Vaccination against Streptococcus pneumoniae with currently used 10- and 13-valent pneumococcal conjugate vaccines (PCV-10 and PCV-13) has been shown to dramatically reduce vaccine-type invasive pneumococcal disease when included in childhood immunization schedules.1–5 Both vaccines are immunogenic and have been shown to induce immune memory.6,7
There is limited information on the interchangeability of PCVs; however; there are potential reasons why it may be advantageous to include PCV-10 in the infant vaccination schedule. Nontypeable Haemophilus influenzae (NTHi) protein D is not included in any concurrent or previously administered vaccine. Its use may, therefore, reduce the risk of immune interference and theoretically increase the immune response to the polysaccharides. Immune interference can occur if the same carrier protein is used in concurrent or sequential vaccinations and can suppress the immune response to the desired antigen.8 In addition, use of NTHi protein D as a carrier protein may confer additional protection against NTHi infection, a common cause of otitis media in children,9 although effectiveness of PCV-10 against NTHi carriage or disease has not been demonstrated in previous vaccine trials.10–12
Ideally, a vaccine should provide long-lasting protection in the form of antibodies, but should also induce immunological memory through the formation of memory B cells (BMEM). A number of investigators have studied this outcome by measuring BMEM frequency in peripheral blood after vaccination.13–16 The majority of BMEM is thought to reside in lymphoid tissue, but small numbers of circulating BMEM can be detected in peripheral blood even years after vaccination. The frequency of these cells in peripheral blood rises shortly after vaccination, particularly after booster doses.17 Re-encounter with antigen triggers the proliferation of BMEM and their differentiation into antibody-secreting plasma cells, giving rise to a rapid and effective secondary immune response. The presence of BMEM may also contribute to the maintenance of circulating antibody by intermittent or continuous differentiation into antibody-secreting plasma cells in response to antigen-dependent or independent stimulation.18
This study evaluated the potential for the use of PCV-10 as a booster after priming with PCV-13 in infancy by assessing noninferiority of the proportions of participants with postbooster IgG ≥ 0.35 μg/mL for PCV-10 serotypes. The results of antibody measurements in this cohort of children have already been published and showed that in PCV-13-primed infants, a booster dose of PCV-10 induces a strong antibody response, which is generally less pronounced than the response after a PCV-13 booster.19 In the analysis presented here, we investigated the frequencies of peripheral blood BMEM before and at 1 and 12 months after a 12-month booster dose of PCV-10 or PCV-13. Using a cultured enzyme-linked immunospot (ELISpot) assay, we studied the BMEM response to 2 different pneumococcal vaccines, of which only one had previously been given to study participants. By comparing the booster response to the 2 vaccines, which differ in the concentrations of pneumococcal polysaccharides, the method of conjugation and the type and concentrations of the carrier proteins, we had the unique opportunity to study the effects of a mixed vaccination schedule on B cell biology.
MATERIALS AND METHODS
Subjects and Vaccines
Healthy children who had been vaccinated with the PCV-13 at 2 and 4 months of age were recruited as previously described19 (Fig., Supplemental Digital Content 1, http://links.lww.com/INF/C656). Ethical approval was obtained from the Oxfordshire Research Ethics Committee (reference number 11/SC/0473), and the study was registered on Clinicaltrials.gov (registration number NCT01443416). After enrolment into the study, these children were randomized to receive either PCV-10 [Synflorix, GSK Biologicals; containing capsular polysaccharides of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F, conjugated to NTHi protein D, tetanus toxoid (18C) or diphtheria toxoid (19F), respectively] or PCV-13 [Prevenar 13, Pfizer; containing serotypes 3, 6A and 19A in addition to PCV-10 serotypes, conjugated to diphtheria toxin mutant or cross reactive material (CRM197)]. Blood samples were taken on 3 visits: at day 0 immediately before vaccination (12 months of age) and at 1 and 12 months after vaccination (13 and 24 months of age).
Peripheral Blood Mononuclear Cell isolation and ELISpot
The cultured ELISpot assay for the detection of antigen-specific BMEM was performed as previously described.20 First, peripheral blood mononuclear cells were isolated from whole blood by density gradient centrifugation with lymphoprep (Axis-Shield). Cells were then cultured for 6 days at 37°C in 5% carbon dioxide and 95% humidity with an antigen stimulation mix containing Staphylococcus aureus Cowan strain (Calbiochem-Novabiochem) at a 1:5000 dilution, poke weed mitogen (Sigma-Aldrich) at a final concentration of 83 ng/mL and CpG oligonucleotide (InvivoGEN) at a final concentration of 1.7 μg/mL. After harvesting, cells were washed and seeded at 2 × 105 viable cells per well onto a 96-well plate with PVDF membranes (Millipore). Membranes were precoated with either pneumococcal polysaccharides (1, 3, 4, 9V, 14 and 19A; LCG Promochem) conjugated to methylated human serum albumin (NIBSC UK), diphtheria toxoid (10 μg/mL, also used to represent the CRM197 carrier protein; Statens Serum Institut), or tetanus toxoid (5 μg/mL, Statens Serum Institut). Pneumococcal serotypes were chosen to reflect serotypes contained in both PCV-10 and PCV-13 (serotypes 1, 4, 9V and 14) or unique to PCV-13 (serotypes 3, 19A). Plates included phosphate-buffered saline wells and polyvalent goat anti-human immunoglobulin (10 μg/mL) wells as negative and positive controls, respectively. After overnight incubation, plates were washed, and bound IgG antibody was detected using a goat anti-human IgG alkaline phosphatase conjugate (Calbiochem) and alkaline phosphatase substrate kit (Bio-Rad). Plates were dried overnight before being read using an automated ELISpot reader (AID ELR03, AID Diagnostika). All plate readings were manually checked to exclude artifacts.
The average number of spots per well was used to calculate the number of antibody-secreting cells per million peripheral blood mononuclear cells. If any spots were observed in the control phosphate-buffered saline wells, the average count across these control wells was subtracted from the average count from each type of antigen-coated well. Wells coated with total immunoglobulin were used as positive controls. Results of zero were assigned the value of 0.625 per million peripheral blood mononuclear cells (half of the lower limit of detection). Geometric mean frequencies (GMF) and corresponding 95% confidence intervals were calculated for each group and study time point. Differences between groups and time points were investigated using 2-sample t tests of logarithmically transformed BMEM frequencies and Satterthwaite’s correction for unequal variances. Correlations between responses at different time points were assessed using the Pearson correlation coefficient.
Out of a total of 434 blood samples across both groups and the 3 study visits from the original serological analysis,19 377 (87%) samples had cells available for BMEM and antibody analysis. The breakdown of samples by visit and vaccine group is shown in Table 1.
Antigen-specific BMEM GMF by Antigen and Vaccine Group
At 12 months of age, BMEM frequencies were low, and no significant differences between the groups were seen in BMEM GMF for all antigens tested (Figure 1; Table, Supplemental Digital Content 2, http://links.lww.com/INF/C657). One month after booster vaccination at 13 month of age, significantly higher BMEM GMF were found for serotypes 1, 4, 9V and 3 in the PCV-13 compared with the PCV-10 group, whereas BMEM responses to tetanus toxoid were statistically higher in PCV-10 than in PCV-13 recipients (Figure 1; Table, Supplemental Digital Content 2, http://links.lww.com/INF/C657). One year after booster at 24 months of age, no significant differences were detected between the groups for any of the antigens tested (Figure 1; Table, Supplemental Digital Content 2, http://links.lww.com/INF/C657).
Antigen-specific BMEM Frequency Fold Changes
Significant increases between BMEM measured at age 12 and 13 months were seen for most pneumococcal serotypes in the PCV-13 group (with the exception of serotype 14, which showed a 1.6-fold increase with a borderline P value of 0.05) and none in the PCV-10 group (Table 2). For antigens representing carrier proteins, a significant rise in BMEM frequencies was seen for both diphtheria and tetanus toxoid in the PCV-10 group but only for diphtheria toxoid in the PCV-13 group (Table 2). When adjusted for baseline BMEM values, age, sex and ethnicity, changes in BMEM frequencies between age 12 and 13 months were significantly greater in the PCV-13 compared with the PCV-10 group for all pneumococcal serotypes and statistically superior in PCV-10 compared with PCV-13 recipients only for tetanus toxoid (Table 2). Frequencies of BMEM were not significantly different between age 12 and 24 months for most serotypes in both groups. In the PCV-10 group, significantly higher BMEM frequencies were seen for serotypes 14 (GMF of 3.83 versus 1.91) and 19A (4.76 versus 2.66) at 24 months compared with 12 months of age, whereas in the PCV-13 group, a significant fold increase was only seen for serotype 3 (2.88 versus 1.61; Table, Supplemental Digital Content 3, http://links.lww.com/INF/C658). However, when comparing these fold changes from age 12 to 24 months between the vaccine groups, no differences were seen when adjusted for baseline values, age, sex and ethnicity (data not shown).
Correlation Between Serotype-specific BMEM and Antibody Responses
Pearson correlation was used to investigate correlations between log-transformed BMEM frequencies at different time points and between BMEM and antibody responses (both IgG concentration and opsonophagocytic assay titers). All correlations and their statistical significance are shown in Figure 2. The most striking correlations were seen between BMEM and antibody, both at age 13 months. Antibody and BMEM were correlated at 12 months and at 24 months of age for some serotypes; however, this relationship was less consistent than that seen at age 13 months. BMEM at 12 and 13 months of age were also predictive of later antibody responses for some serotypes in each group, but again this relationship was not consistent across serotypes.
A significant increase in BMEM frequencies from 12 to 13 months of age against the majority of the tested serotypes was only observed for the PCV-13 group, but the extent of the response was serotype dependent (Table 2). In PCV-13 recipients, the immune response against serotype 3 was associated with a strong increase in peripheral BMEM (Figure 2) and associated with a weak antibody responses compared with other serotypes.19 In contrast, antibody responses against serotype 14 were strong,19 whereas it was associated with only a small increase in postbooster BMEM frequency (Figure 2).
This study provides an assessment of the BMEM responses after booster vaccination with either PCV-13 or PCV-10 in children previously primed with 2 doses of PCV-13. Our results show that PCV-13-primed children do not generate peripheral BMEM in response to a PCV-10 booster. This is a surprising finding because 4 of the 6 serotypes that were evaluated are contained in both vaccines. However, the vaccines differ in the concentrations of pneumococcal polysaccharides, the method of conjugation and the type and concentrations of the carrier proteins, which may result in diverging presentation and processing of antigens.
One recent study assessed the short-term (7–9 days after booster) immunogenicity and the plasma and BMEM response around a booster dose given at 11 months of age after a vaccination series with either PCV-10 or PCV-13.21,22 This study showed that the pneumococcal serotype-specific frequencies of peripheral blood BMEM measured before and shortly after a PCV booster were significantly higher in PCV-13 compared with those in PCV-10 recipients for 3 out of 4 serotypes common to both vaccines.21 No such differences were seen for serotype-specific plasma cell responses,21 but the study also showed statistically superior postbooster IgG responses in the PCV-13 compared with the PCV-10 group to the majority of serotypes common to both vaccines.22 After 4 doses of PCV-10, a significant BMEM booster response was observed21; however, even in that study, PCV-13 appeared to be a more potent inductor of BMEM than PCV-10. Interchangeability of PCVs was not assessed in that study because children received the same booster vaccine as they had received in infancy.
In the present clinical trial, in the PCV-10 group, no change in serotype-specific BMEM frequencies was seen between before booster and 1 month later booster vaccination, whereas a significant increase in BMEM specific for diphtheria and tetanus toxoid was noted between these 2 study time points. Children who were allocated to the PCV-10 group had previously received 2 doses of PCV-13 and were, therefore, already primed with all pneumococcal serotypes contained in PCV-10. However, their immune system had not been exposed to the same conjugates and the carrier protein D derived from NTHi in the form of a vaccine, although some may have encountered it through carriage or disease. Priming and boosting with different carrier proteins has previously been investigated in children vaccinated against meningitis C.23 Children primed with a dose of tetanus toxoid–conjugated polysaccharide (MenC-TT) at 3 months generated better BMEM responses to a MenC-TT booster at 12 months than those primed with either 1 or 2 doses of CRM-conjugated polysaccharide (MenC-CRM). In the present study, BMEM booster responses to serotypes 18C and 19F, which are conjugated to tetanus and diphtheria toxoid, respectively, were not assessed. Investigating 18C and 19F BMEM responses to a booster dose of PCV-10 in PCV-13-primed children may have provided further insight into whether exposure to the carrier protein through routine vaccination (as these children have already received several doses of diphtheria and tetanus toxoid–containing vaccines) is enough to generate pneumococcal B cell memory in these children or whether the carrier protein has to be conjugated to pneumococcal polysaccharides to achieve effective priming. In the meningitis C study, children primed with MenC-CRM generated inferior booster B cell responses to a MenC-TT booster, despite previous exposure to tetanus toxoid in other routine vaccinations,23 suggesting that in children of this age group, priming with a similar carrier protein is essential for BMEM generation. Overall, the findings of the present study suggest that in children previously primed with PCV-13, the protein D–conjugated polysaccharides in a booster dose of PCV-10 appear to evoke a primary rather a secondary immune response. This may involve, in the short term, processing of antigens similar to plain polysaccharides (which do not generate BMEM responses) rather than recognition as T-dependent antigens. Future studies are needed to explore in detail the observed lack of B cell responses after PCV-10 vaccination, especially by investigating responses in children primed with PCV-10 and boosted with PCV-13.
In the group of children who were primed and boosted with PCV-13, the BMEM peak at age 13 months was followed by a return to almost baseline by age 24 months for most serotypes, by which time most cells have probably transited to lymph nodes. In PCV-10 recipients, previously receiving PCV-13 primary vaccination, an increase in BMEM frequencies was seen between 12 and 24 months of age for all serotypes tested, which was significant for serotype 14 and serotype 19A (Table, Supplemental Digital Content 3, http://links.lww.com/INF/C658). These findings suggest that there was a slower generation of BMEM in response to PCV-10 vaccination or ongoing generation through natural exposure via carriage,24,25 resulting in some increase in BMEM frequencies in the peripheral blood 1 year after the booster vaccination.
It is possible that because of differences between polysaccharides in the vaccine and those bound to the ELISpot plates, the assay used in this study was less able to detect BMEM induced by the PCV-10 vaccine, despite their presence in peripheral blood. However, the fact that a late rise in BMEM was detected in the PCV-10 group at age 24 months makes this explanation less likely.
In the PCV-13 group, the most marked BMEM response was seen for serotype 3. However, the immune response to serotype 3 polysaccharide has previously been shown to be atypical, and antibody responses to a booster appear to be particularly impaired.26 Compared with other serotypes, immunogenicity for serotype 3 is attenuated and a serotype 3–containing vaccine has previously failed to show protective efficacy against serotype 3 otitis media,9 although postimplementation surveillance suggests that immunization with PCV-13 does have some efficacy against serotype 3.27 Despite diminished antibody production, the BMEM response to serotype 3 polysaccharide has previously been shown in adults to resemble that of other serotypes.28 In the present analysis, serotype 3 showed both the greatest increase in BMEM frequency and the lowest antibody response of all PCV-13 serotypes (Figure 1),19 suggesting that one reason for impaired serotype 3–specific antibody concentrations after a booster might be that the immune response is driven towards the generation of BMEM rather than antibody-secreting cells but still provides some protection.
A question addressed by a number of studies has been whether baseline BMEM positively correlate with later antibody responses.17 A correlation between BMEM frequency after priming and antibody persistence at 1 year was found in children receiving the meningococcal serotype C conjugate vaccine,16 and for some meningococcal serogroups in children receiving the MenACWY vaccine29; however, no relationship between BMEM and antibody was found in older children receiving a booster dose of the Hib-MenC conjugate vaccine.30 Here we show that BMEM and antibody responses 1 month after booster correlate well for most serotypes, particularly in PCV-13 recipients; however, baseline BMEM are not a consistent predictor of postvaccination BMEM and IgG responses. This may reflect the unmeasured contribution of other antibody-producing cell types, such as long-lived plasma cells.
Here we present the first study assessing the BMEM response to a mixed PCV schedule. We were unable to detect serotype-specific BMEM after a booster dose of PCV-10 given to children who had been primed with PCV-13. In contrast, a strong serotype-specific BMEM response was generated in children primed with PCV-13 after receipt of a PCV-13 booster. These findings suggest that immunizing with a PCV containing polysaccharides conjugated to a novel carrier protein is not sufficient to generate a rapid and strong BMEM response, at least when primary vaccination with PCV-13 is followed by a booster dose of PCV-10. Although the clinical implications of these results are unknown, they still indicate that a vaccination series only using PCV-13 is advantageous over a mixed PCV schedule consisting of a priming series with PCV-13 and boosting with PCV-10.
The authors are grateful to all the study participants, without whom this study would have not been possible. A.J.P. and M.D.S. are Jenner Institute Investigators.
Authors’ contributions: Study concept and design: J.T., M.D.S., and A.J.P. Acquisition of data: J.T. and D.G. Statistical analysis: S.J., J.T., and M.V. Interpretation of the data: J.T., M.D.S, and A.J.P. Drafting of the article: J.T. Critical revision of the article for important intellectual content: all authors.
1. Palmu AA, Jokinen J, Borys D, et al. Effectiveness of the ten-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-CV10) against invasive pneumococcal disease: a cluster randomised trial. Lancet 2013;381:214–222.
2. Miller E, Andrews NJ, Waight PA, et al. Effectiveness of the new serotypes in the 13-valent pneumococcal conjugate vaccine. Vaccine 2011;29:9127–9131.
3. Kaplan SL, Barson WJ, Lin PL, et al. Early trends for invasive pneumococcal infections in children after the introduction of the 13-valent pneumococcal conjugate vaccine. Pediatr Infect Dis J. 2013;32:203–207.
4. Domingues CM, Verani JR, Montenegro Renoiner EI, et al. Effectiveness of ten-valent pneumococcal conjugate vaccine against invasive pneumococcal disease in Brazil: a matched case-control study. Lancet Respir Med. 2014;2:464–471.
5. Moore CE, Paul J, Foster D, et al. Reduction of invasive pneumococcal disease 3 years after the introduction of the 13-valent conjugate vaccine in the Oxfordshire region of England. J Infect Dis. 2014;210:1001–1011.
6. Knuf M, Pankow-Culot H, Grunert D, et al. Induction of immunologic memory following primary vaccination with the 10-valent pneumococcal nontypeable haemophilus influenzae protein d conjugate vaccine in infants. Pediatr Infect Dis J. 2012;31:e31–e36.
7. Quinet B, Laudat F, Gurtman A, et al. Pneumococcal conjugate vaccine-elicited antibody persistence and immunogenicity and safety of 13-valent pneumococcal conjugate vaccine in children previously vaccinated with 4 doses of either 7-valent or 13-valent pneumococcal conjugate vaccine. Pediatr Infect Dis J. 2014;33:1065–1076.
8. Dagan R, Poolman J, Siegrist CA. Glycoconjugate vaccines and immune interference: a review. Vaccine. 2010; 28:5513–5523.
9. Prymula R, Peeters P, Chrobok V, et al. Pneumococcal capsular polysaccharides conjugated to protein D for prevention of acute otitis media caused by both Streptococcus pneumoniae and non-typable Haemophilus influenzae: a randomised double-blind efficacy study. Lancet. 2006;367:740–748.
10. Van Den Bergh MR, Spijkerman J, Swinnen KM, et al. Effects of the 10-valent pneumococcal nontypeable haemophilus influenzae protein d-conjugate vaccine on nasopharyngeal bacterial colonization in young children: a randomized controlled trial. Clin. Infect. Dis. 2013;56:30–39.
11. Vesikari T, Forsten A, Seppä I, et al. Effectiveness of the 10-Valent Pneumococcal Nontypeable Haemophilus influenzae Protein D–Conjugated Vaccine (PHiD-CV) Against Carriage and Acute Otitis Media—A Double-Blind Randomized Clinical Trial in Finland. J Pediatr Infect Dis Soc. 2016;5:237–248.
12. Tregnaghi MW, Sáez-Llorens X, López P, et al. Efficacy of pneumococcal nontypable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) in young Latin American children: a double-blind randomized controlled trial. PLoS Med. 2014;11:e1001657.
13. Blanchard-Rohner G, Pulickal AS, Jol-van der Zijde CM, et al. Appearance of peripheral blood plasma cells and memory B cells in a primary and secondary immune response in humans. Blood. 2009;114:4998–5002.
14. Kelly DF, Snape MD, Perrett KP, et al. Plasma and memory B-cell kinetics in infants following a primary schedule of CRM 197-conjugated serogroup C meningococcal polysaccharide vaccine. Immunology. 2009;127:134–143.
15. Clutterbuck EA, Salt P, Oh S, et al. The kinetics and phenotype of the human B-cell response following immunization with a heptavalent pneumococcal-CRM conjugate vaccine. Immunology. 2006;119:328–337.
16. Blanchard Rohner G, Snape MD, Kelly DF, et al. The magnitude of the antibody and memory B cell responses during priming with a protein-polysaccharide conjugate vaccine in human infants is associated with the persistence of antibody and the intensity of booster response. J Immunol. 2008;180:2165–2173.
17. Mitchell R, Kelly DF, Pollard AJ, et al. Polysaccharide-specific B cell responses to vaccination in humans. Hum Vaccin Immunother. 2014;10:1661–1668.
18. Amanna IJ, Slifka MK. Mechanisms that determine plasma cell lifespan and the duration of humoral immunity. Immunol Rev. 2010;236:125–138.
19. Trück J, Jawad S, Goldblatt D, et al. The antibody response following a booster with either a 10- or 13-valent pneumococcal conjugate vaccine in toddlers primed with a 13-valent pneumococcal conjugate vaccine in early infancy. Pediatr Infect Dis J. 2016;35:787–793.
20. Clutterbuck EA, Lazarus R, Yu LM, et al. Pneumococcal conjugate and plain polysaccharide vaccines have divergent effects on antigen-specific B cells. J Infect Dis. 2012;205:1408–1416.
21. van Westen E, Wijmenga-Monsuur AJ, van Dijken HH, et al. Differential B cell memory around the 11-month booster in children vaccinated with a 10- or 13-valent pneumococcal conjugate vaccine. Clin Infect Dis. 2015;61:342–349.
22. Wijmenga-Monsuur AJ, Van Westen E, Knol MJ, et al. Direct comparison of immunogenicity induced by 10- or 13-valent pneumococcal conjugate vaccine around the 11-month booster in Dutch infants. PLoS One. 2015;10:e0144739.
23. Khatami A, Clutterbuck EA, Thompson AJ, et al. Evaluation of the induction of immune memory following infant immunisation with serogroup C Neisseria meningitidis conjugate vaccines—exploratory analyses within a randomised controlled trial. PLoS One. 2014;9:e101672.
24. Flasche S, van Hoek AJ, Sheasby E, et al. Effect of pneumococcal conjugate vaccination on serotype-specific carriage and invasive disease in England: a cross-sectional study. PLoS Med. 2011;8:14.
25. Tocheva AS, Jefferies JMC, Rubery H, et al. Declining serotype coverage of new pneumococcal conjugate vaccines relating to the carriage of Streptococcus pneumoniae in young children. Vaccine. 2011;29:4400–4404.
26. Poolman J, Kriz P, Feron C, et al. Pneumococcal serotype 3 otitis media, limited effect of polysaccharide conjugate immunisation and strain characteristics. Vaccine. 2009;27:3213–3222.
27. Waight PA, Andrews NJ, Ladhani SN, et al. Effect of the 13-valent pneumococcal conjugate vaccine on invasive pneumococcal disease in England and Wales 4 years after its introduction: an observational cohort study. Lancet Infect Dis. 2015;15:535–543.
28. Trück J, Lazarus R, Clutterbuck EA, et al. The zwitterionic type I Streptococcus pneumoniae polysaccharide does not induce memory B cell formation in humans. Immunobiology. 2013;218:368–372.
29. Blanchard-Rohner G, Snape MD, Kelly DF, et al. The B-cell response to a primary and booster course of MenACWY-CRM197 vaccine administered at 2, 4 and 12 months of age. Vaccine. 2013;31:2441–2448.
30. Perrett KP, Jin C, Clutterbuck E, et al. B cell memory to a serogroup C meningococcal conjugate vaccine in childhood and response to booster: little association with serum IgG antibody. J Immunol. 2012;189:2673–2681.