Total and serotype-specific pneumococcal antibody titres in children with normal and abnormal humoral immunity

Vaccine 24 (2006) 5637–5644

Total and serotype-specific pneumococcal antibody titres in children with normal and abnormal humoral immunity Sharif Uddin a , Ray Borrow b , Mansel R. Haeney c , Andrew Moran c , Rosalind Warrington b , Paul Balmer b , Peter D. Arkwright a,∗ b

a University of Manchester, Booth Hall Children’s Hospital, Charlestown Rd., Manchester, M9 7AA, United Kingdom Health Protection Agency, Clinical Sciences Building II, Manchester Royal Infirmary, Oxford Rd., Manchester, M13 9WL, United Kingdom c Department of Immunology, Hope Hospital, Stott Lane, Manchester, M6 8HD, United Kingdom

Received 18 January 2006; received in revised form 24 March 2006; accepted 28 March 2006 Available online 18 April 2006

Abstract A heptavalent pneumococcal conjugate vaccine (PCV-7) protects children against invasive pneumococcal disease. The aim of this study was to evaluate immunoglobulin subclass and serotype-specific pneumococcal antibody responses to vaccination in children with a history of recurrent or severe bacterial infections. Pneumococcal IgG, IgG1, IgG2 titres were assayed by ELISA, and nine serotype concentrations measured using a nonaplex bead assay in 145 children investigated for recurrent or severe infections. Children mounted an exclusively IgG1 response after vaccination with two doses of PCV-7 and a dose of 23 valent pneumococcal polysaccharide vaccine (PPV-23), with pneumococcal IgG2 antibody titres remaining low to negligible. Measurement of serotype-specific responses demonstrated that although PCV-7 specific serotype responses increased significantly post-vaccination, specific IgG against two of the serotypes not covered by PCV-7 but only by PPV-23 remained low. We conclude that in contrast to antibody response to natural infection with Pneumococcus or pneumococcal polysaccharide vaccines which are often of a IgG2 subclass, responses in children after PCV-7 are of IgG1 subclass. Serotype-specific IgG were useful in determining the protection against specific pneumococcal strains, and showed that the PPV-23 did not broaden protection against non-PCV-7 serotypes. © 2006 Elsevier Ltd. All rights reserved. Keywords: Pneumococcal antibodies; Serotypes; Vaccine; Children

1. Introduction Pneumonia, septicaemia and meningitis secondary to Streptococcus pneumoniae (Pneumococcus) are major causes of morbidity and mortality particularly in young infants, the elderly, and patients with primary immunodeficiency diseases who are unable to mount or maintain an optimal immune response to this bacterium [1–3]. Patients who have serious or recurrent infections with Pneumococcus and other encapsulated organisms should be investigated for their ability to mount effective immune response to these pathogens ∗

Corresponding author. Tel.: +44 161 918 5535; fax: +44 161 224 1013. E-mail address: peter [email protected] (P.D. Arkwright).

0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.03.088

so that underlying primary immunodeficiencies can be diagnosed and treated. A protective immune response to Pneumococcus depends on the production of serotype-specific antibodies which mediate opsonin-dependent phagocytosis [4–6]. About 10 of more than 90 capsular polysaccharide serotypes currently account for the majority of invasive disease [7]. In adults and older children, serotype-specific antibodies after natural exposure to Pneumococcus or vaccination with the pneumococcal polysacscharide vaccine are almost exclusively of the IgG2 subclass, and thus immunity to this bacterium is generally thought of as being IgG2 subclass dependent [8,9]. In contrast antibodies against the protein antigens such as tetanus toxoid are largely of an IgG1 subclass [8,9].

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With the recent licensing and routine use of a heptavalent pneumococcal conjugate vaccine (PCV-7) in the United States from 2000, closely followed by other parts of the world including Europe, it is now important to reassess how pneumococcal antibody responses should be assayed. Measurement of IgG1 rather than IgG2 specific responses may be more appropriate in assessing the humoral response to this conjugate vaccine. Furthermore as PCV-7 only covers seven of the more than 90 pneumococcal serotypes, measurement of responses to these serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) rather than the total IgG pneumococcal titres may be more useful [10]. Serotypes covered by the PCV-7 are presently responsible for 85% of paediatric invasive pneumococcal disease. The vaccine has already reduced the burden of the disease in children in the United States where it is now part of the scheduled immunisation programme for infants [11]. Although in the past the United Kingdom has just recommended the PCV-7 in high risk groups, from April 2006 the United Kingdom will also introduce this vaccine into its routine immunisation schedule at 2, 4 and 13 months. PCV-7 is more immunogenic than the 23-valent pneumococcal polysaccharide vaccine (PPV-23), which is not licensed in young children and infants where the burden of disease is highest because it does not stimulate effective long-lasting immunity at this age [4]. The aim of this study is to compare total pneumococcal IgG antibody, IgG subclass antibody titres and serotypespecific antibody concentrations in children who had received two doses of PCV-7 followed by a dose of PPV-23 in order to determine which assays are most useful in clinical practice. All children had presented with a history of recurrent infections. Pneumococcal antibody responses of a group of children who were subsequently identified as having diseases associated with defective antibody immunity were compared with the rest of the cohort.

2. Materials and methods 2.1. Patients One hundred and forty-five consecutive children 16 years old or less, investigated at the regional paediatric immunology unit for the North West of England for recurrent or severe infections and who had paired classical (IgG, IgG1 and IgG2 titres) and nine serotype-specific (serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F) pneumococcal antibody assays performed on pre- or post-vaccination sera were included in the study. All children were seen between August 2002 and August 2005. Pneumococcal antibody levels were available on 70 (48%) children prior to any pneumococcal vaccination and on 107 (74%) after vaccination, which in all cases had involved giving two doses of the PCV-7 (Prevenar, Wyeth Pharmaceuticals, Herts, United Kingdom) and then one dose of PPV-23 (Pneumovax II, Pasteur Merieux MSD Ltd. Herts,

United Kingdom) at monthly intervals, in line with current recommendations for children over 2 years [12,13]. Eight patients had been excluded from the study (three of whom had only had one dose of PCV-7 and five who had had a dose of PPV-23 but no PCV-7). Paired pre- and post-vaccination samples were available in 37 (26%) of the children. The research project was granted ethical approval by the local research ethics committee and consent was obtained from parents of all patients. Pneumococcal antibody levels were measured using the following assays to determine the response to these immunisations. 2.2. Anti-pneumococcal polysaccharide IgG specific antibody ELISA The method used to measure IgG antibody levels against the 23 serotypes present in PPV-23 was modified from that employed by Hazlewood et al. [14]. Serum samples were pre-absorbed for 1 h at 37 ◦ C with 0.25 mg/ml of cell wall polysaccharide (CWP) (Statens Serum Institut, Copenhagen, Denmark). Microtitre plates were coated overnight at 4 ◦ C with 100 ␮l/well of antigen (the 23-valent pneumococcal vaccine, Pneumovax II (Pasteur Merieux MSD Ltd. Herts, United Kingdom) diluted 1:100 in carbonate buffer pH 9.2. The wells were then sequentially incubated with test and control sera double diluted from 1:10 to 1:1280, murine monoclonal antibody to either human IgG (clone R10), IgG1 (clone NL16) or IgG2 (50:50 mixture of clones GOM1 and HP6014 diluted 1:500) (Recognition Sciences Ltd., Birmingham, United Kingdom) and goat anti-mouse immunoglobulin conjugated to alkaline phosphatase diluted 1:500 for the IgG plates and 1:250 for the IgG1 and IgG2 plates (Dako Ltd., Glostrup, Denmark). Assays was developed with 100 ␮l/well of paranitrophenol phosphate substrate (Sigma Aldrich Co. Ltd., Dorset, United Kingdom) in diethanolamine buffer pH 9.8 (1 mg/ml), and absorbance read at 450 nm. Results were reported as end point titres, determined by the highest dilution giving an optical density of ≥0.2. No national or international calibrant material is available for the determination of pneumococcal specific antibody levels, therefore reference values were generated ‘in house’ by testing control sera from healthy adults (N = 42). Three separate antibody titres in arbitrary units were reported per serum tested, the putative protective levels being shown in brackets; total IgG (≥640), IgG1 (≥10), and IgG2 (≥40). These protective levels were suggested by Hazlewood et al. [14], and antibody levels below these thresholds are considered to provide inadequate protection against pneumococcal infection. Quality of ELISAs was standardised by including high and low control sera of known levels which had been pre-tested in at least three separate assays. The assay is an established part of the clinical immunology laboratory repertoire in approximately 45 laboratories in the UK and is subject to validation by the national external quality assessment scheme (UK-NEQAS, Sheffield, UK).

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2.3. Nonaplex fluorescent bead assay for the quantitative detection of serum IgG antibodies to S. pneumoniae capsular polysaccharides This was a two-step procedure as described by Lal et al. [15]. In the first part, pneumococcal polysaccharide antigens were conjugated to microbeads. Firstly, the polysaccharide is coupled to poly-l-lysine when this complex was attached to the microspheres by a two-step carbodiimide reaction using EDC with sulpho-NHS, cyanuric chloride and poly-l-lysine and purified down a Sephadex column. The assays were performed in micro-wells, using 25 ␮l of the standards and samples. The patient’s samples were diluted at 1 in 100 thus requiring only 5 ␮l of sera. This was, followed by the addition of 5000 beads/region/25 ␮l, and then 100 ␮l of 1:200 dilution of R-phycoerythrin conjugated anti-human IgG in PBS (binding all IgG subclasses) with 20 min incubations (Jackson ImmunoResearch Lab, West Grove, USA). Assays were done in duplicate and read using a Bioplex protein array system (Bio-Rad, Herts, United Kingdom). Results were reported as individual IgG antibody concentrations (␮g/ml) to each of the nine pneumococcal serotypes in the assay. An antibody level ≥0.2 ␮g/ml has been suggested as providing adequate protection against pneumococcal infection in infants [16]. 2.4. Measurement of antibody concentrations to tetanus toxoid and Haemophilus influenzae type b Assessment of specific antibody responses to tetanus toxoid (Biomerieux, Basingstoke, United Kingdom) and H. influenzae type b (Hib) (The Binding Site Ltd., Birmingham, United Kingdom) were assayed using a commercial ELISA kit as previously described [14]. 2.5. Statistical analysis Data were analysed using SPSS 12.0 software package (SPSS Inc., Chicago, USA). Additional clinical information was collected from individual patients’ medical records and from the Pharmacy that provided the Prevenar and Pneumovax vaccines. As much of the data was found to be not normally distributed non-parametric statistics are used throughout. Medians (inter-quartile ranges) are quoted for continuous variables. The Mann–Whitney U-test is used to check for statistical differences between these groups of data. Chi-square statistic is used for discrete data. Because of the number of comparisons, differences between groups were documented as being significant if the P-value < 0.01. 3. Results 3.1. Demographic and clinical characteristics of study cohort (Tables 1 and 2) One hundred and forty-five children aged 2–16 years of whom 87 (60%) were male were included in the study. One

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Table 1 Underlying diagnoses of 12 children classified as having diseases associated with abnormal antibody immunity Disease

Number

Cell-cycle/DNA repair defect Ataxia telangiectasia Nijmegen-breakage syndrome

3 2 1

Dysgammaglobulinemia Evolving common variable immunodeficiency (CVID) B-cell switching disorder Specific antibody deficiency Familial splenic dysfunction syndrome

9 2 3 3 1

hundred and seven (74%) presented with recurrent respiratory tract infections and 25 (17%) with more deep-seated bacterial infections (septicaemia, meningitis, septic arthritis). Twelve (8%) were found to have clinical conditions associated with significant defects in humoral immunity and were classified as having abnormal “antibody” immunity [17,18]. Three children had cell-cycle/DNA repair enzyme defects with waning cellular and humoral immunity and another nine had primary immunodeficiency diseases associated with defective B-lymphocyte function (Table 1). Four (33%) of these 12 children were on prophylactic antibiotics and 8 (67%) required immunoglobulin replacement therapy, which commenced after immunology tests had been taken. The remaining 133 children had normal serum immunoglobulins and normal responses to Hib and pneumococcal vaccines and were classified has having normal antibody immunity. Three of these patients had primary complement deficiencies and two had myeloid dysplasia. Children with abnormal humoral immunity had the same median age as the normal group. They had a 9-fold lower Hib antibody concentrations than those with normal antibody immunity. Tetanus concentrations did not vary significantly between the two groups (Table 2). 3.2. Pneumococcal total and subclass antibody titres before and after vaccination Pneumococcal IgG, IgG1 and IgG2 antibody titres before or after vaccination with two doses of PCV-7 and a dose of PPV-23 at monthly intervals were studied in the two groups of children. 3.2.1. IgG1 titres Children with normal antibody immunity had significantly higher total pneumococcal IgG1 subclass titres post-vaccination (Fig. 1, top panels). The median (inter-quartile range) IgG1 titre increased 4-fold from 40 (10–80) pre-vaccination to 160 (80–640) 1–8 months postvaccination (P < 0.001) (Fig. 2). The IgG1 titre remained significantly elevated (160 (80–320)) for up to the 3-year period the PCV-7 has been available for patients in the study (P < 0.001).

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Table 2 Age, gender, specific antibodies to Haemophilus influenzae type b (Hib) and tetanus toxoid, and treatment of children with normal antibody immunity and diseases associated with abnormal antibody immunity Normal antibody immunity

Abnormal antibody immunity

P-valuea

Number Age (years) Gender (males) Specific antibodies to Hib (␮g/ml) Specific antibodies to tetanus (IU/L)

133 6 (3–11) 80 (60%) 5.3 (1.2–9.0) 1.0 (0.3–4.5)

12 6 (2–13) 5 (42%) 0.6 (0.5–3.2) 1.1 (0.1–3.8)

0.8 0.2 0.04 0.6

Outcome

No medication 113 (85%) Antibiotics 20 (15%) Immunoglobulin 0

No medication 0 Antibiotics 4 (33%) Immunoglobulin 8 (67%)

0.001

Number (percentage); or median (inter-quartile range). a Chi-square test for discrete variables; Mann–Whitney U-test for continuous variables.

Fig. 1. Box-plot showing pneumococcal antibody levels pre-vaccination (pre), 1–8 months post-vaccination (1–8 m post) and >8 months post-vaccination (>8 m post) for children with normal (hatched boxes) and abnormal antibody immunity (unfilled boxes). Vaccination in all cases was with two doses of PCV-7 one month apart followed by a dose of PPV-23 a month later. Median (thick bar) and quartiles (box).

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Fig. 2. Range of pneumococcal titres (IgG, IgG1 and IgG2) in children with normal antibody immunity before (pre) and after (post) vaccination with two doses of PCV-7 and a dose of PPV-23. Y-axis is the percentage of the total cohort with that titre.

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Table 3 Percentage of children with normal and abnormal antibody immunity who had protective levels of pneumococcal antibodies before and after vaccination with two dose of PCV-7 and a dose of PPV-23 Pre-vaccine

1–8 months post-vaccine

Normal antibody immunity n = 65

Normal antibody immunity n = 75

Pneumococcal IgG 640 titre cut-off

Abnormal antibody immunity n = 12

26

47

0

Pneumococcal IgG1 10 titre cut-off 80 titre cut-off

90 38

97 85

67 0

Pneumococcal IgG2 40 titre cut-off

23

16

0

Serotype 1 Serotype 5

29 49

51 64

8 25

Serotype 4 Serotype 6B Serotype 18C Serotype 23F Serotype 9V Serotype 14 Serotype 19F

30 32 37 46 50 60 81

95 89 95 95 93 96 99

58 33 42 42 58 92 75

3.2.2. IgG2 titres In contrast to the IgG1 titres, IgG2 titres were low (10 (0–20)) pre-vaccination and did not increase post-vaccination (10 (0–20)). Children with abnormal antibody immunity had low levels of total, IgG1 and IgG2 titres pre-vaccination, which did not increase post-vaccination (Fig. 1, top panels). 3.3. Pneumococcal serotype-specific concentrations before and after vaccination Pneumococcal serotype-specific IgG concentrations covered (serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) and not covered (serotypes 1 and 5) by PCV-7 were also studied in these same children. IgG antibody levels of serotypes 1 and 5 were low in all children pre-vaccination and did not increase significantly post-vaccination (less than 2-fold increase). There was a large variation in the proportion of children with serotype concentrations >0.2 ␮g/ml pre-vaccination, ranging from 29% for serotype 1 to 81% for serotype 19F (Table 3). Children with normal antibody immunity had a significant increase in all seven serotypes covered by PCV-7 1, to 8 months post-vaccination (4–26-fold increase). Serotype concentrations remained significantly raised for up to 3 years post-vaccination (P < 0.001 for all serotypes) (Fig. 1). Children with abnormal antibody immunity did not show a significant rise in any of the serotypes studied 1–8 months post-vaccination. Pre-vaccination only 22/65 (34%) of children with normal antibody immunity had protective serotype concentrations (>0.2 ␮g/ml) to six or seven of the seven serotypes covered by PCV-7, while 1–8 months post-vaccination 71/75 (95%) of

Fig. 3. Percentage of the seven serotypes covered by PCV-7 which attained protective levels (defined as >0.2 ␮g/ml) before and after pneumococcal vaccination (two doses of PCV-7 and a dose of PPV-23) in children with normal and abnormal antibody immunity (1–8 months post-vaccination only). Bars with increasing shading, range from unshaded (no protective serotypes) to black (all serotypes protective).

children had protective serotypes to six or seven of the seven serotypes (Fig. 3). At >8 months post-vaccination 87% of children tested still had protective serotypes to six or seven of these serotypes. Of the children with impaired antibody immunity, 1–8 months post-vaccination only 2 of 12 (17%) had protective serotype concentrations (>0.2 ␮g/ml) to six or seven of the PCV-7 serotypes. 3.4. Effect of age on children’s response to vaccination In children with normal antibody immunity, age had no significant positive or negative effect on the magnitude of IgG

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and subclass titres, or pneumococcal serotype concentrations post-vaccination.

4. Discussion This study provides novel and clinically useful information as to the relative value of measuring pneumococcal IgG, IgG1 and IgG2 subclass titres and serotype-specific antibody concentrations in the evaluation of children’s immunity to this bacterium. In this study, the IgG1 subclass was the predominant protective antibody subclass produced after vaccination with two doses of PCV-7 and a dose of PPV-23. Only 12 (14%) of 87 children had protective IgG2 titres of ≥40, and specific IgG2 antibodies were of little use in the evaluation of pneumococcal antibody responses. Immune responses to polysaccharides such as pneumococcal capsular antigens are classically thought of as involving T-cell independent pathways [19] and result in production of antibodies of the IgG2 subclass [20], while protein antigens generate a T-cell dependent response with antibodies of predominantly IgG1 subclass [8]. It is important for clinicians to appreciate this difference in order to correctly interpret responses to the newer pneumococcal conjugate, as compared to older pneumococcal polysaccharide vaccines. As well as the type of vaccine, the age of the patient is a further important consideration. Previous studies have shown that although adults are more likely to produce an IgG2 response, infants tend to produce an IgG1 response even to pneumococcal conjugate vaccines [21,22]. Thus, both the type of pneumococcal vaccine and the age of the patient need to be considered in the evaluation of pneumococcal responses. Although IgG1 titres were a good marker of a response to vaccination, our study illustrates the importance of setting optimal limits for protective versus non-protective titres of an antibody assay and making sure they are age-validated. The threshold of in titre of 10 previously set by the laboratory using adult volunteers was obviously inappropriately low for this cohort of children, as even in children with normal immunity who had not been immunised and therefore presumably had limited immunity, only 6% had titres of <10. Examining the distribution of IgG1 titres pre- and post-vaccination a cut-off titre of 80 was a more appropriate threshold for distinguishing children who had been immunised from those who had not. Total IgG titres were less useful, probably because the rise in IgG1 was partly masked by noise from other IgG subclasses. Measurement of serotype responses provided additional information as to children’s natural immunity to Pneumococcus and responses to the pneumococcal vaccines. Interpretation of the nonaplex bead assay is obviously more complex as it requires the analysis of seven to nine separate data points. However, these assays are essential if information on the degree of protection against specific serotypes is required. As expected there was significant variation in the natural

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immunity of children to different serotypes pre-vaccination, which must be related to a combination of exposure to these pneumococcal serotypes as well as the immunogenicity of the different strains. Only 30% of children had protective immunity to serotypes 1, 4 and 6B, while 81% had protective immunity to serotype 19F. Post-vaccination, these assays show that 89–99% of children develop protective concentrations of specific IgG, while serotypes 1 and 5 IgG concentrations which are not covered by PCV-7 remained low. Current recommendations suggest that a dose of PPV-23 should be given as a final booster following the PCV-7 in order to increase the coverage of serotypes the patient is protected against [12,13]. However, we find no objective evidence that the PPV-23 broadens children’s protection to Pneumococcus. This is unlikely to be due to poor immunogenicity of these two serotypes as previous studies in children have found that higher valency pneumococcal conjugate vaccines induce a good response, even after a single dose [23]. Furthermore, there is some evidence from studies with meningococcal vaccines that administration of polysaccharide vaccines may reduce the magnitude and persistence of antibody responses to conjugate vaccines [24]. Further studies are required to investigate these issues with a view to possibly amending current recommendations as to the use of PPS-23 in conjunction with pneumococcal conjugate vaccines. In this cohort of children aged 2–16 years, younger children did not make significantly lower antibody responses than older children, providing additional supportive evidence that vaccination with PCV-7 is associated with the development of protective pneumococcal antibody concentrations across this age range. Furthermore, antibody levels remained elevated up to 3 years post-vaccination with no significant differences in IgG1 titres or serotype concentrations between children having antibody measurements 1–8 months post-vaccination and >8 months post-vaccination. In summary, our study provides a significant advance in our understanding of pneumococcal antibody responses in children with both normal and abnormal humoral immunity. Firstly we show that IgG1 rather than IgG2 antibodies are the predominant subclass of antibody produced in this cohort of children vaccinated with a combination of pneumococcal conjugate and polysaccharide vaccines. The previous notion that pneumococcal immunity is largely IgG2 dependent may need to be modified, as we show that it may vary depending on the method of antigen exposure and the age of the patient. A comparative study of a cohort of adults is planned to look at the age effect in more detail. Secondly, in the post-PCV-7 era, where clinicians are now wondering about changing from assessment of the total pneumococcal titre to measuring serotype-specific responses, although specific-serotype responses are useful in determining a child’s immunity to specific pneumococcal strains and the merits of one vaccine over another, this study suggests that IgG1 titres are a useful simple screening test of which children have normal and abnormal humoral immunity.

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Acknowledgements Conflict of interest statement: There was no external funding for this study. None of the authors declare any conflict of interests.

References [1] Williams BG, Gouws E, Boschi-Pinto C, Bryce J, Dye C. Estimates of world-wide distributions of child deaths from acute respiratory infections. Lancet Infect Dis 2002;2:25–32. [2] Fedson DS, Scott JA. The burden of pneumococcal disease among adults in developed and developing countries: what is and is not known. Vaccine 1999;17(Suppl. 1):S11–8. [3] Picard C, Puel A, Bustamante J, Ku CL, Casanova JL. Primary immunodeficiencies associated with pneumococcal disease. Curr Opin Allergy Clin Immunol 2003;3:451–9. [4] Bogaert D, Hermans PW, Adrian PV, R¨umke HC, de Groot R. Pneumococcal vaccines: an update on current strategies. Vaccine 2004;22:2209–20. [5] Salyers AA, Whitt DD. Streptococcus pneumoniae. In: Bacterial Pathogenesis: A Molecular Approach. 2nd ed. Washington: ASM Press; 2002. p. 279–90. [6] Casal J, Tarrago D. Immunity to Streptococcus pneumoniae: factors affecting production and efficacy. Curr Opin Infect Dis 2003;16:219–24. [7] Hausdorff WP, Feikin DR, Klugman KP. Epidemiological differences among pneumococcal serotypes. Lancet Infect Dis 2005;5:83–93. [8] Barrett DJ, Ayoub EM. IgG2 subclass restriction of antibody to pneumococcal polysaccharides. Clin Exp Immunol 1986;63:127–34. [9] Schauer U, Stemberg F, Rieger CH, Buttner W, Borte M, Schubert S, et al. Levels of antibodies specific to tetanus toxoid, Haemophilus influenzae type b, and pneumococcal capsular polysaccharide in healthy children and adults. Clin Diagn Lab Immunol 2003;10:202–7. [10] Borrow R, Balmer P. Assessment of functional antibody responses. Method Mol Med 2003;87:289–300. [11] Black S, Shinefield H, Baxter R, Austrian R, Bracken L, Hansen J, et al. Postlicensure surveillance for pneumococcal invasive disease after use of heptavalent pneumococcal conjugate vaccine in Northern California Kaiser Permanente. Pediatr Infect Dis J 2004;23:485–9. [12] Advisory Committee on Immunization Practices. Preventing pneumococcal disease among infants and young children. Recommenda-

[13]

[14]

[15]

[16]

[17]

[18] [19] [20]

[21]

[22]

[23]

[24]

tions of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2000;49:1–35. Finn A, Booy R, Moxon R, Sharland M, Heath P. Should the new pneumococcal vaccine be used in high-risk children? Arch Dis Child 2002;87:18–21. Hazlewood M, Nusrat R, Kumararatne DS, Goodall M, Raykundalia C, Wang DG, et al. The acquisition of anti-pneumococcal capsular polysaccharide, Haemophilus influenzae type B and tetanus toxoid antibodies with age in the UK. Clin Exp Immunol 1993;93:157–64. Lal G, Balmer P, Stanford E, Martin S, Warrington R, Borrow R. Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes. J Immunol Methods 2005;296:135–47. J´odar L, Butler J, Carlone G, Dagan R, Goldblatt D, K¨ayhty H, et al. Serological criteria for evaluation and licensure of new pneumococcal conjugate vaccine formulations of the use in infants. Vaccine 2003;1:3265–72. International Union of Immunological Societies. Primary immunodeficiency diseases. Report of an IUIS Scientific Committee. Clin Exp Immunol 1999:118–28. Conley ME, Notorangelo LD, Etzioni A. Diagnostic criteria for primary immunodeficiencies. Clin Immunol 1999;93:190–7. Obukhanych TV, Nussenzweig MC. T-independent type II immune responses generate memory B cells. J Exp Med 2006;203:305–10. Kaniuk AS, Lortan JE, Monteil MA. Specific IgG subclass antibody levels and phagocytosis of serotype 14 pneumococcus following immunisation. Scand J Immunol 1992;11:96–8. Anttila M, Voutilainen M, J¨antti V, Eskola J, K¨ayhty H. Contribution of serotype-specific IgG concentrations, IgG subclass and relative antibody avidity to opsonophagocytic activity against Streptococcus pneumoniae. Clin Exp Immunol 1999;118:402–7. Wuorimaa T, Dagan R, Vakevainen M, Bailleux F, Haikala R, Yaich M, et al. Avidity and subclasses of IgG after immunization of infants with an 11-valent pneumococcal conjugate vaccine with or without aluminum adjuvant. J Infect Dis 2001;184:1211–5. Lucero MG, Puulmalainen T, Ugpo JM, Williams G, Kayhty H, Nohynek H. Similar antibody concentrations in Filipino infants at age 9 months, after 1 or 3 doses of an adjuvanted, 11-valent pneumococcal diphtheria/tetanus-conjugated vaccine: a randomized controlled trial. J Infect Dis 2004;189:2077–84. Southern J, Deane S, Ashton L, Borrow R, Goldblatt D, Andrews N, et al. Effects of prior polysaccharide vaccination on magnitude, duration, and quality of immune responses to and safety profile of a meningococcal serogroup C tetanus toxoid conjugate vaccination in adults. Clin Diagn Lab Immunol 2004;11:1100–4.