The Magnitude of the and Memory Responses during with a Protein-Polysaccharide Conjugate in Human Infants Is Associated with the This information is current as Persistence of Antibody and the Intensity of of September 26, 2021. Booster Response Geraldine Blanchard Rohner, Matthew D. Snape, Dominic F. Kelly, Tessa John, Anita Morant, Ly-Mee Yu, Astrid

Borkowski, Francesca Ceddia, Ray Borrow, Claire-Anne Downloaded from Siegrist and Andrew J. Pollard J Immunol 2008; 180:2165-2173; ; doi: 10.4049/jimmunol.180.4.2165 http://www.jimmunol.org/content/180/4/2165 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Magnitude of the Antibody and Responses during Priming with a Protein-Polysaccharide Conjugate Vaccine in Human Infants Is Associated with the Persistence of Antibody and the Intensity of Booster Response1

Geraldine Blanchard Rohner,2* Matthew D. Snape,* Dominic F. Kelly,* Tessa John,* Anita Morant,* Ly-Mee Yu,† Astrid Borkowski,§ Francesca Ceddia,¶ Ray Borrow,‡ Claire-Anne Siegrist,ʈ and Andrew J. Pollard*

Rapid waning of anti-polysaccharide bactericidal Ab and vaccine effectiveness is observed following infant with the serogroup C meningococcal (MenC) glycoconjugate vaccine. This is despite the demonstrable presence of immunological memory. Downloaded from Persistence of functional Ab, therefore, appears to be the key determinant of MenC conjugate vaccine effectiveness. Ab persistence is thought to depend in the short term on the survival of plasma cells generated during priming and in the longer term on the production of new Ab secreting cells from memory B cells. In this study, we found a strong association between the level of MenC-specific Ab and the frequency of memory B cells measured at 5 mo of age (1 mo after 3-dose primary immunization with MenC conjugate vaccine), and the persistence of functional Ab at one year of age. These findings suggest that these two parameters are good markers of B cell responses to priming and can be used as predictors of long term humoral induced by http://www.jimmunol.org/ glycoconjugate received in early infancy. The Journal of Immunology, 2008, 180: 2165–2173.

eisseria meningitidis is one of the principal causes of only validated correlate of protection is the presence of serum bacterial meningitis and septicaemia in children, with a bactericidal activity (SBA), measured in an in vitro assay using N peak incidence at the age of 6–24 mo. In response to an either rabbit (rSBA) or human SBA complement, with a titer Ն1:8 increase in the number of cases of meningococcal disease caused or 1:4, respectively (2, 6). by serogroup C meningococci during the 1990s, serogroup C me- The introduction of MenCV in the U.K. in 1999 led to a marked ningococcal (MenC)3 conjugate vaccine was introduced into the decline in the number of cases of serogroup C meningococcal dis- by guest on September 26, 2021 U.K. routine infant immunization schedule in 1999 and was of- ease. However, a study of the direct effectiveness of the vaccine fered to all individuals Ͻ19 years in a catch-up campaign (1). reported that, although the vaccine was highly effective during the MenC conjugate vaccine (MenCV) induces bactericidal capsular first year after immunization in all age groups, effectiveness could polysaccharide-specific Abs, the presence of which correlate with not be demonstrated beyond the first year after immunization protection against the disease (2). Several different factors are among those receiving a 3-dose infant schedule (5). Other studies thought to contribute to long term protection after immunization have reported (7, 8) a rapid drop in bactericidal Ab titer by one with conjugate vaccines: herd immunity, persistence of anticapsu- year of age after infant immunization despite the persistence of lar Abs in serum and immunological memory (3–5). However, the immunological memory, which appears to correspond with this decline in effectiveness. In support of the view that functional Ab is necessary for long *Oxford Vaccine Group, Centre for Clinical Vaccinology and Tropical Medicine and †Centre for Statistics in Medicine, University of Oxford, Oxford, United Kingdom; term protection, studies of Haemophilus influenzae type b (Hib) ‡Vaccine Evaluation Unit, Manchester Medical Microbiology Partnership, Manches- and MenC vaccine failures have demonstrated that some children § ter Royal Infirmary, Manchester, United Kingdom; Novartis Vaccines and Diag- developed Hib or meningococcal infections despite the apparent nostics, Marburg, Germany; ¶Novartis Vaccines and Diagnostics, Siena, Italy; and ʈCenter of Vaccinology and Neonatal Immunology, University of Geneva, Ge- presence of immunological memory (9, 10). In these studies, an- neva, Switzerland ticapsular Ab levels and avidity indices were measured in acute Received for publication August 29, 2007. Accepted for publication November and convalescent serum samples obtained from individuals who 26, 2007. had suffered from infection with the encapsulated organism and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance who had either received prior immunization (vaccine failures) or with 18 U.S.C. Section 1734 solely to indicate this fact. were unimmunized. Immunological memory in these studies was 1 This work was supported by Novartis Vaccines. A.J.P. is a Jenner Institute inferred by the presence of significantly higher Ab levels during Investigator. convalescence in the previously vaccinated subjects (vaccine fail- 2 Address correspondence and reprint requests to Dr. Geraldine Blanchard Rohner, ures) than in the unimmunized individuals. Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, OX3 7LJ, U.K. E-mail address: [email protected] Most studies that have examined the kinetics of the Ab response in primed individuals have observed an increase in functional Ab 3 Abbreviations used in this paper: MenC, serogroup C meningococcal; MenCV, MenC conjugate vaccine; SBA, serum bactericidal activity; Hib, Haemophilus influ- by day 5 after immunization (11, 12). However, a recent study (13) enzae type b; PWM, Pokeweed mitogen; SAC, Staphylococcus aureus Cowan bac- of the Ab response to serogroup C polysaccharide vaccine among teria; ASC, Ab-secreting cells. children previously primed with only one dose of MenCV at 12–15 Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 mo of age, reported an increase in bactericidal Ab levels within www.jimmunol.org 2166 B CELL RESPONSE TO MENINGOCOCCAL CONJUGATE VACCINE

2–4 days of reimmunization. These observations imply that im- Anti-N. meningitidis serogroup C bactericidal activity as munological memory by itself is too slow to provide protection measured by rSBA when a primed individual acquires an invasive strain of these encap- A serogroup C N. meningitidis bactericidal assay was performed using sulated bacteria; protection afforded by the secondary rise in Ab fol- baby rabbit complement according to standard protocols (25). In brief, lowing exposure to the organism develops only after several days, two-fold dilutions of heat-inactivated sera were incubated with the C11 whereas the meningococcus invades the blood and disease develops strain of group C N.meningitidis and freshly thawed rabbit complement Ն within hours. As a result of these observations, the current immuni- (PelFreez). The last dilution producing a 50% reduction in colonies (kill- ing) compared with control wells, containing complement and bacteria, zation strategy in the U.K. depends on maintenance of protective Ab was taken as the end point rSBA titer. levels in the blood through use of a booster dose of both Hib and MenC vaccine in the second year of life (www.advisorybodies. Anti-N. meningitidis serogroup C Ab concentration as doh.gov.uk/jcvi/childhoodimmunisationoc05.pdf). measured by ELISA B cells produce Ab and are, therefore, the principal determinant The serogroup C N. meningitidis polysaccharide-specific IgG concentration of long-term protection against encapsulated bacteria after immu- was determined by ELISA following a previously described method (26). nization; indeed, it has been shown that memory B cells persist for In brief, immulon 2 microtiter plates (Thermo Electron Corporation) were ␮ several years after Ag exposure (14). It is not clear whether mem- coated with 5 g/ml serogroup C meningococcal polysaccharide (National Institute for Biological Standards and Control (NIBSC), Hertfordshire, En- ory B cells reside in secondary lymphoid organs (15) in the bone gland) conjugated to 5 ␮g/ml methylated human albumin (NIBSC) in ster- marrow (16) or whether they continuously recirculate between sec- ile PBS. Following blocking, eight twofold dilutions of the reference serum ondary lymphoid organs through the blood (17). It is presumed that (starting dilution 1/150) (CDC 1999) and test sera (starting dilution 1/25)

these memory B cells maintain protective Ab levels in serum by were made directly in the microtiter plate by well-to-well transfer with a Downloaded from multichannel pipette. The reference serum was assayed in triplicate, and continuously differentiating into plasma cells. This phenomenon test sera were assayed in duplicate. Additionally an internal quality control may be Ag-independent and may occur in response to polyclonal (an antimeningococcal adult immune serum) was diluted (1/200, 1/1000, stimuli (derived from microbes or activated T cells) (12, 18, 19). and 1/4000) to yield optical densities approximately on the high, middle, Alternatively, generation of plasma cells could be Ag-dependent and low portions of the reference curve. After overnight incubation at 4°C, ␥ (via cross-reactive Ags, reexposure to the Ag or persisting Ag) (20, microtiter plates were developed with monoclonal-PAN anti-human Fc- peroxidase Ab (diluted in serum/conjugate (S/C) buffer) (Stratech) for 2.5 h 21). Finally, murine studies have shown that a subgroup of plasma at room temperature, followed by the chromogenic substrate tetramethyl- http://www.jimmunol.org/ cells generated after immunization are long-lived and persist in benzidine dihydrochloride monohydrate (Sigma-Aldrich), and the reaction bone marrow and contribute to sustaining long term Ab production was stopped after 30 min with2MH2SO4. The OD of each well was then (22–24). read at 450 nm. Understanding the generation and maintenance of Abs by im- Anti-diphtheria toxoid IgG concentration munization is of central importance in developing methods to en- The diphtheria toxoid- IgG concentration was determined using a Luminex hance long term protection against polysaccharide encapsulated multiplex assay as previously described (27). The results were read on a bacteria in the first few years of life. In this study, we describe the Bio-Plex reader (Bio-Rad) and the data acquired by use of a computer persistence of MenC-specific B cells and Abs at one year of age software package (Bio-Plex Manager; Bio-Rad). after priming with MenCV (a conjugate vaccine containing MenC by guest on September 26, 2021 Preparation of PBMCs polysaccharide chemically conjugated onto CRM 197, a mutant diphtheria toxoid) in early infancy and evaluate the B cell and Ab Up to 5 mls heparinized blood was diluted 1/2 with RPMI 1640 medium response to a booster dose of MenCV given at one year of age. (Sigma-Aldrich) to which penicillin-streptomycin solution (Sigma-Aldrich) and L-glutamine 200 mM (Sigma-Aldrich) had been added at a dilution of Furthermore, we assess the relationship between the postprimary 1/100 (complete medium). The PBMCs were then separated by density generation of memory B cells and Abs, and humoral immunity gradient centrifugation over Lymphoprep (Axis-Shield; Diagnostics). around the 12-mo booster dose of vaccine. The MenC polysaccha- PBMCs were washed once in complete medium before further prepa- ride-specific B cell and Ab responses were compared with the ration for ELISPOT or cell culture. carrier protein (CRM197) responses. Preparation of ELISPOT plates ELISPOT plates (96-well PVDF membrane) (Millipore) were coated with Materials and Methods either 5 ␮g/ml serogroup C meningococcal polysaccharide (NIBSC) con- Subjects and vaccines jugated to methylated human albumin (NIBSC), or 10 ␮g/ml diphtheria toxoid (Statens Serum Institut), or 10 ␮g/ml goat anti-human Ig (Caltag A cohort of 72 children of Ն12 mo of age who had participated in a Laboratories) in sterile PBS. PBS alone was added to the Ag blank wells. previous study assessing their B cell response to the C polysaccharide The ELISpot plates were stored at 4°C until use. component (MenC) during routine infant immunization with MenCV at 2, 3, and 4 mo of age (study M14P5; Eudract number 2004-004962-33) was Detection of plasma cells approached and invited to participate in this “follow-on” study. A total of PBMCs prepared from peripheral blood were washed three times in com- 33 of those invited agreed to participate. After informed consent by the plete medium with 10% FCS (Sigma-Aldrich) and resuspended to a final parents, participants received a dose of 0.5 ml of meningococcal C con- concentration of 2 ϫ 106 PBMCs/ml. A total of 100 ␮l/well of the sus- jugate vaccine (MenCV; Menjugate, Novartis Vaccines and Diagnostics), pension was added to ELISpot plates, previously blocked with complete by i.m. injection into the antero-lateral thigh. The 0.5-ml dose contained 10 medium with 10% FCS, and incubated overnight at 37°C in 5% CO2 and ␮g of the polysaccharide and between 12.5 and 25 ␮g of the conjugate 95% humidity. The cells were washed with PBS-Tween 20 and bound IgG CRM (mutant protein derived from diphtheria toxoid) and was adsorbed 197 Abs detected using a 1/5000 dilution of goat anti-human IgG ␥-chain spe- on aluminum phosphate. At the same time, to boost immunity against Hib cific alkaline phosphatase conjugate (Calbiochem) in complete medium as per the routine U.K. schedule (www.advisorybodies.doh.gov.uk/jcvi/ with 10% FCS, developed using a 5-bromo-4-chloro-3-indolyl phosphate childhoodimmunisationoc05.pdf), a dose of Hib conjugate vaccine (Hi- in NBT dissolved in aqueous dimethylformamide (Bio-Rad). berix) was administered concurrently in the opposite anterolateral thigh. A 6-ml blood sample was obtained from each child before immunization and Detection of memory B cells at day 30 after immunization. The 33 children were split into five sub- groups for an additional blood sample obtained on either day 2, 4, 6, 8, or PBMCs prepared from peripheral blood were resuspended in complete me- 9 after immunization, this allowing investigation of the kinetics of the Ab, dium with 10% FCS at a final concentration of 2 ϫ 106 PBMCs/ml and plasma cell, and memory B cell response after immunization. The protocol added to 96-well round-bottom culture plates (Fisher) in 100 ␮l/well. Stim- was approved by the Oxfordshire’s Research Ethics Committees (approval ulated medium, containing 1/5000 Staphylococcus aureus (Cowan strain no. CO6/Q1604/N41; Eudract no. 2006-000732-28). bacteria; SAC) (Calbiochem), 1/6000, or 83 ng/ml Pokeweed mitogen The Journal of Immunology 2167

Table I. Ab and B cell responses to MenCV at various days following challenge with MenCV at one year of age and at 5 months of age after primary immunizationa

MenC-plasma Cells Diph-plasma cells MenC-memory Diph-memory MenC-rSBA MenC-IgG Diph-IgG Median (5th –95th Median (5th –95th B Cells Median B Cells Median Age GMT (95% CI) GMC (95% CI) GMC (95% CI) Percentile) Percentile) (5th –95th Percentile) (5th –95th Percentile) Visit (Days) [Participants] [Participants] [Participants] [Participants] [Participants] [Participants] [Participants]

Preceding 5 mo 1526 19.8 1.4 N/A N/A 11 10 study (1151, 2023) (16, 24.5) (1.1–1.8) (0, 107) (0, 82.5) ͓n ϭ 33͔͓n ϭ 33͔͓n ϭ 32͔͓n ϭ 22͔͓n ϭ 20͔ Visit 1 0 9 1.1 0.2 0 0 3.5 11.5 12 mo (4, 22) (0.7, 1.7) (0.1–0.2) (0, 0) (0, 0) (0, 9.5) (0, 52.5) ͓n ϭ 22͔͓n ϭ 22͔͓n ϭ 22͔͓n ϭ 23͔͓n ϭ 23͔͓n ϭ 23͔͓n ϭ 23͔ Visit 2 2 5 1 0.1 0 0 0 0 (0, 46) (0.3, 3) (0.1–0.4) (0, 0) (0, 0) (0, 11.5) (0, 14) ͓n ϭ 5͔͓n ϭ 4͔͓n ϭ 4͔͓n ϭ 5͔͓n ϭ 4͔͓n ϭ 4͔͓n ϭ 3͔ 4 128 1.2 0.1 7 8.5 2 0 (18, 901) (0.3, 4.4) (0.1–0.3) (0, 136) (0, 37.5) (0, 6.5) (0, 12.5) ͓n ϭ 5͔͓n ϭ 6͔͓n ϭ 6͔͓n ϭ 7͔͓n ϭ 7͔͓n ϭ 6͔͓n ϭ 4͔ 6 4705 16.3 0.7 112.5 121.5 8 14 (2290, 9667) (10.3, 25.7) (0.2–2.3) (23, 234) (21.5, 170) (0, 47.5) (0, 22.5)

͓n ϭ 5͔͓n ϭ 5͔͓n ϭ 4͔͓n ϭ 6͔͓n ϭ 6͔͓n ϭ 6͔͓n ϭ 6͔ Downloaded from 8 18390 52.9 3.2 19.5 27.5 40 81.5 (4482, 75459) (23, 121.6) (1.2–8) (5.5, 160) (0, 66.5) (1, 658.5) (7.5, 392.5) ͓n ϭ 6͔͓n ϭ 6͔͓n ϭ 6͔͓n ϭ 7͔͓n ϭ 7͔͓n ϭ 4͔͓n ϭ 4͔ 9 13004 46.2 3.3 11 42.5 23 86.5 (4813, 35131) (25.9, 82.7) (2–5.5) (7.5, 344) (0, 156.5) (0, 422.5) (20, 177.5) ͓n ϭ 6͔͓n ϭ 6͔͓n ϭ 5͔͓n ϭ 6͔͓n ϭ 5͔͓n ϭ 5͔͓n ϭ 4͔ Visit 3 30 6087 27.9 1.8 0 0 16.5 52.5 13 mo (4179, 8865) (20.3, 38.4) (1.3–2.5) (0, 5) (0, 22.5) (0, 111) (0, 220) http://www.jimmunol.org/ ͓n ϭ 28͔͓n ϭ 28͔͓n ϭ 28͔͓n ϭ 26͔͓n ϭ 16͔͓n ϭ 28͔͓n ϭ 25͔

a MenC-rSBA GMT, MenC-, and diphtheria toxoid-IgG GMCs (in ␮g/mL and IU/mL); MenC- and diphtheria toxoid-plasma cells (in ASCs per million PBMCs), MenC-, and diphtheria toxoid-memory B cells (in ASCs per million cultured lymphocytes).

(PWM) (Sigma-Aldrich) and 1/40 or 2.5 ␮g/ml CpG oligonucleotide used for all plates and Ags and the operator was blinded to which sample (ODN-2006) (Invitrogen). The cells were incubated at 37°C in 5% CO2 for was being counted. 5.5 days before being re-suspended and washed four times in complete Ab forming spots were large, spherical in size with “fuzzy” granular medium with 10% FCS. The cultured cells were plated onto precoated edges. Spots that did not fit this description were not counted and consid- by guest on September 26, 2021 ELISPOT plates at 2 ϫ 105 cells/well and then incubated and developed as ered as in vitro artifact. for the ex vivo ELISPOT described above. Statistical analysis ELISPOT counting For the purposes of analysis, ELISPOT assays in which fewer than four Spots were counted using an AID ELISPOT Reader ELR02 (AID) and spots were detected were treated as though no Ab-secreting cells (ASCs) ELISPOT software, version 3.2.3 (Cadama Medical). Spot-forming cells were detected. The number of replicate wells used depended on the total were counted and confirmed by visual inspection. Identical settings were number of PBMCs available, which varied between individuals and at the

FIGURE 1. MenC specific- IgG (a), bacteri- cidal Ab (c), and diphtheria toxoid specific-IgG (b) responses to immunization with MenCV at one year of age. a, Using an ELISA, MenC-spe- cific IgG response was measured at 5 mo of age after primary immunization and at various days following immunization with MenCV at one year of age. The horizontal bars represent the GMC at each time point. b, Using a Luminex multiplex assay, diphtheria toxoid-specific IgG response was measured at 5 mo of age after pri- mary immunization and at various days follow- ing immunization with MenCV at one year of age. The horizontal bars represent the GMC at each time point. c, Serum bactericidal activity using the rabbit complement (rSBA) was mea- sured at 5 mo of age after primary immunization and at various days following immunization with MenCV at one year of age. The horizontal bars on the graph represent the GMT at each time point. 2168 B CELL RESPONSE TO MENINGOCOCCAL CONJUGATE VACCINE

FIGURE 2. Ab decline after pri- mary immunization (from 5 to 12 mo of age) and Ab rise after a booster dose of MenCV at one year of age (from 12 to 13 mo). Results are shown for MenC- rSBA titer (a) and MenC- IgG concentration (b). Each line color represents a different child. Downloaded from different time points. For MenC specific B cell assays where at least four (40.9%) children had an rSBA titer equal or above the putative wells of 2 ϫ 105 PBMCs were used the minimum sensitivity of the assay protective threshold of 1:8 (Table I and Fig. 1c). was 5 cells per million PBMCs. For diphtheria toxoid specific B cell assay ϫ 5 By day two, after booster immunization at one year 1/5 children where at least two wells of 2 10 PBMCs were used the minimum Ն Ն sensitivity was 10 cells per million PBMCs. Stata (version 9.1; StataCorp) had an rSBA 1:8, by day four 5/6 had an rSBA titer 1:8 and from day six onwards, all children had protective levels of bacte- was used for the statistical analysis. rSBA titer and ELISA IgG concen- http://www.jimmunol.org/ trations were summarized using geometric means with corresponding 95% ricidal Ab (rSBA GMT Ն1:8). confidence intervals. For B cell numbers medians with 5th - 95th percentile The rSBA GMTs at day 30 postbooster were significantly higher were calculated. Within group comparisons of B cell numbers and Ab ϭ concentrations at varying time points were made using the Wilcoxon than the rSBA GMTs prebooster ( p 0.001) with an average of signed ranks test for paired data with the p values adjusted for multiple 654-fold rise (95% CI: 257-1664) (Table I). The postbooster rSBA comparisons using the false discovery rate method (28). The correlation GMTs were also significantly higher than the rSBA GMT one between the different variables was compared using the Spearman’s rank month postprimary immunization ( p ϭ 0.0003). correlation coefficient, using the log transformed rSBA titer and ELISA IgG concentration but untransformed B cell numbers, with the p values adjusted for multiple comparisons using the false discovery rate method Kinetics of the anti-N. meningitidis serogroup C- and (28). Furthermore, we compared children’s ability to produce memory B by guest on September 26, 2021 cells at 5 mo of age with the persisting protective Ab titer at one year of diphtheria toxoid-IgG responses after immunization age, using the Fisher’s exact test. Because of the small sample size of this At one year of age, before the booster dose of MenCV a MenC- study no multivariate analysis was run. specific IgG concentration Ն2 ␮g/ml was seen in 6/22 children, all Ն Results of whom had a protective rSBA titer ( 1:8). The prebooster con- centrations of MenC-specific IgG and diphtheria toxoid-specific Recruitment IgG were significantly lower than the GMC after 3-dose primary Between May and August 2006, all 33 children completed the immunization ( p ϭ 0.0008 for MenC and diphtheria toxoid). study. However, because of insufficient sample volumes or assay Following challenge with MenCV, levels of Abs specific for failures, not all data points were available for each time point (see MenC and diphtheria toxoid increased rapidly. At 30 days after number of participants for each result of Table I). immunization at one year of age the IgG concentrations were sig- nificantly higher than before the booster dose at 12 mo of age ( p ϭ Kinetics of anti-serogroup C N. meningitidis bactericidal 0.001 for MenC and diphtheria toxoid) with an average fold in- activity after immunization crease of 26.3 (95% CI: 16.2–42.5) for MenC-IgG and 10.7 (95% At one year of age, 8 mo after three doses of MenCV at 2, 3, and CI: 7.2–15.9) for diphtheria toxoid-IgG. Furthermore, 30 days 4 mo of age, the prebooster rSBA geometric mean titer (GMT) had post-MenCV at one year of age, the MenC-IgG concentration was declined significantly from 1526 to 9 ( p ϭ 0.0007) and only 9/22 significantly higher than after primary immunization at 5 mo of

FIGURE 3. MenC- and diphtheria toxoid- specific plasma cell responses to immunization with MenCV at one year of age. The children were immunized at day 0 and the frequency of MenC- (a) and diphtheria tox- oid (b)-specific ASCs was measured at various days after immunization. The horizontal bars represent the median number of specific ASCs at each time point. The minimum sensitivity of the assay is plotted as a broken line on the graph for each figure. In some as- says there were fewer PBMCs available for analysis and the minimum sensitivity was two-fold higher (data not shown on graphs). The zero values have been given the arbitrary value of 1 for illustrative purposes. The Journal of Immunology 2169

FIGURE 4. MenC- and diphtheria toxoid-specific memory B cells at 5 mo and after immunization with MenCV at one year of age. Using an ELISPOT assay after 6 days culture with SAC/CpG/PWM, the MenC- (a) and (b)-diphtheria toxoid-specific memory B cell response was measured at 5 mo of age after primary immunization and at various days following immunization with MenCV at one year of age. The horizontal bars on the graph represent the median number of specific ASCs at each time point. The minimum sensitivity of the assay is plotted as a broken line on the graph for each figure. In some assays there were fewer PBMCs available for analysis and the minimum sensitivity was two-fold higher (data not shown on graphs). The zero values have been given the arbitrary value of 1 for illustrative purposes. Downloaded from age ( p ϭ 0.02). However, the concentration of diphtheria toxoid- toxoid- specific plasma cells above baseline was seen at day 4 with IgG was not significantly higher ( p ϭ 0.06) (Table I and Fig. 1). a peak at day 6 and a rapid decline by day 8–9 (Fig. 3). There were almost no detectable specific plasma cells at day 30 after vacci- Ab decline after primary immunization (from 5 mo to 12 mo of nation. The median frequency of plasma cells specific for MenC age) and Ab rise after a booster dose of MenC vaccine at one and diphtheria toxoid was comparable, but MenC-specific plasma year of age (12 mo to 13 mo) cells decreased more rapidly than diphtheria toxoid-specific http://www.jimmunol.org/ After primary immunization, the decline in MenC-specific Ab plasma cells. In comparison to the response to primary immuni- (measured by rSBA titer and MenC-specific IgG concentration) zation, the peak frequency of plasma cells was higher after the was similar for all children (Fig. 2) from 5 mo (1 mo after priming booster dose of MenCV at one year of age (median of 112.5 with 3 doses of MenCV) to 12 mo of age. Following the booster MenC-plasma cells per million PBMCs and 121.5 diphtheria tox- dose of MenCV at one year of age, there was a tendency that the oid-plasma cells per million PBMCs at day 6 following one year children who had lower Ab levels (measured by rSBA titer and booster dose, compared with 50 MenC-specific plasma cells and IgG concentration) at 12 mo of age had higher increase in Ab (Fig. 83 diphtheria toxoid-specific plasma cells per million PBMCs at 2). Therefore, the rise in Ab was lower in the children who had day 4 following the third dose of MenCV at 4 mo of age) (data not higher Ab levels prebooster, although the postbooster levels in shown). by guest on September 26, 2021 these children were higher (Fig. 2). Further, the children with a rSBA titer at one year of age Ͻ1:8 had an average fold increase of 2353 (n ϭ 10), whereas those with a rSBA Ն1:8 had an average Memory B cells by cultured B cell ELISpot fold increase of 159 (n ϭ 7). The children with an IgG concen- Before the booster dose of MenCV at one year of age 7/23 (30%) tration at one year of age Ͻ2 ␮g/ml had an average of 34.4-fold children had Ն5 MenC-memory B cells per million cultured lym- increase (n ϭ 13), compared with an average fold increase of 10.9 phocytes. The median frequency of MenC-memory B cells for the (n ϭ 4) in those with an IgG concentration Ն2 ␮g/ml. Similar 23 children was 3.5 MenC-memory B cells per million cultured phenomenon were observed for the diphtheria toxoid–IgG lymphocytes. By contrast, at 5 mo of age after primary immuni- responses. zation 8/22 (36%) children had Ͻ5 memory B cells per million cultured lymphocytes. The median frequency of MenC-memory B Plasma cell frequency measured by ex vivo B cell ELISPOT cells for the 22 children was 11 MenC-memory B cells per million Before the booster dose of MenCV at one year of age there were cultured lymphocytes (Table I). The frequency of memory B cells no plasma cells specific for MenC and diphtheria toxoid detected before the one year vaccine dose was significantly lower than the in peripheral blood. The first appearance of MenC- and diphtheria memory B cell frequency reached after primary immunization at 5

Table II. Correlation of MenC-memory B cells and Ab at 5 mo of age with the Ab and memory B cells measured at 5, 12, and 13 mo of agea

Memory B Cells at 5 mo IgG at 5 mo rSBA at 5 mo

rpnrp nrpn

IgG at 5 mo 0.74 0.001 21 rSBA at 5 mo 0.71 0.001 22 0.84 Ͻ0.0001 31 IgG at 12 mo 0.69 0.01 14 0.85 Ͻ0.0001 20 0.72 0.001 22 rSBA at 12 mo 0.65 0.02 14 0.74 0.001 20 0.67 0.002 22 Memory B cells at 12 mo 0.65 0.02 15 0.65 0.003 21 0.67 0.002 23 IgG at 13 mo 0.52 0.04 18 0.67 0.001 20 0.55 0.005 28 rSBA at 13 mo 0.56 0.03 18 0.52 0.01 26 0.61 0.002 28 Memory B cells at 13 mo 0.66 0.003 21 0.61 0.003 26 0.46 0.02 28

a Spearman’s rank correlation coefficient (r) with the p values adjusted for multiple comparisons ( p) and the number of subject tested for each association (n). 2170 B CELL RESPONSE TO MENINGOCOCCAL CONJUGATE VACCINE

FIGURE 5. Correlations between MenC-mem- ory B cells and Ab at 5 mo of age with the persisting Ab at one year of age and postbooster Ab levels, using the Spearman’s correlation method (see Table II for the r and p values of each correlation). Downloaded from

mo of age ( p ϭ 0.01) (Table I). However, the frequency of mem- memory B cells, correlated with the postbooster Ab levels at 13 mo ory B cells specific for diphtheria toxoid had not significantly de- of age (r ϭ 0.6, p ϭ 0.003). clined at one year of age compared with the frequency at 5 mo http://www.jimmunol.org/ (median difference 0, range (Ϫ9, 11), p ϭ 0.6; Table I). Correlation between memory B cells at 5 mo and the rSBA titer Following the booster dose of MenCV at one year of age, the at one year of age using the Fisher exact test first response above baseline was seen by day 8 for MenC- and The presence or absence of detectable MenC specific memory B diphtheria toxoid-specific memory B cells (Fig. 4). At 30 days cells at 5 mo of age (i.e., Ն5 MenC-memory B cells per million after immunization, MenC and diphtheria toxoid-specific memory cultured lymphocytes) was found to have some evidence of cor- B cells were still detectable in blood at a significantly higher fre- relation with the maintenance of a “protective” rSBA titer (Ն1:8) ϭ ϭ quency than before the booster ( p 0.002 for MenC and p 0.02 to 1 year of age ( p ϭ 0.03) (Table III).

for diphtheria toxoid) These frequencies were also significantly by guest on September 26, 2021 higher than those measured 1 mo after primary immunization for Discussion diphtheria toxoid ( p ϭ 0.009) but not for MenC ( p ϭ 0.2). In this study, we observed for the first time that the ability of Correlation between the primary and both children to produce MenC-specific memory B cells and Ab at 5 mo persistence of immunity at 1 year of age and the response to of age during primary immunization allows both determination of a booster dose of MenCV the strength of the immune response to priming and also prediction of the persistence of functional Ab at one year of age and the At 5 mo of age there was a strong evidence of association between intensity of the humoral immune response to a booster dose of the MenC-specific memory B cells and Ab (Table II) but not be- MenCV at one year of age. ϭ tween the diphtheria toxoid-specific memory B cells and Ab (r At one year of age, Ͻ50% of the study children had maintained ϭ 0.3, p 0.2). protective bactericidal Ab levels. These observations support the The level of MenC-specific Ab and memory B cells produced previous evidence that protective Ab levels do not persist well in after priming correlated with the persistence of Ab and memory B children vaccinated with MenCV under 2 years of age (8, 13, 29). cells at one year of age, and with the postbooster Ab and memory By contrast, immunization of older children and adults results in B cell levels (Table II and Fig. 5). Similarly, diphtheria toxoid more persistent protective Ab for some years (5, 11, 30). memory B cells and Ab measured at 5 mo of age correlated with Previous studies (6, 7, 31) demonstrated that immunological the persistence of diphtheria toxoid-antibody at one year of age memory was maintained despite the waning of MenC-specific Abs. ϭ ϭ ϭ ϭ (r 0.8, p 0.003 and r 0.7, p 0.002). However, only the In this study, following secondary immunization with MenCV at diphtheria toxoid-antibody measured at 5 mo of age, and not the one year of age, MenC-specific Abs increased rapidly and by day 6 all children had achieved a protective rSBA titer. This Ab re- sponse to a booster dose of MenCV at one year of age after 3-dose Table III. Correlation between MenC-memory B cells at 5 mo of age and rSBA titer at one year of agea priming in infancy is comparable to the one observed by Tsai et al. (2006) in children primed with a single dose of MenCV at 12–15 mo of age and who were challenged with serogroup C polysac- No Memory B rSBA at 1 Year charide at 24 mo of age (in this study all children had mounted Cells at 5 mo Ͻ8 Ն8 protective bactericidal Ab levels by day 5) (13). Ͻ 550However, the maintenance of functional Ab rather than the im- Ն536 munological memory appears to be the key determinant of long a The presence or absence of detectable MenC specific memory B cells at 5 mo of term protection against invasive diseases caused by encapsulated age (i.e. Ն5 MenC-memory B cells per million cultured lymphocytes) were correlated with the maintenance of a “protective” rSBA titer (Ն1:8) to 1 year of age using the bacteria. It is not clear how long term production of Ab is main- Fisher’s exact test with 1 df,(p ϭ 0.03). tained. It has been suggested (32, 33) that long-lived plasma cells The Journal of Immunology 2171 surviving in bone marrow niches continue to secrete Ab after the The role of B cells generated after priming in the secondary exposure with the Ag. Slifka et al. (23) have demonstrated that immune response has not been studied previously. In this study, these cells could survive for longer than one year in mice. The we observed that the children who achieved the highest Ab level presence of long-lived plasma cells has not been demonstrated in following priming also attain the highest Ab level following the humans. Alternatively, it was suggested that plasma cells would booster dose of MenCV. However, there was a difference in the have to be continuously replaced by the turn over of memory B rise of the Ab response: the children with the lower prebooster Ab cells in humans. This phenomenon may be Ag-independent and level at 12 mo of age had the higher increase in Ab compared with occur by polyclonal stimulation of B cells (12) by microbial prod- the children with higher level at baseline. Similar results have been ucts (which stimulate B cells via toll like receptors), or through observed following other vaccines (40, 41). These findings suggest bystander help (cytokines secreted by T cells activated by that there might be a mechanism of feedback above a certain Ab another Ag) (34). Others (21) have postulated that this event is level and Ag-specific B cells might not increase above a certain Ag-dependent and occurs through cross-reactive Ag or persisting threshold. Similarly, it has been shown that a third dose of MenCV Ag. It has been shown that although a persists for did not induce a higher Ab titer compared with the second dose only 3 wk following immunization, memory B cells continue to (42). It is not clear how this feedback mechanism operates; it could proliferate and to differentiate into plasma cells in the lymphoid result from competition between the pre-existing Ab and the mem- tissues for several months after stimulation in response to the Ag brane-bound B cell receptors for the Ag, impairing the activation persisting on the follicular dendritic cell networks (35). Although of B cells (17). these mechanisms are unclear in humans, studies in mice indicate We used the ex vivo ELISPOT assay to detect cells that spon- that long-lived plasma cells survive in bone marrow niches inde- taneously secrete Ab after PBMCs separation. Clutterbuck et al. Downloaded from pendently of memory B cells and of the Ag (36). (43) have shown that these cells have the phenotype of plasma In the present study, there was a strong correlation between the cells (CD20ϩCD38ϩCD27ϩ or CD20ϪCD38ϩCD27ϩ). In this MenC-specific memory B cells and Abs at 5 mo of age and also study, following immunization with MenCV at one year of age, the between these two parameters and the persistence of Ab at one first appearance of MenC- and diphtheria toxoid- specific plasma year of age. These results suggest that priming with MenCV in cells above baseline was seen at day 4 with a peak at day 6, fol-

early infancy may generate germinal centers of various sizes in lowed by a rapid decline in the frequency to day 8–9. These http://www.jimmunol.org/ different children. Thus, some children produce a higher number of plasma cell kinetics are similar to those observed in previous stud- memory B cells and plasma cells and are therefore able to produce ies with a variety of vaccine Ags in primed adults: MenC poly- higher Ab levels after priming. In contrast, the children who gen- saccharide vaccine and MenCV (30); varied serotypes of the hep- erate small germinal centers will have a smaller B cell pool and tavalent pneumococcal conjugate vaccine and diphtheria toxoid lower Ab response at 5 mo of age (1 mo after primary immuni- (43) and toxoid (12), suggesting that the kinetics of the zation). The immune response is determined by a combination of secondary plasma cell response is common to all Ags (T-depen- genetic and environmental factors but genetic factors predomi- dent and T-independent) and all age groups. nantly determine the Ab response to vaccines given in early life Given the rapidity of the plasma cell response following immu- (37, 38), whereas environmental factors influence Ab persistence nization in previously primed individuals, these cells are likely to by guest on September 26, 2021 and avidity maturation (38). represent plasma cells derived from pre-existing memory B cells. In this study, the decline of the Ab concentrations from 5 mo (1 It was shown in humans that memory B cells respond to Ag re- mo after primary immunization) to 12 mo was similar in all chil- challenge 20–30 h earlier than naive B cells, because of their lower dren. Thus, it is not clear whether B cells have a direct role in activation requirements (44) and their homing to unique Ag-drain- sustaining Ab production after priming or whether the level of ing sites (e.g., mucosal epithelium) (45). Additionally, it was ob- persisting Ab at one year of age only depends on the biological served in mice that most memory B cells give rise to extra-ger- half life of Ab and would, therefore, mostly be determined by the minal center plasma cells upon rechallenge with the Ag (46, 47). level of Ab reached after primary immunization. However, it was It is also possible that, some of these plasma cells are long-lived observed that the anti MenC IgG GMC was 19.7 ␮g/ml at 5 mo of plasma cells, translocated from their bone marrow niches by an age (i.e., post primary immunization), whereas at one year it was unknown signal after re-exposure with the Ag or that the transient 1.1 ␮g/ml. An explanation based solely on Ab half-life (3 wk (39)) appearance of the plasma cells in peripheral blood represents their is insufficient to account for these high levels. This suggests the passage from their site of production to their long term residence existence of other mechanisms determining the long-term persis- in bone marrow (30). tence of Ab. The fate of the plasma cells produced in this day 6/7 burst is Persisting Ab at one year of age correlated more strongly with unknown. The rise in MenC-antibody was also observed from day the level of Ab at 5 mo than with the number of memory B cells 4–6 with a peak by day 8, suggesting that the rapid increase in Ab present at 5 mo. However, when children were separated into those levels following immunization is produced by these newly formed who had detectable and undetectable memory B cells at priming, plasma cells. we found that all those with undetectable memory B cells after A previous larger study (30) found a weak correlation between priming did not maintain protective Ab levels at one year of age. the increase in Ab level and the peak plasma cell response follow- Conversely, 2/3 of the children with detectable memory B cells at ing immunization with polysaccharide conjugate vaccines. As 5 mo had maintained protective rSBA titer at one year. All children mentioned earlier, this could be explained by negative feedback in produced high levels of Ab at 5 mo of age (IgG between 6.2 and Ab production among those with pre-existing raised Ab levels. 47.6 ␮g/ml and rSBA titer between 512 and 8192); therefore, it is Furthermore, in this study there was a moderate correlation be- more difficult to define a cut-off of Ab level after priming to predict tween the memory B cells at 5 mo and the postbooster Ab levels. long term protection. Therefore, even if the role of memory B cells These findings suggest that the rise in Ab from day 0 to day 30 in the long term maintenance of protective Ab levels is unclear, it postbooster may have various different origins (e.g., persisting appears that the measure of memory B cells, rather than the Ab at memory B cells and long-life plasma cells, newly formed plasma 5 mo of age can better discriminate the children who will sustain cells and memory B cells) and depend on several factors (e.g., Th protective levels of Ab. cell, APCs, serum titer of specific antibody). 2172 B CELL RESPONSE TO MENINGOCOCCAL CONJUGATE VACCINE

Polyclonal stimulation with SAC, CpG-DNA, and PWM for 6 are activated to re-circulate by the presence of Ag in periphery days induces polyclonal proliferation of IgGϩ memory B cells and (52). their differentiation into ASC (48). Further, naive B cells are not In summary, this study found that the infants who generate a activated in the absence of specific Ag (they require B cell recep- large germinal center (identified by high Ab and memory B cells tors triggering by anti-Ig) (12) and pre-existing plasma cells do not level) at 5 mo of age (1 mo after primary immunization), best survive more than few days without appropriate stromal cells sup- maintained protective Ab levels at one year of age. However, one port (49). It is therefore likely that the cells detected in this study third of the infants in this study produced a very low number of after culture are derived from B cells that have a memory memory B cells after the initial immunization (Ͻ5 memory B cells phenotype. per million cultured lymphocytes) and did not maintain protective It has been suggested that after their production memory B cells Ab levels by one year of age. An understanding of the factors that continuously recirculate through the blood and secondary lym- determine the production of adequate germinal centers during phoid organs (17, 50) and it was therefore postulated that the mem- priming in early infancy could lead to an improvement in the short ory B cells found in the circulation were representative for the and longer-term protection induced by glycoconjugate vaccines entire B cell pool (17). Previous studies (14, 17) with diphtheria that are so important for the protection of young children against toxoid, tetanus toxoid, and smallpox specific memory B cells have serious diseases caused by polysaccharide encapsulated bacteria. shown their persistence for a prolonged period after immunization, with a frequency in human peripheral blood of 0.01–1% of total Acknowledgments IgG-memory B cells. Similarly, in this study we found that 0.02% We thank all the children who participated in the study, and the research of total IgG memory B cells were MenC specific (median fre- nurses and research doctors of Oxford Vaccine Group (University of Ox- Downloaded from quency) and 0.12% were diphtheria specific (median frequency) by ford, Oxford, U.K.) who undertook the clinical procedures. Elizabeth one year of age (8 mo after the 3-dose priming schedule). The Clutterbuck and Elizabeth Kibwana assisted with the development of frequency of persisting MenC-memory B cells was significantly the ELISPOT and ELISA. lower than that reached 1 mo after primary immunization. How- Disclosures ever, the frequency of diphtheria toxoid-memory B cells was main- A.J.P. has conducted clinical trials on behalf of Oxford University, spon- tained. The factors influencing the maintenance of memory B cells http://www.jimmunol.org/ sored by Wyeth Vaccines, GlaxoSmithKline Vaccines, Sanofi Pasteur, in the circulation after vaccine exposure could be cellular compe- Sanofi Pasteur MSD, and Novartis Vaccines and has received assistance tition (making space for new memory B cells) in secondary lym- from vaccine manufacturers to attend scientific meetings. Industry-sourced phoid organs (14), or the persistence of the Ag in follicular den- honoraria for lecturing or writing are paid directly to an independent char- dritic cell (51). ity or an educational/administrative fund held by the Department of Pae- We found in this study that following immunization at one year diatrics, University of Oxford. M.D.S. has received assistance to attend of age, MenC- and diphtheria toxoid-specific memory B cells were scientific meetings from Wyeth Vaccines and Novartis Vaccines and has detected by the end of the first week after immunization and were had travel and accommodation expenses paid by Novartis Vaccines while still raised above baseline levels at one month (for MenC: 0.26% working in collaboration with Novartis Vaccines in Siena, Italy. of total IgG memory B cells; for diphtheria toxoid 0.67% of total A.B. and R.C. are employers of Novartis Vaccines. by guest on September 26, 2021 IgG memory B cells). 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