Decreased immune response to pneumococcal conjugate vaccine after 23-valent pneumococcal polysaccharide vaccine in children

Vaccine 32 (2014) 417–424

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Decreased immune response to pneumococcal conjugate vaccine after 23-valent pneumococcal polysaccharide vaccine in children夽 Sigurveig Th. Sigurdardottir a,b , Kimberly J. Center c,d , Katrin Davidsdottir e , Vilhjalmur A. Arason e , Bjorn Hjalmarsson e , Ragnheidur Elisdottir e , Gunnhildur Ingolfsdottir a , Robert Northington c , Daniel A. Scott d , Ingileif Jonsdottir a,b,∗ a

Department of Immunology, Landspitali, The National University Hospital of Iceland, Hringbraut, 101 Reykjavik, Iceland Faculty of Medicine, University of Iceland, Reykjavik, Iceland c Pfizer Inc., 500 Arcola Road, Collegeville, PA 19426, USA d Pfizer Inc., 401 North Middletown Road, Pearl River, NY 10965 USA e Centre for Child Health Services, The Primary Health Care of the Reykjavik Capital Area, Alfabakka 16, 109 Reykjavik, Iceland b

a r t i c l e

i n f o

Article history: Received 28 March 2013 Received in revised form 17 September 2013 Accepted 6 November 2013 Available online 2 December 2013 Keywords: Antibody persistence Conjugate vaccine Hyporesponsiveness Memory Pneumococcus Polysaccharide

a b s t r a c t Background: Pneumococcal polysaccharide vaccine (PPV) is used in children at high risk of IPD. PPV is generally not considered to induce immunologic memory, whereas pneumococcal conjugate vaccines (PCVs) elicit protective antibody responses in infants and induce immunologic memory. Little is known about the characteristics of immune responses to PCV in children who previously received PCV and PPV in series. Objective: To characterize immune responses to 13-valent pneumococcal CRM197 conjugate vaccine (PCV13; serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) in children vaccinated in infancy with 9-valent pneumococcal–meningococcal C-CRM197 conjugate combination vaccine (PCV9-MnCC), followed by a toddler dose of PCV9-MnCC or 23-valent pneumococcal polysaccharide vaccine (PPV23). Methods: Children (n = 89) who received PCV9-MnCC in infancy and PPV23 or PCV9-MnCC at age 12 months in a previous (2002–2003) study were vaccinated at age 7.5 years with PCV13; groups PPV23/PCV13 (n = 50) and PCV9/PCV13 (n = 39). Immunoglobulin (Ig)G antibodies, avidity, and opsonophagocytic activity (OPA) were measured before and at 1 and 4 weeks postvaccination. Results: One week postvaccination, IgG levels increased significantly for all serotypes in both groups, and >97% of vaccinees achieved IgG ≥0.35 ␮g/ml 4 weeks after PCV13 vaccination. The PCV9/PCV13 group had higher IgG responses compared with the PPV23/PCV13 group. The upper limits of the 95% confidence intervals of the PPV23/PCV13:PCV9/PCV13 IgG geometric mean concentration ratios were <1.0 for serotypes 1, 4, 5, 9V, 18C, and 23F at 1 week. OPA and avidity results supported these findings. Conclusions: PPV23 vaccination of toddlers may compromise subsequent responses to pneumococcal conjugate vaccines. The clinical relevance of this finding is unclear. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction

夽 ClinicalTrials.gov identifier NCT00853749; EU EudraCT number: 2008-00619433. ∗ Corresponding author at: Department of Immunology, Landspitali, The National University Hospital of Iceland, Hringbraut, 101 Reykjavik, Iceland. Tel.: +354 543 5800; fax: +354 543 4828. E-mail addresses: [email protected] (S.Th. Sigurdardottir), Kimberly.Center@pfizer.com (K.J. Center), [email protected] (K. Davidsdottir), [email protected] (V.A. Arason), [email protected] (B. Hjalmarsson), [email protected] (R. Elisdottir), [email protected] (G. Ingolfsdottir), Robert.Northington@pfizer.com (R. Northington), dan.scott@pfizer.com (D.A. Scott), [email protected] (I. Jonsdottir). 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.11.029

The development of bacterial polysaccharide-protein conjugate vaccines was an important landmark in prevention of lifethreatening diseases in infants and young children, who generally respond poorly, if at all, to pure polysaccharide vaccine antigens. In countries or regions where use of the conjugate Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae (pneumococcus) vaccines in infants and young children has become routine and widespread, serious disease caused by serotypes included in these vaccines has nearly been eradicated [1–4]. The conjugated Neisseria meningitidis serogroup C (meningococcus C) vaccine has also led to rapid and decisive control of outbreaks and of endemic disease in countries where routine use of this vaccine has been implemented

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[5]. Furthermore, while immunization with plain polysaccharide vaccines is generally not considered to induce immunologic memory or boosting with subsequent doses, immunization with conjugate vaccines elicits protective antibody responses in infants and induces immunologic memory, both of which are thought to be of long-lasting duration [6–8]. Little is known, however, about the characteristics of immune responses to further immunologic challenge in children who receive both polysaccharide-protein conjugate and plain polysaccharide vaccines in series. Several reports have described hyporesponsiveness to conjugate vaccines after receipt of polysaccharide vaccines. In Gambian children, a toddler dose of meningococcus C polysaccharide (MCPS) following meningococcal C conjugate (MnCC) vaccination in infancy induced immunological hyporesponsiveness to subsequent MCPS challenge at 5 years of age, indicating that MCPS challenge at 2 years compromised subsequent memory responses [9]. In another study from The Gambia, children previously vaccinated with two doses of MCPS vaccine who were challenged with MCPS at 2 years had significantly lower MCPS antibody levels than children receiving only one dose of MCPS before 6 months of age or unvaccinated controls [10]. The question of hyporesponsiveness after use of the 23-valent pneumococcal polysaccharide vaccine (PPV23) in children who have already received pneumococcal conjugate vaccine (PCV) is clinically important because children who receive PPV23 after PCV are likely to be at increased risk of invasive pneumococcal disease, such as those who are immunocompromised or have certain chronic comorbid conditions [11]. Evidence for hyporesponsiveness was recently reviewed and possible mechanisms, particularly related to the effect of such schedules on memory B-cells, have been proposed [12], but this possible immunocompromising effect of PPV23 vaccination on memory B-cells generated by PCV vaccination has not been studied in children. PPV23 has been used in clinical trials in infants and young children following vaccination with PCV to demonstrate the existence of memory, indicating successful priming by the PCV. Several studies have shown that PPV23 elicits an increase in serotype-specific antibody following PCV priming, which varies between serotypes and studies and when compared with PCV booster [13–15]. However, little information exists regarding the nature of the subsequent immune response to a further PCV challenge after PPV23 administration, i.e. whether PCV can overcome PPV23-induced hyporesponsiveness. Furthermore, there is little information on if and how a PCV-induced response following a PPV23 booster might differ from a PCV-induced response in children naïve to PPV23 vaccine. To evaluate these questions, healthy children approximately 7.5 years of age who participated in an earlier study in which they received a primary infant series of an investigational 9-valent pneumococcal–meningococcal C-CRM197 conjugate combination vaccine (PCV9-MnCC) that contained the 7 serotypes included in 7-valent PCV (PCV7; serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) plus serotypes 1 and 5, followed by a toddler dose of either PCV9-MnCC (PCV9/PCV9 regimen) or PPV23 (PCV9/PPV23 regimen) [15], were recruited to participate in the current study. After subsequent vaccination with 13-valent PCV (PCV13; PCV7 serotypes plus serotypes 1, 3, 5, 6A, 7F, and 19A), immunoglobulin G (IgG) antibody responses, opsonophagocytic activity (OPA), and antibody affinity before and 1 and 4 weeks postvaccination were compared between children who had received PCV9 (PCV9/PCV9 regimen) versus PPV23 booster (PCV9/PPV23 regimen) in their second year of life. The early measurements of IgG antibody levels and avidity at 1 week post-vaccination with PCV13 were included to give additional information about memory responses.

2. Materials and methods 2.1. Participants and study design Participants were children who in 2002–2003 were vaccinated in infancy with two or three doses of PCV9-MnCC and were randomized to receive either the same conjugate vaccine or PPV23 at 12 months of age. In the current study these children, at approximately 7.5 years of age, were vaccinated with a single dose of PCV13 (Fig. 1). Written informed consent was obtained from parent(s)/legal guardian(s) of every subject before enrollment. This study was conducted in accordance with the International Conference on Harmonisation Guideline for Good Clinical Practice and the ethical principles that have their origins in the Declaration of Helsinki.

2.2. Vaccines A total of 218 children were enrolled as infants in the original study during 2002–2003, and were randomly assigned to be vaccinated at either 3 and 5 months or 3, 4, and 5 months of age with PCV9-MnCC (Wyeth; pneumococcal serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F; and meningococcal group C oligosaccharide; pneumococcal serotypes and meningococcal group C oligosaccharide individually conjugated to CRM197 ) concomitantly with routine childhood vaccines (diphtheria, tetanus, acellular pertussis, inactivated poliovirus, and Hib vaccine [DTaP-IPV/Hib]; Sanofi Pasteur MSD) administered at ages 3 and 5 months, according to Icelandic recommendations. Subjects were re-randomized at the toddler dose (age 12 months) to receive either the same PCV9MnCC or PPV23 (Sanofi Pasteur MSD; pneumococcal serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F) given concomitantly with DTaP-IPV/Hib [15]. In the present study, the children were vaccinated with 1 dose of PCV13 (Pfizer Inc, lot number 7-5095-005A); thus the present study comprised two groups based on the booster vaccination at age 12 months with PPV23 or PCV9-MnCC, designated as PPV23/PCV13 or PCV9/PCV13, respectively. PCV13 contains saccharides from pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F individually conjugated to CRM197 . PCV13 is formulated to contain 2.2 ␮g of each saccharide, except for 4.4 ␮g of serotype 6B, per 0.5 mL dose, and 5 mM succinate buffer, with 0.125 mg of aluminum as aluminum phosphate (AlPO4 ) per 0.5 mL dose, and 0.02% polysorbate 80. PCV13 was administered by intramuscular injection (needle size 23G × 1 in.) into the arm.

2.3. Blood samples and measurements Blood samples were obtained immediately before, and 1 and 4 weeks after vaccination. Serotype-specific IgG concentration to all 13 serotypes included in PCV13 was measured by enzyme-linked immunosorbent assay (ELISA) [16–18]. The protocol differs slightly from the currently recommended consensus ELISA protocol [19] (Supplemental text: IgG ELISA methodology). A modified microcolony OPA (mcOPA) assay [20,21] was performed for all vaccine serotypes and at all time-points. The protocol differs slightly from the consensus OPA assay [22] (Supplemental text: mcOPA). Avidity of IgG antibodies to serotypes 1, 5, 6B, 19F, and 23F was measured by ELISA at all three time points [13] and expressed as avidity index (AI) [23]; see Supplemental text: Avidity index methodology.

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Fig. 1. Study design. PCV9-MnCC, 9-valent pneumococcal–meningococcal C-CRM197 conjugate combination vaccine; PCV13, 13-valent pneumococcal conjugate vaccine; PPV23, 23-valent pneumococcal polysaccharide vaccine.

2.4. Safety evaluation

3. Results

Local and systemic reactogenicity and adverse events (AEs) were assessed in both groups. Parent(s)/legal guardian(s) recorded local reactions and systemic events in a paper diary for 4 days following vaccination. Local reactions included tenderness, redness, and swelling at the PCV13 injection site, and were categorized as absent, mild, moderate, or severe based on standard criteria. Systemic events included decreased appetite, irritability, increased and decreased sleep, and fever. Oral temperature was measured daily at bedtime and at any time fever (oral temperature ≥ 38.0 ◦ C) was suspected for 4 days following vaccination. AEs (including serious adverse events [SAEs]) were collected from the day of PCV13 administration to completion of the study. AEs were identified based on any ancillary information reported on the paper diary, clinical evaluation at study visits, or from information obtained by asking parent(s)/legal guardian(s) a nonspecific question.

3.1. Subject disposition and demographics

2.5. Statistical analyses The primary endpoints were the proportion of subjects achieving a serotype-specific IgG concentration ≥0.35 ␮g/ml measured by ELISA (i.e. responders), and the proportion of subjects achieving a mcOPA titer of ≥1:8 for each of the pneumococcal serotypes at 28–42 days after vaccine administration. These endpoints are based on a World Health Organization guideline for the evaluation of antibodies to pneumococcal serotypes [24]. For each serotype, exact, unconditional, 2-sided 95% confidence intervals (CIs) on the proportions were calculated. To assess treatment differences, exact, unconditional, 2-sided 95% CIs on the difference in proportions (PPV23/PCV13 minus PCV9/PCV13) were calculated using the noninferiority procedure [25], using the standardized test statistic and gamma = 0.000001. Within each vaccine group and for each antibody concentration or mcOPA titer, geometric mean concentrations (GMCs) or titers (GMTs), respectively, were calculated. Each concentration and titer was logarithmically transformed for the analysis; 2-sided 95% CIs were constructed by back transformation of the CI for the mean of the logarithmically transformed assay results computed using the Student t distribution. To assess differences between the vaccine groups, 2-sided 95% CIs for the ratio of the GMCs and the GMTs were constructed using the Student t distribution for the mean difference of the measures on the logarithmic scale (PPV23/PCV13 minus PCV9/PCV13). For the avidity assays, descriptive statistics by visit, including geometric means were provided, and 2-sided 95% CIs were calculated.

A total of 89 subjects were enrolled in the present study; 39 in the PCV9/PCV13 group and 50 in the PPV23/PCV13 group (Fig. 1). Two subjects in the PCV9/PCV13 group were excluded from the immunogenicity analysis; 1 failed to return, the other was not eligible. The 2 groups were similar with respect to demographic characteristics. The mean age at PCV13 vaccination for all subjects was 7.6 ± 0.2 years. The numbers of subjects who received 2 versus 3 doses of PCV9 in the original study are 15 and 25, respectively, in the PCV9/PCV13 group, and 27 and 22, respectively, in the PPV23/PCV13 group.

3.2. Pneumococcal immunoglobulin G antibody response Prior to PCV13 vaccination, groups differed in pneumococcal serotype-specific IgG concentrations (Supplemental Table 1. IgG GMCs and 95% CIs). For 10 of the 13 serotypes, the IgG GMC ratio (PPV23/PCV13:PCV9/PCV13) was <1.0, i.e. favoring PCV9/PCV13 (Fig. 2A, Supplemental Table 1). The upper limits of the 95% CIs were <1.0 for 3 serotypes (6B, 9V, and 23F). A single dose of PCV13 was immunogenic in both groups, with >97% of vaccinees achieving pneumococcal IgG concentrations ≥0.35 ␮g/ml [18] for all serotypes 4 weeks after PCV13 vaccination (see Supplemental Fig. S1: Reverse cumulative distribution curves of IgG response). Differences between groups were observed as early as 1 week after vaccination. The PCV9/PCV13 group generally had higher IgG responses than the PPV23/PCV13 group at 1 week and 4 weeks (Supplemental Table 1). Upper 95% CI limits of the PPV23/PCV13:PCV9/PCV13 IgG GMC ratios were <1.0 for 7 serotypes (1, 4, 5, 9V, 14, 18C, and 23F) at 1 week (Fig. 2B), and 6 serotypes (1, 4, 5, 9V, 18C, and 23F) at 4 weeks (Fig. 2C). Notably, for 3 serotypes (1, 4, and 5) in both PCV9-MnCC and PPV23, IgG concentrations were higher at both 1 week (Fig. 2B, Supplemental Table 1) and 4 weeks postvaccination (Fig. 2C; see Supplemental Table 1 and Fig. S1) in the PCV9/PCV13 group than the PPV23/PCV13 group, indicative of a more rapid rise in antibody concentrations in the PCV9/PCV13 group. In contrast, for serotypes not present in PCV9MnCC (serotypes 3, 6A, 7F, and 19A), no marked differences were observed between groups in IgG GMCs (Fig. 2B and C, Supplemental Table 1) or the proportion of IgG responders (Supplemental Fig. S1). A post hoc analysis of the IgG GMCs according to two versus three primary doses within each group showed that at 7 years of age, recall IgG responses at 4 weeks after vaccination with PCV13 were more robust in children who were primed as infants with 3 doses in the PCV9/PCV13 group, but no difference was observed in

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Fig. 2. IgG GMCs and ratios immediately before (A), and 1 week (B), and 4 weeks (C) after PCV13 vaccination. Error bars indicate 95% CIs for GMCs. *Upper limits of the 95% CIs PPV23/PCV13:PCV9/PCV13 IgG GMC ratios were <1.0. † Serotypes 3, 6A, 7F, and 19A are included in PCV13, but not PCV9. CI, confidence interval; GMC, geometric mean concentration; IgG, immunoglobulin G; PCV9, 9-valent pneumococcal conjugate vaccine; PCV13, 13-valent pneumococcal conjugate vaccine; PPV23, 23-valent pneumococcal polysaccharide vaccine.

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the PCV9/PPV23 group (Supplemental Table 2. IgG GMCs and 95% CIs according to two versus three PCV9 doses) [26]. 3.3. Opsonophagocytic activity At baseline, mcOPA GMTs were lower for 8 serotypes in the PPV23/PCV13 group than the PCV9/PCV13 group, consistent with IgG GMCs; the upper limits of the 95% CIs for the ratio of mcOPA GMTs between the groups were <1.0 for serotypes 9V and 23F (Table 1). The increase in OPA GMTs had already reached a maximum by 1 week after PCV13 vaccination in both groups for most of the PCV9 serotypes, and increased somewhat further for some serotypes but decreased for others (most notably for serotype 4); however, the variation in mcOPA was very large for all serotypes. For the additional serotypes not in PCV9, OPA GMTs were significantly increased over baseline in both groups by 1 week postvaccination, and continued to increase by 4 weeks postvaccination; however, the absolute OPA GMT response was lower for serotype 3 than for the other serotypes not in PCV9, although the increase was still significant in both groups. Groups differed in mcOPA response as early as 1 week after vaccination (Table 1). At 1 week the upper limits of the 95% CIs for the GMT ratios (PPV23/PCV13:PCV9/PCV13) were <1.0 for 3 serotypes (1, 5, and 23F). At 4 weeks the upper limits of the 95% CIs for the GMT ratios (PPV23/PCV13:PCV9/PCV13) were <1.0 for 4 serotypes (1, 5, 18C, and 23F). Notably, three out of four serotypes present in PCV13 but not PCV9 had ratios >1.0. Changes in mcOPA titers over time and mcOPA titers at 4 weeks postvaccination were similar between the two groups except for serotypes 1 and 5, which were markedly lower in the PPV23/PCV13 group (Table 1; see Supplemental Fig. S2: Reverse cumulative distribution curves for mcOPA response); however, in both groups >97% of vaccinees achieved mcOPA titers of ≥1:8 for all serotypes 4 weeks after PCV13 vaccination. Post hoc analysis of mcOPA responses according to two versus three primary doses within each group showed that at 7 years of age, mcOPA responses at 4 weeks after vaccination with PCV13 were more robust in children who were primed as infants with 3 doses in the PCV9/PCV13 group, but no difference was observed in the PCV9/PPV23 group (Supplemental Table 3. mcOPA GMTs and 95% CIs, according to two versus three PCV9 doses) [26]. 3.4. Antibody avidity The PCV9/PCV13 group had higher geometric mean AIs than the PPV23/PCV13 group at baseline and at weeks 1 and 4 after PCV13 vaccination for all five serotypes measured (Fig. 3). The upper limits of the 95% CI for the PPV23/PCV13:PCV9/PCV13 ratios were <1.0 for serotypes 1, 5, 6B, and 23F at all time-points. 3.5. Safety An acceptable safety profile was demonstrated with this childhood dose of PCV13 in previously-vaccinated children. The incidence of both local and systemic reactions was comparable in both groups. The most common local reaction was tenderness, reported by most subjects in each group. Fever was reported in 2.0% and 2.8% of subjects in the PPV23/PCV13 and PCV9/PCV13 groups, respectively; all were mild (≥38 ◦ C but ≤39 ◦ C). Most AEs were consistent with childhood illnesses considered common in this age group and were mild in severity, and no SAEs were reported. 4. Discussion A single dose of PCV13 was safe and immunogenic in children approximately 7.5 years of age who received primary vaccination with PCV9-MnCC in infancy, regardless of whether they received

Fig. 3. Geometric mean avidity indices and ratios at baseline (A), 1 week (B), and 4 weeks (C) after PCV13 vaccination. Error bars indicate 95% CIs for geometric means. *Upper limits of the 95% CIs of the PPV23/PCV13:PCV9/PCV13 GMRs were <1.0. CI, confidence interval; GMR, geometric mean ratio; PCV9, 9-valent pneumococcal conjugate vaccine; PCV13, 13-valent pneumococcal conjugate vaccine; PPV23, 23-valent pneumococcal polysaccharide vaccine

PCV9-MnCC or PPV23 as a toddler dose. Prior to the PCV13 vaccination at 7 years of age, IgG GMCs were lower in the PPV23/PCV13 group for 10 out of 13 serotypes, but the upper limit of the 95% CI of the ratio PPV23/PCV:PCV9/PCV13 was <1 only for three serotypes (6B, 9V and 23F), which are all in PCV9. We found significant differences in the immune response profiles between children who received PPV23 following priming with the conjugate vaccine and those who only received conjugate vaccine, which were manifested in lower immune responses among those who had received PPV23, both in the quantity and quality of pneumococcal antibodies elicited even many years after the last vaccination. An effective immune memory was reflected in an increase in mcOPA GMTs. These had already reached a maximum by 1 week after PCV13 vaccination in both groups for most of the PCV9 serotypes, with further increases by 4 weeks for some serotypes, and decreases for others

422 Table 1 mcOPA GMTs (95% CIs) to the PCV13 serotypes before, 7, and 28 days after PCV13 vaccination of children 7 years of age who received PCV9 in infancy and either PPV23 (PPV23/PCV13 group) or PCV9 (PCV9/PCV13 group) at 12 months of age. Serotype

Prior to PCV13 vaccination

Serotypes in PCV9f 8 (5.6–10.9) 1 4 18 (7.7–43.8) 5 4 (3.6–5.4) 6B 1646 (901.7–3003.6) 46 (19.1–108.8) 9V 14 950 (572.0–1576.6) 18C 33 (13.3–82.1) 55 (27.6–111.5) 19F 28 (12.5–62.1) 23F Additional serotypes not in PCV9 3 21 (13.2–34.8) 6A 22 (10.2–47.4) 7F 114 (42.4–305.4) 19A 42 (21.3–81.0)

1 week after PCV13 vaccination PCV9/PCV13 GMTa (95% CIb ) (nc = 26–37)

5 (4.1–6.6) 31 (9.5–99.9) 4 (NE–NE) 2838 (2166.4–3717.5) 254 (109.1–592.4) 619 (302.7–1264.6) 102 (36.4–288.2) 71 (30.8–161.8) 102 (38.8–269.3)

19 (11.0–31.2) 43 (18.7–100.5) 99 (33.9–289.8) 60 (29.9–120.1)

4 weeks after PCV13 vaccination

Ratiod (95% CIe )

PPV23/PCV13 GMTa (95% CIb ) (nc = 38–42)

PCV9/PCV13 GMTa (95% CIb ) (nc = 26–27)

Ratiod (95% CIe )

1.5 (0.98–2.31) 0.6 (0.15–2.43) 1.1 (0.87–1.40) 0.6 (0.28–1.20) 0.2 (0.05–0.61)g 1.5 (0.67–3.54) 0.3(0.08–1.26) 0.8 (0.27–2.29) 0.3 (0.08–0.93)g

332 (250.9–438.4) 4377 (2686.8–7129.2) 419 (255.2–688.5) 8433 (5324.8–13,356.3) 2203 (1455.3–3334.1) 2850 (2110.7–3848.2) 3820 (2567.4–5683.8) 1148 (847.8–1553.3) 1233 (818.3–1858.6)

1085 (607.8–1937.8) 9352 (4304.2–20,319.2) 1006 (589.2–1716.9) 10,097 (6754.5–15,093.6) 2497 (1396.8–4464.4) 2980 (2021.8–4391.1) 6091 (2731.4–13,581.7) 887 (499.2–1575.3) 2134 (1601.1–2844.0)

0.3 (0.17–0.54)g 0.5 (0.20–1.10) 0.4 (0.20–0.87)g 0.8 (0.44–1.59) 0.9 (0.45–1.74) 1.0 (0.59–1.54) 0.6 (0.28–1.40) 1.3 (0.72–2.32) 0.6 (0.34–0.99)g

1.2 (0.57–2.36) 0.5 (0.16–1.58) 1.1 (0.27–4.86) 0.7 (0.27–1.80)

175 (127.8–240.9) 7640 (5237.8–11,142.5) 4982 (4202.7–5905.3) 1149 (849.6–1555.2)

194 (154.5–243.6) 4163 (1928.4–8988.3) 4120 (2218.1–7654.2) 1249 (810.3–1925.9)

0.9 (0.59–1.40) 1.8 (0.86–3.92) 1.2 (0.71–2.05) 0.9 (0.56–1.52)

PCV9/PCV13 GMTa (95% CIb ) (nc = 34–37)

Ratiod (95% CIe )

217 (162.6–290.5) 2374 (1656.3–3404.0) 264 (178.5–389.9) 11,156 (9073.5–13,715.2) 1651 (1067.8–2553.3) 3041 (2311.4–4001.1) 3230 (2616.1–3987.1) 1266 (1013.9–1582.0) 1678 (1212.3–2322.1)

1087 (717.5–1646.3) 3765 (2339.1–6059.8) 719 (500.9–1032.4) 11,477 (8418.5–15,646.8) 1713 (1087.0–2700.0) 3048 (2318.3–4006.9) 5684 (3122.2–10,349.6) 1198 (842.3–1703.9) 2714 (1970.1–3739.5)

0.2 (0.12–0.32)g 0.6 (0.35–1.12) 0.4 (0.21–0.63)g 1.0 (0.68–1.38) 1.0 (0.51–1.81) 1.0 (0.67–1.48) 0.6 (0.33–0.98)g 1.1 (0.72–1.56) 0.6 (0.39–0.99)g

153 (117.2–200.2) 7060 (5352.2–9312.6) 5835 (4160.0–8184.1) 1256 (990.9–1591.9)

188 (146.6–240.8) 5034 (3336.6–7594.4) 7887 (6447.9–9647.0) 1556 (1108.8–2182.2)

0.8 (0.56–1.18) 1.4 (0.88–2.25) 0.7 (0.48–1.14) 0.8 (0.54–1.20)

PPV23/PCV13 GMTa (95% CIb ) (nc = 49–50)

CI, confidence interval; GMT, geometric mean titer; mcOPA, microcolony opsonophagocytic activity; NE, not evaluated; PCV7, 7-valent pneumococcal conjugate vaccine; PCV9, 9-valent pneumococcal conjugate vaccine; PCV13, 13-valent pneumococcal conjugate vaccine; PPV23, 23-valent pneumococcal polysaccharide vaccine. a GMTs were calculated using all subjects with available data for the specified blood draw. b CIs are back transformations of a CI based on the Student t distribution for the mean logarithm of the titers. c n = number of subjects with a determinate antibody titer for the specified serotypes. d Ratio of GMTs: PPV23/PCV13:PCV9/PCV13 reference. e CIs for the ratio are back transformations of a CI based on the Student t distribution for the mean difference of the logarithms of the measures (PPV23/PCV13 minus PCV9/PCV13 reference). f PCV9 contains serotypes 1 and 5 in addition to the PCV7 serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F). g Upper limit of 95% CI for the ratio of GMTs is <1.0.

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PPV23/PCV13 GMTa (95% CIb ) (nc = 39–50)

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(most notably for serotype 4), with considerable variation in mcOPA for all serotypes. Natural induction of memory, as demonstrated by significant increases over baseline by week 1, had also occurred for the additional serotypes not in PCV9, which are prevalent in Iceland; of note, although the mcOPA response was much lower for the low-prevalence serotype 3, it was still significantly increased above baseline in both groups. The effect of PPV23 vaccination was also reflected in reduced avidity at baseline and less increase after PCV13 vaccination in the PPV23/PCV13 group for all serotypes measured, except 19F, but was not necessarily reflected in lower mcOPA responses. This is in agreement with our previous results, showing that antibody avidity did not limit the protective capacity against 6A and 6B pneumococci in vitro and in vivo, when antibody levels were high [23]. These findings suggest that PPV23 vaccination of toddlers may impair antibody persistence and reduce subsequent immune responses to pneumococcal polysaccharides and polysaccharide-based vaccines. In the original study IgG GMCs were lower after vaccination with a PCV9 booster dose at 12 months in subjects primed with 2 doses compared with those primed with 3 doses, although this difference was only significant for serotype 18C [15]. After vaccination with PCV13 at 7 years of age, IgG GMC and mcOPA recall responses were more robust in infants who were primed with 3 doses in the PCV9/PCV13 group, but not in the PCV9/PPV23 group. Thus, not only the type of toddler booster but also the number of infant PCV doses may affect immunological memory responses to PCV13. Several studies have shown that PPV23 elicits increases in serotype-specific antibodies following priming by the conjugate vaccine [12–15]. However, little information exists regarding the nature of the immune response to a further PCV challenge after PPV23 administration or whether this response might differ from that of children naïve to PPV23 [12]. Although results from a previous study in Icelandic infants suggested that an 8-valent PCV priming/PPV23 booster in infancy did not significantly reduce the immune response to a fractional PPV23 dose (1/10th) at 7 years of age [27], another study in Fijian infants primed with PCV7 in early infancy showed that the response to a fractional PPV23 dose at 17 months was significantly reduced in those previously exposed to PPV23 serotypes (each p < 0.001), suggesting that the ultimate outcome of PCV followed by PPV23 could be a reduced response to subsequent PCV administration [14]. These results are in agreement with the MCPS-induced hyporesponsiveness to subsequent MCPS re-challenge in young Gambian children [9,10]. In children who may have reduced immune responsiveness to pneumococcal polysaccharides, either through exposure to circulating S. pneumoniae or active immunization with subsequent doses of PPV as is now recommended following licensure of PCV13 [11], a vaccine schedule including a PPV booster could have important clinical implications. A Dutch study of children 1–7 years of age who had experienced recurrent acute otitis media (AOM) and were vaccinated with PCV7 followed by PPV23, showed lack of efficacy against subsequent AOM, although PCV7 serotype antibodies were increased after PPV23 [28]. As neither avidity nor OPA was reported and there was no age-matched control group without AOM prior to vaccination, only limited conclusions could be drawn; however, these findings may suggest that the functional capacity of the antibodies may have been compromised by PPV23 exposure. Three more recent studies suggest that natural exposure to pneumococcal polysaccharides in infancy may induce serotype-specific hyporesponsiveness to a subsequent immune challenge, as demonstrated by decreased IgG responses to the PCV serotypes that were cultured from the infant’s nasopharynx before or at the time of PCV vaccination [29–31]. Furthermore, studies in other high-risk groups such as the elderly also indicate that PPV23 causes hyporesponsiveness to subsequent PCV vaccination [32–34]. In a study in adults >65 years of age, PCV7 induced significant increases in

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serotype-specific memory B cells, while their frequency was reduced by PPV23. Furthermore, subsequent PCV7 immunization following PPV23 resulted in attenuation of memory B-cell responses and reduction of B1b cells, indicating that PPV23induced depletion of memory and B1b-cell subsets may provide a basis for antibody hyporesponsiveness [35]. In mice primed with PCV as neonates, PPS booster given to infant mice compromises antibody responses, and induces hyporesponsiveness to further PPS administrations, which persists to adulthood [36]. The PPS booster also significantly reduces the frequency of PPS-specific antibody-secreting cells in both the spleen and bone marrow, explaining the reduced PPS antibody [37]. In MnCC-primed neonatal mice, MCPS booster significantly reduced the frequency of newly activated meningococcus Cpolysaccharide-specific B cells, and mainly switched IgG+ memory cells by driving them into apoptosis, demonstrating that apoptosis is at least one major cause of polysaccharide-induced hyporesponsiveness [38].

5. Conclusions A single dose of PCV13 had an acceptable safety profile and was immunogenic in children approximately 7.5 years of age, regardless of previous vaccination with a PCV9-MnCC – only regimen, or PCV9-MnCC followed by PPV23. This was shown by increases in both pneumococcal polysaccharide-specific IgG antibody levels and functional activity measured by OPA for all 13 serotypes. PCV13 vaccination also resulted in increased antibody avidity in both groups. There were, however, differences in the immune response to subsequent immunization with PCV13 between subjects who had previously received PPV23 and those who had previously received only PCV9-MnCC, with evidence of a reduced immune response to PCV13 in subjects previously receiving PPV23, both in serum IgG polysaccharide antibody concentrations and functional antibody responses (OPA and antibody avidity), which were apparent even prior to PCV13 vaccination. The number of subjects in this study was small, reduced responses were not seen for all serotypes, and the degree of these reductions varied among immune parameters and serotypes. However, the totality of the data supports a conclusion that giving PPV23 after priming with conjugate vaccine has a detrimental impact on the ability to respond to subsequent vaccination with PCV13. The clinical relevance of these differences, especially for children at increased risk of pneumococcal disease, remains to be explored.

Acknowledgments We acknowledge the work of our study nurses and secretary at the Center for Child Health Services, The Primary Health Care of the Reykjavik Capital Area, Iceland; and Siggeir F. Brynjolfsson, Sindri F. Eidsson, and Stefania P. Bjarnarson at the Department of Immunology for handling all blood samples and measuring avidity. We thank all the parents and children who participated in the study. We also thank Dr Peter C. Giardina of Pfizer Inc for his support of laboratory assays and review of the manuscript, and Dr William C. Gruber of Pfizer Inc, for his contributions to data interpretation and thoughtful review of the manuscript. Editorial support was provided by Vicki Schwartz, PhD, at Excerpta Medica and was funded by Pfizer Inc. Financial support: This study was sponsored by Wyeth, which was acquired by Pfizer Inc in October 2009. The funding source contributed to the study design; to the collection, analysis, and interpretation of data; and to medical writing support.

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