 and within group 2.

Study population:
Children eligible to receiving the regular vaccinations of the NIP, born after August 2011 (assuring that all children are eligible for and will receive Hepatitis B vaccination)

Intervention:
Children of group 1 will receive the DTaP-IPV-Hib-HepB vaccination according to the NIP; they will receive PCV13 instead of PCV10. All vaccinations will be given by the study team during home visits.
Children of group 2 will receive all vaccinations (DTaP-IPV-Hib-HepB and PCV10) as part of the NIP by a well-baby clinic nurse; this is not part of the trial.

Primary
Pneumococcal serotypes  Cellular immune response (Plasma B cells and memory B cells) immediately before and 7-9 days after the booster at 11-months of age  Humoral immune response (antibody concentrations and geometric mean concentrations (GMT)) at 12 months of age Secondary

Pneumococcal serotypes
 Opsonophagocytoses immediately before and 7-9 days after the booster at 11months of age  Avidity at 5, 8, immediately before and 7-9 days after the booster at 11-months and at  One blood collection of 8 ml (2x 4 ml tubes). The burden and risk is considered low.
The children might find the needle scary and it might be painful but only for a few seconds. A local anaesthetic (Emla® crème, Astra Zeneca) may be used to minimize pain. Blood collection could result in a small bruise at the location of injection, which will disappear within a few days.
Group 1; one heel/finger stick sampling, group 2: 3-4 heel/finger sticks sampling. The burden and risk is considered low.
For group 2 (PCV10 group), the children themselves have no direct benefit in participating in this trial. The trial is aimed to study the immune response after 3+1 PCV10 or PCV13 vaccinations. These children, who have followed the Dutch NIP, are the only possible children that can participate in the trial. Visits will take 10-30 minutes each (depending on the type of blood collection and whether a questionnaire is taken).
Children in group 1 will receive PCV13 vaccinations. The side effects of these vaccinations are expected to be equal to the side effects of PCV10 (which the children would have received as part of the NIP). They will however receive these vaccinations at home to reduce the burden. These children will benefit from the added protection of the three extra serotypes which are not present in the PCV10 vaccination. These children are the only possible study group, since they are eligible for the Dutch NIP. Visits will take maximum 30 minutes each. As of March 2011 all newborns will receive PCV10 vaccinations instead of PCV7. PCV13 showed to be non-inferior to PCV7 for 6 out of 7 serotypes present in PCV7 (except for 6B), when comparing proportions of responders (≥0.35 ug/ml) and geometric mean concentrations (see SPC of PCV13 and (5)). In addition it was shown that PCV13 elicits OPA responses comparable to those elicited by PCV7.

KOKKI, PIM and our current study
An improved understanding of the immune biology of the conjugate vaccines, such as PCV10 and PCV13, is essential to develop the best immunization strategies that provide sustained protection. The results from the cellular immunity and the antibody concentrations will help to decide on the best vaccination strategy for pneumococcal vaccination in the NIP.
The current study in combination with our previous KOKKI and PIM studies will provide more information for the optimization of the pneumococcal immunization program.

Cellular immunity
The aim of our previous study (KOKKI, cellular immunogenicity after PCV7 vaccination We lack information on the memory response to PCV10 and PCV13, therefore our current study aims to determine the development of the cellular immune response (plasma B cells and memory B-cells) for the 3+1 vaccination schedule of PCV10 and PCV13 immediately before and 7-9 days after the booster at 11-months of age. Time points of blood collection are similar to those in the previous KOKKI study. Also the antibody concentrations, avidity and opsonophagocytoses will be determined at these time points. These vaccines differ in number of serotypes and type of conjugate and could therefore differ in their cellular immunity.
A selection of 5-6 serotypes will be made; two of the four serotypes tested in the KOKKI study and three to four serotypes added by PCV10 and PCV13 for the current study.
Selection will depend on serotype circulation just before analyses.

Humoral immunity
The aim of our previous study (PIM, (NL28918.000.09), 2010-2012), was to asses the optimal PCV vaccination schedule for PCV13, based on humoral immunogenicity after four different vaccination schedules.
An alternative timing and reduction of the number of vaccination doses on the serological response directed against the different serotypes of pneumococci was compared to the currently used* 3+1 vaccination schedule. The schedules were two 2+1 vaccination schedule (2, 4 and 11 months of age; 3, 5 and 11 months of age) and two 3+1 vaccination schedules (*2, 3, 4 and 11 months of age; 2, 4, 6 and 11 months of age).
After the start of the study PCV10 was introduced in the NIP and not PCV13. It is therefore important to compare the vaccines for the currently used 3+1 vaccination schedule. Also universal vaccination against Hepatitis B will be introduced for children born after August 2011, and therefore the DTaP-IPV-Hib vaccine will be replaced by DTaP-IPV-Hib-HepB, which could influence the response to PCV13.
The current study will investigate the humoral immunogenicity of PCV10 after the currently used 3+1 vaccination schedule (vaccination at 2, 3, 4 and 11 months of age).
The data will be compared to the 3+1 vaccination schedule of PCV13 (PIM study). To rule out the influence of the shift from DTaP-IPV-Hib to DTaP-IPV-Hib-HepB vaccine we will use data from group 1 (PCV13) of the current study, to bridge both studies.
As in the PIM study blood samples are collected at one month after the primary series (5 months), at 8, 11 and 12 months of age. The 12 months sample is used to compare the schedules (primary endpoint). The 8 month sample is chosen, since the peak incidence of A controlled, randomized, intervention trial with 2 groups (see figure 2 and table 1).
 Children of group 1 will be invited first (around the age of 1 month), since they have to receive all DTaP-IPV-Hib-HepB and PCV13 vaccinations as part of the trial o Vaccinations will be given during home visits o Children will be randomized over 2 sub groups in order to diminish the burden of the 8 ml blood sample (which is collected just before or 7-9 days after the

11-months vaccination)
o For the immune memory, collect 33 children per sub group in order to have 25 evaluable children per sub group (sub group 1a and 1b)  Children of group 2 will be invited around the age of 3.5 months, as to include them in the trial at the age of 5 months o Vaccinations are not part of the trial, since they already receive them during well-baby clinic visits o Children will be randomized over 2 sub groups in order to diminish the burden of the 8 ml blood sample (which is collected just before or 7-9 days after the

11-months vaccination)
o For the immune memory, collect 33 children per sub group in order to have 25 evaluable children per sub group (sub group 2a and 2b) Randomization will be done within group 1 and separately within group 2.
The randomization will not be done for the total study (group 1 and 2 together) since children of the PCV10 group already receive all proper vaccinations in the NIP and randomization would result in a long period between invitation at 1 month of age and inclusion at 5 months of age for the PCV10 group.   In the KOKKI study, for each child blood was collected at one time point only, being either pre-or 7-9 days post-booster. Inclusion and blood collection was during the same visit.

Exclusion criteria
Inclusion in the current study however starts at first vaccination, respectively 9 (group 1) and 6 (group 2) months prior to blood collection for cellular immunity, which can lead to a higher amount of dropout. Taking into account a 10% dropout up to 11 months, we included 33 children in each group. A subsequent estimated failure rate to collect sufficient blood cells of 15% will generate 25 evaluable children.
Antibody concentrations will be compared between the PCV10 group of the current study and the PCV13 group of the PIM study (vaccination schedule 2, 3, 4 and 11 months) at 12 months after vaccination. As significance level we take 0.05 two-sided and as power 80%. The expected variance of log(GMC) is 0.27 (this is for serotype 6B, the serotype with the highest variance). Using the following formula n = 2k 2 σ 2 / δ 2 , we need 47 children per group to detect a 2-fold difference in GMC (=0.30 difference in log(GMC)) between the two groups. A 2-to 2.5-fold difference is considered to represent a true difference in immunogenicity in this type of study (8,9). To account for 20% dropout, 59 Version 4.0 March 16 th , 2012 Page 24/43 children need to be included per group. As we already include 2x33 = 66 children in the cellular immunity part of the study, this group is also large enough to assess antibody concentrations.
The antibody concentrations will be compared for each of the serotypes included in the vaccine. Adjustment for multiple testing is not necessary for these comparisons because the serotypes will not be compared to each other and we are not interested in the overall null hypothesis of no effect (10).
Antibody concentrations will also be compared between the PCV13 group of the current study and the PIM study at 12 months after vaccination to bridge the two studies.
Using the same sample size calculation as above, we need 59 children in this group as well.
Based on previous experience we expect a participation rate of 5% for all groups.
The study burden for group 1 involves only one 8 ml blood collection, vaccinations and one heel/finger stick (100 ul) Vaccinations are given at home, which parents appreciate. Since PCV13 is a registered vaccine and is not expected to give more side effects as PCV10, we assume that the study burden is perceived as relatively low, especially when the additional protection of three serotypes is taken into consideration.
The study burden for group 2 involves an 8 ml blood collection and 3-4 heel/finger sticks samples. Although the participants have no benefit in this trial, we expect that the heel/finger stick samples will not be perceived as a high burden.
Due to the lack of benefit for the children in group 2, recruitment rates after the first mailings amounted to around 2% instead of 5%.
In order to reach enough inclusions we need to invite approximately 1200 children for group 1 and 3500 children for group 2. We plan to enroll the children of group 1 in one month and the children of group 2 in 3.5 months.
The RCP region Noord Holland -Utrecht will be used and we will asses the exact region based on data from the Dutch Central Bureau for the Statistics (http://www.cbs.nl), in the same period of 2010. Due to a hampering recruitment for group 2, the RCP region Noord-Holland-Utrecht recruitment area will be extended with parts of Gelderland and Flevoland.

TREATMENT OF SUBJECTS
All children in group 2 follow the standard NIP (they have or will receive vaccinations with Synflorix®, a ten-valent pneumococcal conjugate vaccine (PCV10) and DTaP-IPV-Hib-HepB, at the age of 2, 3, 4 and 11 months), which is not part of this study.

Investigational product/treatment
All children in group 1 will receive Prevenar-13® instead of PCV10. For the rest they will follow the standard NIP, at the age of 2, 3, 4 and 11 months. All vaccinations (PCV13 and DTaP-IPV-Hib-HepB) are given by the study team in order to prevent vaccine mix-up during well-baby clinic appointments. The DTaP-IPV-Hib-HepB is not part of this study, but will be given by the study team for practical reasons.
There is no placebo group present in this trial.

Use of co-intervention (if applicable)
There is no objection against the use of co-medication or other kinds of interventions against concomitant disorders. The use of painkillers against local pain after vaccination is also not prohibited. Use of immunosuppressive medicines during the trial could in certain cases lead to exclusion of the corresponding blood samples for further laboratory analysis, since that might interfere with the outcomes of the study. Medicine use will be recorded in the questionnaire and the CRF.

Escape medication (if applicable)
For blood collection a local anesthetic (Emla® crème; AstraZeneca) will be used to minimize the pain.
For vaccination normal NIP practices will be followed.
Synflorix®, a ten-valent pneumococcal vaccine (PCV10) is not administered in the trial, since children receive this vaccine as part of the NIP. However, we intent to look at the outcome of these vaccinations and therefore we included the information of PCV10 in chapter 6.1-6.4.

Name and description of investigational medicinal product(s)
PCV13: For the qualitative and quantitative composition see chapter 2 and 6.1 of the SPC.

PCV10:
For the qualitative and quantitative composition see chapter 2 and 6.1 of the SPC. stored in a dedicated study fridge. The transfer from the RCP/IOD to the study team will be documented.

Summary of findings from non-clinical studies
All vaccines taken from the fridge by the study team will be documented. The investigator/study team member is responsible for the correct transport and storage conditions to the location of vaccination. The vaccines are transported in isolated coolers that demonstrate the adequate temperature. The vaccine may not be frozen.
In case of unusable vaccines, e.g. vials that are expired or damaged, the vaccine will be returned to the RIVM (RCP/IOD) and replaced.
Each vaccine delivery has to be accompanied with signed receipt form containing information about quantity, expiry date and batch numbers of the supplied vaccines. The form needs to be dated and signed by the person responsible for the transport and the person receiving the product. The investigator is responsible for the accountability in the fridge. If discrepancies are observed between the number of delivered and used vaccines at the end of the study, a written declaration has to be supplied by the investigator. All

Main study parameter/endpoint
Pneumococcal serotypes  Cellular immune response (Plasma B cells and memory B cells) immediately before and 7-9 days after the booster at 11-months of age  Humoral immune response (antibody concentrations and geometric mean concentrations (GMT)) at 12 months of age

Secondary study parameters/endpoints
Pneumococcal serotypes  Opsonophagocytoses immediately before and 7-9 days after the booster at 11months of age

Other study parameters
Date of birth, gender, duration of pregnancy, birth weight, duration of breast feeding, use of day care, family members and age, smoking habits parents, use of antibiotics last 3 months, current symptoms of cold, ear infections, other disorders. PCV13 groups: painkiller use around vaccination.

Randomisation, blinding and treatment allocation
Two randomization lists will be made, one for group 1 and one for group 2.
Prior to starting the trial (group 1) and prior to the 11 month visit (group 2) envelopes which contain a letter indicating allocation to one of the sub groups will be numbered in random order using a random number generator (www.random.org). The envelopes will be sealed by the principal investigator (who will not be involved in randomization visits) and given to the study team. For group 1 during visit 1, after eligibility has been confirmed and the informed consent form has been signed, the study team member should open the envelope with the lowest number still available. The volunteer will be assigned to the group indicated in the letter. For group 2 the randomization will take place before the 11 months home visits.
In case replacements are warranted due to premature withdrawal of volunteers, after all envelopes have been used, the sponsor will prepare a new set of sealed and randomly numbered envelopes corresponding to the number of required replacements.
For group 1 at the time of randomization of a subject the study team member should take the investigational product vial and vaccinate the subject with the product(s) as indicated.
The treatment number should be recorded in the CRF.
The study will not be blinded.

Study procedures Invitation and enrolment
Based on live birth data from the CBS (http://www.cbs.nl/nl-NL/menu/home/default.htm) the optimal region for the study is assessed. The region will comprise of parts of Utrecht and Noord-Holland and part of Flevoland and Gelderland for group 2. The RIVM-RCP (who send invites for the National vaccination program) uses the region to make an address list and sends this list to the distributor who sends the invites. Invites containing the information leaflet, return card and envelop and a short recommendation letter from the RCP will be send to all addresses on the list. The parents send in the return card, to show their interest in the study. In response an email will be send with the extra information (including the informed consent form and the general patient brochure from the CCMO).
A telephone call will be made to the parent, to inquire if they are still interested, explain the study in case of questions and to make an appointment. Their address will be registered for the home visit and to send the extra information also on paper. The study parameters in the questionnaire are as mentioned in paragraph 7.1.3. The questionnaire is part of the CRF and will be completed by the investigator.

Blood sample
In case of partial or complete failure a second attempt may be performed with consent of the parents. The maximum number of blood collection attempts is 2.
In case of resistance by the child the behaviour code of the Dutch Society of Pediatricians will be followed.
For cellular immunity, an 8 ml blood sample is expected to yield 8-10 x 10 6 PBMC.

IgG antibodies
Blood samples will be transported to the CIB and after centrifugation the serum samples will be stored at -80C. Later, the samples will be tested for all 13 vaccine pneumococcal polysaccharides and DTaP-Hib using the X-map Luminex technology. Only 100 ul of blood is needed for these analyses.

Preparation of PBMCs
Specific B cell frequencies will be measured for five to six serotypes.
In the previous KOKKI study the following serotypes were tested: 6B, 14, 19F and 23F.
They are present in PCV7, PCV10 and PCV13.
For the current study a selection of the 13 serotypes will be made. Probably 6B and 19F from PCV7, one-two serotypes from the three serotypes added by PCV10, and two serotypes added by PCV13 (probably 6A and 19A). The final selection will be based on the carrier frequencies just before evaluation.
Fresh Peripheral Blood Mononuclear Cells (PBMCs) will be separated from heparinized blood, within 24 hours after collection, by density gradient gel centrifugation in CPT tubes

B cell stimulation in vitro (memory B cells)
For the indirect ELISPOT, PBMCs will be resuspended and cultured at a concentration of 2x10 6 cells/ml in AIM-V culture medium in 24-wells plates. PBMCs will be stimulated polyclonally with 3 µg/ml CpG-C, PTO modified (5'-TCG TCG TCG TTC GAA CGA CGT TGA T-3') (Isogen) in the presence of 10 ng/ml IL-2 (strathmann), 10 ng/ml IL-10 (Calbiochem) and 2 ng/ml of pooled polysaccharides (Stathens Serum Institute) for 5 days at 37°C and 5% CO 2 . Cells will be harvested by centrifugation, washed with culture medium and tested in antigen-specific ELISPOT assays.

Plasma B cells
To determine plasma cell frequencies, PBMCs are diluted in culture medium to a concentration of 3x10 6 cells/ml and used in an ELISPOT to examine the number of plasma cells.

ELISPOT assay
Multiscreen Filtration plates were pre-incubated with 35% ethanol for 1 minute, washed

Withdrawal of individual subjects
Subjects can leave the study at any time for any reason if they wish to do so without any consequences. The investigator can decide to withdraw a subject from the study for urgent medical reasons.

Replacement of individual subjects after withdrawal
For sub groups 1a, 1b, 2a and 2b, 33 subjects are collected per sub group (in order to reach 25 evaluable subjects for cellular immunity. The numbers will also enable sufficient samples for antibody concentrations).
Only in case of dropout subject can be replaced:  Due to the vaccinations of group 1 it is impossible to replace these subjects after all two months vaccinations are given  In case of being still feasible, subjects in group 2 can be replaced in case subjects drop out during the first visits.

Follow-up of subjects withdrawn from treatment
Follow-up is only applicable to group 1. When withdrawing before the end of the vaccination schedule, options need to be discussed to make sure that the child receives the necessary vaccinations. A schedule started with PCV13 should be finished with either PCV13 or PCV7 (not by PCV10). Completion of the schedule can be done by the study team, who can give PCV13. If the parents refuse home visits from the study team the well-baby clinic can complete the schedule with PCV7 (since PCV13 is not part of the RVP and the well-baby clinic can therefore not provide PCV13).

Premature termination of the study
The sponsor is entitled to terminate the study at any time if new data on the safety or efficacy of the product under study becomes available during the study, making further use of the product undesirable, even in a controlled situation. The METC will be informed about such a decision.
The study can be discontinued:  If the investigator of that site comes into a situation that impedes the further progress of the study and the investigator cannot be replaced or no other solution can be found.
 In case of repeated unacceptable protocol violations.
In case of premature study termination, options should be discussed to ensure complete vaccination series for the children, see chapter 7.6.

Section 10 WMO event
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects and the reviewing accredited METC if anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly greater than was foreseen in the research proposal. The study will be suspended pending further review by the accredited METC, except insofar as suspension would jeopardise the subjects' health. The investigator will take care that all subjects are kept informed.

Adverse and serious adverse events
Adverse events are defined as any undesirable experience occurring to a subject during the study, whether or not considered related to [the investigational product / the experimental treatment]. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded.
A serious adverse event is any untoward medical occurrence or effect that at any dose: results in death; is life threatening (at the time of the event); requires hospitalisation or prolongation of existing inpatients' hospitalisation; results in persistent or significant disability or incapacity; is a congenital anomaly or birth defect; is a new event of the trial likely to affect the safety of the subjects, such as an unexpected outcome of an adverse reaction, lack of efficacy of an IMP used for the treatment of a life threatening disease, major safety finding from a newly completed animal study, etc.
All SAEs will be reported through the web portal ToetsingOnline to the accredited METC that approved the protocol, within 15 days after the sponsor has first knowledge of the serious adverse reactions.
SAEs that result in death or are life threatening should be reported expedited. The expedited reporting will occur not later than 7 days after the responsible investigator has first knowledge of the adverse reaction. This is for a preliminary report with another 8 days for completion of the report.
SAE's and SUSARs will be reported to the CCMO according to the following: All other unrelated SAEs will be reported semiannual a. Group 2: Registration will taken place during one week after blood collection b. Group 1: i. Registration will take place in the period of trial entry until one month after the primary series (in practice until ~5 months of age) ii. During the period of pre-booster blood collection until one month after the booster vaccination c. The report will contain the following: subject number, vaccination date, SAE start and stop date, diagnosis, severity, relation to vaccination or study procedure

Suspected unexpected serious adverse reactions (SUSAR)
Adverse reactions are all untoward and unintended responses to an investigational product related to any dose administered.
Unexpected adverse reactions are adverse reactions, of which the nature, or severity, is not consistent with the applicable product information (e.g. Investigator's Brochure for an unapproved IMP or Summary of Product Characteristics (SPC) for an authorised medicinal product).
The sponsor will report expedited the following SUSARs through the web portal The expedited reporting of SUSARs through the web portal ToetsingOnline is sufficient as notification to the competent authority.
The sponsor will report expedited all SUSARs to the competent authorities in other Member States, according to the requirements of the Member States.
The expedited reporting will occur not later than 15 days after the sponsor has first knowledge of the adverse reactions. For fatal or life threatening cases the term will be maximal 7 days for a preliminary report with another 8 days for completion of the report.

Annual safety report
Not applicable

Follow-up of adverse events
All adverse events will be followed until they have abated, or until a stable situation has been reached. Depending on the event, follow up may require additional tests or medical procedures as indicated, and/or referral to the general physician or a medical specialist.

Data Safety Monitoring Board (DSMB)
Not applicable. Other more qualitative endpoints, such as avidity and opsonophagosytoses, will be described as such.

Univariate analysis
For group 2 of the currents study and the PCV13 group of the PIM study (vaccination at 2, 3, 4 and 11 months of age), differences in serotype specific antibody concentrations between different schedules will be analyzed. Primary, the antibody concentrations against pneumococcal polysaccharides for each serotype at 12 months in the different study arms will be calculated. GMCs and the degree of protection (the proportion with concentration > 0.35 µg/ml) will be determined.
Secondary, the pneumococcal antibody concentrations at 5, 8, immediately before and 7-9 days after the 11-months booster and at 12 months of age will be calculated. For these endpoints, GMCs and the degree of protection (the proportion with concentration > 0.35 µg/ml) will be determined. The antibody concentrations of the longitudinal samples of each child will be used to assess the kinetics.
Also the DTaP-Hib antibody concentrations at 5, 8, immediately before and 7-9 days after the 11-months booster and at 12 months of age will be calculated. A chi-square test will be used to determine differences in proportions. A T-test or a distribution free variable will be used to test differences in mean or median.

Multivariate analysis
Modifying factors, like family structure etc. will be analyzed for exploratory reasons only, in a multivariate regression analyses with as primary outcome measure antibody concentrations against the 13 serotypes S. pneumoniae.

Recruitment and consent
See 7.3 for a detailed overview of events. Before the first study appointment parents/legal representatives will receive an information leaflet with a response card (Annex 1).
Following a positive response more detailed patient information with informed consent forms will be sent (Annex 2). The parent(s)/legal representative(s) will then be contacted by phone to discuss the study, answer questions and to make an appointment for the first home visit. The parent(s)/legal representative(s) will have at least one week between the phone call and the home visit where they sign the informed consent.
Study procedures will only take place after both parents/legal representative(s) have signed the informed consent (one parent in case of an orphan, or single-parent family).

Objection by minors or incapacitated subjects (if applicable)
Parents are at all times allowed to withdraw the informed consent. The parent and the investigator can at all times decide to end the participation of the child if the child shows resistance to the study procedure. The code of conduct will be followed which is published by the Nederlandse Vereniging voor Kindergeneeskunde 'Gedragscode bij verzet van minderjarigen die deelnemen aan medisch-wetenschappelijk onderzoek' .
The right of the parents to withdraw informed consent at all times is put down in the informed consent letter.

Benefits and risks assessment, group relatedness
Blood collection: one blood collection of 8 ml (2x4 ml tubes). The burden and risk is considered low.
The children might find the needle scary and it might be painful (only for a few seconds).
A local anaesthetic (Emla® crème, Astra Zeneca) may be used to minimize pain. Blood For group 2 (PCV10 group), the children themselves have no direct benefit in participating in this trial. The trial is aimed to study the (cellular) immune response after 4 PCV10 or PCV13 vaccinations. These children, who have followed the Dutch NIP, are the only possible children that can participate in the trial. Visits will take 10-30 minutes each (depending on the type of blood collection and whether a questionnaire is taken).
Children in group 1 will receive PCV13 vaccinations. The side effects of these vaccinations are expected to be equal to the side effects of PCV10 (which the children would have received as part of the NIP). They will however receive these vaccinations at home to reduce the study burden. These children will benefit from the added protection of the three extra serotypes which are not present in the PCV10 vaccination. These children are the only possible study group, since they are eligible for the Dutch NIP. Visits will take maximum 30 minutes each.

Compensation for injury
According to a Ministerial Order, RIVM is excluded from compulsory insurance for clinical research as determined by the Dutch law on Medical Investigations (WMO, section 7, paragraph 6). Participants can recover the loss from RIVM. Any claims will be settled according to the same terms that an insurance company uses.
Normal participants insurance provides cover for damage to research subjects through injury or death caused by the study. The insurance applies to the damage that becomes apparent during the study or within 4 years after the end of the study.

Incentives
All children will receive one or two small presents during some visits. The maximum amount spend on presents will be 15 euro per child.

Handling and storage of data and documents
All children participating in the study will receive a unique subject number, a difference is made between PCV13 and PCV10 groups to allow for prioritizing the serotypes tested in case of low amounts of blood:  Group 1: COP13_001 -COP13_066  Group 25: COP10_101 -COP10_166 Al trial data is recorded using this subject number and is saved for 15 years according to legal requirements.
All recorded data is treated confidential such that data in reports or other publications of the trial can never be traced back to the child or family. Recorded data can only be accessed by competent and qualified research employees, by members of the CCMO or by representatives of the sponsor and the competent authorities.

Amendments
Amendments are changes made to the research after a favourable opinion by the accredited METC has been given. All amendments will be notified to the METC that gave a favourable opinion.
A 'substantial amendment' is defined as an amendment to the terms of the METC application, or to the protocol or any other supporting documentation, that is likely to affect to a significant degree: the safety or physical or mental integrity of the subjects of the trial; the scientific value of the trial; the conduct or management of the trial; or the quality or safety of any intervention used in the trial.
All substantial amendments will be notified to the METC and to the competent authority.
Non-substantial amendments will not be notified to the accredited METC and the competent authority, but will be recorded and filed by the sponsor.

Annual progress report
The sponsor/investigator will submit a summary of the progress of the trial to the accredited METC once a year. Information will be provided on the date of inclusion of the

End of study report
The sponsor will notify the accredited METC and the competent authority of the end of the study within a period of 90 days. The end of the study is defined as the last patient's last visit.
In case the study is ended prematurely, the sponsor will notify the accredited METC and the competent authority within 15 days, including the reasons for the premature termination.
Within one year after the end of the study, the investigator/sponsor will submit a final study report with the results of the study, including any publications/abstracts of the study, to the accredited METC and the Competent Authority.

Public disclosure and publication policy
The study results will be reported in an internal report and submitted for publication in peer-reviewed journals. Publications will be drafted by the sponsor investigators.