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Keywords:

  • B-cell memory;
  • Hepatitis B;
  • Long-lived plasma cells;
  • Serum antibodies;
  • Vaccination

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The immunogenicity of a vaccine is conventionally measured through the level of serum Abs early after immunization, but to ensure protection specific Abs should be maintained long after primary vaccination. For hepatitis B, protective levels often decline over time, but breakthrough infections do not seem to occur. The aim of this study was to demonstrate whether, after hepatitis B vaccination, B-cell memory persists even when serum Abs decline. We compared the frequency of anti-hepatitis-specific memory B cells that remain in the blood of 99 children five years after priming with Infanrix®-hexa (GlaxoSmithKline) (n=34) or with Hexavac® (Sanofi Pasteur MSD) (n=65). These two vaccines differ in their ability to generate protective levels of IgG. Children with serum Abs under the protective level, <10 mIU/mL, received a booster dose of hepatitis B vaccine, and memory B cells and serum Abs were measured 2 wk later. We found that specific memory B cells had a similar frequency in all children independently of primary vaccine. Booster injection resulted in the increase of memory B cell frequencies (from 11.3 in 106 cells to 28.2 in 106 cells, p<0.01) and serum Abs (geometric mean concentration, GMC from 2.9 to 284 mIU/mL), demonstrating that circulating memory B cells effectively respond to Ag challenge even when specific Abs fall under the protective threshold.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Four subsets of B cells can be identified in the peripheral blood according to the expression of surface molecules. Transitional B cells, corresponding to recent bone marrow emigrants, are short-lived CD19posCD24brightCD38bright and express high levels of IgM and IgD. They are unable to generate effective immune responses and in the periphery they differentiate into CD19posCD24posCD38low mature-naïve B cells reducing the expression of IgM and IgD. Mature-naïve B cells have never been exposed to Ag and represent the bulk of circulating and resident follicular B cells. Memory B cells lose the CD38 marker, acquire CD27, and, depending on the presence of surface IgM, are subdivided into IgM memory or switched memory B cells 1. IgM memory B cells are believed to represent a primitive, low-affinity type of cell 2. Switched memory B cells, instead, have been selected in germinal centers and are the effectors of the high-affinity adaptive response 3, 4. B cells have the transitional phenotype in the neonate, whereas the development of mature, IgM, and switched memory B cells starts with Ag exposure 1.

Switched memory B cells are generated in the germinal centers, specialized and complex structures triggered by exposure to infection or vaccination. Here B cells, helped by T cells and Ag-presenting cells, extensively proliferate, introduce somatic hypermutations (SHMs) in their Ig genes, and are selected for their improved affinity to the stimulating Ag. The final products of the germinal center reaction are of two types: (i) Abs immediately used to terminate the infection and clear the pathogen; (ii) memory B cells and long-lived plasma cells that will prevent reinfection 5. Switched memory B cells are the effectors of anamnestic responses, because they have the remarkable ability to re-enact their previous encounters with specific Ags rapidly, producing large amounts of high-affinity Abs 6. It has been suggested that memory B cells may also contribute to preserve the diversity of serum Igs by the constant secretion of small amounts of Abs upon polyclonal stimulation by Toll-like receptor 9 (TLR 9) 3, 7, 8.

Recently it has been demonstrated that another population of B cells, long-lived plasma cells, play an important role in protecting against infections. At the end of the germinal center reaction, long-lived plasma cells migrate to the BM, where they occupy a specialized niche and survive virtually forever 9, continuously producing Igs 10, 11 against previously encountered pathogens. Although both memory B cells and long-lived plasma cells contribute to protective immunity, their separate roles have not been elucidated yet 12. It is also not known whether both populations are equally generated by the immune response and equally survive over the years.

Vaccination prevents infectious disease by generating serum Abs and memory B cells specific for the immunizing Ag. In order to achieve protection against several diseases as soon as possible after birth, hexavalent vaccines have been developed. In Europe, two products, Hexavac® (Sanofi Pasteur MSD) and Infanrix®-hexa (GlaxoSmithKline), were licensed for use in October 2000. Both vaccines protect against diphtheria, tetanus, pertussis, poliomyelitis, Haemophilus influenzae type b, and hepatitis B and were considered to have a comparable immunogenicity at the time of licensure, based on the studies conducted shortly after the completion of primary immunization 13–16.

In September 2005, following the observation of reduced immunogenicity of the hepatitis B component 17 in Hexavac® (Sanofi Pasteur MSD) and potential consequences on long-term protection, the European Medicines Agency (EMA) recommended, as a precautionary measure, the withdrawal of Hexavac® from the market (for the scientific conclusions and grounds for the suspension of the marketing authorisation of Hexavac®http://ema.europe.eu/humandocs/PDFs/EPAR/Hexavac-H-298-Z-28-en.pdf). As Hexavac® has been administered to a large number of children, a multicenter study funded by the Italian Drug Agency was carried out to investigate whether vaccinated children were still able respond to a booster dose of hepatitis B vaccine five years after primary immunization 18. This study had the aim of evaluating whether the decline in Ab titers below the protective threshold (10 mIU/mL), observed most frequently in children receiving Hexavac®, reflects a loss of immune memory. In this case, revaccination would be advisable. The report included 833 children vaccinated with Hexavac® and 710 with Infanrix®-hexa. Specific Ab concentrations had fallen under the protective level in 62% (512/831) of the children that had received Hexavac® and in 17% (119/590) of those vaccinated with Infanrix®-hexa. All children with anti-hepatitis B surface Ag (anti-HBsAg)<10mIU/mL received a booster vaccination with a monovalent hepatitis B recombinant vaccine. The recall response was observed in both groups of children and comparable levels of anti-HBsAg Ab were measured in the sera. Thus, immunological memory persists in children five years after the primary cycle of vaccination even when specific Abs wane in the serum.

Since serology for hepatitis B measured five years after primary immunization was not predictive of the ability to respond to a recall vaccination, we studied the long-term response to hepatitis B in a subgroup of children focusing on B-cell immunity with the aim of discriminating the persistence and function of the two components of immunological memory, i.e. memory B cells and serum Abs.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Thirty-four children primed with Infanrix®-hexa (GlaxoSmithKline) (VacA) and 65 vaccinated with Hexavac® (Sanofi Pasteur MSD) (VacB) were recruited in the central/south of Italy and included in the study (Table 1).

Table 1. Demographic and serologic characterization of the children enrolled in the current study
 Vaccine used for primary immunizationNumber of subjectsSex (F/M)Age (years)HBsAg carrier present in the familySerum GMC (mIU/mL) (95% CI)Number of children with anti-HBsAg IgG (mIU/mL)
     Yes/no <1010–100>100
VacAInfanrix®-hexa3414/206–71/3347.0 (1.9–1122.8)6 (17.6%)14 (41%)14 (41%)
VacBHexavac®6527/386–72/637.62 (0.44–130.2)38 (58.7%)22 (34.9%)5 (6.3%)

As expected, Ab levels were significantly higher in the VacA than in VacB group (geometric mean concentration GMC 47 versus 7.6 mIU/mL, p<0.0001). The frequency of children with anti-HBsAg<10 mIU/mL, considered nonprotective, was 17.6% (6/34) in the VacA group and 58.7% (38/65) in those who received VacB (Table 1 and Fig. 1A). Moreover, although nearly 30% of all children had a level of anti-HBsAg Abs between 10 and 100 mIU/mL (41.2% vaccinated with VacA versus 34.9% vaccinated with VacB), higher Ab concentrations were almost exclusively detected in children who received VacA (41.2% in VacA versus 6.3% in VacB, Table 1 and Fig. 1A). In conclusion, the serologic findings in our cohort of children are very similar to those previously reported 18 and confirm the higher efficacy of Infanrix®-hexa in the maintenance of Ab levels.

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Figure 1. Efficacy of Infanrix®-hexa (VacA, black dots) and Hexavac® (VacB, white dots) vaccines measured by serum Abs. (A) Graph shows the frequency of children grouped according to anti-HBsAg IgG serum concentrations of <10, 10–100, or >100 mIU/mL vaccinated with VacA (black) or VacB (white) vaccines. (B) Graph shows the frequency of VacA and VacB children with anti-HBsAg IgG serum concentrations of <10, 10–100, or >100 mIU/mL 14–20 days boosting.

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Serology after booster dose

Among children who participated in the serology analysis and were recruited for the B-cell immunity study, 45 had anti-HBsAg<10 mIU/mL. In all, 82% of them had a primary vaccination cycle with VacB and 17% with VacA. Consent for booster vaccination was only obtained for 25 children, 20 previously vaccinated with VacB (80%), and 5 (20%) with VacA. A single dose of 10 μg monovalent hepatitis B recombinant vaccine, Engerix-B® (GlaxoSmithKline), was administered and serum Abs were measured 14–20 days after challenge. In 93% (23/25) of the children, anti-HBsAg levels increased. In five children of the VacA group, the GMC increased from 3.5 to 1012.5, whereas in the VacB cohort the GMC increased from 2.9 to 203.3 (Table 2). High anti-HBsAg levels (>100 mIU/mL) were detected in the majority of children after the challenge dose, independently of the type of the vaccine used for priming (Fig. 1B). The difference in the geometric mean titers of the two groups probably depends on the small size of the VacA cohort. Previous reports on a larger group did not show any significant difference between GMCs after boosting 18. The high frequency of children with protective levels of Abs early after a single dose of Engerix® suggests that we are observing a memory response. It has been shown earlier that only 8% of the vaccinees are seroprotected after a single dose 19.

Table 2. Postbooster anti-HBsAg serum concentrations and anti-HBsAg IgG-secreting cells in children primed with Infanrix®-hexa or Hexavac® five years before
Vaccine primary immunizationVaccine boostNumber of subjectsSerum GMC (mIU/mL) (95% CI)Number of children with anti-HBsAg IgG (mIU/mL) after boostNumber of anti-HBsAg IgG spots/106 cells
   PrePost<1010–100>100PrePost
Infanrix®-hexaEngenrix®53.5 (1.43–8.3)1012.5 (199.2–5146.3)01415±1823±25
Hexavac®Engenrix®202.9 (2.2–3.6)203.3 (87.6–471.8)171210±729±13

Our results indicate that the concentration of specific Abs detected in the serum several years after the primary immunization is not sufficient to assess the persistence of specific immunological memory, because the ability to respond to recall immunization remains even when serum Abs fall under the protective threshold.

Memory pool

Recall Ab responses are due to the rapid activation of memory B cells. As discordance between the frequency of memory B cells and the level of serum Abs against hepatitis B has been reported before in HIV patients 20 and in adult-care workers 21, we decided to evaluate the size of the total and specific memory pool in children before and after booster vaccination.

Memory B cells, absent at birth, slowly accumulate with antigenic experience over the years. We analyzed the frequency of CD19pos cells (Supporting Information Fig. 1A) and memory B cells (Supporting Information Fig. 1B) in the two groups of vaccinated children and we did not find significant differences. The frequency of total B cells was 10.3±4.4% in the VacA group and 10±3.5% in VacB. Among B cells, the frequency of memory cells was 26.3±8 and 24.6±7.5% in VacA and VacB recipients, respectively.

In order to explore the specificities of the B-cell memory pool, we polyclonally stimulated memory B cells with unmethylated CpG oligodeoxynucleotides in the presence of IL-21 22 and IL-4. We chose this method because of its ability to specifically stimulate and test the function of memory B cells 3, 23. Our previous experiments performed with sorted cells from blood donors have demonstrated that only memory B cells proliferate and secrete Abs upon CpG stimulation, whereas mature B cells survive without dividing and differentiating 3. We repeated these experiments and tested by ELISpot the frequency and the Ig class of Ab-secreting cells (ASCs) derived from purified mature and memory B cells. We found that mature B cells stimulated with CpG+IL-4+IL-21 for 5 days were unable to generate plasma cells. On the contrary, memory B cells were able to differentiate in ASCs in the same culture conditions, generating 3032±147 IgM- and 2457.5±369 IgG- secreting cells per 106 (Fig. 2A and B). Abs specific for tetanus toxin were detected only in the wells with memory B cells and were of the IgG class (Fig. 2A and B).

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Figure 2. ASC analysis in total peripheral blood lymphocytes, in sorted mature B cells (CD24posCD27neg) and in sorted memory B cells (CD24posCD27pos) using ELISpot. (A) For total IgG and IgM determination, plates were coated with anti-IgG and anti-IgM, respectively, whereas tetanus toxin peptide (C-term) was used for specific anti-tetanus IgG determination. Spots were developed with anti-IgM (first row) or anti-IgG (second and third rows). Images correspond to a representative example of five independent experiments. (B) Bars represent the number of memory B cells (per million of PBLs) secreting either IgM or IgG or specific IgG. The results for total cells (PBLs), FACS-sorted mature, and memory B cells are shown as means+standard deviation of five independent experiments.

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Based on these results, we cultured with CpG+IL-4+IL-21 total PBMCs from children before and after the booster dose in order to measure the size and specificity of the memory pool. After 5 days in bulk culture, cells were counted and distributed in plates precoated with either Abs or Ags and left to secrete for 5 h. Frequency of anti-hepatitis B memory cells was calculated by counting the number of ASCs that upon polyclonal stimulation were able to secrete IgG specific for hepatitis B. As both vaccines used for primary immunization also contained the tetanus Ag, we tested the frequency of anti-tetanus IgG-secreting cells and used it as our internal control (Supporting Information Fig. 2A). We also calculated the total number of IgM and IgG Ab-forming cells in order to determine for each individual the size of the pool of memory B cells that we were able to stimulate in vitro.

The number of IgG and IgM ASCs in VacA and VacB was not statistically different (3870±2564 IgG ASCs/106 cells for VacA and 5277±3416 IgG ASCs/106 for VacB, p=0.08; 2518±1586 IgM ASCs/106 cells for VacA and 3344±2153 IgM ASCs/106 cells for VacB, p=0.147), indicating that the two groups of children were comparable (Fig. 3A). The homogeneity of the two groups was confirmed by the fact that there was no difference in the number of B cells secreting Abs against the tetanus Ag (Fig. 3B) (mean 8.5 ASCs/106 plated cells for VacA and 8.6 ASCs/106 plated cells for VacB, p>0.12). The number of HBsAg-specific spots in 1 million plated cells was 12 for VacA and 10.4 for VacB (p>0.08) (Fig. 3B). Thus, the two vaccines had a similar efficacy in generating memory B cells. In addition, when we grouped the children based on the serum level of Abs, we found no difference in the number of HbsAg-specific ASCs independently of whether the serum level of anti-HBsAg IgG was <10, between 10 and 100 and >100 mIU/mL (Fig. 3C).

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Figure 3. Number of memory B cells, per million PBMCs, secreting Abs after 5 days of stimulation with CpG, IL-4, and IL-21, measured by ELISpot. (A) Box plot represents the number of IgG and IgM Ab-producing cells in 1 million nucleated cells from children vaccinated with Infanrix®-hexa (VacA) and Hexavac® (VacB) respectively. (B) Box plot represents the number of Ag-specific IgG-secreting cells reactive to tetanus toxin peptide (Anti-T.Tox) and HBsAg (Anti-HBsAg) in children vaccinated with Infanrix®-hexa (VacA) or Hexavac® (VacB). (C) Dots represent the number of anti-HBsAg IgG-secreting cells in children vaccinated either with Infanrix®-hexa (VacA, n=34) or with Hexavac® (VacB, n=65) and grouped according to anti-HBsAg IgG serum concentrations of <10, 10–100, or >100 mIU/mL.

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We also calculated the frequency of Ag-specific memory B cells, by dividing the number of specific spots by the number of IgG spots, corresponding to the memory B cells which we are able to stimulate in vitro. The two groups of vaccinated children had the same frequency of anti-HBsAg IgG and of anti-tetanus toxin IgG-secreting cells (Supporting Information Fig. 2A and B). Roughly 4–5 in 1000 of the in vitro-stimulated memory B cells were specific for either tetanus or hepatitis B.

In order to prove that the ASCs detected in vitro derive from functional memory B cells that are also able to react to the Ag in vivo, we measured their frequency 2–3 wk after the administration of a monovalent hepatitis B vaccine. Ab titers increased in the serum in all but one child (VacB group preboost 0.9 mIU/mL and postboost 11 mIU/mL) (Fig. 4A). The number of anti-HBsAg ASCs (from 11.3 to 28.2/106 plated cells, p<0.02) and their frequency were also significantly increased (Fig. 4B and C), (from 0.46 to 0.89%, p<0.02), whereas anti-tetanus ASC frequencies remained stable (Supporting Information Fig. 3, from 0.32 to 0.4%, p>0.4).

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Figure 4. Anti-HBsAg IgG memory B cells and serum Ab analysis before (pre) and after boosting (post) with Engerix-B®. Note that all children (n=25) were selected to have anti-HBsAg IgG serum levels lower than 10 mIU/mL before boosting. (A) Graph shows the rise in serum anti-HBsAg IgG 14–20 days after challenge in children previously vaccinated with Infanrix®-hexa (VacA) or Hexavac® (VacB). (B) Number of anti-HBsAg IgG-secreting cells per million of plated cells before (pre) and 14–20 days after recall (post) a single dose of Engerix-B®. (C) Box plot represents the frequency of anti-HBsAg IgG-producing cells in the pool of IgG-secreting cells in children before (pre) and after boosting (post), p<0.02 (p-value calculated using Student's paired-sample t-test).

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Thus, memory B cells in children without protective Abs were functional, because upon in vivo restimulation they increased in number and generated high Ab titers.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Immunological memory is the extraordinary property of the immune system to store, preserve, and recall previous antigenic experience and to use it in order to prevent or limit reinfection 24. Vaccination, the most effective method to prevent infections, acts by creating an Ag-specific defense system composed of high-affinity Abs and memory cells 25. The combination of preformed Abs and cells able to rapidly react to pathogen is a very effective system to neutralize microbes and prevent the disease. Nonetheless, we do not know whether we would be protected from infection if only serum Abs or only memory B cells were available.

The case of hepatitis B vaccines provides some important elements for discussion and further studies. For hepatitis B, a level of anti-HBsAg Ab above 10 mIU/mL is considered protective 26, but 5 years after vaccination the protective level is often not maintained 27. However, notwithstanding the low levels of serum Abs, breakthrough infections remain rare in vaccinated individuals, even in countries where hepatitis B is still endemic 28, 29. This observation suggests that other mechanisms of defense generated by the vaccination, such as memory B cells, may play an important role in the prevention of overt disease.

In our study, memory B cells specific for the HBsAg were present in virtually all (63/65 for Hexavac® and 31/34 for Infanrix®-hexa) children regardless of the type vaccine received 5 years before and independently of whether the level of Ab was higher or lower than 10 mIU/mL. In the small group of children we were allowed to boost, memory B cells were functional. In response to a challenge with a hepatitis B monovalent vaccine, their frequency increased and high amounts of Abs were detectable in the serum, independently of the type of primary vaccine and of the prechallenge Ab levels.

From our studies two important conclusions can be drawn. Firstly, the frequency of specific memory B cells is not related to the level of serum Abs. Thus, serum Abs cannot be used to predict a subsequent response to a specific challenge. Secondly, vaccine components inducing the production and maintenance of serum Ab are different from those responsible for the generation of memory B cells, because VacA and VacB are equally able to generate memory B cells, but only VacA ensures the maintenance of protective levels of Abs in the serum.

Each experience with Ag leads to the production of specific memory B cells and plasma cells 30. A fraction of the plasma cells is recruited to a dedicated, but yet unidentified, BM niche 31 and survives for a long time, continuously secreting Abs 32–34. These Abs are useful and ready-to-use weapons against pathogen. As plasma cells are a terminal differentiated population that do not express a membrane-bound BCR and lack most of the B-cell-specific signaling elements 35, they cannot sense Ag and are, therefore, unable to respond to vaccination and/or infection. Their role is the maintenance of preformed high-affinity Abs 10, whereas recall responses are carried out by memory B cells.

Our data show that specific memory B cells are rare in the peripheral blood. We found that five memory B cells in 1000 are specific for hepatitis B five years after vaccination. Memory B cells against tetanus are present at the same frequency. As the frequency of cells able to react to any specific Ag has been estimated to be 1 in 1 million in the population of mature naïve B cells, the enrichment due to a previous encounter is significant, especially if we consider that our pool of memory B cells must include all our past immunological experience in order to be effective. The rapid production of Abs upon recall is due to memory B cells dividing and differentiating into new plasma cells, causing a peak of specific Abs in the serum, followed by a progressive decline.

Our data confirm that in children, as in adults, the level of specific Abs in the serum, measured years after vaccination, does not correlate with the frequency of memory B cells 21, 36–38. This observation suggests that vaccine components generating long-lived plasma cells and memory B cells may be different. The comparison between Hexavac® and Infanrix®-hexa strongly confirms this observation, because even a vaccine that has been withdrawn from the market due to its inefficiency in eliciting and maintaining high Ab level generates normal numbers of effective and long-lived memory B cells. The most apparent difference between Hexavac® and Infanrix®-hexa is the amount of HB surface Ag in a vaccine dose, but both vaccines had complex hexavalent formulation. Further studies are necessary to identify the key element/s that modulate the frequency of either long-lived plasma cells or memory B cells.

Although life-long immunity has been demonstrated after infection and vaccination, not all vaccines are able to induce protective Ab levels in the long term. The concept of waning immunity, as an example, has been proposed to explain outbreaks of pertussis in vaccinated children and adolescents 39, 40. For hepatitis B, however, notwithstanding the low levels of serum Abs, breakthrough infections remain rare in vaccinated individuals most probably due to the “reactivation” of the specific memory B cells 29.

The long incubation of hepatitis B may allow sufficient time for the memory B cells to sense Ag, activate, and differentiate into ASCs. In contrast, ready-to-use Ab may be indispensable for the control of infections in order to confine invasive pathogen to the site of entry.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Subjects

Children were eligible for the study if they were born in 2001–2002, received the primary immunization cycle (including three doses administered at 3, 5, and 11 months of life) with Infanrix®-hexa (GlaxoSmithKline) (n=34) or Hexavac® (Sanofi Pasteur MSD) (n=65), did not receive additional doses of hepatitis B vaccine, did not have chronic illness, or congenital, or acquired immune disorder, and their families agreed to have a blood sample drawn and signed an informed consent. The study was approved by the Ethical Committee of the Bambino Gesù Hospital.

Study design

A blood sample was collected at Ospedale Pediatrico Bambino Gesù, Rome from all participants at baseline. Children with anti-HBsAg IgG levels <10 mIU/mL were invited to receive a challenge dose of hepatitis B vaccine (Engerix-B®, Glaxo SmithKline) and an additional blood sample collected 14–20 days after the recall. Ten millilters of peripheral blood in sodium heparin were collected for Ag-specific B memory analysis and plasma to measure the serological response to HBV.

Cell sorting

Healthy donor's peripheral blood was donated and collected at Ospedale Pediatrico Bambino Gesù, Rome. Mononuclear cells were isolated by Ficoll Paque™ Plus (Amersham Pharmacia Biotech) density-gradient centrifugation and stained with the appropriate combination of fluorescent labeled Abs: monoclonal clone ML5 (anti-CD24) and clone M-T271 (anti-CD27) were obtained from BD Biosciences (San Diego, CA, USA). Mature cells, CD24posCD27neg and memory B cells, CD24highCD27pos, were purified using FACSAria II SE (BD Biosciences). Cell purity postsorting was >98%.

Cell cultures

In total, 4×106 PBMCs were stimulated for 5 days in RPMI 1640 (Gibco BRL) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Hyclone Laboratories, Logan, UT, USA), 2% L-glutamine (Gibco BRL), 5×10−5 M 2-mercaptoethanol (Sigma, St. Louis, MO, USA), and 20 mg/mL gentamycin (Gibco BRL), 2.5 μg/mL CpG unmethylated oligodeoxynucleotides (a 24-synthetic oligodeoxynucleotide sequence containing the proper CpG-DNA motif mimicking the immunostimulatory effects of bacterial DNA and the human optimal motif GTCGTT from Hycult Biotechnology, Uden, The Netherlands), 20 ng/mL IL-21 (Peprotech, UK), and 20 ng/mL IL-4 (Peprotech).

ELISpot

Briefly, 96-well plates (MultiScreen-HA, Millipore) were coated overnight with AffiniPure F(ab′)2 Fragment Goat anti-human IgA+IgG+IgM (H+L) (Jackson Immuno Research Laboratories, Pennsylvania, USA), synthetic tetanus toxin peptide (C-term) (Acris-Abs, Germany) and recombinant hepatitis B surface Ag (HBsAg adw), (ProSpec, Rehovot, Israel). After washing with sterile PBS/0.05% Tween-20, plates were blocked for 1 h at 37°C with PBS/gelatine 1%. PBMCs stimulated for 5 days, as described before, were collected, counted, and seeded in the precoated ELISA plates. Plates were left at 37°C, 5% CO2 for 5 h to allow Ab secretion. Serial dilution (1/2) was done starting in the first well with: 2.5×105 cells for IgG and 2×106 cells for specific anti-tetanus IgG and anti-hepatitis B IgG respectively. After incubation, plates were washed with dH2O/0.05% Tween-20 (once) and PBS/0.05% Tween-20 (twice) and incubated overnight with anti-hIgG HRPO (1:2000) (Jackson Immuno Research Laboratories) diluted in PBS+gelatin1%+0.05% Tween-20 (Sigma). After washing twice with PBS/0.05% Tween-20, TMB substrate (MABTECH-Elisaspot plus for human IgG kit) was used according to the manufacture' instructions. Plates were left to dry before counting. Analysis was performed blinded as for the type of vaccine.

Statistical analysis

Results are reported as mean±standard error (SEM). Statistical analyses were carried out using the StatView statistical MacIntosh program (StatView Software, San Diego, CA, USA). For comparing overall differences of results obtained with each assay between children receiving as primary vaccination Infanrix®-hexa or Hexavac®, the Student's paired-sample t-test was performed. p-Values lower than 0.05 were considered to indicate statistical significance, and all reported p-values are two sided.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank all children and their families for their participation in the study as well as the study team and nurses of the Local Health Units whose enthusiastic collaboration made possible this study. This study was funded by AIFA (Agenzia Italiana del farmaco, Bando per la Ricerca Indipendente, anno 2006) with the registered code FAM67NFPN and by the EUROPAD NET (EC Grant No. 201549).

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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