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Kinetics of the immune response following pneumococcal PD conjugate vaccination
Vaccine 25 (2007) 1953–1961
Kinetics of the immune response following pneumococcal PD conjugate vaccination Lode Schuerman a,∗ , Roman Prymula b , Viktor Chrobok c , Ilse Dieussaert a , Jan Poolman a a GlaxoSmithKline Biologicals, Rixensart, Belgium Department of Epidemiology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic Department of Otorhinolaryngology and Head and Neck Surgery, Pardubice, Czech Republic b
c
Received 28 July 2006; received in revised form 27 November 2006; accepted 5 December 2006 Available online 26 December 2006
Abstract Primary vaccination with pneumococcal protein D conjugate vaccine in the first year of life induced clear ELISA and OPA responses, which varied considerably for the different serotypes. Antibody levels declined following primary vaccination but were restored (except for serotype 3) to above post-primary levels by booster vaccination in the second year of life. Antibody levels declined when measured in the fourth year of life, although remaining higher than in the non-immunized children. For some serotypes, antibody levels did not decline indicating exposure to pneumococci or cross-reacting bacteria. Development of natural immunity to several serotypes was evident from the increase in opsonophagocytic activity in the control group between booster and plain polysaccharide vaccination. Vigorous and rapid OPA and ELISA responses were elicited to all vaccine serotypes including serotype 3 following administration of plain polysaccharide vaccine in both the conjugate and control groups, being higher in the conjugate group (Study ID: 104083/NCT00169507). © 2006 Elsevier Ltd. All rights reserved. Keywords: Pneumococcal conjugate vaccine; ELISA; Opsonophagocytic activity
1. Introduction Streptococcus pneumoniae is a major causative agent of bacterial pneumonia, meningitis and acute otitis media (AOM) in children and adults worldwide. It is also one of the leading causes of invasive bacterial diseases. The polysaccharide capsule is the principal virulence factor of S. pneumoniae by virtue of its anti-phagocytic properties. A 23-valent pneumococcal polysaccharide vaccine has been available since the early 80s [1]. However, polysaccharide vaccines are, in general, poorly immunogenic in young children. This is due to the incapacity of the T-cell independent polysaccharides to activate na¨ıve B cells to become antibody producing cells in the period early in life when natural priming has not yet occurred [2].
∗
Corresponding author. Tel.: +32 2 656 7232; fax: +32 2 656 8044 E-mail address: [email protected] (L. Schuerman).
0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.12.007
Pneumococcal vaccines developed for use in young children are based on capsular polysaccharides conjugated to carrier proteins. We have developed a pneumococcal conjugate vaccine containing capsular polysaccharides conjugated individually to a recombinant non-lipidated form of the protein D (PD) from Haemophilus influenzae [3]. In a double-blind randomized controlled efficacy study in children, this pneumococcal PD conjugate vaccine has demonstrated significant protective efficacy against AOM caused by pneumococcal vaccine serotypes and H. influenzae [4]. The number of AOM episodes was sufficient to assess serotype specific efficacy for five of the vaccine pneumococcal serotypes. Significant protective efficacy was seen for individual serotypes 6B, 14, 19F and 23F, no protection however could be demonstrated against AOM caused by vaccine serotype 3 pneumococci. This observation together with the attenuated booster response against serotype 3 [1,4] raised concern about the potential induction of immune tolerance or hyporesponsiveness to serotype 3. To address
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this concern, a follow-up immunization was performed to assess the capacity of PD conjugate vaccinees to respond to the plain serotype 3 pneumococcal polysaccharide contained in the licensed 23-valent polysaccharide vaccine. A subset of children who had received either PD conjugate vaccine or hepatitis A vaccine at 3, 4, 5 and 12–15 months of age in the otitis efficacy study were vaccinated with a single dose of plain 23-valent pneumococcal polysaccharide vaccine in the fourth year of life. In this report, we follow the kinetics of the pneumococcal immune response in this group of children from primary and booster vaccination through to administration of native polysaccharide.
month after completion of the primary series, immediately before and 1 month after the administration of the booster dose. In the follow up study, subjects from this blood sampling subset (51 who have been primed and boosted with 4 doses of PD conjugate and 49 who had received hepatitis A vaccine) were vaccinated in the fourth year of life with a single dose of 23-valent pneumococcal polysaccharide vaccine (Pneumo 23® Aventis Pasteur, containing 25 g of purified capsular polysaccharides of pneumococcal serotypes: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F). Two blood samples were taken from each subject prior to and 10–15 days following vaccination.
2. Materials and methods
2.3. Laboratory methods
2.1. Study design
Serotype-specific total IgG antibodies were measured by ELISA, which included a pre-adsorption step with serotype 22F polysaccharide in order to increase the specificity of the assay [5,6]. The assay cut-off was 0.05 g per milliliter. The antibody concentrations were determined by calibration with the standard reference serum 89-SF (courtesy of Dr. Frasch US FDA) [7]. Opsonophagocytic (OPA) activity was measured using a modification of the HL-60 cell WHO reference method [8,9] which has been validated using sera collected following primary vaccination with the 7-valent CRM197 conjugate [10]. The results are presented as the dilution of serum (opsonic titre) able to sustain 50% killing of live pneumococci under the assay conditions. The cut-off of the assay is an opsonic titre of 8 (serum dilution of 1:8).
The prospective randomized double-blind controlled study conducted between October 2000 and June 2004 was designed to assess the efficacy of the 11-valent PD conjugate vaccine (containing 1 g each of capsular polysaccharide of pneumococcal serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated individually to protein D) in preventing AOM in healthy young children. The control vaccine was the hepatitis A vaccine (HavrixTM , GlaxoSmithKline Biologicals). A detailed account of the study methodology has been published along with the main vaccine efficacy and ELISA results [4,5]. The follow-up study conducted between March and April 2005 was an open study with two parallel groups. It was designed to evaluate the immune responses to plain pneumococcal polysaccharide in general and more specifically the potential induction of immune tolerance for serotype 3 following prior primary and toddler booster vaccination with PD conjugate vaccine. The secondary objective was to assess antibody persistence following primary and booster immunization with the PD conjugate vaccine. The study was conducted on 100 healthy subjects who were vaccinated with PD conjugate or hepatitis A vaccine in the aforementioned efficacy study. Both studies were conducted according to the Declaration of Helsinki (as amended in Somerset West, South Africa 1996). The protocols were reviewed and approved by the appropriate Independent Ethics Committees or Institutional Review Boards. Written informed consent was obtained from a parent or legal guardian of each child before enrollment. 2.2. Vaccinations and blood sampling In the efficacy study, subjects received a primary course of PD conjugate (n = 2489) or hepatitis A vaccine (n = 2479) at approximately 3, 4, 5 months of age followed by a fourth dose of the same vaccine at 12–15 months of age. Blood samples were collected in a subset of subjects in pre-selected centers prior to the administration of the first study vaccine dose, 1
2.4. Analysis of results For the subjects who participated in the follow-up study, ELISA geometric mean concentrations (GMCs) and OPA geometric mean titres (GMTs) against individual pneumococcal vaccine serotypes (and also cross-reactive serotypes 6A and 19A) were calculated for PD conjugate primed and un-primed (hepatitis A vaccinees) groups for all blood sampling points since the beginning of the primary immunization series in the efficacy study. The same calculations were made prior to and following administration of 23-valent plain polysaccharide in the follow-up study. GMCs/GMTs were calculated by taking the antilog of the mean of the log concentration/titer transformations. Antibody concentrations and OPA titers below the assay cut-off were given an arbitrary value of one-half the cut-off for the purpose of GMC/GMT calculation. Ratios of post- to prevaccination antibody concentrations and OPA titres were calculated for each subject and summarized using geometric mean ratios (GMR) with 95% confidence intervals. In addition, post-vaccination antibody GMCs and OPA GMTs for primed and un-primed subjects were compared by computing primed/un-primed GMC and GMT ratios with 95% confidence intervals. Exploratory comparisons of ELISA GMCs
Table 1 ELISA antibody levels against individual pneumococcal serotypes prior to and 2 weeks following administration of 23-valent pneumococcal polysaccharide vaccine in the fourth year of life to pneumococcal PD conjugate vaccine primeda and un-primed subjectsb Un-primed subjectsb (n = 49)
ELISA antibody GMC (g/ml) (95% CI)
Post/Pre 23v
ELISA antibody GMC (g/ml) (95% CI)
Post/Pre 23v
Pre 23v
Post 23v
GMR (95% CI)
Pre 23v
Post 23v
GMR (95% CI)
0.12 (0.09–0.15) 0.34 (0.19–0.60) 0.13 (0.09–0.18) 0.20 (0.15–0.26) 1.59 (0.89–2.84) 0.31 (0.24–0.39) 0.23 (0.15–0.36) 2.05 (1.14–3.71) 0.16 (0.10–0.26) 3.59 (1.72–7.49) 0.92 (0.50–1.69) 0.35 (0.20–0.60) 0.62 (0.37–1.03)
21.62 (18.18–25.70) 7.88 (6.14–10.13) 18.51 (15.00–22.84) 18.48 (14.32–23.86) 10.23 (7.57–13.83) 21.19 (17.19–26.12) 25.13 (19.07–33.11) 38.15 (29.92–48.63) 18.33 (14.77–22.74) 39.13 (27.93–54.81) 14.43 (10.86–19.19) 2.03 (1.27–3.25) 6.57 (4.66–9.27)
185.7 (144.7–238.5) 22.7 (13.2–39.1) 139.2 (93.8–206.6) 93.6 (67.4–129.8) 6.7 (3.7–12.2) 68.3 (52.5–88.8) 113.2 (69.2–185.3) 19.3 (10.0–37.2) 111.4 (70.9–175.1) 11.4 (5–25.9) 14.6 (7.2–29.6) 6.0 (3.4–10.5) 10.7 (6.4–18.1)
0.04 (0.03–0.06) 0.20 (0.10–0.38) 0.05 (0.03–0.06) 0.07 (0.05–0.09) 0.13 (0.09–0.20) 0.04 (0.04–0.06) 0.06 (0.04–0.08) 0.17 (0.10–0.27) 0.04 (0.03–0.06) 0.33 (0.18–0.60) 0.08 (0.05–0.12) 0.09 (0.06–0.14) 0.23 (0.15–0.36)
7.16 (5.40–9.48) 5.01 (4.01–6.26) 7.31 (5.56–9.60) 2.45 (1.66–3.63) 0.52 (0.36–0.75) 4.6 (3.74–5.65) 2.20 (1.57–3.09) 1.14 (0.67–1.95) 1.53 (1.08–2.17) 4.80 (3.41–6.75) 0.60 (0.39–0.92) 0.30 (0.21–0.44) 2.95 (1.81–4.80)
165.4 (112.7–242.9) 25.6 (13.9–47.0) 166.5 (111.5–248.5) 33.8 (23.1–49.4) 3.9 (2.5–6.0) 103.2 (77.4–137.5) 39.8 (26.4–60.0) 6.1 (4.4–8.5) 35.4 (24.2–51.7) 14.6 (7.8–27.5) 7.4 (4.5–12.1) 3.2 (2.1–4.9) 12.7 (9.4–17.2)
Post 23v ratio of primed to un-primed antibody GMCs (95% CI)
3.0 (2.2–4.2) 1.6 (1.1–2.2) 2.5 (1.8–3.6) 7.5 (4.8–11.9) 19.7 (12.4–31.5) 4.6 (3.5–6.2) 11.4 (7.4–17.6) 33.4 (18.8–59.3) 12.0 (8.0–17.9) 8.2 (5.1–13.1) 24.1 (14.6–39.8) 6.8 (3.7–12.3) 2.2 (1.2–4.0)
L. Schuerman et al. / Vaccine 25 (2007) 1953–1961
Serotype 1 Serotype 3 Serotype 4 Serotype 5 Serotype 6B Serotype 7F Serotype 9V Serotype 14 Serotype 18C Serotype 19F Serotype 23F Serotype 6A Serotype 19A
Subjects primeda with PD conjugate vaccine (n = 50)
23v, 23-valent plain polysaccharide vaccine; 95% CI, 95% confidence interval; GMC, geometric mean concentration; GMR, geometric mean ratio; Pre 23v, prior to 23-valent plain polysaccharide vaccine; Post 23v, 15 days following administration of 23-valent plain polysaccharide vaccine. a Subjects were primed with three doses of pneumococcal PD conjugate vaccine at approximately 3, 4, 5 months of age and a fourth dose at 12–15 months of age. b Un-primed, primed with hepatitis A vaccine at approximately 3, 4, 5 months of age and a fourth dose at 12–15 months of age.
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1956 Table 2 Opsonophagocytic (OPA) titres against individual pneumococcal serotypes prior to and 2 weeks following administration of 23-valent pneumococcal polysaccharide vaccine in the fourth year of life to pneumococcal PD conjugate vaccine primeda and un-primed subjectsb Subjects primeda with PD conjugate vaccine (n = 49)
Post 23v ratio of primed to un-primed
OPA GMT dilutionc (95% CI)
Post/pre 23v
OPA GMT dilutionc (95% CI)
Post/pre 23v
Pre 23v
Post 23v
GMR (95% CI)
Pre 23v
Post 23v
GMR (95% CI)
5.0 (4.2–6.0) 14.7 (8.5–25.3) 17.0 (7.4–39.1) 10.2 (7.1–14.5) 829.8 (363.2–1896.0) 243.5 (63.8–929.7) 391.4 (129.4–1183.4) 858.6 (555.6–1326.8) 6.9 (4.6–10.4) 110.9 (48.9–251.7) 625.7 (236.9–1652.4) 105.6 (43.4–257.1) 15.0 (8.0–28.1)
7357.8 (5316.9–10182.0) 865.4 (677.8–1105.1) 18268.3 (14472.9–23058.9) 3288.7 (2415.6–4477.4) 3763.5 (2593.4–5461.5) 36063.3 (26969.6–48223.4) 30558.6 (22517.2–41471.7) 9374.0 (6959.4–12626.4) 2227.4 (1737.3–2855.8) 1889.5 (1162.1–3072.2) 15055.0 (10880.7–20830.7) 551.7 (244.0–1247.4) 1592.5 (1154.0–2197.8)
1620.6 (1173.1–2239.0) 56.6 (29.3–109.3) 1060.0 (404.2–2780.2) 327.2 (223.0–480.3) 4.5 (1.7–12.0) 73.6 (18.3–296.5) 30.3 (4.2–216.3) 12.6 (7.3–21.9) 325.8 (186.9–567.9) 21.2 (6.5–69.7) 23.8 (7.7–73.3) 7.8 (2.8–21.9) 99.6 (36.9–268.6)
4.0 (4.0–4.0) 9.5 (6.0–14.9) 5.3 (3.5–7.8) 4.2 (3.8–4.5) 328.7 (135.8–1078.2) 16.4 (3.2–83.8) 259.4 (80.1–840.7) 347.2 (226.1–533.2) 6.0 (4.2–8.5) 6.8 (4.7–9.8) 184.8 (59.3–575.7) 119.8 (59.8–240.0) 7.8 (5.1–11.9)
1000.2 (675.4–1481.1) 518.3 (407.6–659.2) 13246.1 (10682.8–16424.3) 164.3 (109.2–247.3) 1744.5 (1037.2–2934.2) 23762.4 (16961.0–33291.2) 18338.9 (12438.3–27038.7) 3468.7 (2567.9–4685.4) 865.2 (566.8–1320.8) 277.1 (128.7–596.4) 2954.2 (1305.3–6685.9) 1432.6 (798.9–2568.9) 1344.5 (880.8–2052.2)
250.1 (168.9–370.3) 55.9 (30.4–102.6) 2286.2 (1464.5–3568.9) 40.7 (27.1–61.3) 4.4 (1.3–14.8) 649.5 (75.1–5618.4) 112.8 (33.2–382.9) 10.3 (6.7–15.8) 158.1 (98.0–255.2) 53.3 (22.0–129.5) 17.1 (5.7–51.8) 10.4 (3.5–30.6) 197.3 (120.9–321.9)
GMTs (95% CI)
7.4 (4.4–12.2) 1.7 (1.2–2.3) 1.4 (1.0–1.9) 20.0 (12.0–33.3) 2.2 (1.1–4.1) 1.5 (1.0–2.3) 1.7 (1.0–2.7) 2.7 (1.8–4.1) 2.6 (1.6–4.2) 6.8 (2.7–17.1) 5.1 (2.2–11.9) 0.4 (0.1–1.0) 1.2 (0.7–2.0)
23v, 23-valent plain polysaccharide vaccine; 95% CI, 95% confidence interval; GMT, geometric mean titre; GMR, geometric mean ratio; Pre 23v, prior to 23-valent plain polysaccharide vaccine; Post 23v, 15 days following administration of 23-valent plain polysaccharide vaccine. a Subjects were primed with three doses of pneumococcal PD conjugate vaccine at approximately 3, 4, 5 months of age and a fourth dose at 12–15 months of age. b Un-primed, primed with hepatitis A vaccine at approximately 3, 4, 5 months of age and a fourth dose at 12–15 months of age. c Dilution of serum able to kill 50% of viable pneumococci.
L. Schuerman et al. / Vaccine 25 (2007) 1953–1961
Serotype 1 Serotype 3 Serotype 4 Serotype 5 Serotype 6B Serotype 7F Serotype 9V Serotype 14 Serotype 18C Serotype 19F Serotype 23F Serotype 6A Serotype 19A
Un-primed subjectsb (n = 48)
L. Schuerman et al. / Vaccine 25 (2007) 1953–1961
and OPA GMTs were performed for each serotype and at each timepoint, using the classical ANOVA test or Wilcoxon’s non-parametric test, depending on whether the conditions for the classical analysis of variance were fulfilled or not.
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male) or un-primed (n = 49, age = 3.7 years mean ± 0.48 S.D., 44.9% male) groups vaccinated with 23-valent pneumococcal polysaccharide vaccine were in the same range. 3.2. Immune response
3. Results 3.1. Demographics The demographic characteristics of the PD conjugate primed (n = 51, age = 3.6 years mean ± 0.50 S.D., 52.9%
The detailed results for the response to 23-valent plain polysaccharide vaccination are presented in Table 1 (ELISA) and Table 2 (OPA). The kinetics of the ELISA and OPA responses over the full study period from primary and booster PD conjugate or hepatitis A vaccination in the efficacy study
Fig. 1. Evolution of pneumococcal antibody concentrations (ELISA, GMC g/ml) and opsonophagocytic activity (GMT, dilution of serum able to kill 50% of viable pneumococci) measured before immunization with PD-conjugate vaccine or Hepatitis A control vaccine (Pre), 1 month after the three-dose primary course (Post III), before (Pre IV) and 1 month after the booster dose (Post IV) in the second year of life, and before (Pre 23v) and 2 weeks after a 23-valent plain polysaccharide vaccine (Post 23v) administered at 3.5 years of age. * P < 0.05; ns = not statistically significant (classical ANOVA test or Wilcoxon’s non-parametric test). (⇓) Limited or no decline of IgG antibody levels in the conjugate group. (⇑) Notable increase in OPA and to a lesser extent ELISA IgG levels in the un-primed group.
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Fig. 1. (Continued )
to 23-valent plain polysaccharide vaccination in the followup study is illustrated in Fig. 1. Data are presented for all 11 vaccine serotypes and also the cross-reacting serotypes 6A and 19A. The three dose PD conjugate primary vaccination induced clear ELISA and OPA responses (Fig. 1, Post III) for all vaccine serotypes when compared to vaccination with the hepatitis A control vaccine. Both ELISA and OPA antibody levels decreased in the 6–9 months between post-primary and pre-booster blood sampling (Fig. 1, Pre IV). Following booster vaccination (Fig. 1, Post IV) however both ELISA and OPA levels were restored to above post-primary level for all vaccine serotypes (for detailed data see references [4] and [11]) except serotype 3 where the ELISA antibody level (2.84 g/ml)
remained below that measured at post-primary (3.78 g/ml). The booster dose induced both ELISA and OPA responses for cross-reactive serotypes 6A and 19A. Antibody levels against most serotypes declined again in the approximately 2 year period between post-booster sampling (Fig. 1, Post IV) and pre 23-valent plain polysaccharide sampling, although remaining significantly higher in the conjugate versus control group (Table 1, Fig. 1, Pre 23v). Limited or no decline of IgG antibody levels in the conjugate group was observed for serotypes 6A, 6B, 14, 19A, 19F and 23F (see (⇓) in Fig. 1). During this period there was also a notable increase in OPA and to a lesser extent ELISA IgG levels in the un-primed group for some serotypes (6A, 6B, 9V, 14 and 23F, see (⇑) in Fig. 1). The ELISA persistence results in the primed subjects and the Pre 23v-polysaccharide OPA results
L. Schuerman et al. / Vaccine 25 (2007) 1953–1961
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Fig. 1. (Continued ).
in the un-primed subjects both indicate natural exposure of subjects to some pneumococcal serotypes or cross-reacting bacteria. Vigorous ELISA and OPA responses were elicited in primed subjects following administration of the 23-valent plain polysaccharide vaccine (Tables 1 and 2 and Fig. 1, Post 23v). The strongest response in terms of post/pre 23valent polysaccharide vaccination GMR was observed for
serotype 1 (186-fold for ELISA and 1621-fold for OPA) while the weakest response was observed for those serotypes that maintained the higher antibody levels Pre 23v (for example, 6.7-fold increase for ELISA and 4.5-fold for OPA for serotype 6B). Notably, the response to serotype 3 was comparable (23fold for ELISA and 57-fold for OPA) to some of the other vaccine serotypes although the actual post-vaccination antibody GMCs and OPA GMTs remained the lowest among
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all serotypes. The magnitude of the response for serotype 6A (not included in the 23-valent vaccine) reflected that of 6B. A strong OPA response was induced to 19A (which is included in the 23-valent vaccine) while the ELISA response was weaker. The 23-valent vaccine also elicited both ELISA and OPA responses to all 11 serotypes in subjects who had not been primed with PD conjugate vaccine. The magnitude of the responses however as measured by post-vaccination antibody GMCs or OPA GMTs was significantly higher in primed subjects for most serotypes (see (*) in Fig. 1).
4. Discussion This report followed the kinetics of the immune response from primary and booster pneumococcal PD conjugate vaccination as compared to non-immunized controls in the first 15 months of life through to vaccination with pneumococcal polysaccharides in the fourth year of life. Determination of serotype-specific IgG levels by ELISA following administration of three primary doses has become the standard measurement of response to pneumococcal conjugate vaccines in young children. As shown here and elsewhere [1,4] three doses of PD conjugate vaccine elicits a clear ELISA IgG response for all vaccine serotypes but the antibody levels induced vary considerably for the different serotypes. This variation was also observed previously for the licensed 7-valent CRM-conjugate vaccine [12,13]. Due to differences in the ELISA methods used, it is not possible to directly compare these antibody concentrations to those induced by the PD conjugate vaccine, but it is interesting to note that the post-primary ranking of the antibody concentrations against the seven shared serotypes is similar for both vaccines (14 > 19F > 4 > 18C > 9V > 23F > 6B). The similarity of the pattern of serotype response for different conjugates has been noted by others [14] who concluded that the magnitude of the ELISA response is likely to be an inherent property of the individual polysaccharides. The functionality of pneumococcal antibodies can be directly measured by assessing their opsonophagocytic activity. However, to be used as a primary read-out, OPA assays require validation and technical development to allow a highthroughput application. This means that OPA measurements have mainly been used on a limited number of samples to characterize immune responses. In the PD conjugate efficacy and follow-up studies a validated high-throughput OPA assay was employed [10]. Following primary vaccination, a clear OPA immune response for all the PD conjugate vaccine serotypes was observed indicating the induction of functionally active antibodies. As with ELISA there was a considerable variation in OPA levels however, the ranking of serotypes in terms of magnitude of post-primary OPA GMTs did not necessarily reflect the post-primary ELISA GMC ranking. For example, the ELISA responses to 6B and 23F were among the lowest while the OPA responses
were among the highest and vice versa for serotype 3. Such serotype-specific differences are not understood, but may relate to differences in complement deposition on the surface of different serotypes. In addition to the induction of satisfactory antibody responses in infancy, another important characteristic of conjugate vaccines is the ability to induce immunological memory. As demonstrated here and elsewhere [12,14] antibody levels gradually diminish after the primary series of immunizations. However, induction of immune memory implies that subsequent exposure to the pathogen or another dose of vaccine allows a vigorous response due to the presence of an expanded pool of memory B cells. A fourth dose of PD conjugate was shown to induce enhanced ELISA (except for serotype 3) and OPA responses, indicative of the induction of immune memory. Induction of B cell memory was previously demonstrated by the strong antibody response following administration of native polysaccharide in the second year of life as a booster to children primed with PD conjugate vaccine [3]. Post-licensure experience with the 7-valent CRM-conjugate vaccine in the US has shown that protection against IPD was maintained despite many children not receiving the recommended booster dose. This lends support to the hypothesis that immune memory following primary conjugate vaccination confers long term protection, at least following the USA schedule of 2–4–6 months for primary immunization, although herd immunity may also have played a role [15]. The pattern of antibody persistence in the period between the conjugate booster dose in the second year of life and the polysaccharide booster 2 years later was clearly serotype dependent. For the majority of serotypes, a sharp decline in antibody levels was observed, while for serotypes 6B, 14, 19F and 23F, higher levels were maintained. During this period there was also a notable increase in antibody levels in the un-primed group for the same serotypes indicating that the higher antibody levels against these serotypes were probably the result of natural exposure to the circulating pneumococci. The natural immunity appeared to be more evident when measured by IgG ELISA in the conjugate primed group and when measured by OPA in the un-primed group. This probably reflects a predominantly IgG memory response in the conjugate primed subjects and a predominantly IgM early primary response in the un-primed children. The presence of polysaccharide-specific B cell memory following PD-conjugate primary and booster vaccination was demonstrated by the vigorous response to native polysaccharide. Given the age at which children received the 23-valent polysaccharide booster in this study (in their fourth year of life) an immune response could be expected even in unprimed children. For most serotypes a significant difference between conjugate primed versus un-primed children could however be observed for both ELISA and OPA immune responses following polysaccharide vaccination, and this can therefore be considered as evidence of a sustained conjugate vaccine induced immune memory.
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A significant observation in the study was that the administration of native polysaccharide succeeded in eliciting both ELISA and OPA responses to serotype 3 in the PD conjugate vaccinees. Although the absolute post-vaccination serotype 3 antibody levels were the lowest of all vaccine serotypes in the primed group, they were at least as high as the serotype 3 antibody levels induced in the un-primed group (P = 0.0079 in favour of the conjugate group). This indicates that the capacity of children vaccinated with serotype 3 PD conjugate to respond to native serotype 3 polysaccharide, and thus to natural infection, is not impaired. Therefore, although there may be a relatively narrow window of tolerance following primary vaccination with serotype 3 PD conjugate, this does not seem to be sustained over time. It has been demonstrated that conjugate vaccines can provide some degree of protection against vaccine-related pneumococcal serotypes, except for serotype 19A [16]. The cross-reactivity between serotypes 19F and 19A is known to be limited, and the existing 23-valent plain polysaccharide vaccine contains both 19A and 19F. This contrasts to serotype 6A and 6B for which cross-protection has been demonstrated [16]. In this report, a conjugate induced booster response in the second year of life could be demonstrated by OPA and ELISA against serotypes 6A and 19A, despite the absence of a primary response against these serotypes (with the exception of a clear OPA response against 6A that could be measured following primary immunization). OPA activity for 6A and 19A following PD conjugate vaccination has also been measured by Finnish investigators [17]. It could therefore be speculated that the PD conjugate vaccine might indeed provide some protection against these cross-reactive serotypes, as already suggested by the AOM efficacy results, although these did not reach statistical significance [4]. In conclusion, pneumococcal PD conjugate primary vaccination in young children stimulates the production of functional serotype-specific antibodies and induces antigenspecific memory B cells for lasting immunity.
Acknowledgements The authors would like to thank all the infants and their families as well as the study investigational team in the Czech Republic and Slovak Republic. The authors are indebted to Patricia Lommel for statistical support, Isabelle Henckaerts for serological assays, Joelle Onkelinx for central study coordination (GlaxoSmithKline Biologicals, Rixensart, Belgium) and Miriam Hynes (independent, UK) for writing assistance in preparation of the manuscript. This study was supported by GlaxoSmithKline Biologicals, Rixensart, Belgium (Study ID: 104083/NCT00169507). HavrixTM is a trademark of GlaxoSmithKline Group of Companies. Pneumo 23® is a registered trademark of Aventis Pasteur.
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References [1] Schwartz JS. Pneumococcal vaccine: clinical efficacy and effectiveness. Ann Intern Med 1982;96:208–20. [2] Dintzis RZ. Rational design of conjugate vaccines. Pediatr Res 1992;32:376–85. [3] Nurkka A, Joensuu J, Henckaerts I, Peeters P, Poolman J, Kilpi T, et al. Immunogenicity and safety of the eleven valent pneumococcal polysaccharide-protein D conjugate vaccine in infants. Pediatr Infect Dis J 2004;11:1008–14. [4] Prymula R, Peeters P, Chrobok V, Kriz P, Novakova E, Kaliskova E, et al. Pneumococcal capsular polysaccharides conjugated to protein D provide protection against otitis media caused by both Streptococcus pneumoniae and nontypable Haemophilus influenzae: a randomized double blind efficacy study. Lancet 2006;367:740–8. [5] Henckaerts I, Goldblatt D, Ashton L, Poolman J. Critical differences between pneumococcal polysaccharide enzyme-linked immunosorbent assays with or without 22F inhibition at low antibody concentrations in pediatric sera. Clin Vaccine Immunol 2006;13:356–60. [6] Concepcion N, Frasch NE. Pneumococcal type 22F polysaccharide absorption improves the specificity of a pneumococcal-polysaccharide enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol 2001;8:266–72. [7] Quataert SA, Kirch CS, Quackenbush Wiedl LJ, Phipps DC, Strohmeyer S, Cimino CO, et al. Assignment of weight-based antibody units to a human antipneumococcal standard reference serum, lot 89-S. Clin Diagn Lab Immunol 1995;2:590–7. [8] Centers for Disease Control and Prevention and Emory University. Streptococcus pneumoniae opsonophagocytosis using differentiated HL-60 cells (Promyelocytic Leukemia Cell Line). Laboratory protocol. Available at http://www.vaccine.uab.edu. Accessed 17 February 2006. [9] Romero-Steiner S, Frash C, Concepcion N, Goldblatt D, Kayhty H, Vakevainen M, et al. Multi-laboratory evaluation of a viability assay for measurement of opsonophagocytic antibodies specific to the capsular polysaccharides of Streptococcus pneumoniae. Clin Diagn Lab Immunol 2003;10(6):1019–24. [10] Henckaerts I, Durant N, De Grave D, Schuerman L, Poolman J. Validation of a routine opsonophagocytosis assay to predict invasive pneumococcal disease efficacy of conjugate vaccine in children. Vaccine 2006, doi:10.1016/j.vaccine.2006.09.029. [11] Schuerman L, Prymula R, Henckaerts I, Poolman J. ELISA IgG concentrations and opsonophagocytic activity following pneumococcal protein D conjugate vaccination and relationship to efficacy against acute otitis media. Vaccine 2007;25:1962–8. [12] Black S, Shinefield H, Fireman B, Lewis E, Ray P, Hansen JR, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Pediatr Infect Dis J 2000;19:187–95. [13] Eskola J, Kilpi T, Palmu A, Jokinen J, Haapakoski J, Herva E, et al. Efficacy of a pneumococcal conjugate vaccine against acute otitis media. N Engl J Med 2001;344:403–9. [14] Eskola J, Anttila M. Pneumococcal conjugate vaccines. Pediatr Infect Dis J 1999;18:543–51. [15] 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. Pediatric Infect Dis J 2004;23(6):485–9. [16] Yu X, Gray B, Chang S, Ward JI, Edwards KM, Nahm MH. Immunity to cross reactive serotypes induced by pneumococcal conjugate vaccines in infants. J Infect Dis 1999;180:1569–76. [17] Nurkka A, Lehtonen H, Vuorela A, Ekstr¨om H, K¨ayhty H. Functionality of antibodies against serotypes 6A and 19A induced by three different pneumococcal conjugate vaccines (PCV) in infants. In: Proceedings of the Program and Abstracts of the 5th International Symposium on Pneumococci and Pneumococcal Diseases. 2006 [Abstract PO10.08].
Kinetics of the immune response following pneumococcal PD conjugate vaccination Lode Schuerman a,∗ , Roman Prymula b , Viktor Chrobok c , Ilse Dieussaert a , Jan Poolman a a GlaxoSmithKline Biologicals, Rixensart, Belgium Department of Epidemiology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic Department of Otorhinolaryngology and Head and Neck Surgery, Pardubice, Czech Republic b
c
Received 28 July 2006; received in revised form 27 November 2006; accepted 5 December 2006 Available online 26 December 2006
Abstract Primary vaccination with pneumococcal protein D conjugate vaccine in the first year of life induced clear ELISA and OPA responses, which varied considerably for the different serotypes. Antibody levels declined following primary vaccination but were restored (except for serotype 3) to above post-primary levels by booster vaccination in the second year of life. Antibody levels declined when measured in the fourth year of life, although remaining higher than in the non-immunized children. For some serotypes, antibody levels did not decline indicating exposure to pneumococci or cross-reacting bacteria. Development of natural immunity to several serotypes was evident from the increase in opsonophagocytic activity in the control group between booster and plain polysaccharide vaccination. Vigorous and rapid OPA and ELISA responses were elicited to all vaccine serotypes including serotype 3 following administration of plain polysaccharide vaccine in both the conjugate and control groups, being higher in the conjugate group (Study ID: 104083/NCT00169507). © 2006 Elsevier Ltd. All rights reserved. Keywords: Pneumococcal conjugate vaccine; ELISA; Opsonophagocytic activity
1. Introduction Streptococcus pneumoniae is a major causative agent of bacterial pneumonia, meningitis and acute otitis media (AOM) in children and adults worldwide. It is also one of the leading causes of invasive bacterial diseases. The polysaccharide capsule is the principal virulence factor of S. pneumoniae by virtue of its anti-phagocytic properties. A 23-valent pneumococcal polysaccharide vaccine has been available since the early 80s [1]. However, polysaccharide vaccines are, in general, poorly immunogenic in young children. This is due to the incapacity of the T-cell independent polysaccharides to activate na¨ıve B cells to become antibody producing cells in the period early in life when natural priming has not yet occurred [2].
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Corresponding author. Tel.: +32 2 656 7232; fax: +32 2 656 8044 E-mail address: [email protected] (L. Schuerman).
0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.12.007
Pneumococcal vaccines developed for use in young children are based on capsular polysaccharides conjugated to carrier proteins. We have developed a pneumococcal conjugate vaccine containing capsular polysaccharides conjugated individually to a recombinant non-lipidated form of the protein D (PD) from Haemophilus influenzae [3]. In a double-blind randomized controlled efficacy study in children, this pneumococcal PD conjugate vaccine has demonstrated significant protective efficacy against AOM caused by pneumococcal vaccine serotypes and H. influenzae [4]. The number of AOM episodes was sufficient to assess serotype specific efficacy for five of the vaccine pneumococcal serotypes. Significant protective efficacy was seen for individual serotypes 6B, 14, 19F and 23F, no protection however could be demonstrated against AOM caused by vaccine serotype 3 pneumococci. This observation together with the attenuated booster response against serotype 3 [1,4] raised concern about the potential induction of immune tolerance or hyporesponsiveness to serotype 3. To address
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this concern, a follow-up immunization was performed to assess the capacity of PD conjugate vaccinees to respond to the plain serotype 3 pneumococcal polysaccharide contained in the licensed 23-valent polysaccharide vaccine. A subset of children who had received either PD conjugate vaccine or hepatitis A vaccine at 3, 4, 5 and 12–15 months of age in the otitis efficacy study were vaccinated with a single dose of plain 23-valent pneumococcal polysaccharide vaccine in the fourth year of life. In this report, we follow the kinetics of the pneumococcal immune response in this group of children from primary and booster vaccination through to administration of native polysaccharide.
month after completion of the primary series, immediately before and 1 month after the administration of the booster dose. In the follow up study, subjects from this blood sampling subset (51 who have been primed and boosted with 4 doses of PD conjugate and 49 who had received hepatitis A vaccine) were vaccinated in the fourth year of life with a single dose of 23-valent pneumococcal polysaccharide vaccine (Pneumo 23® Aventis Pasteur, containing 25 g of purified capsular polysaccharides of pneumococcal serotypes: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F). Two blood samples were taken from each subject prior to and 10–15 days following vaccination.
2. Materials and methods
2.3. Laboratory methods
2.1. Study design
Serotype-specific total IgG antibodies were measured by ELISA, which included a pre-adsorption step with serotype 22F polysaccharide in order to increase the specificity of the assay [5,6]. The assay cut-off was 0.05 g per milliliter. The antibody concentrations were determined by calibration with the standard reference serum 89-SF (courtesy of Dr. Frasch US FDA) [7]. Opsonophagocytic (OPA) activity was measured using a modification of the HL-60 cell WHO reference method [8,9] which has been validated using sera collected following primary vaccination with the 7-valent CRM197 conjugate [10]. The results are presented as the dilution of serum (opsonic titre) able to sustain 50% killing of live pneumococci under the assay conditions. The cut-off of the assay is an opsonic titre of 8 (serum dilution of 1:8).
The prospective randomized double-blind controlled study conducted between October 2000 and June 2004 was designed to assess the efficacy of the 11-valent PD conjugate vaccine (containing 1 g each of capsular polysaccharide of pneumococcal serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated individually to protein D) in preventing AOM in healthy young children. The control vaccine was the hepatitis A vaccine (HavrixTM , GlaxoSmithKline Biologicals). A detailed account of the study methodology has been published along with the main vaccine efficacy and ELISA results [4,5]. The follow-up study conducted between March and April 2005 was an open study with two parallel groups. It was designed to evaluate the immune responses to plain pneumococcal polysaccharide in general and more specifically the potential induction of immune tolerance for serotype 3 following prior primary and toddler booster vaccination with PD conjugate vaccine. The secondary objective was to assess antibody persistence following primary and booster immunization with the PD conjugate vaccine. The study was conducted on 100 healthy subjects who were vaccinated with PD conjugate or hepatitis A vaccine in the aforementioned efficacy study. Both studies were conducted according to the Declaration of Helsinki (as amended in Somerset West, South Africa 1996). The protocols were reviewed and approved by the appropriate Independent Ethics Committees or Institutional Review Boards. Written informed consent was obtained from a parent or legal guardian of each child before enrollment. 2.2. Vaccinations and blood sampling In the efficacy study, subjects received a primary course of PD conjugate (n = 2489) or hepatitis A vaccine (n = 2479) at approximately 3, 4, 5 months of age followed by a fourth dose of the same vaccine at 12–15 months of age. Blood samples were collected in a subset of subjects in pre-selected centers prior to the administration of the first study vaccine dose, 1
2.4. Analysis of results For the subjects who participated in the follow-up study, ELISA geometric mean concentrations (GMCs) and OPA geometric mean titres (GMTs) against individual pneumococcal vaccine serotypes (and also cross-reactive serotypes 6A and 19A) were calculated for PD conjugate primed and un-primed (hepatitis A vaccinees) groups for all blood sampling points since the beginning of the primary immunization series in the efficacy study. The same calculations were made prior to and following administration of 23-valent plain polysaccharide in the follow-up study. GMCs/GMTs were calculated by taking the antilog of the mean of the log concentration/titer transformations. Antibody concentrations and OPA titers below the assay cut-off were given an arbitrary value of one-half the cut-off for the purpose of GMC/GMT calculation. Ratios of post- to prevaccination antibody concentrations and OPA titres were calculated for each subject and summarized using geometric mean ratios (GMR) with 95% confidence intervals. In addition, post-vaccination antibody GMCs and OPA GMTs for primed and un-primed subjects were compared by computing primed/un-primed GMC and GMT ratios with 95% confidence intervals. Exploratory comparisons of ELISA GMCs
Table 1 ELISA antibody levels against individual pneumococcal serotypes prior to and 2 weeks following administration of 23-valent pneumococcal polysaccharide vaccine in the fourth year of life to pneumococcal PD conjugate vaccine primeda and un-primed subjectsb Un-primed subjectsb (n = 49)
ELISA antibody GMC (g/ml) (95% CI)
Post/Pre 23v
ELISA antibody GMC (g/ml) (95% CI)
Post/Pre 23v
Pre 23v
Post 23v
GMR (95% CI)
Pre 23v
Post 23v
GMR (95% CI)
0.12 (0.09–0.15) 0.34 (0.19–0.60) 0.13 (0.09–0.18) 0.20 (0.15–0.26) 1.59 (0.89–2.84) 0.31 (0.24–0.39) 0.23 (0.15–0.36) 2.05 (1.14–3.71) 0.16 (0.10–0.26) 3.59 (1.72–7.49) 0.92 (0.50–1.69) 0.35 (0.20–0.60) 0.62 (0.37–1.03)
21.62 (18.18–25.70) 7.88 (6.14–10.13) 18.51 (15.00–22.84) 18.48 (14.32–23.86) 10.23 (7.57–13.83) 21.19 (17.19–26.12) 25.13 (19.07–33.11) 38.15 (29.92–48.63) 18.33 (14.77–22.74) 39.13 (27.93–54.81) 14.43 (10.86–19.19) 2.03 (1.27–3.25) 6.57 (4.66–9.27)
185.7 (144.7–238.5) 22.7 (13.2–39.1) 139.2 (93.8–206.6) 93.6 (67.4–129.8) 6.7 (3.7–12.2) 68.3 (52.5–88.8) 113.2 (69.2–185.3) 19.3 (10.0–37.2) 111.4 (70.9–175.1) 11.4 (5–25.9) 14.6 (7.2–29.6) 6.0 (3.4–10.5) 10.7 (6.4–18.1)
0.04 (0.03–0.06) 0.20 (0.10–0.38) 0.05 (0.03–0.06) 0.07 (0.05–0.09) 0.13 (0.09–0.20) 0.04 (0.04–0.06) 0.06 (0.04–0.08) 0.17 (0.10–0.27) 0.04 (0.03–0.06) 0.33 (0.18–0.60) 0.08 (0.05–0.12) 0.09 (0.06–0.14) 0.23 (0.15–0.36)
7.16 (5.40–9.48) 5.01 (4.01–6.26) 7.31 (5.56–9.60) 2.45 (1.66–3.63) 0.52 (0.36–0.75) 4.6 (3.74–5.65) 2.20 (1.57–3.09) 1.14 (0.67–1.95) 1.53 (1.08–2.17) 4.80 (3.41–6.75) 0.60 (0.39–0.92) 0.30 (0.21–0.44) 2.95 (1.81–4.80)
165.4 (112.7–242.9) 25.6 (13.9–47.0) 166.5 (111.5–248.5) 33.8 (23.1–49.4) 3.9 (2.5–6.0) 103.2 (77.4–137.5) 39.8 (26.4–60.0) 6.1 (4.4–8.5) 35.4 (24.2–51.7) 14.6 (7.8–27.5) 7.4 (4.5–12.1) 3.2 (2.1–4.9) 12.7 (9.4–17.2)
Post 23v ratio of primed to un-primed antibody GMCs (95% CI)
3.0 (2.2–4.2) 1.6 (1.1–2.2) 2.5 (1.8–3.6) 7.5 (4.8–11.9) 19.7 (12.4–31.5) 4.6 (3.5–6.2) 11.4 (7.4–17.6) 33.4 (18.8–59.3) 12.0 (8.0–17.9) 8.2 (5.1–13.1) 24.1 (14.6–39.8) 6.8 (3.7–12.3) 2.2 (1.2–4.0)
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Serotype 1 Serotype 3 Serotype 4 Serotype 5 Serotype 6B Serotype 7F Serotype 9V Serotype 14 Serotype 18C Serotype 19F Serotype 23F Serotype 6A Serotype 19A
Subjects primeda with PD conjugate vaccine (n = 50)
23v, 23-valent plain polysaccharide vaccine; 95% CI, 95% confidence interval; GMC, geometric mean concentration; GMR, geometric mean ratio; Pre 23v, prior to 23-valent plain polysaccharide vaccine; Post 23v, 15 days following administration of 23-valent plain polysaccharide vaccine. a Subjects were primed with three doses of pneumococcal PD conjugate vaccine at approximately 3, 4, 5 months of age and a fourth dose at 12–15 months of age. b Un-primed, primed with hepatitis A vaccine at approximately 3, 4, 5 months of age and a fourth dose at 12–15 months of age.
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1956 Table 2 Opsonophagocytic (OPA) titres against individual pneumococcal serotypes prior to and 2 weeks following administration of 23-valent pneumococcal polysaccharide vaccine in the fourth year of life to pneumococcal PD conjugate vaccine primeda and un-primed subjectsb Subjects primeda with PD conjugate vaccine (n = 49)
Post 23v ratio of primed to un-primed
OPA GMT dilutionc (95% CI)
Post/pre 23v
OPA GMT dilutionc (95% CI)
Post/pre 23v
Pre 23v
Post 23v
GMR (95% CI)
Pre 23v
Post 23v
GMR (95% CI)
5.0 (4.2–6.0) 14.7 (8.5–25.3) 17.0 (7.4–39.1) 10.2 (7.1–14.5) 829.8 (363.2–1896.0) 243.5 (63.8–929.7) 391.4 (129.4–1183.4) 858.6 (555.6–1326.8) 6.9 (4.6–10.4) 110.9 (48.9–251.7) 625.7 (236.9–1652.4) 105.6 (43.4–257.1) 15.0 (8.0–28.1)
7357.8 (5316.9–10182.0) 865.4 (677.8–1105.1) 18268.3 (14472.9–23058.9) 3288.7 (2415.6–4477.4) 3763.5 (2593.4–5461.5) 36063.3 (26969.6–48223.4) 30558.6 (22517.2–41471.7) 9374.0 (6959.4–12626.4) 2227.4 (1737.3–2855.8) 1889.5 (1162.1–3072.2) 15055.0 (10880.7–20830.7) 551.7 (244.0–1247.4) 1592.5 (1154.0–2197.8)
1620.6 (1173.1–2239.0) 56.6 (29.3–109.3) 1060.0 (404.2–2780.2) 327.2 (223.0–480.3) 4.5 (1.7–12.0) 73.6 (18.3–296.5) 30.3 (4.2–216.3) 12.6 (7.3–21.9) 325.8 (186.9–567.9) 21.2 (6.5–69.7) 23.8 (7.7–73.3) 7.8 (2.8–21.9) 99.6 (36.9–268.6)
4.0 (4.0–4.0) 9.5 (6.0–14.9) 5.3 (3.5–7.8) 4.2 (3.8–4.5) 328.7 (135.8–1078.2) 16.4 (3.2–83.8) 259.4 (80.1–840.7) 347.2 (226.1–533.2) 6.0 (4.2–8.5) 6.8 (4.7–9.8) 184.8 (59.3–575.7) 119.8 (59.8–240.0) 7.8 (5.1–11.9)
1000.2 (675.4–1481.1) 518.3 (407.6–659.2) 13246.1 (10682.8–16424.3) 164.3 (109.2–247.3) 1744.5 (1037.2–2934.2) 23762.4 (16961.0–33291.2) 18338.9 (12438.3–27038.7) 3468.7 (2567.9–4685.4) 865.2 (566.8–1320.8) 277.1 (128.7–596.4) 2954.2 (1305.3–6685.9) 1432.6 (798.9–2568.9) 1344.5 (880.8–2052.2)
250.1 (168.9–370.3) 55.9 (30.4–102.6) 2286.2 (1464.5–3568.9) 40.7 (27.1–61.3) 4.4 (1.3–14.8) 649.5 (75.1–5618.4) 112.8 (33.2–382.9) 10.3 (6.7–15.8) 158.1 (98.0–255.2) 53.3 (22.0–129.5) 17.1 (5.7–51.8) 10.4 (3.5–30.6) 197.3 (120.9–321.9)
GMTs (95% CI)
7.4 (4.4–12.2) 1.7 (1.2–2.3) 1.4 (1.0–1.9) 20.0 (12.0–33.3) 2.2 (1.1–4.1) 1.5 (1.0–2.3) 1.7 (1.0–2.7) 2.7 (1.8–4.1) 2.6 (1.6–4.2) 6.8 (2.7–17.1) 5.1 (2.2–11.9) 0.4 (0.1–1.0) 1.2 (0.7–2.0)
23v, 23-valent plain polysaccharide vaccine; 95% CI, 95% confidence interval; GMT, geometric mean titre; GMR, geometric mean ratio; Pre 23v, prior to 23-valent plain polysaccharide vaccine; Post 23v, 15 days following administration of 23-valent plain polysaccharide vaccine. a Subjects were primed with three doses of pneumococcal PD conjugate vaccine at approximately 3, 4, 5 months of age and a fourth dose at 12–15 months of age. b Un-primed, primed with hepatitis A vaccine at approximately 3, 4, 5 months of age and a fourth dose at 12–15 months of age. c Dilution of serum able to kill 50% of viable pneumococci.
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Serotype 1 Serotype 3 Serotype 4 Serotype 5 Serotype 6B Serotype 7F Serotype 9V Serotype 14 Serotype 18C Serotype 19F Serotype 23F Serotype 6A Serotype 19A
Un-primed subjectsb (n = 48)
L. Schuerman et al. / Vaccine 25 (2007) 1953–1961
and OPA GMTs were performed for each serotype and at each timepoint, using the classical ANOVA test or Wilcoxon’s non-parametric test, depending on whether the conditions for the classical analysis of variance were fulfilled or not.
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male) or un-primed (n = 49, age = 3.7 years mean ± 0.48 S.D., 44.9% male) groups vaccinated with 23-valent pneumococcal polysaccharide vaccine were in the same range. 3.2. Immune response
3. Results 3.1. Demographics The demographic characteristics of the PD conjugate primed (n = 51, age = 3.6 years mean ± 0.50 S.D., 52.9%
The detailed results for the response to 23-valent plain polysaccharide vaccination are presented in Table 1 (ELISA) and Table 2 (OPA). The kinetics of the ELISA and OPA responses over the full study period from primary and booster PD conjugate or hepatitis A vaccination in the efficacy study
Fig. 1. Evolution of pneumococcal antibody concentrations (ELISA, GMC g/ml) and opsonophagocytic activity (GMT, dilution of serum able to kill 50% of viable pneumococci) measured before immunization with PD-conjugate vaccine or Hepatitis A control vaccine (Pre), 1 month after the three-dose primary course (Post III), before (Pre IV) and 1 month after the booster dose (Post IV) in the second year of life, and before (Pre 23v) and 2 weeks after a 23-valent plain polysaccharide vaccine (Post 23v) administered at 3.5 years of age. * P < 0.05; ns = not statistically significant (classical ANOVA test or Wilcoxon’s non-parametric test). (⇓) Limited or no decline of IgG antibody levels in the conjugate group. (⇑) Notable increase in OPA and to a lesser extent ELISA IgG levels in the un-primed group.
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Fig. 1. (Continued )
to 23-valent plain polysaccharide vaccination in the followup study is illustrated in Fig. 1. Data are presented for all 11 vaccine serotypes and also the cross-reacting serotypes 6A and 19A. The three dose PD conjugate primary vaccination induced clear ELISA and OPA responses (Fig. 1, Post III) for all vaccine serotypes when compared to vaccination with the hepatitis A control vaccine. Both ELISA and OPA antibody levels decreased in the 6–9 months between post-primary and pre-booster blood sampling (Fig. 1, Pre IV). Following booster vaccination (Fig. 1, Post IV) however both ELISA and OPA levels were restored to above post-primary level for all vaccine serotypes (for detailed data see references [4] and [11]) except serotype 3 where the ELISA antibody level (2.84 g/ml)
remained below that measured at post-primary (3.78 g/ml). The booster dose induced both ELISA and OPA responses for cross-reactive serotypes 6A and 19A. Antibody levels against most serotypes declined again in the approximately 2 year period between post-booster sampling (Fig. 1, Post IV) and pre 23-valent plain polysaccharide sampling, although remaining significantly higher in the conjugate versus control group (Table 1, Fig. 1, Pre 23v). Limited or no decline of IgG antibody levels in the conjugate group was observed for serotypes 6A, 6B, 14, 19A, 19F and 23F (see (⇓) in Fig. 1). During this period there was also a notable increase in OPA and to a lesser extent ELISA IgG levels in the un-primed group for some serotypes (6A, 6B, 9V, 14 and 23F, see (⇑) in Fig. 1). The ELISA persistence results in the primed subjects and the Pre 23v-polysaccharide OPA results
L. Schuerman et al. / Vaccine 25 (2007) 1953–1961
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Fig. 1. (Continued ).
in the un-primed subjects both indicate natural exposure of subjects to some pneumococcal serotypes or cross-reacting bacteria. Vigorous ELISA and OPA responses were elicited in primed subjects following administration of the 23-valent plain polysaccharide vaccine (Tables 1 and 2 and Fig. 1, Post 23v). The strongest response in terms of post/pre 23valent polysaccharide vaccination GMR was observed for
serotype 1 (186-fold for ELISA and 1621-fold for OPA) while the weakest response was observed for those serotypes that maintained the higher antibody levels Pre 23v (for example, 6.7-fold increase for ELISA and 4.5-fold for OPA for serotype 6B). Notably, the response to serotype 3 was comparable (23fold for ELISA and 57-fold for OPA) to some of the other vaccine serotypes although the actual post-vaccination antibody GMCs and OPA GMTs remained the lowest among
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all serotypes. The magnitude of the response for serotype 6A (not included in the 23-valent vaccine) reflected that of 6B. A strong OPA response was induced to 19A (which is included in the 23-valent vaccine) while the ELISA response was weaker. The 23-valent vaccine also elicited both ELISA and OPA responses to all 11 serotypes in subjects who had not been primed with PD conjugate vaccine. The magnitude of the responses however as measured by post-vaccination antibody GMCs or OPA GMTs was significantly higher in primed subjects for most serotypes (see (*) in Fig. 1).
4. Discussion This report followed the kinetics of the immune response from primary and booster pneumococcal PD conjugate vaccination as compared to non-immunized controls in the first 15 months of life through to vaccination with pneumococcal polysaccharides in the fourth year of life. Determination of serotype-specific IgG levels by ELISA following administration of three primary doses has become the standard measurement of response to pneumococcal conjugate vaccines in young children. As shown here and elsewhere [1,4] three doses of PD conjugate vaccine elicits a clear ELISA IgG response for all vaccine serotypes but the antibody levels induced vary considerably for the different serotypes. This variation was also observed previously for the licensed 7-valent CRM-conjugate vaccine [12,13]. Due to differences in the ELISA methods used, it is not possible to directly compare these antibody concentrations to those induced by the PD conjugate vaccine, but it is interesting to note that the post-primary ranking of the antibody concentrations against the seven shared serotypes is similar for both vaccines (14 > 19F > 4 > 18C > 9V > 23F > 6B). The similarity of the pattern of serotype response for different conjugates has been noted by others [14] who concluded that the magnitude of the ELISA response is likely to be an inherent property of the individual polysaccharides. The functionality of pneumococcal antibodies can be directly measured by assessing their opsonophagocytic activity. However, to be used as a primary read-out, OPA assays require validation and technical development to allow a highthroughput application. This means that OPA measurements have mainly been used on a limited number of samples to characterize immune responses. In the PD conjugate efficacy and follow-up studies a validated high-throughput OPA assay was employed [10]. Following primary vaccination, a clear OPA immune response for all the PD conjugate vaccine serotypes was observed indicating the induction of functionally active antibodies. As with ELISA there was a considerable variation in OPA levels however, the ranking of serotypes in terms of magnitude of post-primary OPA GMTs did not necessarily reflect the post-primary ELISA GMC ranking. For example, the ELISA responses to 6B and 23F were among the lowest while the OPA responses
were among the highest and vice versa for serotype 3. Such serotype-specific differences are not understood, but may relate to differences in complement deposition on the surface of different serotypes. In addition to the induction of satisfactory antibody responses in infancy, another important characteristic of conjugate vaccines is the ability to induce immunological memory. As demonstrated here and elsewhere [12,14] antibody levels gradually diminish after the primary series of immunizations. However, induction of immune memory implies that subsequent exposure to the pathogen or another dose of vaccine allows a vigorous response due to the presence of an expanded pool of memory B cells. A fourth dose of PD conjugate was shown to induce enhanced ELISA (except for serotype 3) and OPA responses, indicative of the induction of immune memory. Induction of B cell memory was previously demonstrated by the strong antibody response following administration of native polysaccharide in the second year of life as a booster to children primed with PD conjugate vaccine [3]. Post-licensure experience with the 7-valent CRM-conjugate vaccine in the US has shown that protection against IPD was maintained despite many children not receiving the recommended booster dose. This lends support to the hypothesis that immune memory following primary conjugate vaccination confers long term protection, at least following the USA schedule of 2–4–6 months for primary immunization, although herd immunity may also have played a role [15]. The pattern of antibody persistence in the period between the conjugate booster dose in the second year of life and the polysaccharide booster 2 years later was clearly serotype dependent. For the majority of serotypes, a sharp decline in antibody levels was observed, while for serotypes 6B, 14, 19F and 23F, higher levels were maintained. During this period there was also a notable increase in antibody levels in the un-primed group for the same serotypes indicating that the higher antibody levels against these serotypes were probably the result of natural exposure to the circulating pneumococci. The natural immunity appeared to be more evident when measured by IgG ELISA in the conjugate primed group and when measured by OPA in the un-primed group. This probably reflects a predominantly IgG memory response in the conjugate primed subjects and a predominantly IgM early primary response in the un-primed children. The presence of polysaccharide-specific B cell memory following PD-conjugate primary and booster vaccination was demonstrated by the vigorous response to native polysaccharide. Given the age at which children received the 23-valent polysaccharide booster in this study (in their fourth year of life) an immune response could be expected even in unprimed children. For most serotypes a significant difference between conjugate primed versus un-primed children could however be observed for both ELISA and OPA immune responses following polysaccharide vaccination, and this can therefore be considered as evidence of a sustained conjugate vaccine induced immune memory.
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A significant observation in the study was that the administration of native polysaccharide succeeded in eliciting both ELISA and OPA responses to serotype 3 in the PD conjugate vaccinees. Although the absolute post-vaccination serotype 3 antibody levels were the lowest of all vaccine serotypes in the primed group, they were at least as high as the serotype 3 antibody levels induced in the un-primed group (P = 0.0079 in favour of the conjugate group). This indicates that the capacity of children vaccinated with serotype 3 PD conjugate to respond to native serotype 3 polysaccharide, and thus to natural infection, is not impaired. Therefore, although there may be a relatively narrow window of tolerance following primary vaccination with serotype 3 PD conjugate, this does not seem to be sustained over time. It has been demonstrated that conjugate vaccines can provide some degree of protection against vaccine-related pneumococcal serotypes, except for serotype 19A [16]. The cross-reactivity between serotypes 19F and 19A is known to be limited, and the existing 23-valent plain polysaccharide vaccine contains both 19A and 19F. This contrasts to serotype 6A and 6B for which cross-protection has been demonstrated [16]. In this report, a conjugate induced booster response in the second year of life could be demonstrated by OPA and ELISA against serotypes 6A and 19A, despite the absence of a primary response against these serotypes (with the exception of a clear OPA response against 6A that could be measured following primary immunization). OPA activity for 6A and 19A following PD conjugate vaccination has also been measured by Finnish investigators [17]. It could therefore be speculated that the PD conjugate vaccine might indeed provide some protection against these cross-reactive serotypes, as already suggested by the AOM efficacy results, although these did not reach statistical significance [4]. In conclusion, pneumococcal PD conjugate primary vaccination in young children stimulates the production of functional serotype-specific antibodies and induces antigenspecific memory B cells for lasting immunity.
Acknowledgements The authors would like to thank all the infants and their families as well as the study investigational team in the Czech Republic and Slovak Republic. The authors are indebted to Patricia Lommel for statistical support, Isabelle Henckaerts for serological assays, Joelle Onkelinx for central study coordination (GlaxoSmithKline Biologicals, Rixensart, Belgium) and Miriam Hynes (independent, UK) for writing assistance in preparation of the manuscript. This study was supported by GlaxoSmithKline Biologicals, Rixensart, Belgium (Study ID: 104083/NCT00169507). HavrixTM is a trademark of GlaxoSmithKline Group of Companies. Pneumo 23® is a registered trademark of Aventis Pasteur.
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